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Posts Tagged ‘post translational modifications’


Lipids link to breast cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Lipids Found Critical to Breast Cancer Cell Proliferation

http://www.genengnews.com/gen-news-highlights/lipids-found-critical-to-breast-cancer-cell-proliferation/81252572/

http://www.genengnews.com/Media/images/GENHighlight/112523_webApr6_2016_IRBBarcelona_BreastTumorsLIPD3119618425.jpg

 

Scientists in Spain report finding that breast cancer cells need to take up lipids from the extracellular environment so that they can continue to proliferate. The main protein involved in this process is LIPG, an enzyme found in the cell membrane and without which tumor cell growth is arrested. Analyses of more than 500 clinical samples from patients with various kinds of breast tumors reveal that 85% have high levels of LIPG expression.

The research (“FoxA and LIPG Endothelial Lipase Control the Uptake of Extracellular Lipids for Breast Cancer Growth”) is published in Nature Communications.

In Spain, breast cancer is the most common tumor in women and the fourth most common type in both sexes (data from the Spanish Society of Medical Oncology, 2012), registering more than 25,000 new diagnoses each year. According to figures from the World Health Organization, every year 1.38 million new cases of breast cancer are diagnosed and 458,000 people die from this disease (International Agency for Research on Cancer Globocan, 2008).

It was already known that cancer cells require extracellular glucose to grow and that they reprogram their internal machinery to produce greater amounts of lipids. The relevance of this study is that it reveals for the first time that tumor cells must import extracellular lipids to grow.

“This new knowledge related to metabolism could be the Achilles heel of breast cancer,” explains ICREA researcher and Institute for Research in Biomedicine–Barcelona group leader Roger Gomis, Ph.D., co-leader of the study together with Joan J. Guinovart, Ph.D., director of IRB Barcelona and professor at the University of Barcelona. Using animal models and cancer cell cultures, the scientists have demonstrated that blocking of LIPG activity arrests tumor growth.

“What is promising about this new therapeutic target is that LIPG function does not appear to be indispensable for life, so its inhibition may have fewer side effects than other treatments,” explains the first author of the study, Felipe Slebe, a Ph.D. Fellow at IRB Barcelona.

According to Dr. Guinovart, “because LIPG is a membrane protein, it is potentially easier to design a pharmacological agent to block its activity.”

“If a drug were found to block its activity, it could be used to develop more efficient chemotherapy treatments that are less toxic than those currently available,” adds Dr. Gomis.

The scientists are now looking into international collaborations for developing LIPG inhibitors.

FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

Felipe SlebeFederico RojoMaria Vinaixa,…Joan AlbanellJoan J. Guinovart & Roger R. Gomis

Nature Communications7,Article number:11199      http://dx.doi.org:/10.1038/ncomms11199

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids.

FoxA1 and FoxA2 in BCa growth

The importance of FoxA1 in BCa cells differentiation and its contribution to controlling the expression of metabolic genes in several other tissues makes this transcription factor a highly attractive target to explain the metabolic alterations reported in BCa. For these reason, we decided to ascertain the metabolic processes controlled by FoxA1 in BCa. We first confirmed the association between high FoxA1 expression (mRNA and protein) and luminal subtype (Fig. 1a). To this end, we used two cohorts of primary breast tumours with annotated clinical features and follow-up. The MSKCC/EMC BCa data set is based on gene expression profiles from an original series of 560 cases10, whereas the Spanish BCa data set (n=439) is a tissue microarray of formalin-fixed paraffin-embedded stage I–III breast tumour specimens11 (details provided in Methods Section). High FoxA1 gene expression significantly correlated with high expression of well-established luminal markers, such as GATA3 and ESR1, in primary tumours (Supplementary Fig. 1a). Next we explored FoxA1 expression beyond the luminal subtype. Lower FoxA1 expression was observed in non-luminal tumours (Fig. 1a,b); however, a subset also expressed higher FoxA1 levels (Supplementary Fig. 1b and Supplementary Table 1). Given that FoxA2, in conjunction with FoxA1, is also involved in the regulation of several metabolic pathways, we determined the expression of this factor in BCa samples. Unfortunately, no FoxA2 probes in the Affymetrix platform used in the MSKCC/EMC data set provided a reliable interpretation. To overcome this limitation, we used tissue arrays of early BCa samples (Spanish BCa set). Histological examination of FoxA2-stained tissue microarray slides from the Spanish BCa set revealed the expression of this factor in six non-luminal samples, which were scored as FoxA1 (examples in Fig. 1b and summarized inSupplementary Table 1). Collectively, the number of FoxA+ BCa samples detected by immunohistochemistry accounted for 81.3% of all samples in the Spanish BCa set (Supplementary Table 1), which represent a significant proportion of BCa and point to the participation of FoxA in this disease, beyond to its involvement in differentiation and control of hormonal responses.

Figure 1: FoxA1 and FoxA2 in BCa growth.

http://www.nature.com/ncomms/2016/160405/ncomms11199/images_article/ncomms11199-f1.jpg

(a, top) FoxA1 mRNA expression in the MSKCC/EMC set. BCa samples were stratified in Luminal A, Luminal B, Her2, triple negative and unknown subgroups. The unknown group represents specimens that were not classified in any group. (bottom) FoxA1 protein levels by IHC staining in Luminal, Her2 and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (b) FoxA1 and FoxA2 IHC staining in FFPE human specimens representative of the different BCa subtypes. Six independent cases are depicted. FoxA1 and FoxA2 are expressed mainly in the nuclei of tumour cells. Scale bar, 50μm. (c) FoxA1 and FoxA2 mRNA expression analysis by qRT-PCR and protein expression by western blot in human BCa cell lines compared with HMECs. T-test was used. Data are average±s.e.m.; n= 3. Of note, MDA435 are of melanoma origin. (d) FoxA1 and FoxA2 expression in MCF7, MDA231 and their derivatives cells by qRT-PCR and western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of FoxA2 in MCF7 cells or FoxA1 in MDA231 cells. Cell populations were cultured in the presence or absence of doxycycline for 6 days. P value is the result of T-test. Data are average±s.e.m.;n=3. *P≤0.05, ***P≤0.001 (e, left) Schematic representation of MDA231 and MCF7 cells grown without doxycycline and inoculated in Balb/c nude mice treated with or without doxycycline to induce the expression of the indicated FoxA short hairpins. All tumour cell lines have GFP constitutive expression, and tRFP concomitantly with the short hairpin were expressed in doxycycline treated tumours. (right) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05,**P≤0.01, ***P≤0.001. FFPE, formalin-fixed paraffin-embedded.

Next, we extended our analysis to BCa cell lines for further mechanistic studies. We compared FoxA1 and FoxA2 mRNA expression in four estrogen receptor positive (ER+) (MCF7, T47D, BT474 and ZR75) and four estrogen receptor negative (ER−) (SKBR3, MDA468, BT20 and MDA231) BCa cell lines, a cell line of melanoma origin (MDA435), and human mammary epithelial cells (HMECs). Of note, two of the BCa lines tested were HER2+ (BT474 and SKBR3) (Fig. 1c). All ER+ BCa cells (MCF7, T47D, BT474 and ZR75), the ER−/HER2+ SKBR3 and both triple negative-like MDA468 and BT20 cell lines expressed FoxA1. Interestingly, MDA231 triple negative-like cells expressed high levels of FoxA2 but not FoxA1, and the non-tumour HMECs did not express these factors (Fig. 1c). No BCa cells co-expressed these two proteins (Fig. 1c). Our results suggest that the expression of FoxA transcription factors is a common feature of breast tumours, as well as of BCa cell lines. This notion implies that FoxA factors play a major role in BCa growth, independently of luminal fate specification.

To examine the molecular basis of the contribution of FoxA1 and FoxA2 to BCa growth, we engineered constitutive GFP-luciferase-expressing MCF7 and MDA231 cells with a doxycycline-inducible short-hairpin RNA (sh-RNA) vector targeting either FoxA1 or FoxA2. Doxycycline addition to the cell culture media decreased FoxA expression in both cell lines compared with control cells (ShControl (Dox+) and Sh FoxA1 or Sh FoxA2 (Dox−))(Fig. 1d), with the concomitant expression of tRFP (Supplementary Fig. 1c). Of note, there was no gain of expression of FoxA2 in FoxA1-depleted cells or vice versa (Fig. 1d). Interestingly, cancer cell proliferation was impaired in vitroupon depletion of either FoxA1 or FoxA2 in MCF7 and MDA231 cells, respectively (Supplementary Fig. 1d,e). Similarly, when Balb/c nude mice implanted with xenograft tumours from the above described cellular populations were treated with doxycycline and the short hairpins were induced, striking differences in tumour growth were observed. FoxA1-depleted MCF7 and FoxA2-depleted MDA231 tumour growth was blunted (Fig. 1e and additional controls in Supplementary Fig. 1f. Experimental details in the Supplementary Methods Section). Collectively, these observations confirm that FoxA1 or FoxA2 expression is required for BCa growth.

Previous studies indicate that FoxA1 and FoxA2 transcriptionally regulate common genes in the liver and pancreas that are central to development and metabolism. We therefore hypothesized that crossed expression of FoxA factors could rescue tumour growth by restoring the expression of essential metabolic genes. To this end, we engineered doxycycline-driven shFoxA1 MCF7 cells to express exogenous FoxA2 and doxycycline-driven shFoxA2 MDA231 cells to express exogenous FoxA1 (Fig. 1d). Interestingly, when these BCa modified cells were implanted in Balb/c nude mice and FoxA depletion was induced with doxycycline, the sustained expression of another FoxA factor (FoxA2 in MCF7 and FoxA1 in MDA231 cells) was sufficient for tumours to continuously grow (Fig. 1e and additional controls in Supplementary Fig. 1f). Quantitative real-time PCR (qRT-PCR) analysis confirmed FoxA expression in the distinct tumour populations ex-vivo (Supplementary Fig. 1g). These results showed that retention of minimal levels of FoxA1 or FoxA2 expression is necessary for BCa cell growth.

FoxA1- and FoxA2-regulated transcripts for BCa growth

Figure 2: A genomic approach to identify FoxA1- and FoxA2-regulated transcripts in MCF7 and MDA231 cells.

http://www.nature.com/ncomms/2016/160405/ncomms11199/images_article/ncomms11199-f2.jpg

(a) FACS profiling of MCF7 and MDA231 cells derived from tumours isolated from mice on the basis of the expression of GFP+ and RFP− (control group) or GFP+ and tRFP+ (knockdown and rescue groups). (b) Representation of the transcripts up- and downregulated by FoxA in MCF7 and MDA231 cells isolated from tumours. Up- and downregulated transcripts present a Bayesian false discovery rate below 5% and fold change >2.5. (c) LIPG, Bcl2 and Cdh11OB mRNA levels of the indicated genetically modified MCF7 and MDA231 tumour xenografts analysed by qRT-PCR. P value is the result of T-test. Data are average±s.e.m.; n= 5–8 tumours. *P≤0.05, ***P≤0.001. (d) LIPG protein expression in constitutive shFoxA1 MCF7 or shFoxA2 MDA231 cells. (e) Promoter reporter assay in HEK 293 cells. Cells were transfected with LIPG promoter reporter and FoxA1 or FoxA2 expressing vectors when indicated. P value is the result of T-test. Data are average±s.e.m.; n=3. ****P≤0.0001.

LIPG expression in BCa

Next, we showed that LIPG expression in primary tumours was specific to BCa tumour cells and not to other stroma cellular entities (Fig. 3a). Subsequently, we tested LIPG expression in normal breast epithelia and interrogated 20 samples from mammoplasty reductions. Normal breast epithelial cells showed a lower expression of LIPG than cells from tumour specimens (Fig. 3b). Similar results were obtained for LIPG protein levels in a panel from BCa lines compared with HMEC cells. Of the cellular populations tested, the eight BCa cell lines expressing FoxA1 or FoxA2 had very high levels of LIPG protein compared with the melanoma MDA435 cell line and the human epithelial cell (Fig. 3c). Consistent with this observation, 83.8% of BCa samples in the Spanish tumour cohort were LIPG+ (Fig. 3d and Supplementary Table 3), and LIPG expression correlated with FoxA expression (Spearman correlation; r=0.477, P=0.000001; Fig. 3e). Further analysis showed that LIPG expression levels in primary tumours do not have the capacity to stratify patients for differential risk of overall or disease-free survival (Supplementary Fig. 2a) and are not dependent on estrogen signalling (Supplementary Fig. 2b), thus reinforcing the notion that LIPG is essential for BCa growth.

Figure 3: LIPG contributes to BCa growth.

http://www.nature.com/ncomms/2016/160405/ncomms11199/images_article/ncomms11199-f3.jpg

a) Representative LIPG IHC staining on primary BCa tissues (cohort of 439 BCa patients). LIPG is expressed in the cytoplasm of tumour cells. Faint staining is also detected in the extracellular area. Scale bar, 50μm. (b) Representative LIPG IHC staining in normal breast tissue from mammoplasty reductions. Weak LIPG expression occurs in epithelial cells from ducts and lobuli. Scale bar, 50μm. (c) LIPG protein expression in human cancer cell lines compared with HMECs. Actin was used as loading control.*Unspecific band. Of note, MDA435 are of melanoma origin. (d) LIPG protein levels by IHC staining in Luminal, Her2, and triple negative samples in the Spanish BCa set (cohort of 439 BCa patients). Data is average±s.d. (e) Spearman correlation (P=0.000001) between FoxA and LIPG IHC staining intensities in Spanish BCa set (cohort of 439 BCa patients). (f) Left panel, in vitro proliferation curves of MCF7 and MDA231 cells transduced with a control or a LIPG short hairpin. Data are average±s.e.m.; n=3. (right) LIPG protein expression in shLIPG MCF7 and shLIPG MDA231 cells. The blot shown is representative of three independent experiments. P value is the result of T-test.**P≤0.01, ***P≤0.001. (g) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points.P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05.

LIPG is a phospholipase located in the cytosol and cellular membrane and has been shown to hydrolyse extracellular phospholipids from high-density lipoprotein that are afterwards incorporated into intracellular lipid species thus providing lipid precursors of cell metabolism17, 18. Thus we questioned whether LIPG regulates essential lipid intake in BCa and whether it is necessary for proliferation. To validate this hypothesis, we genetically downregulated the expression of this protein in MCF7 and MDA231 cells by means of sh-RNA (Fig. 3f and Supplementary Fig. 2c). LIPG depletion blunted BCa cell capacity to proliferate in vitro (Fig. 3f), as previously observed in FoxA-depleted cells (Supplementary Fig. 1d,e), and caused a reduction in invasion and self-renewal properties (Supplementary Fig. 3a–d). Similarly, LIPG-depleted cells were unable to grow tumours in vivo (Fig. 3g).

LIPG induces BCa cells lipid metabolic reprograming

Figure 4: LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.

LIPG regulates the uptake of lipids in BCa cells inducing a lipid metabolic reprograming.

http://www.nature.com/ncomms/2016/160405/ncomms11199/images_article/ncomms11199-f4.jpg

(a) Schematic representation of LIPG action. (b) Heat map representation of the downregulated (blue) lipids identified by MS/MS in the cell homogenates of MCF7 or MDA231 LIPG-depleted cells compared with shControl cells. Depicted lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-testn=5. (c) Downregulated lipid species (previously identified in b) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (d) Heat map representation of the upregulated (red) lipids identified by MS/MS in the media of MCF7 or MDA231 LIPG-depleted cells compared with the corresponding shControl cells. Characterized lipids have a fold change >1.5 and P value<0.05 using the Welch’s t-test n=5. (e) Upregulated lipid species in the media (previously identified in d) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG cells (blue box). P values are <0.05 and calculated using Welch’s t-test, n=5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. (f) Heat map representation of the MS/MS downregulated (blue) lipids in the cell media of MCF7/MDA231 LIPG-depleted or shControl cells (as described in d) compared with fresh medium (without cell incubation). Depicted lipid species have a log2 fold change>1.5 and P value<0.05 using the Welch’s t-test n=5. (g) MDA231 and MCF7 cell growth for 48h in complete medium: medium containing 10% FBS 10%); lipoprotein-free medium: medium containing 10% free lipoprotein FBS; and LPC (18:0): medium containing 10% free lipoprotein FBS and 20μM of LPC (18:0). P value is the result of T-test. Data are average±s.e.m.; n=3. **P≤0.01, ***P≤0.001, ****P≤0.0001. (h) Above, schematic representation of the experimental protocol used. (bottom) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice treated with high-fat diet (HFD) are determined at the indicated time points. P value is the result of T-test. Data are average±s.e.m.; n= 6–8 tumours. *P≤0.05, **P≤0.01. Inside graph, plasma cholesterol levels of animals treated with standard diet (SD) or HFD. P value is the result of T-test. Data are average±s.e.m.; n= 4 animals per group. **P≤0.01, ***P≤0.001.

LIPG location has been shown to be functional on the outer face of the cellular membrane (Fig. 4a)18, thus we postulated the possibility that BCa cells are dependent on LIPG function to access extracellular lipids to support their growth needs. To test this notion, we profiled the media of control and LIPG-depleted MCF7 and MDA231 cells following the same liquid chromatography-mass spectrometry-based untargeted lipidomic approach as for cell homogenates. LIPG depletion prevented the absorption of particular lipids from the media (Supplementary Fig. 4a). The structural identification of the lipids by MS/MS confirms the absence of degradation of glycerophospholipids belonging to the LPC class in both MCF7 and MDA231 cells, which is depicted by higher levels in the media of these species in LIPG-depleted when compared with control cells (Fig. 4d,e). Interestingly when we analysed the LPCs species in the media of control and LIPG-depleted cells and compared with fresh media (without cells), all LPC species from control cell media were decreased. This reduction was weaker in the media of Sh LIPG cells, indicating that LIPG-depleted cells have a defect in processing and importing of pre-existing lipid species from the medium (Fig. 4f).

Finally, we evaluated which of the commonly identified potential substrates of LIPG sustains BCa cell proliferation. Initially, we confirmed that the growth of MCF7 and MDA231 cells is impaired when grown in vitro in lipoprotein-depleted media (Fig. 4g). Next we tested the capacity of LPC (18:0) to rescue BCa cell growth in the absence of lipoproteins and confirmed that this lysophosphatidylcholine was able to restore the cells’ capacity to proliferate (Fig. 4g). In accordance, this process was dependent on LIPG expression (Fig. 4g). Similarly, LIPG-depleted cells were not able to grow in vivo in animals fed with high-fat diet (Fig. 4h) indicating that LIPG is indispensable to process the extracellular lipids and mediate their uptake by the cells, irrespectively of the concentration of lipid substrates in circulation, a phenotype also observed in FoxA-depleted cells (Fig. 4h).

LIPG activity supports BCa growth

Figure 5: LIPG activity is essential for BCa growth.

LIPG activity is essential for BCa growth.

http://www.nature.com/ncomms/2016/160405/ncomms11199/images_article/ncomms11199-f5.jpg

(a, top) Homology 3D structural model of LIPG (backbone coloured according to the QMEANlocal parameter values; red residues with low error). The heavy atoms of the three catalytic residues are shown explicitly and the residue mutated in this study is shown in green (Asp 193). (b) FoxA1, FoxA2 and LIPG protein expression in MCF7, MDA231 and their derivative cells determine by western blot. FoxA1 and FoxA2 depletion was achieved with a doxycycline-inducible short hairpin vector. FoxA-depleted cells were rescued by expression of a WT or Inactive LIPG. Cell populations were cultured in the presence or absence of doxycycline for 6 days. *blots represent different exposition times. (c) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice are determined at the indicated time points. Pvalue is the result of T-test. Data are average±s.e.m.; n=5–8 tumours. *P≤0.05, **P≤0.01. (d) MDA231 and MCF7 cell growth for 48h treated with DMSO (control), FAS inhibitor (C75) and/or lipase inhibitor (Orlistat). For MDA231 cells C75 was used at a final concentration of 10μgml−1 and for MCF7 cells 8μgml−1. Orlistat was used at a final concentration of 30 or 10μgml−1 in MCF7 or MD231 respectively. Pvalue is the result of T-test. Data are average±s.e.m.; n=3.*P≤0.05, **P≤0.01, ***P≤0.001. (e) Forty-eight hours cell growth of MDA231 or MCF7 cells overexpressing exogenous WT or Inactive LIPG. Cells were treated with DMSO (control) and FAS inhibitor (C75) at a final concentration of 20μgml−1. P value is the result of T-test. Data are average±s.e.m.; n=3.***P≤0.001, ****P≤0.0001 (f) Schematic representation showing how FoxA controls LIPG and lipid metabolism to support tumour growth.

As previous reports showed that de novo lipid metabolism is necessary for BCa growth3, 22, we next questioned whether this lipid synthesis was sufficient or, instead, whether exogenous sources are also required to support BCa cell growth and proliferation, as suggested by our experimental data. To this end, we inhibited the activity of fatty acid synthase (FAS) in BCa cells by means of the chemical inhibitor C75 (ref. 23). FAS activity is crucial for de novo lipid synthesis in cancer cells3,22. To test the complementarity of both de novo and/or exogenous lipid supplies, we used a C75 concentration causing a 50% reduction in BCa cell growth in vitro 48h post incubation (Fig. 5d andSupplementary Fig. 5d). Similarly, we tested the contribution of LIPG inhibition by means of treatment with a lipase inhibitor, Orlistat21. A specific dose causing a 50% reduction in the growth of each BCa cell line was further used (Fig. 5d and Supplementary Fig. 5d). Interestingly, concomitant treatment with FAS and LIPG inhibitors caused an additive effect, blunting BCa cell growth (Fig. 5d). Next, we evaluated whether LIPG activity was sufficient to rescue the chemical inhibition of FAS. To this end, we overexpressed WT and inactive LIPG and grew MCF7 and MDA231 cells in the presence or absence of a high dose of C75 (20mgml−1), which blocks cell growth (Supplementary Fig. 5d). Complete blockade of FAS was not rescued by LIPG (Fig. 5e). Collectively, our results suggest that both exogenous lipid precursors provided by means of LIPG activity and de novo lipid synthesis mediated by FAS are necessary for BCa cell growth.

 

Here we reveal that FoxA factors provide a central metabolic growth function by specifically regulating LIPG expression, thereby allowing the acquisition of indispensable extracellular lipids for BCa tumour proliferation. FoxA family of transcription factors are expressed in the vast majority of BCa and FoxA1 is expressed across various BCa subtypes. Moreover we show that, in some cases, its absence is associated with the expression of FoxA2. Interestingly, in addition of FoxA1 contribution to luminal commitment24, 25, 26, 27 the factor may drive BCa growth by specifically regulating LIPG levels.

The catalytic activity of LIPG generates extracellular lipid precursors that are imported to fulfill the intracellular production of lipid species (Fig. 5f). LIPG downregulation blocks BCa cell growth, thereby indicating that the import of extracellular lipid precursors is important for the proliferation of these cells. This is a striking observation given that it is generally believed that de novo fatty acid synthesis is the main driver of tumour growth22. Indeed, our experimental data with LIPG-depleted BCa cells revealed a massive decrease of most intracellular glycerolipid intermediates in the synthesis of TG (PC, PE, PG and DG) and their derivatives (LPC and LPE). Accordingly, certain lipid species (LPC) in the media were not decreased in LIPG-depleted cells as much as in control cells, thus indicating that extracellular lipids are the substrates for intracellular lipid production. In particular, we demonstrate the relevance of extracellular LPC (18:0) for BCa cell proliferation in a lipoprotein-depleted medium, a process dependent on LIPG. In this context, a high-fat diet was shown to rescue the absence of a critical intracellular lipase, Monoacylglycerol lipase, for cancer pathogenesis given cancer cells ability to uptake lipids from the extracellular compartment was functional19. Herein, we showed that this rescue mechanism is not functional in BCa cells in the absence of FoxA2 or LIPG. In support of this notion, it is worth noting that extracellular LIPG activity releases fatty acids from high-density lipoprotein phospholipids and these acids are further employed for intracellular lipid production in the human hepatic cell line HepG2 (refs 28, 29).

In conclusion, BCa cells are dependent on a mechanism to supply precursors derived from extracellular sources for intracellular lipid production, and LIPG fulfills this function. Therefore, LIPG stands out as an important component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. Our results also suggest thatde novo lipid synthesis is necessary but not sufficient to support lipid production for BCa tumour growth. Accordingly, recent clinical studies demonstrate the association between lipids and lipoproteins in circulation and risk of BCa in women with extensive mammographic density. This observation implies that interventions aimed to reduce them may have effect on BCa risk30. All together, these observations make LIPG activity an Achilles heel of luminal and, more importantly, of triple negative/basal-like breast tumours, for which limited therapeutic options are currently available.

In normal cells, the glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting, enzyme in de novo lipogenesis. ACL is a cytosolic enzyme that catalyzes the generation of acetyl CoA from citrate. Inhibition of ACL results in a loss of B-cell growth and cell viability [10] .
The plasma membrane and its constituent phosphoinositides form the basis of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway, which is crucial for cell proliferation and survival. Phosphatase and tensin-homolog deleted on chromosome 10 (PTEN) is a tumor-suppressor protein that regulates phosphatidylinositol 3-kinase (PI3-K) signaling by binding to the plasma membrane and hydrolyzing the 3′ phosphate from phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) to form phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Several loss-of-function mutations in PTEN that impair lipid phosphatase activity and membrane binding are oncogenic, leading to the development of a variety of cancers. Of these three residues, R335 was observed to interact with the membrane to the greatest extent across all of the simulations. R335L, in common with several other germline mutations, has been associated with the inherited cancer [11] .
ACLY is up-regulated or activated in several types of cancers, and its inhibition is known to induce proliferation arrest in cancer cells both in vitro and in vivo. The last studies were showed that BCR-mediated signaling is regulated in part by the amount of membrane cholesterol. It was observed that statins (Lovostatin), the pharmacological inhibitors of cholesterol synthesis, induce apoptosis of CLL cells in vitro and in vivo. Also the ectopic expression of CD5 in a B-cell line stimulates the transcription of genes involved in the synthesis of cholesterol [12] .

[10] Zaidi N, Swinnen JV, Smans K. ATP-citrate lyase: a key player in cancer metabolism Cancer Res; 2012 (11): 3709-14.

[11] Craig N, Mark S.P. Sansom. Defining the Membrane-Associated State of the PTEN Tumor Suppressor Protein. Biophys J 2013; 5; 104(3: 613–21.

[12] Tomowiak C, Kennel A, Gary-Gouy, Hadife N. High Membrane Cholesterol in CLL B-Cells and Differential Expression of Cholesterol Synthesis Genes in IG GENE Unmutated vs Mutated Cells. British Journal of Medicine & Medical Research 2012; 2(3): 313-26.

 

Cancer’s Vanguard

Exosomes are emerging as key players in metastasis.

By Catherine Offord | April 1, 2016   http://www.the-scientist.com/?articles.view/articleNo/45577/title/Cancer-s-Vanguard/

http://www.the-scientist.com/images/April2016/AprLongLit_640px.jpg

PREPARING THE TURF: Before tumor cells arrive at their metastatic destination, part of the site is readied for them. One recent study of liver metastasis in mice found that resident macrophages called Kupffer cells take up exosomes from the original tumor (1). Additionally, macrophages from the bone marrow show up upon the release of fibronectin by other liver cells called stellate cells (2). A current proposal for additional steps in metastatic niche development includes the recruitment of epithelial cells and fibroblasts, which contribute to angiogenesis, and, finally, the arrival of tumor cells themselves (3).© IKUMI KAYAMA/STUDIO KAYAMA

In 2005, David Lyden noticed something unexpected. He and his colleagues at Weill Cornell Medical College had been researching metastasis—the spread of cancer from one part of the body to another. The team had shown that bone marrow–derived cells (BMDCs) were recruited to future metastatic sites before the arrival of tumor cells, confirming that metastasis occurred after a habitable microenvironment, or “premetastatic niche,” had been prepared.1

But carefully studying images of this microenvironment in the lung tissue of mice, Lyden saw something else. Amongst the BMDCs, the micrographs showed tiny specks, far too small to be cells, gathering at the future site of metastasis. “I said, ‘What are these viruses doing here?’” recalls Lyden. “I had no idea about exosomes, microvesicles, and microparticles.”

Those specks, Lyden would come to realize, were in fact primary tumor–derived exosomes. These membrane-enclosed vesicles packed full of molecules are now attracting growing attention as important mediators of intercellular communication, particularly when it comes to cancer’s insidious capacity to spread from one organ to another.

Preparing the ground

Tumors require a community of support cells, including fibroblasts, BMDCs, and endothelial cells, to provide functional and structural assistance and to modulate immune system behavior. Bringing together the first members of this community before the arrival of tumor cells is all part of cancer’s survival strategy, says Joshua Hood, a cancer researcher at the University of Louisville.

“It wouldn’t be efficient for tumor cells to strike out on their own, and just say, ‘Oh, here we are!’” he says. “They would run the risk of being destroyed.” Preparing a “nest” in advance makes the process much safer. “Then the tumor can just efficiently come along and set up shop without ever having to fight much of a battle with the immune system.”

But although Lyden’s group had shown that this preparation was taking place, it remained unclear how such a process might be regulated. For the next few years, many cancer researchers believed that tumor cells must communicate with the premetastatic niche primarily through tumor-secreted signaling molecules such as cytokines.

Meanwhile, research into extracellular vesicles, previously considered biological garbage bags, was revealing new modes of intercellular communication. In 2007, a group of scientists in Sweden discovered that exosomes, tiny vesicles measuring just 30 nanometers to 100 nanometers across, transport mRNA and microRNAs intercellularly, with the potential to effect changes in protein synthesis in recipient cells.2 A new means for tumors to regulate distant cellular environments came into focus, and research on exosomes exploded. In 2011, Hood and his colleagues showed that exosomes facilitate melanoma metastasis through the lymphatic system.3 The following year, Lyden’s group demonstrated that tumor-derived exosomes can direct BMDCs to one of melanoma’s most common sites of metastasis, the lung.4 Exosomes, it seemed, had been underestimated.

Tiny terraformers

Armed with the knowledge that exosomes are involved in multiple stages of melanoma metastasis, Lyden’s lab went searching for the vesicles’ potential role in the metastasis of other cancers. Turning to pancreatic ductal adenocarcinoma (PDAC)—one of the most lethal cancers in humans—postdoctoral researcher Bruno Costa-Silva led a series of exhaustive in vitro and in vivo experiments in mouse models to detail the process of premetastatic niche formation in the liver, PDAC’s most common destination. The team’s results, published last May, reveal an intricate series of sequential steps—mediated by PDAC-derived exosomes (Nature Cell Biol, 17:816-26, 2015).

Using fluorescence labeling, Lyden’s group observed that PDAC-derived exosomes are taken up by Kupffer cells, specialized macrophages lining the outer walls of blood vessels in the liver. There, the exosomes trigger the cells’ secretion of transforming growth factor β (a type of cytokine involved in cell proliferation), plus the production of fibronectin by neighboring hepatic stellate cells, and the recruitment of BMDCs.

The researchers also showed that this cascade of events could be inhibited by depleting exosomal macrophage migratory inhibitory factor (MIF), an abundant protein in PDAC exosomes. “If you target the specific proteins of exosomes, you can reduce metastasis,” explains coauthor Héctor Peinado, leader of the microenvironment and metastasis group at the Spanish National Cancer Research Center.

For Hood, the findings add to a developing picture of exosomes’ vital role as “vanguard” in the progression of cancer. “It’s like the colonization of a new planet,” he says. “They’re terraforming the environment to make it hospitable.”

MOLECULAR & CELLULAR BIOLOGY

THE PAPERS

  • B. Costa-Silva et al., “Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver,”Nature Cell Biol, 17:816-26, 2015.
  • A. Hoshino et al., “Tumour exosome integrins determine organotropic metastasis,” Nature, 527:329-35, 2015.
  • L. Zhang et al., “Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth,”Nature, 527:100-04, 2015.

Internal mail

Although research was revealing the steps involved in forming premetastatic sites, it was less clear how these sites were being selected. “This has always been a great mystery in cancer,” says Ayuko Hoshino, a research associate in Lyden’s lab. “Why do certain cancers metastasize to certain organs?”

One theory, proposed in 1928 by pathologist James Ewing, suggested that anatomical and mechanical factors explained organ specificity in metastasis. The premetastatic niche, then, might form wherever exosomes are likely to land. But this couldn’t be the whole story, says Hoshino. “For instance, there’s eye melanoma. Thinking about that site, you could imagine it metastasizing to the brain. But actually, it almost only metastasizes to the liver.”

Because exosomes arrive at metastatic sites before tumor cells, the team reasoned, perhaps the exosomes themselves were organotropic (i.e., attracted to particular organs or tissues). Sure enough, Lyden says, when Hoshino and Costa-Silva began injecting tumor-derived exosomes into mice, “their preliminary findings were that wherever they injected the exosomes, the pancreatic cancer ones were ending up in the liver and the breast metastasis exosomes would end up in the lung.”

Using mass spectrometry, the researchers analyzed the protein content of exosomes from lung-tropic, liver-tropic, and brain-tropic tumors. They found that the composition of exosomes’ integrins—membrane proteins involved in cell adhesion—was destination-specific (Nature, 527:329-35, 2015). Exosomes bearing integrin α6β4, for example, were directed to the lung, where they could prepare a premetastatic niche potent enough even for normally bone-tropic tumor cells to colonize. Integrin αvβ5, meanwhile, directed metastasis to the liver.

The researchers also showed that exosomal integrins didn’t necessarily correspond to the parent-cell proteins, making exosomes potentially better indicators of where a cancer will spread than the tumor cells themselves. “We can show that an integrin that’s high in the tumor cell might be completely absent in the tumor exosome or vice versa,” says Lyden, adding that, taken together, the results point to a role for exosomes in “dictating the future sites of metastasis.”

“It’s a beautiful story,” says Dihua Yu, a molecular and cellular oncologist at the University of Texas MD Anderson Cancer Center. “This is a very novel finding that gives really good indicators for potential strategies to intervene in metastasis.”

Metastatic crosstalk

In the same month that Lyden’s group published its work on organotropism, Yu’s own lab published a different exosome study—one that told another side of the story.

Yu and her colleagues had found that when tumor cells in mice metastasized to the brain, they downregulated expression of a tumor suppressor gene called PTEN, and became primed for growth at the metastatic site. When the tumor cells were taken out of the microenvironment and put in culture, however, they restored normal PTEN expression.

The researchers demonstrated that a microRNA from astrocytes—star-shape glial cells in the brain—reversibly downregulated the levels of PTEN transcripts in the tumor cells, but they couldn’t figure out how the microRNA was getting into the tumor. Blocking “obvious signaling pathways,” such as gap junctions, failed to have an effect, Yu says.

Scrutinizing astrocyte-conditioned media using electron microscopy, the researchers identified spherical vesicles between 30 nanometers and 100 nanometers in diameter—the defining size of exosomes. Exposing mouse tumor cells to these vesicles increased cell microRNA content and reduced PTENexpression (Nature, 527:100-04, 2015). The study revealed yet another role for exosomes in the communication between tumors and their microenvironment.

The findings were a surprise, says Yu, not least because they showed a different perspective from the bulk of recent research. “We’re talking about astrocytes in the brain secreting exosomes to give welcome help to the cancer cells,” she says.

“I find it an extremely interesting paper because it shows that the astrocytes can change the whole phenotype of the tumor in the brain,” says Lyden. He adds that the results underline the importance of studying the mutational status of tumors at various sites. “All this work in exosomes, it adds to the complexity,” he says. “We can’t just target tumor cells at the primary site. We’ll have to understand all the details of metastasis if we’re really going to tackle it.”

What’s next?

The discovery of multiple roles for exosomes in metastasis has generated excitement about the potential for their use in diagnostics and treatment. As protective containers of tumor-derived genetic material, exosomes could provide information about the status of cancer progression. And as mediators of premetastatic niche formation, they make obvious targets for inhibition. (See “Banking on Blood Tests,”here.)

Exosomes might even be useful as vehicles to deliver drugs because they’re patient-matched and “naturally designed to function in a biocompatible way with living systems,” says Hood. “You could take them out of people, and at some point down the road try to have patients be their own nanofactory, using their own particles for treatment purposes.”

Pancreatic cancer exosomes initiate pre-metastatic nihe formation in the liver

Bruno Costa-SilvaNicole M. AielloAllyson J. Ocean, et al.   Nature Cell Biology 2015; 17,816–826   http://dx.doi.org:/10.1038/ncb3169

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Ayuko HoshinoBruno Costa-SilvaTang-Long ShenGoncalo RodriguesAyako HashimotoMilica Tesic Mark, et al. Nature Nov 2015; 527,329–335  http://dx.doi.org:/10.1038/nature15756

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  1. Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 8, 98101 (1989)
  2. Hart, I. R. & Fidler, I. J. Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res. 40, 22812287 (1980)
  3. Müller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature410, 5056 (2001)
  4. Weilbaecher, K. N., Guise, T. A. & McCauley, L. K. Cancer to bone: a fatal attraction. Nature Rev. Cancer 11, 411425 (2011)
  5. Zhou, W. et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25, 501515 (2014)
  6. Chang, Q. et al. The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.Neoplasia 15, 848862 (2013)
  7. Lu, X. & Kang, Y. Organotropism of breast cancer metastasis. J. Mammary Gland Biol. Neoplasia 12, 153162 (2007)

…….

Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth

Lin ZhangSiyuan ZhangJun YaoFrank J. LoweryQingling ZhangWen-Chien Huang, et al.  Nature  Nov 2015; 527,100–104   http://dx.doi.org:/10.1038/nature15376

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells’ adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites1. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs2. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth3, 4. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

  1. Quail, D. F. & Joyce, J.A. Microenvironmental regulation of tumor progression and metastasis. Nature Med. 19, 14231437 (2013)
  2. Park, E. S. et al. Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis. Proc. Natl Acad. Sci. USA 108, 1745617461 (2011)
  3. Joyce, J. A. & Pollard, J. W. Microenvironmental regulation of metastasis. Nature Rev. Cancer 9, 239252 (2009)
  4. Vanharanta, S. & Massagué, J. Origins of metastatic traits. Cancer Cell 24, 410421 (2013)
  5. Gray, J. Cancer: genomics of metastasis. Nature 464, 989990 (2010)
  6. Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 9921009 (2011)

Banking on Blood Tests

How close are liquid biopsies to replacing current diagnostics?

By Jyoti Madhusoodanan | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45584/title/Banking-on-Blood-Tests/

No matter where a tumor lurks in the body, its secrets circulate in the blood. Stray tumor cells begin metastatic migrations by slipping into the vasculature. Vesicles secreted by cancer cells and free-floating DNA are also released into the bloodstream. Because these bits of cellular debris are a grab-bag of biomarkers that could both signal a cancer’s presence and predict its progression and response to treatment, the use of blood-based tests, or liquid biopsies, to detect and evaluate them is now drawing significant commercial interest.

Last year, San Diego–based Pathway Genomics began advertising a screen “for the early detection of up to 10 different cancer types in high-risk populations.” But the screen had only been tested in already-diagnosed patients, not in at-risk individuals, and within weeks of making it commercially available, the company received an FDA notice to provide more information about their promotional claims before further marketing. “We . . . have not found any published evidence that this test or any similar test has been clinically validated as a screening tool for early detection of cancer in high risk individuals,” the agency wrote.

The Forces of Cancer

A tumor’s physical environment fuels its growth and causes treatment resistance.

By Lance L. Munn and Rakesh K. Jain | April 1, 2016   http://www.the-scientist.com/?articles.view/articleNo/45603/title/The-Forces-of-Cancer/

Ahelium balloon tugs gently at the end of its string. The tension in the string resists the buoyant force of the helium, and the elastic nature of the balloon’s rubber contains the helium gas as it tries   to expand. Cutting the string or poking the rubber with a pin reveals the precarious balance between the forces, upsets the equilibrium, and sets the system into motion.

Some biological tissues also exist in such a state of offsetting forces. The most familiar example is the balance between blood pressure and the elastic tension in the cardiovascular system that contains and conveys blood without bursting or collapsing. And in tumors, both solid and fluid forces are generated that make the cancerous tissue a lot like that helium balloon: cut a tumor with a scalpel and it rapidly swells and deforms as pent-up forces break free from structural elements that are severed.1

One force that is notably higher in tumors than in healthy tissues is fluid pressure, resulting from hyperpermeable, leaky blood vessels and a dearth of draining lymphatic vessels. Researchers have known since the 1950s that tumors exhibit elevated fluid pressure, but the implications for tumor progression and drug delivery were not realized until the late 1980s. That was when we (R.K.J. and colleagues) used a mathematical model to predict—and subsequently validate in animal and human tumors—that a precipitous drop in fluid pressure at the tumor–normal tissue interface causes interstitial fluid to ooze out of the tumor.2 This seeping fluid pushes drugs, growth factors, and cancer cells into the surrounding tissue and lymphatics, reducing drug delivery and facilitating local tumor invasion and distant metastasis.

Based on this insight, we suggested in 2001 that anti-angiogenic drugs could be used to lower a tumor’s fluid pressure and improve treatment outcome.3 This hypothesis changed the thinking about how existing anti-angiogenesis therapies actually work and spurred research into other physical forces acting in cancer.4 In the last 15 years, researchers have identified diverse sources of increased pressure in tumors, which may serve as possible targets for cancer therapy.5 For example, solid forces exerted by the extracellular matrix can be reduced by treatment with drugs approved by the US Food and Drug Administration (FDA) for controlling hypertension (angiotensin blockers) or diabetes (metformin). Retrospective clinical studies have found improved survival in cancer patients who were treated with these agents, which are now being tested in prospective trials for a variety of solid tumors.6,7

Tumors under pressure

In vitro experiments showing that cancer cells actively migrate in response to fluid flow have supported the hypothesis that fluid escaping from the boundary of a tumor may guide the invasive migration of cancer cells toward lymphatic or blood vessels, potentially encouraging metastasis. There remains controversy over how the fluid forces induce the migration; the cells may respond to chemical gradients created by the cells and distorted by the flowing fluid,8 or the fluid may activate cell mechanosensors.9 Because of the potential for new therapeutic interventions, the transduction of mechanical fluid forces into biochemical signals by cell mechanosensors is an active area of investigation. In a more direct manner, the fluid flow can physically carry cancer cells to lymph nodes.

Fluid forces may also promote tumor progression by recruiting blood vessels into the cancerous mass.10 Because tumor blood vessels are leaky, plasma can pass freely between vessels that have different pressures. When this happens at the periphery of a tumor, where angiogenic growth factors are prevalent, there can be synergistic induction of new vessel sprouts.

UNDER PRESSURE    See full infographic: WEB | PDF© N.R.FULLER, SAYO-ART LLC

And fluid pressure is just one of the many forces in a tumor that can influence its development and progression. Tumors also develop increased solid pressure, as compared with normal tissue, stemming from the uncontrolled division of cancer cells and from the infiltration and proliferation of stromal and immune cells from the surrounding tissue and circulation. High-molecular-weight polysaccharides known as hydrogels found in the extracellular matrix (ECM) also add pressure on a tumor. The most well-studied of these hydrogels is hyaluronan; when the polysaccharide absorbs water, it swells, pressing on surrounding cells and structural elements of the tissue.

The ECM contains a highly interconnected network of collagen and other fibers and is normally very good at resisting and containing such tension. It also has support from infiltrating myofibroblasts, which detect areas where the ECM density or tension is not normal and initiate actomyosin-based contraction of collagen and elastin matrix structures to restore tensional homeostasis. But while this repair effort is typically effective in healthy tissues, uncooperative tumor cells interfere with these efforts, both by themselves generating pressure and by hyperactivating cancer-associated fibroblasts to produce more ECM and thus produce even more force.11

Because cell growth and ECM composition are not spatially uniform in cancer, tumors are subjected to multiple, dispersed sources of pressure associated with matrix “containers” of various sizes. This solid pressure from within the tumor deforms the surrounding normal tissue, potentially facilitating the metastatic escape of cancer cells. The physical forces also compress blood vessels and lymphatic vessels in the tumor and adjacent normal tissue,12 increasing the fluid pressure in the tumor13  and interrupting the delivery of nutrients, removal of waste, and entry of tumor-targeted drugs via the blood.4 Insufficient blood flow also results in poor oxygenation, which has been linked to immunosuppression, inflammation, invasion, and metastasis, as well as lowered efficacy of chemo-, radio-, and immunotherapies.4 These are all indirect consequences of solid stresses in and on tumors.

Such forces can also have direct effects on cancer cells, and may serve as independent triggers for tumor invasion. Mechanical forces are central to many of our sense systems, such as hearing, touch, and pain, and to tissue maintenance programs, such as bone regeneration and blood vessel remodeling. In these systems, mechanical forces are transduced by mechanosensors to activate downstream biochemical and genetic pathways. (See “Full Speed Ahead,” The Scientist, December 2009.) Cancer cells may similarly be able to sense and respond to dynamic forces in tumors. We have shown, for example, that metastatic cancer cells exposed to compressive stresses in a culture dish undergo a phenotypic transformation to become more invasive,14 and others have shown that compressive forces applied in vivo can also induce oncogenes in normal epithelium of the mouse colon.15

It is thus becoming quite clear that the physical environment can influence a tumor’s development and spread, and it may even be possible for physical forces to kick-start cancerous growth.

…..

Full Speed Ahead

Physical forces acting in and around cells are fast—and making waves in the world of molecular biology.

By Jef Akst | December 1, 2009    http://www.the-scientist.com/?articles.view/articleNo/27816/title/Full-Speed-Ahead/

When it comes to survival, few things are more important than being able to respond quickly to a change of circumstances. And when it comes to fast-acting indicators, it turns out that signals induced by physical forces acting in and around cells, appropriately dubbed biomechanical signals, are the champions of the cellular world.

“If you look at this mechanical signaling, it’s about 30 meters per second—that’s very fast,” says bioengineer Ning Wang of the University of Illinois at Urbana-Champaign. That’s faster than most family-owned speedboats, and second only to electrical (e.g., nerve) impulses in biological signaling. By comparison, small chemicals moving by diffusion average a mere 2 micrometers per second—a speed even the slowest row boater could easily top.

Indeed, when the two signal types were pitted against each other in a cellular race last year, the mechanical signals left chemical signals in their wake, activating proteins at distant sites in the cytoplasm in just a fraction of a second, at least 40 times faster than their growth factor opponent.1 Mechanical signals are so fast, Wang adds, they are “beyond our resolution,” meaning that current imaging techniques cannot capture the very first cellular changes that result from mechanical stress, which occur within nanoseconds.

For centuries, scientists have scrutinized the molecular inner workings of the body, with little or no regard to the physical environment in which these biological reactions take place. But the growing realization that physical forces have a pervasive presence in physiology (operating in a variety of bodily systems in thebone, blood, kidney, and ear, for instance), and act with astonishing speed, has caused many to consider the possibility that mechanical signaling may be just as important as chemical communication in the life of a cell.

“Biologists have traditionally ignored the role of mechanics in biology,” says biomechanical engineer Mohammad Mofrad of the University of California, Berkley, “[but] biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”

The wave within: Mechanical forces acting inside the cell

Once believed to be little more than sacks of chemically active goop, cells didn’t seem capable of transmitting physical forces into their depths, and researchers largely limited their search for molecules or structures that respond to physical forces, or mechanosensors, to the plasma membrane.

Mechanical signaling may be just as important as chemical communication in the life of a cell.

In the late 1990s, however, closer examination revealed that the cell’s interior is in fact a highly structured environment, composed of a network of filaments.2 Pull on one side of the cell, and these filaments will transmit the force all the way to other side, tugging on and bumping into a variety of cellular structures along the way—similar to how a boat’s wake sends a series of small waves lapping up on a distant and otherwise peaceful shoreline. Scientists are now realizing the potential of such intracellular jostling to induce molecular changes throughout the cell, and the search for mechanosensing molecules has escalated dramatically in scope, including, for example, several proteins of the nucleus.

It’s a search that will likely last a while, predicts cell biologist Donald Ingber, director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. “To try to find out what’s the mechanosensor is kind of crazy at this point,” he says. As scientists are now learning, “the whole cell is the mechanosensor.”

A key player, most agree, is the cytoskeleton, which is comprised of a variety of microfilaments, including rigid actin filaments and active myosin motors—the two principle components of muscle. Activation of the so-called nonmuscle myosins causes the cytoskeleton to contract, much like an arm muscle does when it lifts a heavy object.

The first intimation that the cytoskeleton could go beyond its established inner-cell duties (molecule transport and cell movement and division) came in 1997, when Ingber did the logical (in hindsight, at least) experiment of pulling on the cells to see what happened inside.2 Using a tiny glass micropipette coated in ligands, Ingber and his team gently probed the surface proteins known as integrins, which secure the cell to the extracellular matrix. When they quickly pulled the micropipette away, they saw an immediate cellular makeover: cytoskeletal elements turned 90 degrees, the nucleus distorted, and the nucleolus—a small, dense structure within the nucleus that functions primarily in ribosome assembly—aligned itself with the direction of the applied force.

“That kind of blew people away,” Ingber recalls. “It revealed that cells have incredible levels of structure not only in the cytoplasm but in the nucleus as well.”

Wang (once a postdoc in Ingber’s lab at the Harvard School of Public Health) and other collaborators combined a similar technique with fluorescent imaging technology to visualize how these forces were channeled within the cell’s interior. Upping the resolution and further refining these techniques, Wang began mapping these intracellular forces as they made their way through the cell. In 2005, the maps confirmed the physical connection between the cell-surface integrins and the nucleus, and showed that these external forces follow a nonrandom path dictated by the tension of the cytoskeletal elements.3

“Biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”
–Mohammad Mofrad

The end point of these mechanical pathways is likely a mechanosensitive protein, which changes shape in response to the force, thereby exposing new binding areas or otherwise changing the protein’s function. In mitochondria, for example, mechanical forces may trigger the release of reactive oxygen species and activation of signaling molecules that contribute to inflammation and atherosclerosis.

Similarly, proteins on the nuclear membrane may pass mechanical signals into the nucleus by way of a specialized structure known as LINC (linker of nucleoskeleton and cytoskeleton), which physically links the actin cytoskeleton to proteins important in nuclear organization and gene function. To determine if mechanical forces directly affect gene expression, last year scientists began exploiting the increasingly popular fluorescence resonance energy transfer (FRET) technology,1 in which energy emitted by one fluorescent molecule can stimulate another, resulting in a visible energy transfer that can track enzymatic activities in live cells. By combining FRET technology with the techniques that apply physical forces to specific cell membrane proteins, scientists can visualize entire mechanochemical transduction pathways, Wang says.

“The big issue right now in the field of mechanotransduction is whether the genes in the nucleus can be directly activated by forces applied to the cell surface,” Wang explains. While the physical maps of the cytoskeleton tentatively sketch out a path that supports this possibility, confirmatory data is lacking. This combination of new technologies will be “tremendously” helpful in answering that question, he says, and “push the field” towards a more complete understanding of how mechanical forces can influence cellular life.

An early start: Mechanical forces in development

In the world of developmental biology, the cytoskeleton’s role in biomechanics really comes into its own. As the embryo develops, the cells themselves are the force generators, and by contracting at critical times, the cytoskeleton can initiate many key developmental steps, from invagination and gastrulation to proliferation and differentiation, and overall cellular organization.

The idea that physical forces play a role in development is not a new one. In the early 20th century, back when Albert Einstein was first developing the molecular basis of viscosity and scientists were realizing molecules are distinct particles, biologist and mathematician D’Arcy Thompson of the University of Dundee in Scotland suggested that mechanical strain is a key player in morphogenesis. Now, nearly a century later, biologists are finally beginning to agree.

Because Thompson “couldn’t measure [the forces] at that time, that kind of thinking got pushed to the wayside as genetic thinking took over biology,” says bioengineer Christopher Chen of the University of Pennsylvania. That is, until 2003, when Emmanuel Farge of the Curie Institute in France squeezedDrosophila embryos to mimic the compression experienced during early development and activated twist—a critical gene in the formation of the digestive tract.4 These results gave weight to Thompson’s idea that stress in the embryo stimulates development and growth, and inspired developmental scientists to begin considering mechanical effects, Chen says. “Now we’re at the stage where there’s a lot of interest and willingness to consider the fact that mechanical forces are not only shaping the embryo, but are linked to the differentiation programs that are going on.”

Again, the cytoskeleton is a key player in this process. In fruit flies and frogs, for example, nonmuscle myosins contract the actin filaments to generate the compressive forces necessary for successful gastrulation—the first major shape-changing event of development. Myosins similarly influence proliferation in the development of the Drosophila egg chamber, with increased myosin activity resulting in increased cell division.

Cytoskeleton contractility also appears to direct stem cell differentiation. In 2006, Dennis Discher of the University of Pennsylvania demonstrated that the tension of the substrate on which cells are grown in culture is important for determining what type of tissue the cells will form.5 Cells grown on soft matrices that mimic brain tissue tended to grow into neural cells, while cells grown on stiffer matrices grew into muscle cell precursors, and hard matrices yielded bone. In this case, it seems that stiffer substrates increased the expression of nonmuscle myosin, generating greater tension in the actin cytoskeleton and affecting differentiation. (Altering or inhibiting myosin contraction can also affect differentiation.)

“To try to find out what’s the mechanosensor is kind of crazy at this point. As scientists are now learning, the whole cell is the mechanosensor.”
–Donald Ingber
……..
Shaping a tumor

In addition to the influence of physical forces on cancer growth and invasion, forces can alter a tumor’s mechanical properties, and vice versa. Tumors are more rigid, or stiffer, than surrounding tissues, usually because they contain excess collagen in the ECM,5 and this can contain and amplify local forces produced by proliferating cancer cells. On the other hand, tumor rigidity can be further enhanced if the cells exert tension on ECM collagen fibers by pulling on them, or by stretching them, as occurs when tumors grow uncontrollably. Fluid forces can also influence the assembly of collagen fibers within and around tumors,8potentially increasing stiffness.

Importantly, tumor stiffness tends to be associated with poor prognoses, though the reasons for this are not fully understood. Cells are known to differentiate into different lineages depending on the local rigidity;16 for example, stem cells differentiate into bone on stiff substrates, but make adipose (fat) cells on softer substrates. Similar mechanisms are thought to affect tumor progression when the ECM changes rigidity, inducing cancer cells to become more invasive as well as more likely to metastasize. Indeed, longer collagen fibers in the matrix are associated with increased invasion and metastasis, as well as reduced survival, in mice.17

In addition, the abnormal ECM in tumors can affect cancer progression by activating normal stromal cells, such as macrophages and fibroblasts, that accelerate tumor growth and treatment resistance. These activated stromal cells further strengthen and stretch the ECM, causing a snowball effect.

The biochemical composition and organization of the ECM also influences tumor biology. Dysregulation of normal matrix signals can lead to tumor progression, characterized by excessive cell proliferation, immortality, enhanced migration, changes in metabolism, and evasion of the immune response. More research is needed to dissect the relationships between the ECM’s mechanical properties, forces, and cell signaling pathways.

Targeting the ECM

Because unchecked proliferation of cancer cells increases solid stress in the tumor, anticancer therapies should decrease the compressive forces in tumors and reopen collapsed blood and lymphatic vessels.11 This is exactly what happens when tumors are treated with certain doses of paclitaxel or docetaxel, two widely used cancer drugs. Shrinking tumors increases blood flow and allows more efficient fluid movement through the extravascular space, lowering the tumor interstitial fluid pressure in mouse models and in patients with breast cancer.5 However, cancer cells invariably develop resistance to treatment and begin to regrow, increasing solid stress again. As a result, other targets for reducing solid stresses are needed.

Because of its role in containing and concentrating the forces in a tumor, the collagen matrix within and around the tumor is another potential target for relieving tumor-related stresses. Indeed, solid stress in tumors can be reduced by drugs that selectively reprogram activated fibroblasts or modify the assembly of matrix components such as collagen and hyaluronan. In rodent studies, targeting these force-altering components in the tumor microenvironment has been shown to decrease solid stress, improve blood perfusion and drug delivery, and improve tumor response to chemotherapy and animal survival.6 We have found, for example, that injecting tumors with a collagen-digesting enzyme increases the diffusion of antibodies and viral particles and improves drug penetration in the tumor. Similarly, treatments that target transforming growth factor–beta (TGF-β), which controls the production of collagen by myofibroblasts, increase perfusion, improve the delivery of drugs of all sizes in mammary tumors, and improve treatment outcomes in mice.5

A class of drugs that is widely used to control blood pressure in hypertensive patients also blocks the TGF-β pathway. These drugs, known as angiotensin receptor 1 blockers, can reduce collagen production in and around the tumor by reducing the activity of TGF-β, as well as by blocking the function of connective tissue growth factor (CTGF), which is involved in stabilizing collagen and inducing resistance to chemotherapy.6Losartan and other angiotensin inhibitors reduce levels of collagen in various experimental models of fibrosis, and decrease renal and cardiac fibrosis in hypertensive patients. When given to mice with one of four different types of tumors characterized by high levels of cancer-associated fibroblasts (CAFs) and excess extracellular matrix—pancreatic ductal adenocarcinoma, breast cancer, sarcoma, and melanoma—losartan treatment caused a decrease in collagen content in a dose-dependent manner, enhanced penetration of nanoparticles into the tumor, and improved efficacy of diverse anticancer drugs. This is supported by a number of retrospective studies in patients with pancreatic, lung, and kidney cancers.6Researchers at Massachusetts General Hospital are now running a Phase 1/2 clinical trial to test losartan in pancreatic cancer patients.

http://www.the-scientist.com/images/April2016/forces_cancer_2.jpg

THE TUMOR ENVIRONMENT: The extracellular matrix and stromal cells within a tumor’s microenvironment influence the physical forces a tumor experiences. Left: The immunofluorescent image shows stromal cells (red and green) surrounding tumor cells (red cluster with blue nuclei); the cells were isolated from a mouse model of lung adenocarcinoma. Right: In this immunofluorescent image of triple-negative breast cancer, tumor cells (blue) are in close contact with matrix collagen (purple). Immune cells are labeled in red and green.VASILENA GOCHEVA, JACKS LAB, KOCH INSTITUTE AT MIT; DONGMEI ZUO, LABORATORY DR. MORAG PARK

Another potential cancer treatment target is hyaluronan, which is abundant in 20 percent to 30 percent of human tumors, most notably breast, colon, and prostate cancers. In addition to its role as a pressure-creating gel, hyaluronan can sequester growth factors and inhibit interstitial fluid movement within the tumor. Hyaluronidase, an enzyme that digests hyaluronan, reduces mechanical stress in tumors grown in mice.1 And San Diego–based Halozyme Therapeutics’s PEGPH20, a formulation of hyaluronidase coated with polyethylene glycol to enhance bioavailability, can decompress blood vessels and improve treatment outcome in genetically engineered mouse models of pancreatic ductal adenocarcinoma. Based on these studies, Halozyme researchers are now testing PEGPH20 in a randomized clinical trial of pancreatic cancer patients. Another matrix-altering drug is the widely-prescribed antidiabetic drug metformin, which has been shown to decrease collagen and hyaluronan levels in pancreatic tumors in obese mice and patients.7 Metformin is currently being tested in more than 200 clinical trials worldwide as a treatment for different types of cancer.

Clearly, tumors should be studied not only in light of their biochemical processes and genetic underpinnings, but also for the specific physical forces and mechanical properties that may influence progression. Understanding the physical microenvironment of tumors, as well as its interplay with the biochemical environment, is necessary to improve cancer detection, prevention, and treatment.

  1. T. Stylianopoulos et al., “Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors,” PNAS, 109:15101-08, 2012.
  2. R.K. Jain, L.T. Baxter, “Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: Significance of elevated interstitial pressure,” Cancer Res, 48:7022-32, 1988.
  3. R.K. Jain, “Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy,”Science, 307:58-62, 2005.
  4. R.K. Jain, “Antiangiogenesis strategies revisited: From starving tumors to alleviating hypoxia,”Cancer Cell, 26:605-22, 2014.
  5. R.K. Jain et al., “The role of mechanical forces in tumor growth and therapy,” Annu Rev Biomed Eng, 16:321-46, 2014.
  6. V.P. Chauhan et al., “Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels,” Nat Commun, 4:2516, 2013.
  7. J. Incio et al., “Metformin reduces desmoplasia in pancreatic cancer by reprogramming stellate cells and tumor-associated macrophages,” PLOS ONE, 10:e0141392, 2015.
  8. M.A. Swartz, A.W. Lund, “Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity,” Nat Rev Cancer, 12:210-19, 2012.
  9. H. Qazi et al., “Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion,”Integr Biol, 5:1334-43, 2013.
  10. J.W. Song, L.L. Munn, “Fluid forces control endothelial sprouting,” PNAS, 108:15342-47, 2011.
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brown adipocyte protein CIDEA promotes lipid droplet fusion

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

 

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding

Parker, Nicholas T Ktistakis, Ann M Dixon, Judith Klein-Seetharaman, Susan Henry, Mark Christian Dirk Dormann, Gil-Soo Han, Stephen A Jesch, George M Carman, Valerian Kagan, et al.

eLife 2015;10.7554/eLife.07485     http://dx.doi.org/10.7554/eLife.07485

 

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

 

Evolutionary pressures for survival in fluctuating environments that expose organisms to times of both feast and famine have selected for the ability to efficiently store and release energy in the form of triacyclglycerol (TAG). However, excessive or defective lipid storage is a key feature of common diseases such as diabetes, atherosclerosis and the metabolic syndrome (1). The organelles that are essential for storing and mobilizing intracellular fat are lipid droplets (LDs) (2). They constitute a unique cellular structure where a core of neutral lipids is stabilized in the hydrophilic cytosol by a phospholipid monolayer embedding LD-proteins. While most mammalian 46 cells present small LDs (<1 Pm) (3), white (unilocular) adipocytes contain a single giant LD occupying most of their cell volume. In contrast, brown (multilocular) adipocytes hold multiple LDs of lesser size, increasing the LD surface/volume ratio which facilitates the rapid consumption of lipids for adaptive thermogenesis (4).

The exploration of new approaches for the treatment of metabolic disorders has been stimulated by the rediscovery of active brown adipose tissue (BAT) in adult humans (5, 6) and by the induction of multilocular brown-like cells in white adipose tissue (WAT) (7). The multilocular morphology of brown adipocytes is a defining characteristic of these cells along with expression of genes such as Ucp1. The acquisition of a unilocular or multilocular phenotype is likely to be controlled by the regulation of LD growth. Two related proteins, CIDEA and CIDEC promote LD enlargement in adipocytes (8-10), with CIDEA being specifically found in BAT. Together with CIDEB, they form the CIDE (cell death-inducing DFF45-like effector) family of LD-proteins, which have emerged as important metabolic regulators (11).

Different mechanisms have been proposed for LD enlargement, including in situ neutral lipid synthesis, lipid uptake and LD-LD coalescence (12-14). The study of CIDE 62 proteins has revealed a critical role in the LD fusion process in which a donor LD progressively transfers its content to an acceptor LD until it is completely absorbed (15). However, the underlying mechanism by which CIDEC and CIDEA facilitate the interchange of triacylglycerol (TAG) molecules between LDs is not understood. In the present study, we have obtained a detailed picture of the different steps driving this LD enlargement process, which involves the stabilization of LD pairs, phospholipid binding, and the permeabilization of the LD monolayer to allow the transference of lipids.

 

CIDEA expression mimics the LD dynamics observed during the differentiation of brown adipocytes

Phases of CIDEA activity: LD targeting, LD-LD docking and LD growth

A cationic amphipathic helix in C-term drives LD targeting

The amphipathic helix is essential for LD enlargement

LD-LD docking is induced by the formation of CIDEA complexes

CIDEC differs from CIDEA in its dependence on the N-term domain

CIDEA interacts with Phosphatidic Acid

PA is required for LD enlargement

 

The Cidea gene is highly expressed in BAT, induced in WAT following cold exposure (46), and is widely used by researchers as a defining marker to discriminate brown or brite adipocytes from white adipocytes (7, 28). As evidence indicated a key role in the LD biology (47) we have characterized the mechanism by which CIDEA promotes LD enlargement, which involves the targeting of LDs, the docking of LD pairs and the transference of lipids between them. The lipid transfer step requires the interaction of CIDEA and PA through a cationic amphipathic helix. Independently of PA-binding, this helix is also responsible for anchoring CIDEA in the LD membrane. Finally, we demonstrate that the docking of LD pairs is driven by the formation of CIDEA complexes involving the N-term domain and a C-term interaction site.

CIDE proteins appeared during vertebrate evolution by the combination of an ancestor N-term domain and a LD-binding C-term domain (35). In spite of this, the full process of LD enlargement can be induced in yeast by the sole exogenous expression of 395 CIDEA, indicating that in contrast to SNARE-triggered vesicle fusion, LD fusion by lipid transference does not require the coordination of multiple specific proteins (48). Whereas vesicle fusion implicates an intricate restructuring of the phospholipid bilayers, LD fusion is a spontaneous process that the cell has to prevent by tightly controlling their phospholipid composition (23). However, although phospholipid-modifying enzymes have been linked with the biogenesis of LDs (49, 50), the implication of phospholipids in physiologic LD fusion processes has not been previously described.

Complete LD fusion by lipid transfer can last several hours, during which the participating LDs remain in contact. Our results indicate that both the N-term domain and a C-term dimerization site (aa 126-155) independently participate in the docking of LD pairs by forming trans interactions (Fig. 7). Certain mutations in the dimerization sites that do not eliminate the interaction result in a decrease on the TAG transference efficiency, reflected on the presence of small LDs docked to enlarged LDs. This suggests that in addition to stabilizing the LD-LD interaction, the correct conformation of the 409 CIDEA complexes is necessary for optimal TAG transfer. Furthermore, the formation of stable LD pairs is not sufficient to trigger LD fusion by lipid transfer. In fact, although LDs can be tightly packed in cultured adipocytes, no TAG transference across neighbour LDs is observed in the absence of CIDE proteins (15), showing that the phospholipid monolayer acts as a barrier impermeable to TAG. Our CG-MD simulations indicate that certain TAG molecules can escape the neutral lipid core of the LD and be integrated within the aliphatic chains of the phospholipid monolayer. This could be a transition state 416 prior to the TAG transference and our data indicates that the docking of the amphipathic helix in the LD membrane could facilitate this process. However, the infiltrated TAGs in LD membranes in the presence of mutant helices, or even in the absence of docking, suggests that this is not enough to complete the TAG transference.

To be transferred to the adjacent LD, the TAGs integrated in the hydrophobic region of the LD membrane should cross the energy barrier defined by the phospholipid polar heads, and the interaction of CIDEA with PA could play a role in this process, as suggested by the disruption of LD enlargement by the mutations preventing PA-binding (K167E/R171E/R175E) and the inhibition of CIDEA after PA depletion. The minor effects observed with more conservative substitutions in the helix, suggests that the presence of positive charges is sufficient to induce TAG transference by attracting anionic phospholipids present in the LD membrane. PA, which requirement is indicated by our PA-depletion experiments, is a cone-shaped anionic phospholipid which could locally destabilize the LD monolayer by favoring a negative membrane curvature incompatible with the spherical LD morphology (51). Interestingly, while the zwitterion PC, the main component of the monolayer, stabilizes the LD structure (23), the negatively charged PA promote their coalescence (29). This is supported by our CD-MD results which resulted in a deformation of the LD shape by the addition of PA. We propose a model in which the C-term amphipathic helix positions itself in the LD monolayer and interacts with PA molecules in its vicinity, which might include trans interactions with PA in the adjacent LD. The interaction with PA disturbs the integrity of the phospholipid barrier at the LD-LD interface, allowing the LD to LD transference of TAG molecules integrated in the LD membrane (Fig. 7). Additional alterations in the LD composition could be facilitating TAG transference, as differentiating adipocytes experience a reduction in saturated fatty acids in the LD phospholipids (52), and in their PC/PE ratio (53) which could increase the permeability of the LD membranes, and we previously observed that a change in the molecular structures of TAG results in an altered migration pattern to the LD surface (32).

During LD fusion by lipid transfer, the pressure gradient experienced by LDs favors TAG flux from small to large LDs (15). However, the implication of PA, a minor component of the LD membrane, could represent a control mechanism, as it is plausible that the cell could actively influence the TAG flux direction by differently regulating the levels of PA in large and small LDs, which could be controlled by the activity of enzymes such as AGPAT3 and LIPIN-1J (13, 30). This is a remarkable possibility, as a switch in the favored TAG flux direction could promote the acquisition of a multilocular phenotype and facilitate the browning of WAT (24). Interestingly, Cidea mRNA is the LD protein- encoding transcript that experiences the greatest increase during the cold-induced process by which multilocular BAT-like cells appear in WAT (24). Furthermore, in BAT, cold exposure instigates a profound increase in CIDEA protein levels that is independent of transcriptional regulation (54). The profound increase in CIDEA is coincident with elevated lipolysis and de novo lipogenesis that occurs in both brown and white adipose tissues after E-adrenergic receptor activation (55). It is likely that CIDEA has a central role in coupling these processes to package newly synthesized TAG in LDs for subsequent lipolysis and fatty acid oxidation. Importantly, BAT displays high levels of glycerol kinase activity (56, 57) that facilitates glycerol recycling rather than release into the blood stream, following induction of lipolysis (58), which occurs in WAT. Hence, the reported elevated glycerol released from cells depleted of CIDEA (28) is likely to be a result of decoupling lipolysis from the ability to efficiently store the products of lipogenesis in LDs and therefore producing a net increase in detected extracellular glycerol. This important role of CIDEA is supported by the marked depletion of TAG in the BAT of Cidea null mice following overnight exposure to 4 °C (28) and our findings that CIDEA-dependent LD enlargement is maintained in a lipase negative yeast strain.

Cidea and the genes that are required to facilitate high rates of lipolysis and lipogenesis are associated with the “browning” of white fat either following cold exposure (46) or in genetic models such as RIP140 knockout WAT (59). The induction of a brown- like phenotype in WAT has potential benefits in the treatment and prevention of metabolic disorders (60). Differences in the activity and regulation of CIDEC and CIDEA could also be responsible for the adoption of unilocular or multilocular phenotypes. In addition to their differential interaction with PLIN1 and 5, we have observed that CIDEC is more resilient to the deletion of the N-term than CIDEA, indicating that it may be less sensitive to regulatory posttranslational modifications of this domain. This robustness of CIDEC activity together with its potentiation by PLIN1, could facilitate the continuity of the LD enlargement in white adipocytes until the unilocular phenotype is achieved. In contrast, in brown adipocytes expressing CIDEA the process would be stopped at the multilocular stage for example due to post-translational modifications that modulate the function or stability of the protein or alteration of the PA levels in LDs.

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PostTranslational Modification of Proteins

 

Author and Curator: Larry H Bernstein, MD, FCAP 

 

Posttranslational modification of proteins: expanding nature’s inventory.

Walsh, Christopher T.
Roberts & Company Publishers   2006
Englewood, Colo.: xxi, 490

For students of protein structure, metabolism, and cellular signaling, Walsh (biological chemistry, molecular pharmacology, Harvard Medical School), a leading enzymologist, examines major classes of posttranslational modifications (PTMs) that account for the diversity of protein structure and function in living cells. He contributes to emerging knowledge,
relevant to pharmaceutical intervention,

of the enzymes involved in generating PTMs, i.e.,

changes that occur after messenger RNA code has been translated into the amino acid sequence code of nascent proteins.

The text contains numerous examples of the role PTMs play in signal transduction and metabolism, and crisp color illustrations.

The Quarterly Review of Biology, Vol. 83, No. 4. (1 December 2008), pp. 403-403,    http://dx.doi.org/10.1086/596250        Key: citeulike:3682226

 

Peptidylglycine alpha-amidating monooxygenase: A multifunctional protein with catalytic, processing, and routing domains

by Betty A. Eipper, Sharon L. Milgram, E. Jean Husten, Hye-Young Yun, Richard E. Mains

Protein Science 1993; 2(4): pp. 489-497,    http://dx.doi.org10.1002/pro.5560020401

Overview of Post-Translational Modifications (PTMs) Analysis:

PTMs(hereafter): Phosphorylation (pS/T, pY), Methylation, Deamidation, Oxidation, Nitration, N-glycosylation, Amino acid mutation, Unnatural amino acid, Chemical modifications, Palmitoylation, Glycosylation, Ubiquitination, SUMOylation, Dimethylation, Acetylation, Decarboxylation, etc..

Protein post-translational modification (PTM) increases the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits or degradation of entire proteins. These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis. Therefore, identifying and understanding PTMs is critical in the study of cell biology and disease treatment and prevention.

 

1) Significance:

Protein post-translational modifications play a key role in many cellular processes such as cellular differentiation (Grotenbreg and Ploegh, 2007), protein degradation (Geiss-Friedlander and Melchior, 2007), signaling and regulatory processes (Morrison, et al 2002), regulation of gene expression, and protein-protein interactions. These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis. Therefore, identifying and understanding PTMs is critical in the study of cell biology and disease treatment and prevention.

PTM modifications

PTM modifications

 

 

 

 

 

 

 

 

 

 

2) Post-translational modifications are key mechanisms to increase proteomic diversity

While the human genome comprises 20-25,000 genes, the proteome is estimated to encompass over 1 million proteins. Changes at the transcriptional and mRNA levels increase the size of the transcriptome relative to the genome, and the myriad of different post-translational modifications exponentially increases the complexity of the proteome relative to both the transcriptome and genome.

a)       Some Modifications (Phosphorylations, etc.) are easier to find than others. We can look for specific modifications or unknown modifications.

b)       As a general rule, any post-translational modification (PTM) could be searched for in your protein as long as we know the mass added by the modification and the potentially modified amino acid (e.g. in the case of phosphorylation: +80 Da on a Serine, Threonine or Tyrosine).

PTM (Post-Translational Modification) Analysis  http://www.creative-proteomics.com/protein-post-translational-modification-analysis.htm#1._Overview_of_Post-Translational_Modifications_%28PTMs%29_Analysis

 

Jose Eduardo de Salles Roselino:

The easy way to look at protein is to present it as a by-product of DNA. However, protein must be viewed as central macromolecule in biology since; even DNA is made from building blocks by protein activity. DNA are the reservoir of genetic information that establishes amino acid in proteins.
In normal living beings, normality defined by general health parameters whose values are inside an acceptable range of variation. Normal here is a statistical idea, as it must be and not as presented in recent years, as a living being that has a genome that does not have “glitches”, or a genome that would be defined as an ideal or a perfect genome.
In line with this idea, protein receives the information that determines its amino acid sequence from DNA but have its conformation, activity and function derived from its ability to change its conformation in response to changes in its microenvironment and environment. These changes in conformation are in a form adequate to keep those parameters mentioned above inside the range that define the idea of normality in accordance with the condition in which the living being is, both in time (development) as well as in space.
Therefore, post-translational must indicate a clear cut in the domain of DNA influence and not something, which is also derived from this DNA-centric view. This distortion of biochemistry has led to the never-ending genetics of non-genetic diseases. Genetics appears in inborn errors that are not acquired and show its effects in defects of proteins that could be established by a change in the DNA. Normality, or lack of abnormal genetic defect are perceived in all genomes that are able to maintain inside the normality range those parameters that define normal under defined circumstances. When this view is taken into account, DNA is take into account only when genetic diseases are considered. For the majority of the cases the scheme here presented must be made for each kind of cell, in each organ or system and the posttranslational changes thus, presented as function of development and/or a required fast regulatory change necessary to keep a cell and the organisms in general inside the normal range.

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Post-Translational Modifications

Author and Curator: Larry H Bernstein, MD, FCAP 

 

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry.

Jensen ON.
Curr Opin Chem Biol. 2004 Feb; 8(1):33-41.   PMID: 15036154

 

Post-translational modifications generate tremendous

  • diversity,
  • complexity and
  • heterogeneity of gene products, and

their determination is one of the main challenges in proteomics research.

Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the

  • dynamics and
  • spatio-temporal control of protein activities
    • by post-translational modifications, and
    • reveal their roles in biological processes and pathogenic conditions.

Combinations of

  1. affinity-based enrichment and extraction methods,
  2. multidimensional separation technologies and
  3. mass spectrometry

are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.

 

PTM modifications

PTM modifications

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What is the Future for Genomics in Clinical Medicine?


What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Other Related articles on this Open Access Online Scientific Journal, include the following:

https://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

https://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

https://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

https://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

https://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

https://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

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Author and Curator: Ritu Saxena, Ph.D.

 

Introduction

Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer

Bcl2

Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.

p53

p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.

HIF-1a

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.

PTEN

Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.

C-Src

C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.

Reference:

Muntané J and la Mata MD. Nitric oxide and cancer. World J Hepatol. 2010 Sep 27;2(9):337-44. http://www.ncbi.nlm.nih.gov/pubmed/21161018

Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9. http://www.ncbi.nlm.nih.gov/pubmed/22425962

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.http://www.ncbi.nlm.nih.gov/pubmed/20167555

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34. http://www.ncbi.nlm.nih.gov/pubmed/11518674

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A Protease for ‘Middle-down’ Proteomics

Author and Reporter: Ritu Saxena, Ph.D.

Neil Kelleher and his research team at Northwestern University have developed a method for enzymatic proteolysis large peptides for mass spectrometry–based proteomics using a protease OmpT. The method was published in a recent issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Proteomics is defined as the study of the structure and function of proteins. Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-medicine/current-topics/proteomics.page Proteomics, although refers to the study of the structure and function of proteins, it is often specifically used for protein purification and mass spectrometry.

‘Bottom-up’ and ‘Top-down’ are the two main strategies for proteomic studies using mass spectrometry. In Bottom-up proteomics referred to as the more common method, proteins are broken down into smaller pieces through enzymatic digestion followed by characterization into amino acid sequences and post translational modifications prior to analysis by mass spectrometry. By identifying and sequencing these smaller pieces, researchers can then determine the identity of the protein they make up. In Top-down proteomics, on the other hand, the process of proteolysis is skipped and it focuses on complete characterization of intact proteins and their post-translational modifications (PTMs).

“Although both the top-down and bottom-up approaches continue to mature, they each have limitations. The tryptic peptides used in the bottom-up approach are the primary unit of measurement, but their relatively small size (typically ~8–25 residues long) leads to problems such as sample complex­ity, difficulties in assigning peptides to specific gene products rather than protein groups, and loss of single and combinato­rial PTM information. The top-down approach handles these issues by characterizing intact proteins, but its success declines in the high-mass region. Therefore, a hybrid approach based on 2–20 kDa peptides could unite positive aspects of both bottom-up and top-down proteomics” says Kelleher et al in the research article.

The hybrid approach, referred to as ‘middle-down’ proteomics would enable the analysis of complex mixtures pre-sorted by protein size. Previously research efforts ‘middle-down’ proteomics included exploring the restricted proteolysis with enzyme alternatives to Trypsin and chemical methods (such as microwave-assisted acid hydrolysis), However, these methods generated peptides that were marginally longer than those produced by trypsin digestion. For the current study, Kelleher adds “We established an OmpT-based middle-down platform to analyze complex mixtures pre-sorted by protein size. After inte­grating the data from the middle-down workflow that was applied to ~20–100-kDa proteins fractionated from the HeLa cell proteome, we identified 3,697 unique peptides (average size: 6.3 kDa) from 1,038 unique proteins (26% average sequence coverage) at an esti­mated 1% false discovery rate”.

OmpT, a protease derived from Escherichia coli K12 outer membrane belongs to the novel omptin protease family10 and is known to cleave between two consecutive basic amino acid residues (Lys/Arg-Lys/Arg). The authors developed OmpT into an efficient rea­gent to generate >2-kDa peptides for middle-down proteomics, thus, utilizing OmpT to achieve robust, yet restricted, proteolysis of a complex genome. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Researcher Kelleher and his team have been in news earlier for their work on ‘top-down’ proteomics when his team developed a new method that could separate and identify thousands of protein molecules quickly. In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. The study was published in last year in the October issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed?term=22037311

Thus, Kelleher and his group was able to demonstrate that OmpT-based proteomic approach has a robust and restricted proteolysis capacity making it an attractive option for mass-spectrometry-based analysis of primary structure of protein.

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