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Archive for the ‘Inflammasome’ Category


Targeting Cancer Neoantigens and Metabolic Change in T-cells

Curator: Larry H. Bernstein, MD, FCAP

 

Updated 5/28/2016

Updtaed 6/1/2016

Fighting Cancer with Borrowed Immunity

http://www.genengnews.com/gen-news-highlights/fighting-cancer-with-borrowed-immunity/81252754/

Outsource a part of the T cell’s immune value chain, propose cancer immunotherapy researchers, from patient T cells to donor T cells. The novel allogeneic approach could rely on T-cell receptor gene transfer to generate broad and tumor-specific T-cell immune responses. [NIAID]

A new cancer immunotherapy approach could essentially outsource a crucial T-cell function. This function, T-cell reactivity to specific cancer antigens, is sometimes lacking in cancer patients. Yet, according to a new proof-of-principle study, these patients could benefit from T cells provided by healthy donors. Specifically, the healthy donors’ T cells could be used to broaden the T-cell receptor repertoires of the cancer patients’ T cells.

Ultimately, this approach relies on a cancer immunotherapy technique called T-cell receptor (TCR) transfer, or the genetic transfer of TCR chains. TCR transfer can be used to outsource the T cell’s learning function, the process by which a T cell acquires the ability to recognize foreign antigens—in this case, the sort of proteins that can be expressed on the surface of cancer cells. Because cancer cells harbor faulty proteins, they can also display foreign protein fragments, also known as neoantigens, on their surface, much in the way virus-infected cells express fragments of viral proteins.

The approach was detailed in a paper that appeared May 19 in the journal Science, in an article entitled, “Targeting of Cancer Neoantigens with Donor-Derived T Cell Receptor Repertoires.” This article, by scientists based at the Netherlands Cancer Institute and the University of Oslo, describes a novel strategy to broaden neoantigen-specific T-cell responses. Such a strategy would be useful in overcoming a common limitation seen in the immune response to cancer: Neoantigen-specific T-cell reactivity is generally limited to just a few mutant epitopes, even though the number of predicted epitopes is large.

“We demonstrate that T cell repertoires from healthy donors provide a rich source of T cells that specifically recognize neoantigens present on human tumors,” the study’s authors wrote. “Responses to 11 epitopes were observed, and for the majority of evaluated epitopes, potent and specific recognition of tumor cells endogenously presenting the neoantigens was detected.”

First, the researchers mapped all possible neoantigens on the surface of melanoma cells from three different patients. In all three patients, the cancer cells seemed to display a large number of different neoantigens. But when the researchers tried to match these to the T cells derived from within the patient’s tumors, most of these aberrant protein fragments on the tumor cells went unnoticed.

Next, the researchers tested whether the same neoantigens could be seen by T cells derived from healthy volunteers. Strikingly, these donor-derived T cells could detect a significant number of neoantigens that had not been seen by the patients’ T cells.

“Many of the T cell reactivities [among donor T cells] involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes,” the authors of the Science article continued. “T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such ‘outsourced’ immune responses in cancer immunotherapy.”

“In a way, our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neoantigens,” said Ton Schumacher, Ph.D., a principal investigator at the Netherlands Cancer Institute. “The receptor that is used by these donor T cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”

“Our study shows that the principle of outsourcing cancer immunity to a donor is sound,” added Johanna Olweus, M.D., Ph.D., who heads a research group at the University of Oslo. “However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput.”

“We are currently exploring high-throughput methods to identify the neoantigens that the T cells can ‘see’ on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”

 

Targeting of cancer neoantigens with donor-derived T cell receptor repertoires

Erlend Strønen1,2Mireille Toebes3Sander Kelderman3,…., Fridtjof Lund-Johansen2,5Johanna Olweus1,2,*,Ton N. Schumacher3,*,   + Author Affiliations
Science  19 May 2016:                         http://dx.doi.org:/10.1126/science.aaf2288

Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen specific T cell responses are therefore attractive. Here, we demonstrate that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11/57 predicted HLA-A2-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.

Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.

Verbist KC1, Guy CS1, Milasta S1, Liedmann S1, Kamiński MM1, Wang R2, Green DR1
Nature. 2016 Apr 21; 532(7599):389-93.   http://dx. doi.org:/10.1038/nature17442. Epub 2016 Apr 11

Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.

AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5′ AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL.

Glutamine Modulates Macrophage Lipotoxicity.

He L1,2, Weber KJ3,4, Schilling JD5,6,7
Nutrients. 2016 Apr 12;8(4). pii: E215.   http://dx.doi.org:/10.3390/nu8040215
Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α

Sangbin Lim1Hao Liu1,2,*Luciana Madeira da Silva1Ritu Arora1,…., Gary A. Piazza1Oystein Fodstad1,4,*, and Ming Tan1,5,*
C
ancer Res April 5, 2016    http://dx.doi.org:/10.1158/0008-5472.CAN-15-1538

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo. We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3–expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species–dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 1–12. ©2016 AACR.

 

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

Salerno F1, Guislain A2, …, Wolkers MC2.
J Immunol. 2016 May 1;196(9):3695-705.   http://dx.doi.org:/10.4049/jimmunol.1501997. Epub 2016 Mar 25.
CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.
 Immunometabolism of regulatory T cells 

Newton RPriyadharshini B & Laurence A Turk
Nature Immunology 2016;17:618–625
  http://dx.doi.
doi.org:/10.1038/ni.3466

The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.

Figure 1: Proposed model for the metabolic signatures of various Treg cell subsets.

Proposed model for the metabolic signatures of various Treg cell subsets.

(a) Activated CD4+ T cells that differentiate into the Teff cell lineage (green) (TH1 or TH17 cells) are dependent mainly on carbon substrates such as glucose and glutamine for their anabolic metabolism. In contrast to that, pTreg cells…
T-bet is a key modulator of IL-23-driven pathogenic CD4+ T cell responses in the intestine
Krausgruber TSchiering CAdelmann K & Harrison OJ.
Nature Communications 7; Article number:11627    http://dx.doi.org:/10.1038/ncomms11627

IL-23 is a key driver of pathogenic Th17 cell responses. It has been suggested that the transcription factor T-bet is required to facilitate IL-23-driven pathogenic effector functions; however, the precise role of T-bet in intestinal T cell responses remains elusive. Here, we show that T-bet expression by T cells is not required for the induction of colitis or the differentiation of pathogenic Th17 cells but modifies qualitative features of the IL-23-driven colitogenic response by negatively regulating IL-23R expression. Consequently, absence of T-bet leads to unrestrained Th17 cell differentiation and activation characterized by high amounts of IL-17A and IL-22. The combined increase in IL-17A/IL-22 results in enhanced epithelial cell activation and inhibition of either IL-17A or IL-22 leads to disease amelioration. Our study identifies T-bet as a key modulator of IL-23-driven colitogenic responses in the intestine and has important implications for understanding of heterogeneity among inflammatory bowel disease patients.

Th17 cells are enriched at mucosal sites, produce high amounts of IL-17A, IL-17F and IL-22, and have an essential role in mediating host protective immunity against a variety of extracellular pathogens1. However, on the dark side, Th17 cells have also been implicated in a variety of autoimmune and chronic inflammatory conditions, including inflammatory bowel disease (IBD)2. Despite intense interest, the cellular and molecular cues that drive Th17 cells into a pathogenic state in distinct tissue settings remain poorly defined.

The Th17 cell programme is driven by the transcription factor retinoid-related orphan receptor gamma-t (RORγt) (ref. 3), which is also required for the induction and maintenance of the receptor for IL-23 (refs 4, 5). The pro-inflammatory cytokine IL-23, composed of IL-23p19 and IL-12p40 (ref. 6), has been shown to be a key driver of pathology in various murine models of autoimmune and chronic inflammatory disease such as experimental autoimmune encephalomyelitis (EAE)7, collagen induced arthritis8 and intestinal inflammation9, 10, 11, 12. Several lines of evidence, predominantly derived from EAE, suggest that IL-23 promotes the transition of Th17 cells to pathogenic effector cells9, 10, 11, 12. Elegant fate mapping experiments of IL-17A-producing cells during EAE have shown that the majority of IL-17A+IFN-γ+ and IL-17A−IFN-γ+ effector cells arise from Th17 cell progeny13. This transition of Th17 cells into IFN-γ-producing ‘ex’ Th17 cells required IL-23 and correlated with increased expression of T-bet. The T-box transcription factor T-bet drives the Th1 cell differentiation programme14 and directly transactivates the Ifng gene by binding to its promoter as well as multiple enhancer elements15. Indeed, epigenetic analyses have revealed that the loci for T-bet and IFN-γ are associated with permissive histone modifications in Th17 cells suggesting that Th17 cells are poised to express T-bet which could subsequently drive IFN-γ production16, 17.

A similar picture is emerging in the intestine where IL-23 drives T-cell-mediated intestinal pathology which is thought to be dependent on expression of T-bet18 and RORγt (ref. 19) by T cells. In support of this we have recently shown that IL-23 signalling in T cells drives the emergence of IFN-γ producing Th17 cells in the intestine during chronic inflammation20. Collectively these studies suggest a model whereby RORγt drives differentiation of Th17 cells expressing high amounts of IL-23R, and subsequently, induction of T-bet downstream of IL-23 signalling generates IL-17A+IFN-γ+ T cells that are highly pathogenic. Indeed, acquisition of IFN-γ production by Th17 cells has been linked to their pathogenicity in several models of chronic disease13, 21, 22, 23, 24 and a population of T cells capable of producing both IL-17A and IFN-γ has also been described in intestinal biopsies of IBD patients25, 26.

However, in the context of intestinal inflammation, it remains poorly defined whether the requirement for RORγt and T-bet reflects a contribution of Th17 and Th1 cells to disease progression or whether Th17 cells require T-bet co-expression to exert their pathogenic effector functions. Here, we use two distinct models of chronic intestinal inflammation and make the unexpected finding that T-bet is dispensable for IL-23-driven colitis. Rather the presence of T-bet serves to modify the colitogenic response restraining IL-17 and IL-22 driven pathology. These data identify T-bet as a key modulator of IL–23-driven colitogenic effector responses in the intestine and have important implications for understanding of heterogeneous immune pathogenic mechanisms in IBD patients.

Figure 1: IL-23 signalling is required for bacteria-driven T-cell-dependent colitis and the emergence of IL-17A+IFN-γ+ T cells.
C57BL/6 WT and Il23r−/− mice were infected orally with Hh and received weekly i.p. injections of IL-10R blocking antibody. Mice were killed at 4 weeks post infection and assessed for intestinal inflammation. (a) Colitis scores. (b) Typhlitis sores. (c) Representative photomicrographs of colon and caecum (× 10 magnification; scale bars, 200μM). (d) Representative flow cytometry plots of colonic lamina propria gated on viable CD4+ T cells. (e) Frequencies of IL-17A+ and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from two independent experiments (n=12 for WT, n=10 for Il23r−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, ***P<0.001 as calculated by Mann–Whitney U test.

 

IL-23 signals are dispensable for T-bet and RORγt expression 

RORγt but not T-bet is required for T cell transfer colitis

Figure 2: RORγt but not T-bet expression by CD4+ T cells is required for the development of T cell transfer colitis.

C57BL/6 Rag1−/− mice were injected i.p. with 4 × 105 CD4+CD25CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/− T cells developed clinical signs of disease (4–6 weeks) and assessed for intestinal inflammation. (a) Colitis scores. (b) Representative photomicrographs of proximal colon sections (× 10 magnification; scale bars, 200μM). (c) Concentration of cytokines released from colon explants into the medium after overnight culture. Data represent pooled results from two independent experiments (n=14 for WT, n=11 for Rorc−/−, n=14 forTbx21−/−). Bars are the mean and each symbol represents an individual mouse. Bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.

T-bet is dispensable for IL-17A+IFN-γ+ intestinal T cells

Figure 3: T-bet expression by CD4+ T cells is not required for the emergence of IL-17A+IFN-γ+ T cells.

C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of IL-17A and IFN-γ expression in colonic CD4+ T cells. (b) Frequencies of IL-17A+ and/or IFN-γ+ cells among colonic CD4+ T cells. (c) Total numbers of IL-17A+and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from three independent experiments (n=20 for WT, n=18 for Tbx21−/−, n=12 for Rorc−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.

T-bet deficiency promotes an exacerbated Th17-type response

Our transfer of Tbx21−/− T cells revealed a striking increase in the frequency of IL-17A+IFN-γcells (Fig. 3) and we reasoned that T-bet-deficiency could impact on Th17 cell cytokine production. Therefore, we transferred WT or Tbx21−/− CD4+ T cells into Rag1−/− recipients and measured the expression of RORγt, IL-17A, IL-17F and IL-22 by CD4+ T cells isolated from the colon. In agreement with our earlier findings, Tbx21−/− T cells gave rise to significantly increased frequencies of RORγt-expressing T cells capable of producing IL-17A (Fig. 4a). Furthermore, T-bet deficiency also led to a dramatic expansion of IL-17F and IL-22-expressing cells, which constituted only a minor fraction in WT T cells (Fig. 4a,b). By contrast, the frequency of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ producing cells was significantly reduced in T-bet-deficient T cells as compared with WT T cells. When analysed in more detail we noted that the production of IL-17A, IL-17F and IL-22 increased specifically in T-bet-deficient IL-17A+IFN-γ+ T cells as compared with WT T cells whereas IFN-γ production decreased overall in the absence of T-bet as expected (Supplementary Fig. 4A). Similarly, GM-CSF production was also generally reduced in Tbx21−/− CD4+ T cells further suggesting a shift in the qualitative nature of the T cell response.

 

Figure 4: T-bet-deficient CD4+ T cells promote an exacerbated Th17-type inflammatory response.

C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25CD45RBhi T cells from C57BL/6 WT orTbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of cytokines and transcription factors in WT or Tbx21−/− colonic CD4+ T cells. (b) Frequency of IL-17A+, IL-17F+, IL-22+, GM-CSF+ or IFN-γ+ colonic T cells in WT orTbx21−/−. (c) quantitative reverse transcription PCR (qRT-PCR) analysis of mRNA levels of indicated genes in colon tissue homogenates. (d) Total number of neutrophils (CD11b+ Gr1high) in the colon. (e) Primary epithelial cells were isolated from the colon of steady state C57BL/6 Rag1−/− mice and stimulated with 10ngml−1 cytokines for 4h after which cells were harvested and analysed by qRT-PCR for the indicated genes. Data in bd represent pooled results from two independent experiments (n=14 for WT, n=11 for Tbx21−/−). Bars are the mean and error bars represent s.e.m. Data in e are pooled results from four independent experiments, bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01,***P<0.001 as calculated by Mann–Whitney U test.

………

T-bet-deficient colitis depends on IL-23, IL-17A and IL-22

In the present study we show that bacteria-driven colitis is associated with the IL-23-dependent emergence of IFN-γ-producing Th17 cells co-expressing RORγt and T-bet. Strikingly, while RORγt is required for the differentiation of IFN-γ-producing Th17 cells and induction of colitis, T-bet is dispensable for the emergence of IL-17A+IFN-γ+ T cells and intestinal pathology. Our results show that instead of a mandatory role in the colitogenic response, the presence of T-bet modulates the qualitative nature of the IL-23-driven intestinal inflammatory response. In the presence of T-bet, IL-23-driven colitis is multifunctional in nature and not functionally dependent on either IL-17A or IL-22. By contrast, in the absence of T-bet a highly polarized colitogenic Th17 cell response ensues which is functionally dependent on both IL-17A and IL-22. T-bet-deficient T cells are hyper-responsive to IL-23 resulting in enhanced STAT3 activation and downstream cytokine secretion providing a mechanistic basis for the functional changes. These data newly identify T-bet as a key modulator of IL-23-driven colitogenic CD4+ T cell responses.

Contrary to our expectations T-bet expression by CD4 T cells was not required for their pathogenicity. In keeping with the negative effect of T-bet on Th17 differentiation40, 41, 42, we observed highly polarized Th17 responses in T-bet-deficient intestinal T cells. Early studies demonstrated that IFN-γ could suppress the differentiation of Th17 cells40 and thus the reduced IFN-γ production by Tbx21−/−T cells could facilitate Th17 cell generation. However, our co-transfer studies revealed unrestrained Th17 differentiation of Tbx21−/− T cells even in the presence of WT T cells, suggesting a cell autonomous role for T-bet-mediated suppression of the Th17 programme. Indeed, the role of T-bet as a transcriptional repressor of the Th17 cell fate has been described recently. For example, T-bet physically interacts with and sequesters Runx1, thereby preventing Runx1-mediated induction of RORγt and Th17 cell differentiation43. In addition, T-bet binds directly to and negatively regulates expression of many Th17-related genes15, 34 and we identified IL23r to be repressed in a T-bet-dependent manner. In line with this we show here that T-bet-deficient intestinal T cells express higher amounts of Il23r as well as Rorc. This resulted in enhanced IL-23-mediated STAT3 activation and increased production of IL-17A and IL-22. It has also been suggested that T-bet activation downstream of IL-23R signalling is required for pathogenic IL-23-driven T cell responses43, 44. However, we did not find a role for IL-23 in the induction and/or maintenance of T-bet expression and colitis induced by T-bet-deficient T cells was IL-23 dependent. Collectively, these findings demonstrate that T-bet deficiency leads to unrestrained expansion of colitogenic Th17 cells, which is likely mediated through enhanced activation of the IL-23R-STAT3 pathway.

The observation that T-bet-deficient T cells retain their colitogenic potential is in stark contrast to earlier studies. Neurath et al.18 convincingly showed that adoptive transfer of Tbx21−/− CD4+ T cells into severe combined immunodeficiency (SCID) recipients failed to induce colitis and this correlated with reduced IFN-γ and increased IL-4 production. Another study revealed that IL-4 plays a functional role in inhibiting the colitogenic potential of Tbx21−/− T cells, as recipients ofStat6−/−Tbx21−/− T cells developed severe colitis37. Importantly, the intestinal inflammation that developed in recipients of Stat6−/−Tbx21−/− T cells could be blocked by administration of IL-17A neutralizing antibody, suggesting that the potent inhibitory effect of IL-4/STAT6 signals on Th17 differentiation normally prevent colitis induced by Tbx21−/− T cells37. Various explanations could account for the discrepancy between our study and those earlier findings. First, in contrast to the published reports, we used naïve Tbx21−/− CD4+ T cells from C57BL/6 mice instead of BALB/c mice. An important difference between Tbx21−/− CD4+ T cells from these genetic backgrounds appears to be their differential susceptibility to suppression by IL-4/STAT6 signals. We found that transfer of Tbx21−/− T cells induced IL-17A-dependent colitis despite increased frequencies of IL-4-expressing cells in the intestine. This discrepancy may be due to higher amounts of IL-4 produced by activated CD4+ T cells from BALB/c versus C57BL/6 mice45, leading to the well-described Th2-bias of the BALB/c strain45. Second, differences in the composition of the intestinal microbiota between animal facilities can have a substantial effect on skewing CD4+ T cells responses. In particular, the Clostridium-related segmented filamentous bacteria (SFB) have been shown to drive the emergence of IL-17 and IL-22 producing CD4+ T cells in the intestine46. Importantly, the ability of naïve CD4+ T cells to induce colitis is dependent on the presence of intestinal bacteria, as germ-free mice do not develop pathology upon T cell transfer47. In line with this, we previously described that colonization of germ-free mice with intestinal microbiota containing SFB was necessary to restore the development of colitis47. Since our Rag1−/− colony is SFB+ and the presence of SFB was not reported in the previous studies, it is possible that differences in SFB colonization status contributed to the observed differences in pathogenicity ofTbx21−/− T cells.

It is important to note that T-bet-deficient T cells did not induce more severe colitis than WT T cells but rather promoted a distinct mucosal inflammatory response. Colitis induced by WT T cells is characterized by a multifunctional response with high amounts of IFN-γ and GM-CSF and a lower IL-17A and IL-22 response. Consistent with this, we have shown that blockade of GM-CSF abrogates T cell transfer colitis48 as well as bacteria-driven intestinal inflammation49 in T-bet sufficiency whereas blockade of IL-17A or IL-22 fails to do so. By contrast T-bet deficiency leads to production of high amounts of IL-17A and IL-22 in the colon and neutralization of either was sufficient to reduce intestinal pathology. Our in vitro experiments suggest that IL-17A and IL-22 synergise to promote intestinal epithelial cell responses, which may in part explain the efficacy of blocking IL-17A or IL-22 in colitis induced by T-bet-deficient T cells. A similar synergistic interplay has been described in the lung where IL-22 served a tissue protective function in homeostasis but induced airway inflammation in the presence of IL-17A (ref. 50). This highlights the complexity of the system in health and disease, and the need for a controlled production of both cytokines. We describe here only one mechanism of how IL-17A/IL-22 induce a context-specific epithelial cell response that potentially impacts on the order or composition of immune cell infiltration. Overall, these results provide a new perspective on T-bet, revealing its role in shaping the qualitative nature of the IL-23-driven colitogenic T cell response.

We also describe here the unexpected finding that a substantial proportion of T-bet-deficient intestinal T cells retain the ability to express IFN-γ. To investigate the potential mechanisms responsible for T-bet-independent IFN-γ production by intestinal CD4+ T cells we focused on two transcription factors, Runx3 and Eomes. Runx3 has been shown to promote IFN-γ expression directly through binding to the Ifng promoter38 and Eomes is known to compensate for IFN-γproduction in T-bet-deficient Th1 cells37. We found IL-23-mediated induction of Runx3 protein in WT and Tbx21−/− T cells isolated from the intestine, thus identifying Runx3 downstream of IL-23R signalling. By contrast, we could only detect Eomes protein and its induction by IL-23 in T-bet-deficient but not WT T cells. Thus, Runx3 and Eomes are activated in response to IL-23 in T-bet-deficient cells and are likely to be drivers of T-bet-independent IFN-γ production. In support of this we found that the majority of T-bet-deficient IL-17AIFN-γ+ T cells expressed Eomes. However, only a minor population of IL-17A+IFN-γ+ T cells stained positive for Eomes, suggesting the existence of alternative pathways for IFN-γ production by Th17 cells. Intriguingly, a recent study identified Runx3 and Runx1 as the transcriptional regulators critical for the differentiation of IFN-γ-producing Th17 cells51. The author’s demonstrated that ectopic expression of Runx transcription factors was sufficient to induce IFN-γ production by Th17 cells even in the absence of T-bet. These findings, combined with our data on Runx3 activation downstream of IL-23R signalling strongly suggest that Runx3 rather than Eomes is driving IFN-γ expression by intestinal Th17 cells.

We have not formally addressed the role of IFN-γ in colitis driven by T-bet-deficient T cells. A recent report by Zimmermann et al.52 found that antibody-mediated blockade of IFN-γ ameliorates colitis induced by WT or T-bet-deficient T cells suggesting IFN-γ also contributes to the colitogneic response mediated by T-bet-deficient T cells as originally described for WT T cells53, 54. By contrast with our results the Zimmerman study found that IL-17A blockade exacerbated colitis following transfer of Tbx21−/− T cells. The reason for the differential role of IL-17A in the two studies is not clear but it is notable that the Zimmerman study was performed in the presence of co-infection with SFB and Hh, and this strong inflammatory drive may alter the pathophysiological role of particular cytokines. Together the data indicate that T-bet deficiency in T cells does not impede their colitogenic activity but that the downstream effector cytokines of the response are context dependent.

In conclusion, our data further underline the essential role for IL-23 in intestinal inflammation and demonstrate that T-bet is an important modulator of the IL–23-driven effector T cell response. The colitogenic T cell response in a T-bet sufficient environment is multifunctional with a dominant GM-CSF and IFN-γ response. By contrast T-bet-deficient colitogenic responses are dominated by IL-17A and IL-22-mediated immune pathology. These results may have significant bearing on human IBD where it is now recognized that differential responsiveness to treatment may reflect considerable disease heterogeneity. As such, identification of suitable biomarkers such as immunological parameters, that allow stratification of patient groups, is becoming increasingly important55. Genome-wide association studies have identified polymorphisms in loci related to innate and adaptive immune arms that confer increased susceptibility to IBD. Among these are Th1 (STAT4, IFNG and STAT1) as well as Th17-related genes (RORC, IL23R and STAT3) (refs56, 57). Thus, detailed profiling of the T cell response in IBD patients may help identify appropriate patient groups that are most likely to benefit from therapeutic blockade of certain effector cytokines. Finally, our studies highlight the importance of IL-23 in the intestinal inflammatory hierarchy and suggest that IL-23 could be an effective therapeutic target across a variety of patient groups.

 

Yale study: How antibodies access neurons to fight infection

Yale scientists have solved a puzzle of the immune system: how antibodies enter the nervous system to control viral infections. Their finding may have implications for the prevention and treatment of a range of conditions, including herpes and Guillain-Barre syndrome, which has been linked to the Zika virus.

Many viruses — such as West Nile, Zika, and the herpes simplex virus — enter the nervous system, where they were thought to be beyond the reach of antibodies. Yale immunobiologists Akiko Iwasaki and Norifumi Iijima used mice models to investigate how antibodies could gain access to nerve tissue in order to control infection.

In mice infected with herpes, they observed a previously under-recognized role of CD4 T cells, a type of white blood cell that guards against infection by sending signals to activate the immune system. In response to herpes infection, CD4 T cells entered the nerve tissue, secreted signaling proteins, and allowed antibody access to infected sites. Combined, CD4 T cells and antibodies limited viral spread.

“This is a very elegant design of the immune system to allow antibodies to go to the sites of infection,” said Iwasaki. “The CD4 T cells will only go to the site where there is a virus. It’s a targeted delivery system for antibodies.”

 

Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help

Norifumi Iijima & Akiko Iwasaki
Nature 533,552–556 (26 May 2016)
    http://dx.
doi.org:/10.1038/nature17979

 

Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood–brain barrier1 and blood–nerve barrier2, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

 

T Cells Help Reverse Ovarian Cancer Drug Resistance

http://www.genengnews.com/gen-news-highlights/t-cells-help-reverse-ovarian-cancer-drug-resistance/81252753/

T cells (red) attack ovarian cancer cells (green). [University of Michigan Health System]

Researchers at the University of Michigan have recently published the results from a new study that they believe underscores why so many ovarian tumors develop resistance to chemotherapy. The tumor microenvironment is made up of an array of cell types, yet effector T cells and fibroblasts constitute the bulk of the tissue. The investigators believe that understanding the interplay between these two cell types holds the key to how ovarian cancer cells develop resistance.

The new study suggests that the fibroblasts surrounding the tumor work to block chemotherapy, which is why nearly every woman with ovarian cancer becomes resistant to treatment. Conversely, the scientists published evidence that T cells in the microenvironment can reverse the resistance phenotype—suggesting a whole different way of thinking about chemotherapy resistance and the potential to harness immunotherapy drugs to treat ovarian cancer.

“Ovarian cancer is often diagnosed at late stages, so chemotherapy is a key part of treatment,” explained co-senior study author J. Rebecca Liu, M.D., associate professor of obstetrics and gynecology at the University of Michigan. “Most patients will respond to it at first, but everybody develops chemoresistance. And that’s when ovarian cancer becomes deadly.”

Dr. Liu continued, stating that “in the past, we’ve thought the resistance was caused by genetic changes in tumor cells. But we found that’s not the whole story.”

The University of Michigan team looked at tissue samples from ovarian cancer patients and separated the cells by type to study the tumor microenvironment in vitro and in mice. More importantly, the scientists linked their findings back to actual patient outcomes.

The results of this study were published recently in Cell through an article entitled “Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.”

Ovarian cancer is typically treated with cisplatin, a platinum-based chemotherapy. The researchers found that fibroblasts blocked platinum. These cells prevented platinum from accumulating in the tumor and protected tumor cells from being killed off by cisplatin.

Diagram depicting how T cells can reverse chemotherapeutic resistance. [Cell, Volume 165, Issue 5, May 19, 2016]

“We show that fibroblasts diminish the nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy,” the authors wrote. “We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance.”

T cells, on the other hand, overruled the protection of the fibroblasts. When researchers added the T cells to the fibroblast population, the tumor cells began to die off.

“CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts,” the authors explained. “CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xccystine and glutamate antiporter via the JAK/STAT1 pathway.”

By boosting the effector T cell numbers, the researchers were able to overcome the chemotherapy resistance in mouse models. Moreover, the team used interferon, an immune cell-secreted cytokine, to manipulate the pathways involved in cisplatin.

“T cells are the soldiers of the immune system,” noted co-senior study author Weiping Zou, M.D., Ph.D., professor of surgery, immunology, and biology at the University of Michigan. “We already know that if you have a lot of T cells in a tumor, you have better outcomes. Now we see that the immune system can also impact chemotherapy resistance.”

The researchers suggest that combining chemotherapy with immunotherapy may be effective against ovarian cancer. Programmed death ligand 1 (PD-L1) and PD-1 pathway blockers are currently FDA-approved treatments for some cancers, although not ovarian cancer.

“We can imagine re-educating the fibroblasts and tumor cells with immune T cells after chemoresistance develops,” Dr. Zou remarked.

“Then we could potentially go back to the same chemotherapy drug that we thought the patient was resistant to. Only now we have reversed that, and it’s effective again,” Dr. Liu concluded.

 

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer

Weimin Wang, Ilona Kryczek, Lubomír Dostál, Heng Lin, Lijun Tan, et al.
Cell May 2016;  165, Issue 5:1092–1105.   http://dx.doi.org/10.1016/j.cell.2016.04.009
Highlights
  • Fibroblasts diminish platinum content in cancer cells, resulting in drug resistance
  • GSH and cysteine released by fibroblasts contribute to platinum resistance
  • T cells alter fibroblast GSH and cystine metabolism and abolish the resistance
  • Fibroblasts and CD8+ T cells associate with patient chemotherapy response

 

Summary

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8+ T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

 

Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation. Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α..
The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.
CD28 co-stimulation further activates the PI3K/Akt/mTOR pathway in particular, and provides a signal for Glut1 expression and cell surface localization.
Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway. The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism.
Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions

 

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Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP

 

 

Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP

Perspectives

The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.

 

  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294
http://dx.doi.org:/10.1146/annurev.biochem.75.103004.142738

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

 

  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u1.jpg

Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.

 

Box 2

Nuclear stress bodies  

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u2.jpg

The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif

 

Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.

 

HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.

 

Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.

 

HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.

 

Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.

 

HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.

 

Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.

 

  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).

 

Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg

 

Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).

 

  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040  http://195.111.72.71/docs/pcs/03exger.pdf

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).

 

Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.

 

  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.

 

  1. Role of heat shock and the heat shock response in immunity and cancer

 

Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).

 

HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.

 

HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342006/bin/fimmu-03-00075-g001.jpg

Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).

 

Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.

Conclusion

  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.

 

  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*
https://www.researchgate.net/profile/David_Rubinsztein/publication/24447892_Effects_of_heat_shock_heat_shock_protein_40_(HDJ2)_and_proteasome_inhibition_on_protein_aggregation_in_cellular_models_of_Huntington’s_disease/links/00b7d528b80aab69bb000000.pdf

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.

 

  1. Hsp70 in blood cell differentiation.

 

Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Introduction

Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

 

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

 

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

 

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61

 

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80

 

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

 

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.

https://elife-publishing-cdn.s3.amazonaws.com/12821/elife-12821-fig7-v3-480w.jpg

Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016

 

Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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Natural Killer Cell Response: Treatment of Cancer

Curator: Larry H. Bernstein, MD, FCAP

 

Molecular mechanisms of natural killer cell activation in response to cellular stress

C J Chan1,2,3, M J Smyth1,2,3,4,5 and L Martinet1,2,4,5        Edited by M Piacentini

Cell Death and Differentiation (2014) 21, 5–14;    http://www.nature.com/cdd/journal/v21/n1/full/cdd201326a.htm

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.

 

  • Stress induces specific intrinsic and extrinsic physiological mechanisms within cells that lead to their identification as functionally abnormal
  • Sources of cellular stress can be nutritional, physical, pathogenic, or oncogenic
  • Intrinsic responses to cellular stress include activation of the DNA-damage response, tumor-suppressor genes, and senescence
  • The extrinsic response to cellular stress is activation of the immune system, such as natural killer cells
  • Intrinsic responses to cellular stress can directly upregulate factors that can activate the immune system, and the immune system been shown to be indispensable for the efficacy of some chemotherapy

Further critical determinants of intrinsic responses to stress and cell death that can activate the immune system must be identified

  • Identification of the different cellular pathways and molecular determinants controlling the immunogenicity of different cancer therapies is required
  • How can we harness the ability of therapeutic agents to activate both the intrinsic and extrinsic responses to cellular stress to achieve more specific and safer approaches to cancer treatment?

Any insult to a cell that leads to its abnormal behavior or premature death can be defined as a source of stress. As the turnover and maintenance of cells in all multi-cellular organisms is tightly regulated, it is essential that stressed cells be rapidly identified to avoid widespread tissue damage and to maintain tissue homeostasis. Various intrinsic cellular mechanisms exist within cells that become activated when they are exposed to stress. These include activation of DNA-damage response proteins, senescence programs, and tumor-suppressor genes.1 Extrinsic mechanisms also exist that combat cellular stress, through the upregulation of mediators that can activate different components of the immune system.2 Although frequently discussed separately, much recent evidence has indicated that intrinsic and extrinsic responses to cellular stress are intimately linked.3

As the link between cell intrinsic and extrinsic responses to stress have been uncovered, these observations are now being harnessed therapeutically, particularly in the context of cancer.4 Indeed, various chemotherapeutic agents and radiotherapy are critically dependent on the immune system to elicit their full therapeutic benefit.5, 6 The mechanisms by which this occurs may be twofold: (i) the induction of intrinsic cellular stress mechanisms activates innate immunity and (ii) the release and presentation of tumor-specific antigens engages an inflammatory adaptive immune response.

NK cells are the major effector lymphocyte of innate immunity found in all the primary and secondary immune compartments as well as various mucosal tissues.7 Through their ability to induce direct cytotoxicity of target cells and produce pro-inflammatory cytokines such as interferon-gamma, NK cells are critically involved in the immune surveillance of tumors8, 9, 10 and microbial infections.11, 12 The major mechanism that regulates NK cell contact-dependent functions (such as cytotoxicity and recognition of targets) is the relative contribution of inhibitory and activating receptors that bind to cognate ligands.

Under normal physiological conditions, NK cell activity is inhibited through the interaction of their inhibitory receptors with major histocompatibility complex (MHC) class I.13, 14 However, upon instances of cellular stress that are frequently associated with viral infection and malignant transformation, ligands for activating receptors are often upregulated and MHC class I expression may be downregulated. The upregulation of these activating ligands and downregulation of MHC class I thus provides a signal for NK cells to become activated and display effector functions. Activating receptors are able to provide NK cells with a strong stimulus in the absence of co-stimulation due to the presence of adaptor molecules such as DAP10, DAP12, FcRγ, and CD3ζ that contain immunoreceptor tyrosine-based activating motifs (ITAMs).15, 16,17 By contrast, inhibitory receptors contain inhibitory motifs (ITIMs) within their cytoplasmic tails that can activate downstream targets such as SHP-1 and SHP-2 and directly antagonize those signaling pathways activated through ITAMs.18, 19, 20 The specific details of individual classes of inhibitory and activating receptors and their ligands are summarized in Figure 1 and have been extensively reviewed elsewhere.14, 21 Instead, this review will more focus on the relevant activating receptors that are primarily involved in the direct regulation of NK cell-mediated recognition of cellular stress: natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1).

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorNK cell receptors and their cognate ligands. Major inhibitory and activating receptors on NK cells and their cognate ligands on targets are depicted. BAT3, human leukocyte antigen (HLA)-B-associated transcript 3; CRTAM, class I-restricted T-cell-associated molecule; HA, hemagglutinin; HLA-E, HLA class I histocompatibility antigen, alpha chain E; IgG, immunoglobulin G; LFA-1, leukocyte function-associated antigen-1; LLT1, lectin-like transcript 1; TIGIT, T cell immunoglobulin and ITIM domain

Full figure and legend (185K)

NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

NKG2D is a lectin-like type 2 transmembrane receptor expressed as a homodimer in both mice and humans by virtually all NK cells.22, 23 Upon interaction with its ligands, NKG2D can trigger NK cell-mediated cytotoxicity against their targets. The ligands for NKG2D are self proteins related to MHC class I molecules.24 In humans, these ligands consist of the MHC class I chain-related protein (MIC) family (e.g., MICA and MICB) and the UL16-binding protein (ULBP1-6) family.25, 26 In mice, ligands for NKG2D include the retinoic acid early inducible (Rae) gene family, the H60 family, and mouse ULBP-like transcript-1 (MULT-1).27, 28, 29 NKG2D ligands are generally absent on the cell surface of healthy cells but are frequently upregulated upon cellular stress associated with viral infection and malignant transformation.3, 30 Indeed, NKG2D ligand expression has been found on many transformed cell lines, and NKG2D-dependent elimination of tumor cells expressing NKG2D ligands has been well documented in vitro and in tumor transplant experiments.25, 30, 31, 32, 33 In humans, NKG2D ligands have been described on different primary tumors34, 35 and specific NKG2D gene polymorphisms are associated with susceptibility to cancer.36 Finally, blocking NKG2D through gene inactivation or monoclonal antibodies leads to an increased susceptibility to tumor development in mouse models,37, 38demonstrating the key role played by NKG2D in immune surveillance of tumors. NKG2D can also contribute to shape tumor immunogenicity, a process called immunoediting, as demonstrated by the frequent ability of tumor cells to avoid NKG2D-mediated recognition through NKG2D ligand shedding, as discussed later in this review.38, 39, 40

DNAM-1 is a transmembrane adhesion molecule constitutively expressed on T cells, NK cells, macrophages, and a small subset of B cells in mice and humans.41, 42, 43 DNAM-1 contains an extracellular region with two IgV-like domains, a transmembrane region and a cytoplasmic region containing tyrosine- and serine-phosphorylated sites that is able to initiate downstream activation cascades.41, 44 There is accumulating evidence showing that DNAM-1 not only promotes adhesion of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets.41, 45, 46, 47, 48, 49, 50 The ligands for DNAM-1 are the nectin/nectin-like family members CD155 (PVR, necl-5) and CD112 (PVRL2, nectin-2).45, 46 Like NKG2D ligands, DNAM-1 ligands are frequently expressed on virus-infected and transformed cells.51, 52DNAM-1 ligands, especially CD155, are overexpressed by many types of solid and hematological malignancies and blocking DNAM-1 interactions with its ligands reduces the ability of NK cells to kill tumor cells in vitro.41, 49, 53, 54, 55, 56, 57 Further evidence of the role of DNAM-1 in tumor immune surveillance is provided by studies using experimental and spontaneous models of cancer in vivo showing enhanced tumor spread in the absence of DNAM-1.47, 48, 49, 50, 58

As NKG2D and DNAM-1 ligands are frequently expressed on stressed cells, many studies have sought to determine the mechanisms that underpin these observations. The guiding hypothesis for these studies is that cell-intrinsic responses to stress are directly linked to cell-extrinsic responses that can trigger rapid NK cell surveillance and elimination of stressed cells. Indeed, major cell-intrinsic responses to cellular stress can directly lead to NK cell-activating ligand upregulation and are outlined in the following sections.

The DNA-Damage Response

Cellular stress caused by the activation of the DNA-damage response leads to downstream apoptosis or cell-cycle arrest. The activation of DNA-damage checkpoints occurs when there are excessive DNA strand breaks and replication errors, thereby representing an important tumorigenesis barrier that can slow or inhibit the progression of malignant transformation.59, 60 Two major transducers of the DNA-damage response are the PI3-kinase-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM and ATR can modulate numerous signaling pathways such as checkpoint kinases (Chk1 and Chk2, which inhibit cell-cycle progression and promote DNA repair) and p53 (which mediates cell-cycle arrest and apoptosis).61

In addition to the induction of cell-cycle arrest and apoptosis, activation of the DNA-damage response has been shown to promote the expression of several activating ligands that are specific for NK cell receptors, primarily those of the NKG2D receptor. These findings have shown a critical direct link between cellular transformation, apoptosis, and surveillance by the immune system.62 The first evidence of this link between DNA damage and immune cell activation was provided by Raulet and colleagues who showed that NKG2D ligands were upregulated by genotoxic stress and stalled DNA replication conditions known to activate either ATM or ATR.63 These observations have now been extended by several other studies that have defined further DNA-damaging conditions (e.g., genotoxic drugs/chemotherapy, deregulated proliferation, or oxidative stress) that can promote NKG2D ligand upregulation.64, 65, 66, 67

The role of the DNA-damage response in controlling NKG2D ligand expression and subsequent NK cell activation has also been demonstrated in the context of anti-viral immunity, specifically in Abelson murine leukemia virus infection.68 This pathogen was shown to induce activation-induced cytidine deaminase (AID) expression outside the germinal center, resulting in generalized hypermutation, DNA-damage checkpoint activation, and Chk1 phosphorylation. The genotoxic activity of virally induced AID not only restricted the proliferation of infected cells but also induced the expression of NKG2D ligands. More recently, another member of APOBEC-AID family of cytidine deaminases, A3G, has been shown to promote the recognition of HIV-infected cells by NK cells after DNA-damage response activation.69 In this study, viral protein Vpr-mediated repair processes, which generate nicks, gaps, and breaks of DNA, activate an ATM/ATR DNA-damage response that leads to NKG2D ligand expression.

The DNA-damage sensors ATM and ATR have also been shown to regulate other key NK cell-activating ligands such as the DNAM-1 ligand, CD155.58, 65, 70 For example, in the Eμ-myc spontaneous B-cell lymphoma model, activation of the DNA-damage response leads to the upregulation of CD155 in the early-stage transformed B cells, subsequently activating spontaneous tumor regression in an NK cell- and T-cell-dependent manner.58 The DNA-damage response can also regulate the expression of the death receptor DR5.71 The engagement of DR5 by the effector molecule TRAIL, which is expressed by NK cells and T cells, can induce apoptosis of target cells and has been shown to have a key role in immune surveillance against tumors.72 Collectively, these results suggest that the detection of DNA damage, primarily through ATM and ATR, may represent a conserved protection mechanism governing the immunogenicity of infected or transformed cells, leading to direct recognition by NK cells (Figure 2).

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorOverview of the molecular pathways leading to NK cell recognition of intrinsic cellular stress. Oncogenic transformation and viral infection can activate intrinsic cellular responses to stress. These responses include activation of the DNA-damage response, senescence, tumor suppressors, and the presentation and/or release of HSPs that, in turn, can activate NK cells through various receptor–ligand interactions. Senescent cells can also release pro-inflammatory cytokines that can recruit NK cells and other innate immunity, such as macrophages. CCL2, C-C motif chemokine ligand 2; CXCL11, C-X-C motif chemokine ligand 11; DR, death receptor 5; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

Full figure and legend (146K)

As a result of these studies, many therapeutic agents known to induce DNA damage have been evaluated for their ability to increase the immunogenicity of cancer cells for a more targeted therapeutic approach using NK cells.64, 65 For example, treatment of multiple myeloma cells with doxorubicin, melphalan, or bortezomib can lead to DNAM-1 and NKG2D ligand upregulation.65Indeed, many chemotherapeutic agents commonly used, especially in hematological malignancies, can trigger the DNA-damage pathway. Therefore, it is reasonable to speculate that there is a general role of ATM and ATR in the induction of NK cell activation as a therapeutic effect of these agents.

Senescence

Cellular senescence is generally defined as a growth-arrest program in mammalian cells that limits their lifespan.73 The major type of cellular senescence is replicative senescence that occurs due to telomere shortening. However, it is now generally accepted that premature senescence can also occur due to oncogene activation (oncogene-induced senescence) and/or the loss/gain of tumor-suppressor gene function, in the absence of telomere shortening.74 Thus, premature senescence is an important barrier against malignant transformation.59 Upon engagement of the senescence program, although cells are in growth arrest, they remain metabolically active and can produce many pro-inflammatory cytokines, as well as upregulate adhesion molecules and activating ligands to alert the immune system.75, 76, 77Activation of the immune system, in particular innate immunity, has a critical role in the clearance of senescent cells.78, 79, 80, 81More specifically, in a model of hepatocellular carcinoma, it has been shown that reactivation of p53 can induce a senescence program, resulting in tumor regression through the activation of NK cells, macrophages, and neutrophils. Of note, intercellular adhesion molecule (ICAM)-1, which can trigger both adhesion and cytotoxicity of NK cells,82 and interleukin-15, a cytokine that can promote NK cell effector function,83 were both upregulated in senescent tumors. More recently, the potential contribution of NK cells was also shown in the clearance of senescent hepatic stellate cells, a mechanism important in limiting liver fibrosis in response to a fibrogenic agent.80 ICAM-1, NKG2D ligands (MICA and ULPB2), and DNAM-1 ligands (CD155) were all upregulated on senescent hepatic stellate cells.

The specific mechanisms linking the senescence program to immune activation are not yet fully understood. However, the intracellular molecular mechanisms that govern induction of senescence may provide possible indications. Both replicative senescence and premature senescence (e.g., oncogene-induced senescence) have been shown to have common molecular determinants, such as the activation of the DNA-damage response pathway (e.g., ATM and ATR) and downstream activation of p53 and p16INK4A.1, 59, 84, 85, 86 Activation of the DNA-damage response would presumably initiate the upregulation of NK cell-activating ligands as previously discussed. However, how senescence may be linked to the induction of pro-inflammatory cytokine release is a more compelling question and requires further investigation (Figure 2). Nevertheless, induction of pro-inflammatory cytokines is an important protective mechanism in order to recruit immune cells that can rapidly recognize and remove senescent cells. Interestingly, activation of NK cells by senescent cells has been observed in a clinical context when multiple myeloma cells were treated with chemotherapy and genotoxic agents.65 In this setting, NKG2D and DNAM-1 ligands were both upregulated through a mechanism that required activation of the DNA-damage pathway initiated by ATM and ATR.65

Tumor Suppressors: p53

p53 is a potent tumor suppressor and central regulator of apoptosis, DNA repair, and cell proliferation, that is activated in response to DNA damage, oncogene activation, and other cellular stress.87 The number of identified cellular functions that p53 regulates has greatly increased over the past few years, and there is now a vast array of evidence that shows that p53 can be induced by viral infection88 to limit pathogen spread by inducing apoptosis.89, 90 Furthermore, p53 not only acts as an intrinsic barrier against tumorigenesis or pathogenic spread but can also lead to increased cellular immunogenicity. For example, p53 reactivation in a hepatocellular carcinoma can promote tumor regression mediated by innate immunity.78 A direct link between p53 expression and immune cell recognition was recently provided by Textor et al.91 where expression of p53 in lung cancer cell lines strongly upregulated the NKG2D ligands ULBP1 and 2, resulting in NK cell activation. Subsequently, p53-responsive elements were found to directly regulate ULBP1 and 2 expression, the deletion of which abolished the capacity of p53 to mediate ULBP1 and 2 upregulation. Another recent report that used a pharmacological activator of p53 confirmed the ability of p53 to directly induce ULBP2 expression that was independent of ATM/ATR.92 However, it has also been shown that miR34a and miR34C microRNAs (miRNAs) induced by p53 can target ULBP2 mRNA and reduce its cell-surface expression, suggesting that p53 may have a dual role in regulating ULBP2 expression.93 Finally, early work showed that NKG2D ligands can be upregulated by ATR/ATM in the total absence of p53 in tumor cell lines,62, 63 suggesting the existence of ATM/ATR-dependent and p53-independent pathways that regulate NKG2D ligand expression in response to cellular stress.

In addition to regulating NK cell ligand expression, genetic reactivation of p53 in tumors can also induce a wide array of pro-inflammatory mediators ranging from adhesion receptor (ICAM-1) expression to the production of various chemokines (CXCL11 and monocyte chemoattractant protein-1) and cytokines (interleukin-15).78 Furthermore, recent studies in anti-viral immunity indicate that several interferon-inducible genes and Toll-like receptor-3 expression are direct transcriptional targets of p53 and that p53 contributes to production of type I interferon by virally infected cells.94, 95, 96 All together, these studies suggest that p53 accumulation could represent a key determinant of the immunogenicity of stressed cells that are infected or undergoing malignant transformation through its ability to regulate innate immune activation.

Oncogenes

Malignant transformation is a complex process that frequently involves the activation of one or more oncogenes in addition to the inactivation or mutation of tumor-suppressor genes (e.g., p53). Oncogene activation is a powerful inducer of cellular stress that is able to activate intrinsic cellular programs that lead to cell apoptosis or senescence (e.g., activation of the DNA-damage response and p53).1 In addition, many recent reports have also shown that major oncogenes can activate extrinsic responses to cellular stress through inducing the upregulation of NK cell-activating ligands.63, 97, 98 This suggests that oncogene activation can represent a key cellular event in alerting the immune system to ongoing cellular transformation (Figure 3).

Figure 3.

Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorMolecular mechanisms that regulate the cell surface expression of NKG2D ligands. The major group of NK cell-activating ligands that are upregulated by intrinsic cellular responses to stress are those that bind the NKG2D receptor. Activation of the DNA-damage response, senescence, oncogenes, tumor suppressors, or sensing of deregulated proliferation can induce NKG2D ligand gene transcription and increase mRNA translation, leading to extracellular protein expression. MMP, matrix metalloproteases

Full figure and legend (183K)

The enhanced expression of the proto-oncogene Myc has been described as a critical event leading to cellular transformation and is a frequently found genetic alteration in cancer.99 In a recent study, again using the Eμ-myc model, Medzhitov and colleagues demonstrated the ability of c-Myc to alert NK cells to early oncogenic transformation through the upregulation of Rae-1.97 In this study, the induction of Rae-1 was dependent on the direct regulation of Rae-1 transcription by Myc through its interaction with the Raet1 epsilon gene. Collectively, these results provide a possible direct molecular mechanism to explain the increased susceptibility of NKG2D gene-targeted mice to lymphoma development in the Eμ-myc model.38

Recent evidence suggests that several oncogenic mutations of Ras (H-Ras, N-Ras, and K-Ras) can also regulate NKG2D ligand expression in both mice and humans.98 Interestingly, in this case, NKG2D ligands were regulated through MAPK/MEK and PI3K pathways downstream of oncogenic H-RasV12. The activation of PI3K pathways, and more particularly the p110α subunits by virus-encoded proteins, has also been shown to induce the Rae-1 family of ligands.100 As many viruses can manipulate the PI3K pathway101 and tumors often bear Ras and p110α oncogene mutations,102 collectively, this data suggests that there is the existence of a common molecular mechanism by which NK cells sense cellular stress mediated by PI3K-dependent regulation of NKG2D ligands.

Interestingly, whereas Myc was involved in the transcriptional regulation of NKG2D ligands, PI3K can increase NKG2D ligand expression by increasing the translation of Rae-1 mRNA.98 This involved the induction of eIF4E, a protein that enhances the translation of mRNA.103 As number of tumors and viruses can upregulate host translation initiation machinery through the overexpression of eIF4E,104, 105 this may represent an important means by which NK cells can discriminate tumor- and virus-infected cells from normal cells.

Heat-Shock Proteins (HSPs)

HSPs are highly conserved intracellular chaperone molecules that are present in most prokaryotic and eukaryotic cells that mediate protection against cellular damage under conditions of stress. HSPs are distributed in most intracellular compartments of cells where they support the correct folding of nascent polypeptides, prevent protein aggregation, and assist in protein transport across membranes.106 Many tumors display overexpression of HSPs as a response to cellular stress induced by oncogenic transformation.107, 108 HSPs can also be mobilized to the plasma membrane, or even released from cells, under conditions of stress.109

Although intracellular HSPs can promote cell survival by interfering with different apoptosis components, many studies have reported that membrane-bound or soluble HSPs can directly stimulate innate immunity.110 A major immunostimulatory function of HSPs is to promote the presentation of tumor-specific antigens by MHC class I to CD8 T cells.111, 112, 113 Soluble and membrane-bound HSPs can also induce antigen-presenting cell maturation and the resultant secretion of pro-inflammatory cytokines.114, 115, 116Finally, HSPs may directly activate NK cells as HSP70, when overexpressed on tumor cells, can induce a selective dose-dependent increase in NK cell-mediated cytotoxicity in vitro.117 NK cells may directly recognize HSP70 through a 14-amino-acid oligomer (TKD) that is localized in the C-terminal domain of the protein through CD94.118, 119 Tumor-specific HSP70 that is either presented at the cell surface or secreted on exosomes can also enhance NK cell activity against diverse types of cancer in vivo.120, 121 Most importantly, hepatocellular carcinoma cells that are treated with various chemotherapeutic agents can become more susceptible to NK cell-mediated cytotoxicity through their release of HSP-containing exosomes, giving the aforementioned findings a therapeutic context.122 Collectively, these results suggest that HSP translocation to the plasma membrane or secretion during cellular stress may represent a potent danger signal that can stimulate NK cell activity, particularly in the context of cancer.

 

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Protein profiling in cancer and metabolic diseases

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Deep Protein Profiling Key

Company has encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment.

http://www.technologynetworks.com/Proteomics/news.aspx?ID=190145

Proteome Sciences plc has strongly encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment. These highlight the growing need for more detailed, personal assessment of protein profiles to improve the management of cancer treatment.

In the first study two groups from University College London and Cancer Research UK demonstrated that genetic mutations in cancer can lead to changes in the proteins on the cell surface1. These are new sequences which are seen as foreign by the body’s immune system and, with appropriate immunotherapy, the level of response in lung cancer was greatly enhanced.

However many of the patients with these types of mutations unfortunately still did not respond which highlighted the need for deeper analysis of the protein expression in tumours in order to better appreciate the mechanisms that contribute to treatment failure.

The second study, led by Professor Nigel Bundred of Manchester University, reported that use of two drugs that act on the same breast cancer target, an over-expressing protein called Her-2, were able to eradicate detectable tumours in around 10% of those treated in just 11 days, with 87% of those treated having a proteomic change indicating cells had stopped growing and/or cell death had increased2.

Whilst these results appear very promising it is worth noting that the over-expressing Her-2 target is only present in about 20% of breast tumours meaning this combination therapy was successful in clearing tumours in just 2% of the total breast cancer population.

Dr. Ian Pike, Chief Operating Officer of Proteome Sciences commented, “Both these recent studies should rightly be recognised as important steps forward towards better cancer treatment. However, in order to overcome the limitations of current drug therapy programs, a much deeper and more comprehensive analysis of the complex protein networks that regulate tumour growth and survival is required and will be essential to achieve a major advance in the battle to treat cancer.

“Our SysQuant® workflows provide that solution. As an example, in pancreatic cancer3 we have successfully mapped the complex network of regulatory processes and demonstrate the ability to devise personalised treatment combinations on an individual basis for each patient. A retrospective study with SysQuant® to predict response to the targeted drug Sorafenib in liver cancer is in process and we are planning further prospective trials to guide personalised treatment selection in liver cancer.

“We are already delivering systems-wide biology solutions through SysQuant® and TMTcalibrator™ programs to our clients that are generating novel biological data and results using more sensitive profiling that are helping them to better understand their drug development programs and to provide new biomarkers for tracking patient response in clinical trials.

“We are strongly positioned to deliver more comprehensive analysis of proteins and cellular pathways across other areas of disease and in particular to extend the use of SysQuant® with other leading cancer research groups in liver and other cancers.”

Proteome Sciences has also expanded its offering in personalised medicine through the use of its TMTcalibrator™ technology to uniquely identify protein biomarkers that reveal active cancer and other disease processes in body fluid samples. The importance of these ‘mechanistic’ biomarkers is that they are essential to monitor that drugs are being effective and that they can be used as early biomarkers of disease recurrence.

Using SysQuant® and TMTcalibrator™, Proteome Sciences can deliver more comprehensive analysis and provide unparalleled levels of sensitivity and breadth of coverage of the proteome, enabling faster, more efficient drug development and more accurate disease diagnosis.

 

Discovering ‘Outlier’ Enzymes

Researchers at TSRI and Salk Institute have discovered ‘Outlier’ enzymes that could offer new targets to treat type 2 diabetes and inflammatory disorders.

A team led by scientists at The Scripps Research Institute (TSRI) and the Salk Institute for Biological Studies have discovered two enzymes that appear to play a role in metabolism and inflammation—and might someday be targeted with drugs to treat type 2 diabetes and inflammatory disorders. The discovery is unusual because the enzymes do not bear a resemblance—in their structures or amino-acid sequences—to any known class of enzymes.

The team of scientists nevertheless identified them as “outlier” members of the serine/threonine hydrolase class, using newer techniques that detect biochemical activity. “A huge fraction of the human ‘proteome’ remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure,” said co-senior author Benjamin F. Cravatt, chair of TSRI’s Department of Chemical Physiology.

“In this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases,” said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute. The study, which appeared as a Nature Chemical Biology Advance Online Publication on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine/threonine hydrolases using fluorophosphonate (FP) probes—molecules that selectively bind and, in effect, label the active sites of these enzymes.

Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP. “Neither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all,” said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study.

Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. “It looks like they have an active site that is novel—it had never been described in the literature,” said Parsons. Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases—enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work?

At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation.

“[Ben Cravatt’s] lab was screening panels of lipids to find the ones that their new enzymes work on,” said Saghatelian, who is a former research associate in the Cravatt laboratory. “We suggested they throw FAHFAs in there—and these turned out to be very good substrates.” The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes’ functions in vitro and in cultured cells.

Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab. The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid. In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies.

“Our prediction,” said Saghatelian, “is that if FAHFAs do what we think they’re doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity.” The two labs are now collaborating on further studies of the new enzymes—and the potential benefits of inhibiting them—in mouse models of diabetes, inflammation and autoimmune disease.

“One of the neat things this study shows,” said Cravatt, “is that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology.”

Other co-authors of the study, “AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs,” were Siddhesh S. Kamat, Armand B. Cognetta III, Jonathan J. Hulce and Enrique Saez, of TSRI; and co-senior author Barbara B. Kahn of Beth Israel Deaconess Medical Center and Harvard Medical School

 

New Weapon Against Breast Cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says.

Harvard Stem Cell Institute (HSCI) researchers at Dana-Farber Cancer Institute (DFCI) and collaborators at Brigham and Women’s Hospital (BWH) have identified a molecular marker in normal breast tissue that can predict a woman’s risk for developing breast cancer, the leading cause of death in women with cancer worldwide.

The work, led by HSCI principal faculty member Kornelia Polyak and Rulla Tamimi of BWH, was published in an early online release and in the April 1 issue of Cancer Research.

The study builds on Polyak’s earlier research finding that women already identified as having a high risk of developing cancer — namely those with a mutation called BRCA1 or BRCA2 — or women who did not give birth before their 30s had a higher number of mammary gland progenitor cells.

In the latest study, Polyak, Tamimi, and their colleagues examined biopsies, some taken as many as four decades ago, from 302 participants in the Nurses’ Health Study and the Nurses’ Health Study II who had been diagnosed with benign breast disease. The researchers compared tissue from the 69 women who later developed cancer to the tissue from the 233 women who did not. They found that women were five times as likely to develop cancer if they had a higher percentage of Ki67, a molecular marker that identifies proliferating cells, in the cells that line the mammary ducts and milk-producing lobules. These cells, called the mammary epithelium, undergo drastic changes throughout a woman’s life, and the majority of breast cancers originate in these tissues.

Doctors already test breast tumors for Ki67 levels, which can inform decisions about treatment, but this is the first time scientists have been able to link Ki67 to precancerous tissue and use it as a predictive tool.

“Instead of only telling women that they don’t have cancer, we could test the biopsies and tell women if they were at high risk or low risk for developing breast cancer in the future,” said Polyak, a breast cancer researcher at Dana-Farber and co-senior author of the paper.

“Currently, we are not able to do a very good job at distinguishing women at high and low risk of breast cancer,” added co-senior author Tamimi, an associate professor at the Harvard T.H. Chan School of Public Health and Harvard Medical School. “By identifying women at high risk of breast cancer, we can better develop individualized screening and also target risk reducing strategies.”

To date, mammograms are the best tool for the early detection, but there are risks associated with screening. False positive and negative results and over-diagnosis could cause psychological distress, delay treatment, or lead to overtreatment, according to the National Cancer Institute (NCI).

Mammography machines also use low doses of radiation. While a single mammogram is unlikely to cause harm, repeated screening can potentially cause cancer, though the NCI writes that the benefits “nearly always outweigh the risks.”

“If we can minimize unnecessary radiation for women at low risk, that would be good,” said Tamimi.

Screening for Ki67 levels would “be easy to apply in the current setting,” said Polyak, though the researchers first want to reproduce the results in an independent cohort of women.

 

AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs

William H ParsonsMatthew J Kolar, …., Barbara B KahnAlan Saghatelian & Benjamin F Cravatt

Nature Chemical Biology 28 March 2016                    http://dx.doi.org:/10.1038/nchembio.2051

Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.

Figure 1: Discovery and characterization of AIG1 and ADTRP as FP-reactive proteins in the human proteome.

 

http://www.nature.com/nchembio/journal/vaop/ncurrent/carousel/nchembio.2051-F1.jpg

(a) Competitive ABPP-SILAC analysis to identify FP-alkyne-inhibited proteins, in which protein enrichment and inhibition were measured in proteomic lysates from SKOV3 cells treated with FP-alkyne (20 μM, 1 h) or DMSO using the FP-biotin…

 

  1. Willems, L.I., Overkleeft, H.S. & van Kasteren, S.I. Current developments in activity-based protein profiling. Bioconjug. Chem. 25, 11811191 (2014).
  2. Niphakis, M.J. & Cravatt, B.F. Enzyme inhibitor discovery by activity-based protein profiling.Annu. Rev. Biochem. 83, 341377 (2014).
  3. Berger, A.B., Vitorino, P.M. & Bogyo, M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am. J. Pharmacogenomics 4,371381 (2004).
  4. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. USA 96, 1469414699 (1999).
  5. Simon, G.M. & Cravatt, B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J. Biol. Chem. 285, 1105111055 (2010).
  6. Bachovchin, D.A. et al. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 2094120946 (2010).
  7. Jessani, N. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2, 691697 (2005).
  8. Higa, H.H., Diaz, S. & Varki, A. Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes. Biochem. Biophys. Res. Commun. 144, 10991108 (1987).

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesteras-1 inhibitors

Proc Natl Acad Sci U S A. 2011 Apr 26; 108(17): 6811–6816.    doi:  10.1073/pnas.1015248108
National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

Protein phosphorylation is a pervasive and dynamic posttranslational protein modification in eukaryotic cells. In mammals, more than 500 protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues on proteins (1). A much more limited number of phosphatases are responsible for reversing these phosphorylation events (2). For instance, protein phosphatase 2A (PP2A) and PP1 are thought to be responsible together for > 90% of the total serine/threonine phosphatase activity in mammalian cells (3). Specificity is imparted on PP2A activity by multiple mechanisms, including dynamic interactions between the catalytic subunit (C) and different protein-binding partners (B subunits), as well as a variety of posttranslational chemical modifications (2, 4). Within the latter category is an unusual methylesterification event found at the C terminus of the catalytic subunit of PP2A that is introduced and removed by a specific methyltransferase (leucine carbxoylmethyltransferase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A carboxymethylation (hereafter referred to as “methylation”) has been proposed to regulate PP2A activity, at least in part, by modulating the binding interaction of the C subunit with various regulatory B subunits (810). A predicted outcome of these shifts in subunit association is the targeting of PP2A to different protein substrates in cells. PME-1 has also been hypothesized to stabilize inactive forms of nuclear PP2A (11), and recent structural studies have shed light on the physical interactions between PME-1 and the PP2A holoenzyme (12).

There were several keys to the success of our probe development effort. First, screening for inhibitors of PME-1 benefited from the fluopol-ABPP technology, which circumvented the limited throughput of previously described substrate assays for this enzyme. Second, we were fortunate that the NIH compound library contained several members of the ABL class of small molecules. These chiral compounds, which represent an academic contribution to the NIH library, occupy an unusual portion of structural space that is poorly accessed by commercial compound collections. Although at the time of their original synthesis (23) it may not have been possible to predict whether these ABLs would show specific biological activity, their incorporation into the NIH library provided a forum for screening against many proteins and cellular targets, culminating in their identification as PME-1 inhibitors. We then used advanced chemoproteomic assays to confirm the remarkable selectivity displayed by ABLs for PME-1 across (and beyond) the serine hydrolase superfamily. That the mechanism for PME-1 inhibition involves acylation of the enzyme’s conserved serine nucleophile (Fig. 3) suggests that exploration of a more structurally diverse set of ABLs might uncover inhibitors for other serine hydrolases. In this way, the chemical information gained from a single high-throughput screen may be leveraged to initiate probe development programs for additional enzyme targets.

Projecting forward, this research provides an example of how public small-molecule screening centers can serve as a portal for spawning academic collaborations between chemical biology and synthetic chemistry labs. By continuing to develop versatile high-throughput screens and combining them with a small-molecule library of expanding structural diversity conferred by advanced synthetic methodologies, academic biologists and chemists are well-positioned to collaboratively deliver pharmacological probes for a wide range of proteins and pathways in cell biology.

 

New weapon against breast cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says

April 6, 2016 | Popular
BRC_Cancer605

 

New Group of Aging-Related Proteins Discovered

http://www.genengnews.com/gen-news-highlights/new-group-of-aging-related-proteins-discovered/81252599/

Scientists have discovered a group of six proteins that may help to divulge secrets of how we age, potentially unlocking new insights into diabetes, Alzheimer’s, cancer, and other aging-related diseases.

The proteins appear to play several roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading investigators to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, M.D., dean and professor of the University of Southern California Leonard Davis School of Gerontology, identified the proteins and observed their origin from mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Dr. Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria. Dr. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body. Of particular interest is SHLP 2, according to Dr. Cohen.  The protein appears to have insulin-sensitizing, antidiabetic effects as well as neuroprotective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

Proteins That May Protect Against Age Related Illnesses Discovered

 

The cell proliferation antigen Ki-67 organises heterochromatin

 Michal Sobecki, 

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

 

A protein called Ki-67 is only produced in actively dividing cells, where it is located in the nucleus – the structure that contains most of the cell’s DNA. Researchers often use Ki-67 as a marker to identify which cells are actively dividing in tissue samples from cancer patients, and previous studies indicated that Ki-67 is needed for cells to divide. However, the exact role of this protein was not clear. Before cells can divide they need to make large amounts of new proteins using molecular machines called ribosomes and it has been suggested that Ki-67 helps to produce ribosomes.

Now, Sobecki et al. used genetic techniques to study the role of Ki-67 in mice. The experiments show that Ki-67 is not required for cells to divide in the laboratory or to make ribosomes. Instead, Ki-67 alters the way that DNA is packaged in the nucleus. Loss of Ki-67 from mice cells resulted in DNA becoming less compact, which in turn altered the activity of genes in those cells.

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Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45616/title/Microbes-Meet-Cancer

 © ISTOCK.COM/KATEJA_FN; © ISTOCK.COM/FRANK RAMSPOTT  http://www.the-scientist.com/images/April2016/feature1.jpg

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.1 The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.2

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.3 So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.4 Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF
© AL GRANBERG

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.6 That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.8

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.9 The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.11

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.12

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.13The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

 

 

© DR. GARY GAUGLER/SCIENCE SOURCE  http://www.the-scientist.com/images/April2016/immune3.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.14

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski.  “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”

http://www.the-scientist.com/images/April2016/immune4.jpg

 

Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

  • Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
  • Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
  • Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.14

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.16

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.

GENETIC CONNECTION

The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”

References

  1. N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
  2. S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
  3. J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
  4. B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
  5. J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
  6. S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
  7. J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
  8. S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
  9. J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
  10. G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
  11. J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
  12. J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
  13. V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
  14. A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
  15. M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.

……..

 

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou,  Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell    12 Jan 2015; Volume 27, Issue 1, p27–40  http://dx.doi.org/10.1016/j.ccell.2014.11.009
Figure thumbnail fx1
  • TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
  • IL-17 drives malignant progression in IL-6-unresponsive tumors
  • TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
  • A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

see also… Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment.

By Kate Yandell | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45644/title/Immune-Influence

Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame. Here are some of the hypotheses that have come out of recent research in rodents for how gut bacteria shape immunity and influence cancer.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Gut bacteria can also produce factors that lower inflammation and slow tumor growth. Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg

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Selye’s Riddle solved

Larry H. Bernstein, mD, FCAP, Curator

LPBI

 

Mathematicians Solve 78-year-old Mystery

Mathematicians developed a solution to Selye's riddle which has puzzled scientists for almost 80 years.
Mathematicians developed a solution to Selye’s riddle which has puzzled scientists for almost 80 years.

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted. In 1938, Hans Selye introduced “adaptation energy” and found strong experimental arguments in favor of this hypothesis. However, this term has caused much debate because, as it cannot be measured as a physical quantity, adaptation energy is not strictly energy.

 

Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death

Alexander N. Gorbana, , Tatiana A. Tyukinaa, Elena V. Smirnovab, Lyudmila I. Pokidyshevab,

Highlights

•   We formalize Selye׳s ideas about adaptation energy and dynamics of adaptation.
•   A hierarchy of dynamic models of adaptation is developed.
•   Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation.
•   The optimal distribution of resources for neutralization of harmful factors is studied.
•   The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted.       

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted.

In 1938, Selye proposed the notion of adaptation energy and published ‘Experimental evidence supporting the conception of adaptation energy.’ Adaptation of an animal to different factors appears as the spending of one resource. Adaptation energy is a hypothetical extensive quantity spent for adaptation. This term causes much debate when one takes it literally, as a physical quantity, i.e. a sort of energy. The controversial points of view impede the systematic use of the notion of adaptation energy despite experimental evidence. Nevertheless, the response to many harmful factors often has general non-specific form and we suggest that the mechanisms of physiological adaptation admit a very general and nonspecific description.

We aim to demonstrate that Selye׳s adaptation energy is the cornerstone of the top-down approach to modelling of non-specific adaptation processes. We analyze Selye׳s axioms of adaptation energy together with Goldstone׳s modifications and propose a series of models for interpretation of these axioms. Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation. The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted on the base of the dynamical models of adaptation. Natural selection plays a key role in the evolution of mechanisms of physiological adaptation. We use the fitness optimization approach to study of the distribution of resources for neutralization of harmful factors, during adaptation to a multifactor environment, and analyze the optimal strategies for different systems of factors.

In this work, an international team of researchers, led by Professor Alexander N. Gorban from the University of Leicester, have developed a solution to Selye’s riddle, which has puzzled scientists for almost 80 years.

Alexander N. Gorban, Professor of Applied Mathematics in the Department of Mathematics at the University of Leicester, said: “Nobody can measure adaptation energy directly, indeed, but it can be understood by its place already in simple models. In this work, we develop a hierarchy of top-down models following Selye’s findings and further developments. We trust Selye’s intuition and experiments and use the notion of adaptation energy as a cornerstone in a system of models. We provide a ‘thermodynamic-like’ theory of organism resilience that, just like classical thermodynamics, allows for economics metaphors, such as cost and bankruptcy and, more importantly, is largely independent of a detailed mechanistic explanation of what is ‘going on underneath’.”

Adaptation energy is considered as an internal coordinate on the “dominant path” in the model of adaptation. The phenomena of “oscillating death” and “oscillating remission,” which have been observed in clinic for a long time, are predicted on the basis of the dynamical models of adaptation. The models, based on Selye’s idea of adaptation energy, demonstrate that the oscillating remission and oscillating death do not need exogenous reasons. The developed theory of adaptation to various factors gives the instrument for the early anticipation of crises.

Professor Alessandro Giuliani from Istituto Superiore di Sanità in Rome commented on the work, saying: “Gorban and his colleagues dare to make science adopting the thermodynamics style: they look for powerful principles endowed with predictive ability in the real world before knowing the microscopic details. This is, in my opinion, the only possible way out from the actual repeatability crisis of mainstream biology, where a fantastic knowledge of the details totally fails to predict anything outside the test tube.1

Citation: Alexander N. Gorban, Tatiana A. Tyukina, Elena V. Smirnova, Lyudmila I. Pokidysheva. Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death. Journal of Theoretical Biology, 2016; DOI:10.1016/j.jtbi.2015.12.017. Voosen P. (2015) Amid a Sea of False Findings NIH tries Reform, The Chronicle of Higher Education.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Cardiomyocytes from mesenchmal stem cells?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Introduction: A just published article from the Gladstone Institute establishes that cardiac muscle can be generated from inducible explandable  cardiovascular progenitor cells.  However, while the study has validity, it leaves much to be explained, especially in light of the references to many previous studies to generate cardiomycytes for heart failure.

Skin Cells Opening the Door to the Possibility of Personalized Medicine for Heart Attack Patients

iceC-Figure6

https://beyondthedish.files.wordpress.com/2016/03/iecpcs-give-rise-to-cms-ecs-and-smcs-in-vivo-and-improve-cardiac-function-after-mi.jpg?w=652

 

ieCPCs Give Rise to CMs, ECs, and SMCs In Vivo and Improve Cardiac Function after MI

(A–E) Immunofluorescence analyses of RFP and CM (A), EC (B and C), and SMC (D and E) markers in tissue sections collected 2 weeks after transplanting RFP-labeled ieCPCs at passage 10 into infarcted hearts of immunodeficient mice. Scale bars represent 100 μm.

(F and G) Ejection fraction and fractional shortening of the left ventricle (LV) quantified by echocardiography. Results from two independent experiments were shown. D, days; W, weeks.

(H–J) Cardiac fibrosis was evaluated at eight levels (L1–L8) by Masson’s trichrome staining 12 weeks after coronary ligation. The ligation site is marked as X. Sections of representative hearts are shown in (I) with quantification in (J). Scar tissue (%) = (the sum of fibrotic area or length at L1–L8/the sum of LV area or circumference at L1–L8) × 100. Scale bars represent 500 μm.

(K) Quantification of LV circumference of mouse hearts 12 weeks after transplantation of 2nd MEFs or ieCPCs. Data were summarized from 48 sections for each group. Data are mean ± SE. p < 0.05.

“Cardiac progenitor cells could be ideal for heart regeneration,” said senior author Sheng Ding, PhD, a senior investigator at Gladstone. “They are the closest precursor to functional heart cells, and, in a single step, they can rapidly and efficiently become heart cells, both in a dish and in a live heart. With our new technology, we can quickly create billions of these cells in a dish and then transplant them into damaged hearts to treat heart failure.”

 

Discussion:  The study raises some important questions.

  1. How are the cultured cells different than those used in previous studies?
  2. Cardiomyocytes and fibroblasts are both of mesodermal origin.  What determines which way the stem cell line will differentiate?
  3. What is the difference, if any, between the cell culture environment and the in vivo environment into which they are placed?
  4. There is a difference between chronic hypoxemia with congestive heart failure and acute coronary syndrome.  The experiment performed would be more apt to apply to post-ACS than to chronic heart failure.

 

Functional heart muscle regenerated in decellularized human hearts

March 11, 2016    http://snip.ly/txc6j#http://medicalxpress.com/news/2016-03-functional-heart-muscle-regenerated-decellularized.html

A partially recellularized human whole-heart cardiac scaffold, reseeded with human cardiomyocytes derived from induced pluripotent stem cells, being cultured in a bioreactor that delivers a nutrient solution and replicates some of the environmental conditions around a living heart. Credit: Bernhard Jank, MD, Ott Lab, Center for Regenerative Medicine, Massachusetts General Hospital

 

Massachusetts General Hospital (MGH) researchers have taken some initial steps toward the creation of bioengineered human hearts using donor hearts stripped of components that would generate an immune response and cardiac muscle cells generated from induced pluripotent stem cells (iPSCs), which could come from a potential recipient. The investigators described their accomplishments – which include developing an automated bioreactor system capable of supporting a whole human heart during the recellularization process—earlier this year in Circulation Research.

“Generating functional cardiac tissue involves meeting several challenges,” says Jacques Guyette, PhD, of the MGH Center for Regenerative Medicine (CRM), lead author of the report. “These include providing a structural scaffold that is able to support cardiac function, a supply of specialized cardiac , and a supportive environment in which cells can repopulate the scaffold to form mature tissue capable of handling complex cardiac functions.”

The research team is led by Harald Ott, MD, of the MGH CRM and the Department of Surgery, senior author of the paper. In 2008, Ott developed a procedure for stripping the living cells from a donor organ with a detergent solution and then repopulating the remaining extracellular matrix scaffold with organ-appropriate types of cells. Since then his team has used the approach to generate functional rat kidneys and lungs and has decellularized large-animal hearts, lungs and kidneys. This report is the first to conduct a detailed analysis of the matrix scaffold remaining after decellularization of whole human hearts, along with recellularization of the cardiac matrix in three-dimensional and whole-heart formats.

The study included 73 human hearts that had been donated through the New England Organ Bank, determined to be unsuitable for transplantation and recovered under research consent. Using a scaled-up version of the process originally developed in rat hearts, the team decellularized hearts from both brain-dead donors and from those who had undergone . Detailed characterization of the remaining cardiac scaffolds confirmed a high retention of matrix proteins and structure free of cardiac cells, the preservation of coronary vascular and microvascular structures, as well as freedom from human leukocyte antigens that could induce rejection. There was little difference between the reactions of organs from the two donor groups to the complex decellularization process.

Instead of using genetic manipulation to generate iPSCs from , the team used a newer method to reprogram skin cells with messenger RNA factors, which should be both more efficient and less likely to run into regulatory hurdles. They then induced the  to differentiate into or cardiomyocytes, documenting patterns of gene expression that reflected developmental milestones and generating cells in sufficient quantity for possible clinical application. Cardiomyocytes were then reseeded into three-dimensional matrix tissue, first into thin matrix slices and then into 15 mm fibers, which developed into spontaneously contracting tissue after several days in culture.

The last step reflected the first regeneration of human heart muscle from within a cell-free, human whole-heart matrix. The team delivered about 500 million iPSC-derived cardiomyocytes into the left ventricular wall of decellularized hearts. The organs were mounted for 14 days in an automated bioreactor system developed by the MGH team that both perfused the organ with nutrient solution and applied environmental stressors such as ventricular pressure to reproduce conditions within a living heart. Analysis of the regenerated tissue found dense regions of iPSC-derived cells that had the appearance of immature cardiac muscle tissue and demonstrated functional contraction in response to electrical stimulation.

“Regenerating a whole heart is most certainly a long-term goal that is several years away, so we are currently working on engineering a functional myocardial patch that could replace cardiac tissue damaged due a heart attack or heart failure,” says Guyette. “Among the next steps that we are pursuing are improving methods to generate even more – recellularizing a whole heart would take tens of billions—optimizing bioreactor-based culture techniques to improve the maturation and function of engineered cardiac tissue, and electronically integrating regenerated tissue to function within the recipient’s heart.”

Team leader Ott, an assistant professor of Surgery at Harvard Medical School, adds, “Generating personalized functional myocardium from patient-derived cells is an important step towards novel device-engineering strategies and will potentially enable patient-specific disease modeling and therapeutic discovery. Our team is excited to further develop both of these strategies in future projects.”

Explore further: A tool for isolating progenitor cells from human heart tissue could lead to heart repair

More information: Jacques P. Guyette et al. Bioengineering Human Myocardium on Native Extracellular MatrixNovelty and Significance, Circulation Research (2016). DOI: 10.1161/CIRCRESAHA.115.306874

 

Stem cell study in mice offers hope for treating heart attack patients

February 15, 2012  http://medicalxpress.com/news/2012-02-stem-cell-mice-heart-patients.html

 

Stem cell study in mice offers hope for treating heart attack patients

Cardiac stem cells, pictured here, give hope to patients who have suffered a heart attack. Credit: UCSF

A UCSF stem cell study conducted in mice suggests a novel strategy for treating damaged cardiac tissue in patients following a heart attack. The approach potentially could improve cardiac function, minimize scar size, lead to the development of new blood vessels – and avoid the risk of tissue rejection.

In the investigation, reported online in the journal PLoS ONE, the researchers isolated and characterized a novel type of cardiac stem cell from the tissue of middle-aged mice following a .

Then, in one experiment, they placed the in the culture dish and showed they had the ability to differentiate into cardiomyocytes, or “beating heart cells,” as well as endothelial cells and smooth muscle cells, all of which make up the heart.

In another, they made copies, or “clones,” of the cells and engrafted them in the tissue of other of the same genetic background who also had experienced heart attacks. The cells induced angiogenesis, or blood vessel growth, or differentiated, or specialized, into endothelial and smooth muscle cells, improving .

“These findings are very exciting,” said first author Jianqin Ye, PhD, MD, senior scientist at UCSF’s Translational Cardiac Stem Cell Program. First, “we showed that we can isolate these cells from the heart of middle-aged animals, even after a heart attack.” Second, he said, “we determined that we can return these cells to the animals to induce repair.”

Importantly, the stem cells were identified and isolated in all four chambers of the heart, potentially making it possible to isolate them from patients’ hearts by doing right ventricular biopsies, said Ye. This procedure is “the safest way of obtaining cells from the heart of live patients, and is relatively easy to perform,” he said.

“The finding extends the current knowledge in the field of native cardiac progenitor cell therapy,” said senior author Yerem Yeghiazarians, MD, director of UCSF’s Translational Cardiac Stem Cell Program and an associate professor at the UCSF Division of Cardiology. “Most of the previous research has focused on a different subset of cardiac progenitor cells. These novel cardiac precursor cells appear to have great therapeutic potential.”

The hope, he said, is that patients who have severe heart failure after a heart attack or have cardiomyopathy would be able to be treated with their own cardiac stem cells to improve the overall health and function of the heart. Because the cells would have come from the patients, themselves, there would be no concern of cell rejection after therapy.

The cells, known as Sca-1+ stem enriched in Islet (Isl-1) expressing cardiac precursors, play a major role in cardiac development. Until now, most of the research has focused on a different subset of cardiac progenitor, or early stage, cells known as, c-kit cells.

The Sca-1+ cells, like the c-kit cells, are located within a larger clump of cells called cardiospheres.

The UCSF researchers used special culture techniques and isolated Sca-1+ cells enriched in the Isl-1expressing cells, which are believed to be instrumental in the heart’s development. Since Isl-1 is expressed in the cell nucleus, it has been difficult to isolate them but the new technique enriches for this cell population.

The findings suggest a potential treatment strategy, said Yeghiazarians. “Heart disease, including heart attack and heart failure, is the number one killer in advanced countries. It would be a huge advance if we could decrease repeat hospitalizations, improve the quality of life and increase survival.” More studies are being planned to address these issues in the future.

An estimated 785,000 Americans will have a new heart attack this year, and 470,000 who will have a recurrent attack. Heart disease remains the number one killer in the United States, accounting for one out of every three deaths, according to the American Heart Association.

Medical costs of cardiovascular disease are projected to triple from $272.5 billion to $818.1 billion between now and 2030, according to a report published in the journal Circulation.

 

Sca-1+ Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and Improve Cardiac Function after Myocardial Injury

Jianqin Ye , Andrew Boyle , Henry Shih , Richard E. Sievers , Yan Zhang , William Grossman , Harold S. Bernstein , Yerem Yeghiazarians
http://dx.doi.org:/10.1371/journal.pone.0030329

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1+CD45 cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1+CD45 cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45 cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance  

These studies demonstrate that cloned Sca-1+CD45 cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.

 

Incorporation of Mg particles into PDLLA regulates mesenchymal stem cell and macrophage responses

Sandra C. Cifuentes1, Fátima Bensiamar2,3, Amparo M. Gallardo-Moreno3,4, Tim A. Osswald5, José L. González-Carrasco1,3, et al.
J Biomed Materials Res Part A  104(4), pages 866–878, April 2016                    http://dx.doi.org:/10.1002/jbm.a.35625

In this work, we investigated a new approach to incorporate Mg particles within a PDLLA matrix using a solvent-free commercially available process. PDLLA/Mg composites were manufactured by injection moulding and the effects of Mg incorporated into PDLLA on MSC and macrophage responses were evaluated. Small amounts of Mg particles (≤1 wt %) do not cause thermal degradation of PDLLA, which retains its mechanical properties. PDLLA/Mg composites release hydrogen, alkaline products and Mg2+ ions without changing pH of culture media. Mg-containing materials provide a noncytotoxic environment that enhances MSC viability. Concentration of Mg2+ ions in extracts of MSCs increases with the increment of Mg content in the composites. Incorporation of Mg particles into PDLLA stimulates FN production, ALP activity, and VEGF secretion in MSCs, an effect mediated by degradation products dissolved from the composites. Degradation products of PDLLA induce an increase in MCP-1, RANTES, and MIP-1α secretion in macrophages while products of composites have minimal effect on these chemokines. Regulation of MSC behavior at the biomaterial’s interface and macrophage-mediated inflammatory response to the degradation products is related to the incorporation of Mg in the composites. These findings suggest that including small amounts of Mg particles into polymeric devices can be a valuable strategy to promote osseointegration and reduce host inflammatory response. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 866–878, 2016.

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