Transformed Mouse Embryonic Fibroblasts Research Articles

PO-404 Using functional genetic screens to understand cancer immune evasion

IntroductionWith the emerge of immunotherapy as an effective cancer therapy, there is also a growing need to identify new biomarkers of response as well as novel therapeutic targets. Functional CRISPR screens have proven to be an important tool to identify key genes in complex biological processes. We use an in vitro and in vivo CRISPR screening strategy to better understand how tumours evade the immune system.Material and methodsWe collected pre and post treatment biopsies from melanoma and lung cancer patients treated with an anti PD-1 inhibitor. Upon performing whole exome sequencing on biopsies from patients who progressed on therapy, we identified a list of candidate genes. We included genes that fell into one of the three categories: were mutated in more than one patient, were mutated only at relapse and had mutations in both alleles or had a heterozygous mutation at baseline and homozygous on progression.In addition, we also generated an adenovirus transformed mouse embryonic fibroblast cell line, which does not form tumours in the immunocompetent mice due to T-cell killing. We use this system do perform an in vivo CRISPR screen, investigating which genes are crucial for immune evasion.Results and discussionsWe have successfully generated a target gene list from sequences of patient biopsies. We have constructed a focused CRISPR library consisting of multiple gRNAs targeting each of those genes. This library will be used in a genetic screen in melanoma cells co-cultured with matched T-cells that recognise and eliminate the melanoma cells. Moreover, we will also use our in vivo adenoviral system to perform genome wide screens.ConclusionAfter the screening, we can cross validate hits from both platforms to identify robust hits. Furthermore, we can validate them in a bigger patient cohort to determine their potential as predictive biomarkers for immunotherapy response.

RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation.

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.

Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition.

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.

Defective heat shock factor 1 inhibits the growth of fibrosarcoma derived from simian virus 40/T antigen‑transformed MEF cells.

Heat shock factor 1 (Hsf1) serves an important role in regulating the proliferation of human tumor cell lines in vitro and tissue specific tumorigenesis in certain mouse models. However, its role in viral-oncogenesis remains to be fully elucidated. In the current study, the role of Hsf1 in fibroblastoma derived from simian virus 40/T antigen (SV40/TAG)-transformed mouse embryonic fibroblast (MEF) cell lines was investigated. Knockout of Hsf1 inhibited MEF cell proliferation in vitro and fibroblastoma growth and metastasis to the lungs in vivo in nude mice. Knockout of Hsf1 increased the protein expression levels of p53 and phosphorylated retinoblastoma protein (pRb), however reduced the expression of heat shock protein 25 (Hsp25) in addition to the expression of the angiogenesis markers vascular endothelial growth factor, cluster of differentiation 34 and factor VIII related antigen. Furthermore, immunoprecipitation indicated that knockout of Hsf1 inhibited the association between SV40/TAG and p53 or pRb. These data suggest that Hsf1 is involved in the regulation of SV40/TAG-derived fibroblastoma growth and metastasis by modulating the association between SV40/TAG and tumor suppressor p53 and pRb. The current study provides further evidence that Hsf1 may be a novel therapeutic target in the treatment of cancer.

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

P62/SQSTM1 synergizes with autophagy for tumor growth in vivo.

Autophagy is crucial for cellular homeostasis and plays important roles in tumorigenesis. FIP200 (FAK family-interacting protein of 200 kDa) is an essential autophagy gene required for autophagy induction, functioning in the ULK1-ATG13-FIP200 complex. Our previous studies showed that conditional knockout of FIP200 significantly suppressed mammary tumorigenesis, which was accompanied by accumulation of p62 in tumor cells. However, it is not clear whether FIP200 is also required for maintaining tumor growth and how the increased p62 level affects the growth in autophagy-deficient FIP200-null tumors in vivo. Here, we describe a new system to delete FIP200 in transformed mouse embryonic fibroblasts as well as mammary tumor cells following their transplantation and show that ablation of FIP200 significantly reduced growth of established tumors in vivo. Using similar strategies, we further showed that either p62 knockdown or p62 deficiency in established FIP200-null tumors dramatically impaired tumor growth. The stimulation of tumor growth by p62 accumulation in FIP200-null tumors is associated with the up-regulated activation of the NF-κB pathway by p62. Last, we showed that overexpression of the autophagy master regulator TFEB(S142A) increased the growth of established tumors, which correlated with the increased autophagy of the tumor cells. Together, our studies demonstrate that p62 and autophagy synergize to promote tumor growth, suggesting that inhibition of both pathways could be more effective than targeting either alone for cancer therapy.

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway.

Epigenetic Regulator Smchd1 Functions as a Tumor Suppressor

SMCHD1 is an epigenetic modifier of gene expression that is critical to maintain X chromosome inactivation. Here, we show in mouse that genetic inactivation of Smchd1 accelerates tumorigenesis in male mice. Loss of Smchd1 in transformed mouse embryonic fibroblasts increased tumor growth upon transplantation into immunodeficient nude mice. In addition, loss of Smchd1 in Eμ-Myc transgenic mice that undergo lymphomagenesis reduced disease latency by 50% relative to control animals. In premalignant Eμ-Myc transgenic mice deficient in Smchd1, there was an increase in the number of pre-B cells in the periphery, likely accounting for the accelerated disease in these animals. Global gene expression profiling suggested that Smchd1 normally represses genes activated by MLL chimeric fusion proteins in leukemia, implying that Smchd1 loss may work through the same pathways as overexpressed MLL fusion proteins do in leukemia and lymphoma. Notably, we found that SMCHD1 is underexpressed in many types of human hematopoietic malignancy. Together, our observations collectively highlight a hitherto uncharacterized role for SMCHD1 as a candidate tumor suppressor gene in hematopoietic cancers.

Sp1 is involved in regulation of cystathionine γ-lyase gene expression and biological function by PI3K/Akt pathway in human hepatocellular carcinoma cell lines

Hydrogen sulfide (H2S) has been found to play an important role as a novel gasotransmitter involved in many biological processes. The regulatory role of endogenous H2S-producing enzyme on cancer cell survival is complex and unclear. According to the data that cystathionine γ-lyase (CSE) gene, catalyzed H2S production in trans-sulfuration pathway, was upregulated in Akt stably transformed mouse embryonic fibroblast cells, the mechanisms that elevated CSE expression by PI3K/Akt signaling pathway and its biological functions in cell survival were studied. In the present study, firstly, the results showed that PI3K/Akt positively correlated with CSE expression levels in human hepatocellular carcinoma cell lines. CSE expression was decreased by the PI3K inhibitor or Akt deletion, while upregulated with the activating of Akt. Based on dual-luciferase reporter assay, the −592/+139 gene fragment represented the CSE core promoter, and the PI3K/Akt pathway regulated CSE expression on transcriptional level. Sp1 was the critical transcription factor in regulation of CSE expression via the mutation of transcription factor binding sites on the promoter. Furthermore, we proved that Sp1 could directly bind to CSE promoter by ChIP assay. In addition, we explored that the endogenous H2S production was connected with the regulated CSE expression, and CSE/H2S promoted human hepatocellular carcinoma cell proliferation via cell cycle progression regulation. In summary, we have, for the first time, demonstrated that PI3K/Akt pathway regulates the CSE expression via Sp1, which is particularly important to understand the effect of PI3K/Akt and CSE on the tumorigenesis.

Abstract B207: Darinaparsin is an anti-solid tumor cytotoxin in vivo.

Abstract Background: Darinaparsin (DPS; provided by ZIOPHARM Oncology, Inc.) is a novel organic arsenical with promising anticancer activity and a favorable systemic toxicity profile. Compared to the inorganic arsenical anti-cancer drug, arsenic trioxide (ATO), DPS has higher in vitro potency against solid tumor cells under both normoxic (NO) and hypoxic (HO) conditions. Under HO, an important feature of the solid tumor microenvironment, DPS activity was not dependent on the generation of reactive oxygen species and subsequent oxidative stress. Here we report results of studies of the in vivo anti-tumor effect of DPS in xenograft solid tumor models and patient-derived prostate cancer tumor grafts, as well as mechanistic studies including cDNA microarray and other experiments to study mechanisms of action of DPS. Materials and Methods: To test the in vivo efficacy of DPS, we used mouse models of human prostatic [hormone-independent (HI) LAPC-4] and pancreatic [PANC-1] xenograft tumors implanted subcutaneously in immunodeficient mice. In addition, patient-derived prostate cancer tissues were precision-cut and implanted as tissue slice grafts (TSGs) under the renal capsule in immunodeficient mice. Both tumor models were treated with DPS (100 mg/kg, IP, 3 times/wk × 4 wks), or saline as a control. The TSG tumor growth was assessed by immunohistochemistry (AMACR/p63) staining, and the results were represented as the total tumor area in TSG sections. The effects of DPS on JNK and HIF1 signaling pathways were determined by western blot and RT-qPCR. Using cDNA microarray and subsequent pathway analyses, we studied the effect of DPS and ATO on global gene expression in HI-LAPC-4 cells. Normal and transformed Mouse Embryonic Fibroblasts (MEFs) overexpressing the oncogene HRAS and E1A were used to study the effect of DPS on these oncogenes that are important for susceptibility to cell killing. Results: In both HI-LAPC-4 and PANC-1 tumor models, DPS significantly inhibited tumor growth (P < 0.0001). The average tumor volume doubling time increased from 3.95 days (95% CI: 4.22 − 3.72) to 11.8 days (95% CI: 11.2 − 12.5) in HI-LAPC-4 tumors (P < 0.001), and 3.83 days (95% CI: 3.65 − 4.02) to 6.82 (95% CI: 6.59 − 7.07) in PANC-1 tumors (P < 0.001). In the TSG model, DPS decreased the tumor growth by 50% in 4 of the 6 TSGs as compared with the paired control (P = 0.139). DPS-treated mice exhibited decreased physical activity and early body weight loss (∼5–10% in the 1st wk), but no other significant systemic toxicities as assessed by organ histology and blood biochemistry. As previously reported in leukemia cells, JNK was activated by DPS under both NO and HO; however, the JNK inhibitor SP600125 did not inhibit DPS activity. HIF1α, a major regulator of cell survival under HO, was not affected by DPS. Microarray analysis revealed a significant effect of DPS (but not ATO) on RAS and MYC oncogenic pathways. Compared to normal MEFs, oncogene (RAS/E1A) transformed MEFs were markedly more sensitive to DPS-induced apoptosis under both NO and HO. Conclusions: DPS had significant cytotoxic activity in vivo in the two tumor models studied (HI-LAPC-4 and PANC-1) in mice at a dose that was well-tolerated. This may be due, in part, to DPS inhibition of mutant and dysregulated oncogenic pathways that are required for survival of tumor, but not normal cells. Since DPS is in early clinical development as a single agent, these findings may have near term translational potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B207.

Abstract 1633: C-terminal binding proteins are novel drug targets

Abstract The CtBP transcriptional corepressors have been identified as proto-oncogenes. CtBP represses pro-apoptotic genes and promotes cancer cell migration and invasion in a redox sensitive manner through a regulatory dehydrogenase domain. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and induces apoptosis in the human colorectal cancer cell line HCT116. MTOB induced apoptosis is dependent upon expression of the BH3 only protein BIK, a previously established CtBP target. Treatment of both native and transformed Mouse Embryonic Fibroblasts revealed a wide therapeutic index for CtBP inhibition. In human colon cancer cell peritoneal xenografts, MTOB treatment reduced tumor growth and induced apoptosis in vivo without notable toxicity. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Thus, CtBP is a viable therapeutic target in human cancer. In order to identify novel CtBP inhibitors, a simple dehydrogenase assay was developed that utilizes the spectrophotometric measurement of enzymatic conversion of NADH to NAD+ by the CtBP dehydrogenase activity. This assay successfully detects MTOB substrate and inhibitor activity, as well as inhibition of CtBP activity by the related compound 4-methylthio-2-hydroxybutyric acid (MTHB). This assay will be adapted for high throughput screening of small molecule libraries for novel CtBP inhibitors that can be further characterized in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2011-1633

The PI3K‐Akt Pathway Regulates Calpain 6 Expression, Proliferation, and Apoptosis

ObjectiveCalpain 6 Expression Regulated by the PI3K‐Akt Pathway and its mechanism.ResultsAccording to the data that CAPN6 was up‐regulated in the Akt transformed mouse embryonic fibroblast cells by cDNA chip, the mechanisms underlying elevated CAPN6 expression by PI3K‐Akt signaling pathway and its biological functions were studied. The results showed that CAPN6 was down‐regulated on transcriptional and post‐transcriptional levels by the PI3K inhibitor or Akt deletion. CAPN6 protein was stabilized by PI3K‐GSK‐3β pathway. Deleted CAPN6 promoters activity were assessed by dual‐luciferase reporter system, and the founding indicated that −93/+200 DNA fragment was the core promoter of it. Transcription factor binding sites in the CAPN6 promoter were mutated and the results showed that AP1, Oct‐1, and FoxD3 were the critical transcription factors in regulation of CAPN6 expression. In addition, CAPN6 promoted cancer cell proliferation and inhibited its apoptosis.ConclusionThe finding demonstrates that CAPN6 is regulated by the PI3K‐Akt signaling pathway and provides evidence that it may be a therapeutic target of cancer.

The PI3K-Akt pathway regulates calpain 6 expression, proliferation, and apoptosis

The calpains are a family of cysteine proteases involved in some biological processes whose activities are highly dependent on Ca2+. Calpain 6 (CAPN6), one member of the family, is unique in that it lacks the active-site cysteine residues for protease activity. According to the data that CAPN6 was up-regulated in the Akt transformed mouse embryonic fibroblast cells by cDNA chip, the mechanisms underlying elevated CAPN6 expression by PI3K-Akt signaling pathway and its biological functions were studied. The results showed that CAPN6 was down-regulated on transcriptional and post-transcriptional levels by the PI3K inhibitor or Akt deletion. CAPN6 protein was stabilized by PI3K-GSK-3β pathway. Deleted CAPN6 promoters activity were assessed by dual-luciferase reporter system, and the founding indicated that −93/+200 DNA fragment was the core promoter of it. Transcription factor binding sites in the CAPN6 promoter were mutated and the results showed that AP1, Oct-1, and FoxD3 were the critical transcription factors in regulation of CAPN6 expression. In addition, CAPN6 promoted cancer cell proliferation and inhibited its apoptosis. The finding demonstrates that CAPN6 is regulated by the PI3K-Akt signaling pathway and provides evidence that it may be a therapeutic target of cancer.

Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand

CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3′-OH and 5′-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibited no effect on the expression of the nicked DNA in human fibroblasts immortalised by SV40 large T-antigen but caused a very strong decrease of gene expression in spontaneously transformed mouse embryonic fibroblasts (MEFs). Compared to the corresponding CSB-proficient MEFs, the effect was on average 6.7-fold stronger for a defined SSB located in the non-transcribed DNA strand, but only 2.4-fold for a SSB in the transcribed strand and 1.7-fold for a SSB located in the non-genic region. At the same time, CSB deficiency did not compromise the overall efficiency of repair of SSBs generated by treatment of the cells with hydrogen peroxide. The gene expression data thus indicate that CSB prevents irreversible transcription failures at the sites of DNA damage, acting preferentially at SSBs located in the non-transcribed DNA strand of the transcribed genes. We further conclude that SSBs in the non-transcribed DNA strand are commonly more harmful for transcription than those situated in the transcribed strand.

Cell cultivation on microspheres coupled with histones

We have studied the application of histones for the modification of a cell-culture surface. Experiments were conducted on 293 cell lines (human embryonic kidney cells transformed by Ad5 adenovirus) and BALB/3T3 clone A31 (spontaneously transformed mouse embryonic fibroblasts). The cell’s interactions with various types of histons placed on a hydrophobic glass surface or 1.0 μm dextran microspheres was assessed. It was found that all histones studied exhibited adhesive properties but their complexes, such as the total and core histones, were the most adhesive and had improved cell morphology. Cross-linked conjugates of histone complexes immobilized on microshperes facilitated cell network formation by cellular interactions and cell contacts with several microspheres. For the 293 line, unlike BALB/3T3 clone, the A31 cells significantly increased their proliferative activity on microspheres coated with cross-linked conjugates of histone complexes. The study showed that the substrate may be applied for 3D pore matrices designed for tissue-like cell structures in vitro.

Reduced Eukaryotic Initiation Factor 2Bε-Subunit Expression Suppresses the Transformed Phenotype of Cells Overexpressing the Protein

Eukaryotic initiation factor 2B (eIF2B), a five-subunit guanine nucleotide exchange factor, plays a key role in the regulation of mRNA translation. Expression of its epsilon-subunit is specifically up-regulated in certain conditions associated with increased cell growth. Therefore, the purpose of the present study was to examine the effect of repressing eIF2Bepsilon expression on growth rate, protein synthesis, and other characteristics of two tumorigenic cell lines that display up-regulated expression of the epsilon-subunit. Experiments were designed to compare spontaneously transformed fibroblasts to transformed mouse embryonic fibroblasts infected with a lentivirus containing a short hairpin RNA directed against eIF2Bepsilon. Cells expressing the short hairpin RNA displayed a reduction in eIF2Bepsilon abundance to 30% of the value observed in uninfected transformed mouse embryonic fibroblasts, with no change in the expression of any of the other four subunits. The repression of eIF2Bepsilon expression was accompanied by reductions in guanine nucleotide exchange factor activity and global rates of protein synthesis. Moreover, repressed eIF2Bepsilon expression led to marked reductions in cell growth rate in culture, colony formation in soft agar, and tumor progression in nude mice. Similar results were obtained in MCF-7 human breast cancer cells in which eIF2Bepsilon expression was repressed through transient transfection with a small interfering RNA directed against the epsilon-subunit. Overall, the results support a role for eIF2Bepsilon in the regulation of cell growth and suggest that it might represent a therapeutic target for the treatment of human cancer.

Impaired DNA double-strand break repair contributes to chemoresistance in HIF-1 -deficient mouse embryonic fibroblasts

A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1alpha-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1alpha. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of gammaH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1alpha. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1alpha protein in cancer cells with a partial HIF-1alpha mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1alpha might contribute to HIF-dependent tumor therapy resistance.

Repressed eIF2B‐epsilon expression delays tumor growth in transformed mouse embryonic fibroblasts (TMEF)

eIF2B is an essential translation initiation factor that mediates both global and specific regulation of mRNA translation. One mechanism for increasing eIF2B activity is upregulated expression of the catalytic ε-subunit of the protein. In this regard, in many transformed cells, expression of eIF2Bε, but not the other four subunits of the protein, is increased. Furthermore, eIF2Bε mRNA expression is elevated in 80% of tumors examined. In the present study, the hypothesis that increased eIF2B activity due to elevated expression of eIF2Bε leads to cellular transformation and tumor progression was examined using spontaneously transformed MEF's. A lentiviral shRNA system was generated that repressed eIF2Bε protein expression by 60% with no change in expression of any of the other four eIF2B subunits. Repression of eIF2Bε expression in TMEFs resulted in a ~30% reduction in eIF2B activity and global rates of protein synthesis, and a marked reduction in growth rate. Repression of eIF2Bε expression also attenuated the transformed phenotype as assessed both by markedly decreased colony formation in soft agar and dramatically delayed tumor appearance and progression in nude mice. The studies help solidify the role of eIF2B, and specifically eIF2Bε, as an important regulator of overall cell growth and proliferation, and may highlight a novel cancer drug target. (Supported by NIH grant DK-15658)

Inhibition of Fatty Acid Synthase Induces Endoplasmic Reticulum Stress in Tumor Cells

Fatty acid synthase (FAS), the cellular enzyme that synthesizes palmitate, is expressed at high levels in tumor cells and is vital for their survival. Through the synthesis of palmitate, FAS primarily drives the synthesis of phospholipids in tumor cells. In this study, we tested the hypothesis that the FAS inhibitors induce endoplasmic reticulum (ER) stress in tumor cells. Treatment of tumor cells with FAS inhibitors induces robust PERK-dependent phosphorylation of the translation initiation factor eIF2alpha and concomitant inhibition of protein synthesis. PERK-deficient transformed mouse embryonic fibroblasts and HT-29 colon carcinoma cells that express a dominant negative PERK (DeltaC-PERK) are hypersensitive to FAS inhibitor-induced cell death. Pharmacologic inhibition of FAS also induces the processing of X-box binding protein-1, indicating that the IRE1 arm of the ER stress response is activated when FAS is inhibited. Induction of ER stress is further confirmed by the increased expression of the ER stress-regulated genes CHOP, ATF4, and GRP78. FAS inhibitor-induced ER stress is activated prior to the detection of caspase 3 and PARP cleavage, primary indicators of cell death, whereas orlistat-induced cell death is rescued by coincubation with the global translation inhibitor cycloheximide. Lastly, FAS inhibitors cooperate with the ER stress inducer thapsigargin to enhance tumor cell killing. These results provide the first evidence that FAS inhibitors induce ER stress and establish an important mechanistic link between FAS activity and ER function.

Hypoxia-induced radioresistance is independent of hypoxia-inducible factor-1A in vitro

We sought to determine whether hypoxia-induced radioresistance is mediated by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). We used 2 mouse embryonic fibroblast cell lines transformed with H-ras and TAg, 1 HIF-1alpha+/+ and the other HIF-1alpha-/-. Cell were exposed to either 95% air and 5% CO2 (normoxic conditions) or 0.2% O2, 94.8% N2, and 5% CO2 (hypoxic conditions) for 4 hours. Cells were then irradiated and subjected to clonogenic survival assays. Whereas neither +/+ ras/TAg nor -/- ras/TAg cells expressed HIF-1alpha under normoxic conditions, hypoxia induced expression of HIF-1alpha only in +/+ ras/TAg cells, confirming the absence of HIF-1alpha in -/- ras/TAg cells. Clonogenic survival curves for +/+ ras/TAg and -/- ras/TAg cells under normoxia and hypoxia demonstrated that hypoxia increased radioresistance in both cell lines to the same degree. At 1-log cell kill, the +/+ ras/TAg and -/- ras/TAg cells had an identical oxygen enhancement ratio of 1.28 +/- 0.09 and nearly identical oxygen enhancement ratios at 2-log cell kill. In our system of transformed mouse embryonic fibroblasts, hypoxia-mediated radiation resistance is independent of HIF-1alpha.