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Posts Tagged ‘oncogenes’


How cancer metastasis occurs

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

How Cancer Cells Slide along Narrow Path to Metastasis

http://www.genengnews.com/gen-news-highlights/how-cancer-cells-slide-along-narrow-path-to-metastasis/81252674/

In tumors, abnormal protein-fiber environments and genetic perturbations conspire to give rise to metastatic behavior. In this looking-glass world, cells that bump into each other do not halt and reverse direction, as they ordinarily would. Instead, they slide around each other, enhancing migratory potential and bringing to mind the portmanteau “slithy,” which Lewis Carroll invented to describe the behavior of some of his imaginary creatures.

Slithy cancer cells do gyre and gimble in the tumor microenvironment, a looking-glass world in which abnormal protein-fiber scaffolds and genetic perturbations coincide, creating conditions that promote metastasis. Some cancer cells manage to circumnavigate or slide around other cells on protein fibers, and these cells can take relatively straightforward paths out of a primary tumor. Other cells, however, are more likely to turn back upon encountering other cells. They exit tumors less efficiently.

To understand how some cancer cells migrate more efficiently than others, researchers based at Northeastern University undertook a biophysical study. They developed a model environment that mimics protein fibers. First they stamped stripes of a protein called fibronectin on glass plates, making sure to represent various widths. Then they deposited the cells—alternately hundreds of breast cancer cells and hundreds of normal cells—on these fiber­like stripes and used a microscope with time-lapse capabilties to observe and quantify their behavior.

On fibers that were 6 or 9 microns wide—the typical size of fibers in tumors—half the breast cancer cells elongated and slid around the cells they collided with. Conversely, 99% of the normal breast cells did an about face.

To under­stand what gave the cancer cells this remarkable agility, the Northeastern researchers, led by Anand Asthagiri, explored the influence of fiber widths and genetic perturbations. They presented their results April 26 in the Biophysical Journal, in an article entitled, “Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.”

“Downregulating the cell–cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB–231 cells increases the micropattern dimension at which they slide,” wrote the article’s authors.

This finding led the Northeastern team to consider the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement.

“Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD,” the article’s authors continued. “Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior.”

Asthagiri’s system is relatively easy to construct and suited for rapid imaging—two qualities that make it an excellent candidate for screening new cancer drugs. Pharmaceutical companies could input the drugs along with the cancer cells and mea­sure how effectively they inhibit sliding.

In the future, the system could also alert cancer patients and clinicians before metastasis starts. Studies with patients have shown that the structure of a tumor’s protein-fiber scaffolding can indicate how far the disease has progressed. The researchers found that certain aggressive genetic mutations enabled cells to slide on very narrow fibers, whereas cells with milder mutations would slide only when the fibers got much wider. Clinicians could biopsy the tumor and mea­sure the width of the fibers to see if that danger point were approaching. “We can start to say, ‘If these fibers are approaching X microns wide, it’s urgent that we hit certain path­ways with drugs,” said Asthagiri.

Questions, of course, remain. Do other types of cancer cells also have the ability to slide? What additional genes play a role?

Next steps, says Asthagiri, include expanding their fiber­like stripes into three-dimensional models that more closely represent the fibers in actual tumors and testing cancer and normal cells together. “There are so many types of cells in a tumor environment—immune cells, blood cells, and so on,” he noted. “We want to better emulate what’s hap­pening in the body rather than in isolated cells interacting on a platform.”

 

Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.

sjwilliamspa

There was a nice paper a few years ago by Dr. Edna Cukerman from Fox Chase showing how tumor cells slid down on fiber tracks generated from tumor stromal cells and how this pattern of movement is not as random as one would think. if extracellular matrix was generated from normal stromal cells you would not find athis type of coordinated movement.

 

 

Fatty acid oxidation disruption: a therapeutic alternative for triple negative breast cancer

Hormone therapy is ineffective against triple negative breast cancers (TNBC) as they lack HER2, Estrogen, and Progesterone receptors. Therefore new targetable pathways are needed to halt the cancer’s progression. Researchers at UCSF have outlined a means of treating TNBC through disruption of fatty acid oxidation (FAO). The pathway was first revealed as a potential target through metabolomics and gene signatures, identifying upregulated FAO intermediates in MYC-overexpressing TNBC samples. Considering the location, in the proximity of adipose-rich mammary glands, breast cancer FAO dependence pathway seemed to be a logical pathway. Subsequent inhibition of FAO with etomixir , an inhibitor of a major enzyme carnitine palmitoyltransferase 1 (CPT1) in the FAO pathway, lead to dramatic decreases in ATP production in MYC-overexpressing cell lines. Although a decrease in proliferation of cells in culture was observed viability remained unchanged. However, further testing of etomixir in vivo within patient derived xenograft models increased success of FAO disruption with a 4 to 6-fold decrease in relative tumor volume. The differential performance between in vitro and in vivo treatments indicates a need to recapitulate the actual tumor environment when studying metabolic manipulation regimens.

Camarda, et. al. Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.  Nature Medicine   

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

Roman CamardaAlicia Y ZhouRebecca A Kohnz,….,Daniel K Nomura & Andrei Goga
Nature Medicine22,427–432(2016)
       
              http://dx.doi.org:/10.1038/nm.4055

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor–, progesterone receptor– or human epidermal growth factor 2 receptor–positive (RP) breast cancer1, 2. We and others have shown that MYC alters metabolism during tumorigenesis3, 4. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient–derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer.

 

3 Dimensional Ex-Vivo for In-situ Tumor Growth

Brain tumors are both difficult to treat and hard to study because of the organ they affect. The structure of the brain is extremely sensitive to alterations. Until recently the study of architectural alterations and their effects was mostly restricted to in vivo experiments. Typical culturing of brain tissue requires disaggregation and manipulation into a 2-dimensional format, losing any anatomically relevant structure. To study the in situ brain structure, a new technique has been described by researchers from the University of Erlangen-Nürnberg. By carefully sectioning the brains of 4 day-old mice and placing them on a 0.4 uM pore-size transwell membrane 6 well plate insert within required culture medium, they were able to study the endogenous structure under varying conditions. They injected astrocytes or glioma cells with a micropipette into the slices, and investigated the structural changes brain tumors effect in their environment. Termed the Vascular Organotypic Glioma Impact Model (VOGIM), it revealed all the characteristic pathological alterations normally associated with the disease in vivo such as tumor size and borders, vessel length, vessel junctions, and vessel branches, microglia, cell survival, and neuronal modifications. As this method allows for live cell fluorescent observation, they employed the technique to observe cultures treated with the chemotherapeutic Temozolamide (TMZ, Temodal/Temcad®). Indeed they found reduced tumor growth in treatment groups vs controls, but also revealed surprising reduction in microglial cells in the peritumoral region. Additionally, they were able to observe the lack of response TMZ elicited from microglial in healthy regions of the tissue, despite its overall reduction in vascularization towards normal levels. The VOGIM technique allows for ex vivo study of brain tissue requiring three dimensional measurements, but may also be extended to other tissues with unique morphology such as kidney, liver, and intestine.

Ghoochani, et al. (December, 2015) A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain ONCOTARGET

 

A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain

Ali Ghoochani1, Eduard Yakubov1, Tina Sehm1, Zheng Fan1, Stefan Hock1, Michael Buchfelder1, Ilker Y. Eyüpoglu1,*, Nicolai Savaskan1,*
http://dx.doi.org:/10.18632/oncotarget.6550      PDF |  HTML

Primary brain tumors are hallmarked for their destructive activity on the microenvironment and vasculature. However, solely few experimental techniques exist to access the tumor microenvironment under anatomical intact conditions with remaining cellular and extracellular composition. Here, we detail an ex vivo vascular glioma impact method (VOGIM) to investigate the influence of gliomas and chemotherapeutics on the tumor microenvironment and angiogenesis under conditions that closely resemble the in vivo situation. We generated organotypic brain slice cultures from rats and transgenic mice and implanted glioma cells expressing fluorescent reporter proteins. In the VOGIM, tumor-induced vessels presented the whole range of vascular pathologies and tumor zones as found in human primary brain tumor specimens. In contrast, non-transformed cells such as primary astrocytes do not alter the vessel architecture. Vascular characteristics with vessel branching, junctions and vessel length are quantitatively assessable as well as the peritumoral zone. In particular, the VOGIM resembles the brain tumor microenvironment with alterations of neurons, microglia and cell survival. Hence, this method allows live cell monitoring of virtually any fluorescence-reporter expressing cell. We further analyzed the vasculature and microglia under the influence of tumor cells and chemotherapeutics such as Temozolamide (Temodal/Temcad®). Noteworthy, temozolomide normalized vasculare junctions and branches as well as microglial distribution in tumor-implanted brains. Moreover, VOGIM can be facilitated for implementing the 3Rs in experimentations. In summary, the VOGIM represents a versatile and robust technique which allows the assessment of the brain tumor microenvironment with parameters such as angiogenesis, neuronal cell death and microglial activity at the morphological and quantitative level.

 

 

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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

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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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Significance of Oncogenes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Epigenetics and Cancer Causing Genes

    Mar 13, 2015   https://www.linkedin.com/pulse/epigenetics-cancer-causing-genes-oncogenes-kleon-tona

 

Epigenetics and Cancer Causing Genes (Oncogenes)

 

I. Cancer Causing Genes?
I have read an article recently that explained cancer as just plain “bad luck.” I do not believe this to be the case. There are harmful initiators of cancer and it can be associated with unfortunate timing, exposures, poor protections, faulty adaptation and circumstances beyond control. There are approximately 2,619 known genes that are associated with cancer expression and I am confident that this number will change as more research continues. These types of genes are also called oncogenes. These genes can be directly or indirectly associated with cancer initiation, progression, expression, survival and demise.

II. Epigenetics?
Epigenetics plays an important role in the turn on or off signaling of these genes by influencing DNA protection mechanisms without altering the DNA sequencing. Epigenetics refers to high-level information residing above the genetic code. While each cell in the body is equipped with the same genetic manual, epigenetic instructions tell cells how to make a difference. These instructions determine the access to pages with genetic information by directing the way the DNA is packaged into chromatin. DNA organized in loose chromatin is readily available for gene expression. Conversely, DNA tightly packed into dense chromatin has the letters of genetic code effectively buried and unavailable for reading and transcription. Distinct epigenetic marks decide which sets of genes may be expressed and which genes are kept silent.

III. DNA Methylation Hypomethylation and Hypermethylation?
In previous articles (blogs), we discussed the methylation process of DNA. Simply, methylation is the mechanism by which DNA is in homeostasis (healthy sequencing) or in harmful dysfunctional sequencing. Hypomethylation (decreased or low) and Hypermethylation (increased or high) are the two extremes that initiate harmful consequences upon DNA and ultimately lead to and result in unfavorable conditions and change in DNA sequencing performance. Ultimately, this leads to unfavorable function and potentially bad signals altering cell health and survival, predisposing new cell lines to “bad” mutations.

DNA methylation patterns undergo complex changes in cancer. The total amount of methylated cytosine is usually decreased resulting in global (extensive) hypomethylation. Decreased cytosine methylation typically affects satellite DNA, repetitive sequences, and CpG sites. In genetics, CpG is a site where cytosine (C) lies next to guanine (G) in the DNA sequence. (The p indicates that C and G are connected by a phosphodiester bond.) Methylation of DNA occurs at any CpG site. The cause of reduced amount of methylcytosine observed in human tumors has not been determined and remains under investigation. Despite global hypomethylation, high activity of DNA methyltransferases has been detected in multiple human tumor types. This increase may be related to higher proliferation rate of malignant cells.
Besides global (extensive) hypomethylation, most cancers also show focal hypermethylation in distinct subsets of promoter-associated CpG islands as well. Affected genes are permanently silenced, since methylation marks are propagated through mitosis and are maintained in the malignant clone. Aberrant (diverging from normal) hypermethylation occurring in transformed cells serves as an alternative mechanism for inactivation of tumor suppressor genes. Hundreds to thousands of genes can be epigenetically silenced by CpG island hypermethylation in human cancer suggesting a general disturbance of epigenetic memory. Methylation affects individual cancer patients with varying extent. While some patients have minimal changes, others show concordant hypermethylation of multiple genes. This phenomenon was first described as CpG island methylator phenotype (CIMP) in colorectal cancer and confirmed in many other types of cancer and leukemia. Epigenetic DNA methylation changes in cancer appear to be considerably more frequent events than genetic mutations. Mass sequencing of more than 20,000 transcripts in breast and colorectal cancers revealed about 80 harmless and less than 15 potentially oncogenic mutations per tumor. Balanced methylation and protective epigenetics are essential in minimizing cancer-causing mutations.

IV. Not Just Bad Luck!
Cancer is, in fact, a scary diagnosis and it is unfortunate when it occurs. However, I would not term it “bad luck,” as this implies a powerless and hopeless end. The gift of life is filled with miraculous movements that defy the laws of nature. Protective epigenetics can be considered miraculous and spontaneous and is what becomes necessary to move toward favorable adaptations that increase health, longevity and survival. Moreover, it is the mechanism by which succeeding familial generations can benefit in protections against cancer. It’s not just about our own protective actions on gene expression but also for the protective adaptability of future generations. There is hope and record of individuals having spontaneous regression of cancer. I have treated many in my clinical career! Encourage the expression of your healthy genes by protective epigenetics!

Your Genetic Solution

V. For Your Interest and Exploration (2,619)

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