Fas Promoter Research Articles

Pirin Inhibits FAS-Mediated Apoptosis to Support Colorectal Cancer Survival.

Resistance to immunotherapy in colorectal cancer (CRC) is associated with obstruction of FAS (Apo-1 or CD95)-dependent apoptosis, a hallmark of cancer. Here it is demonstrated that the upregulation of pirin (PIR) protein in colon cancers promotes tumorigenesis. Knockout or inhibition of PIR dramatically increases FAS expression, FAS-dependent apoptosis and attenuates colorectal tumor formation in mice. Specifically, NFκB2 is a direct transcriptional activator of FAS and robustly suppressed by PIR in dual mechanisms. One is the disruption of NFκB2 complex (p52-RELB) association with FAS promoter, the other is the inhibition of NIK-mediated NFκB2 activation and nuclear translocation, leading to the inability of active NFκB2 complex toward the transcription of FAS. Furthermore, PIR interacts with FAS and recruits it in cytosol, preventing its membrane translocation and assembling. Importantly, knockdown or knockout of PIR dramatically sensitizes cells to FAS mAb- or active CD8+ T cells-triggered cell death. Taken together, a PIR-NIK-NFκB2-FAS survival pathway is established, which plays a key role in supporting CRC survival.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing

BackgroundElevated TCRαβ+CD4-CD8- double-negative T-cells (DNT) and serum biomarkers help identifying FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified upon standard exon sequencing (ALPS-undetermined: ALPS-U). ObjectiveWe aimed to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. MethodsGenetic analysis included whole FAS gene sequencing, copy number variation analysis and sequencing of FAS cDNA and other FAS pathway related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss-of-heterozygosity (sLOH). ResultsNine of 16 ALPS-U patients lacked FAS expression on CD57+DNT predicting heterozygous “loss of expression” FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7/9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT, one patient showed a FAS exon duplication. Three patients had reduced FAS expression, two of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the four ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. ConclusionA combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing. Clinical implicationDetection of complex FAS pathway gene alterations by extended genetic analysis allows targeted therapy with sirolimus.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Lipid Nanoparticle Delivery of Fas Plasmid Restores Fas Expression to Suppress Melanoma Growth In Vivo.

Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-Tcell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to Tcell cytotoxicity. DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas+ tumor cells and suppresses established melanoma growth in immune-competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein levels on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted Tcells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data demonstrate that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

Simple SummaryA key feature of human colorectal tumor is loss of FAS expression. FAS is the death receptor for FASL of activated T cells. Loss of FAS expression therefore may promote tumor cell immune escape. We aimed at determining whether restoring FAS expression is sufficient to suppress colorectal tumor growth. Mouse and human FAS cDNA was synthesized and encapsulated into cationic lipid nanoparticle DOTAP-Cholesterol to formulate DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Restoring FAS expression in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro and suppressed colon-tumor growth and progression in tumor-bearing mice in vivo. Restoring FAS expression induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells in vitro. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. Tumor-selective delivery of FAS DNA nanoparticle is potentially an effective therapy for human colorectal cancer.A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting FAS signaling.

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between HOTTIP lncRNA and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-seq (HOTTIP binding in genome-wide), ChIP-seq, and ATAC-seq, we found that some miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP−/− AML cells. HOTTIP bound to the miR-196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival of mice compared to wild-type cells transplanted into mice. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, and consequently represses tumor suppressors and promotes leukemogenesis.

ARID1A Controls a Novel Transcriptional Network Regulating FAS in Follicular Lymphoma

Follicular lymphoma (FL) is a clinically and genetically heterogeneous disease. Somatic gene mutations contribute to the heterogeneous clinical course of FL. ARID1A, which encodes for a subunit of the SWI/SNF chromatin remodeling complex, is among the most commonly mutated genes in FL (up to 15% of cases). These mutations are mostly disruptive and are predicted to result in protein haplodeficiency. While we have previously shown that ARID1A mutations are predictive of treatment outcome (Pastore, 2015), the underlying biology of ARID1A loss in FL is unclear.A functional genome-wide in vitro screen showed that ARID1A loss rescued a number of cancer cell lines from FAS-L induced apoptosis (Luo, 2008). FAS-L induced apoptosis plays a critical role in normal B-cell development and homeostasis. Thus, FAS/FAS-L deficiency could contribute to FL development and disease biology. Therefore, we studied the role of ARID1A loss in FAS expression and regulation.We first tested FAS-L induced apoptosis in established lymphoma cell lines that harbor the FL-hallmark translocation t(14;18)[BCL2/IGH] plus ARID1A mutations (Karpas422, WSU-FSCCL) or no ARID1A mutations (OCI-Ly1, OCI-Ly8, SU-DHL16). ARID1A mutant (mut) cells were indeed markedly less sensitive to FAS-L (300 ng/mL/24 hrs) compared to ARID1A wild type (WT) cells (98% vs 52% mean viability by Annexin-V). FAS receptor expression on mutant cells was reduced by almost half compared to WT cells by FACS analysis (N=3, P=0.0004).To test if reduced FAS expression was directly linked to ARID1A loss, we generated single-cell derived clones (from OCI-Ly1 and OCI-Ly8) with either heterozygous (het) loss or complete knock-out (KO) of ARID1A by CRISPR/Cas9. ARID1A loss was validated by Sanger sequencing and Western blot. We consistently observed significantly reduced FAS-L induced apoptosis in het and KO clones (exemplary shown for OCI-Ly8 in Fig A). Remarkably, re-expressed of ARID1A in het cells (het+ARID1A) rescued sensitivity to FAS-L induced apoptosis (Fig A). We confirmed reduced FAS expression on mutant clones by FACS, while re-expression of ARID1A rescued its expression (Fig B). Furthermore, FAS mRNA expression was significantly reduced by qPCR in mut vs WT clones (N=4, P<0.05), while FAS mRNA levels were rescued to WT levels in het+ARID1A cells.To understand the molecular mechanism that links ARID1A loss and reduced FAS expression, we performed ATAC sequencing (Seq) and RNA Seq on 15 single-cell derived clones (9 mut and 6 WT from OCI-Ly1 and OCI-Ly8). RNA Seq confirmed significantly lower ARID1A and FAS mRNA levels (adj p<0.001 each) in the mut clones. We first hypothesized that ARID1A loss could directly affect chromatin accessibility at the FAS promoter. However, we did not observe different chromatin accessibility at the FAS promoter. Next, we searched our data for all known FAS-regulating transcription factors (TFs) (https://dorothea.opentargets.io/#/), but could not identify candidates that were both differentially accessible and differentially expressed. Finally, we searched our data for transcriptional networks, i.e. hubs of all recognized FAS-regulating TFs and their known and predicted interacting partners (https://string-db.org/). Through this, we identified RUNX3, a predicted Co-TF of ETS1, to be both less accessible (“closed chromatin”) and less expressed upon ARID1A loss (Fig C), suggesting a novel ARID1A-dependent FAS-regulatory network.To functionally validate our model, we first confirmed reduced RUNX3 expression in ARID1A mutant clones by qPCR and Western blot, and showed that ETS1 levels were unaffected by ARID1A loss. Then, we stably overexpressed RUNX3 in ARID1A mutant clones by lentiviral transduction and could indeed show rescue of FAS surface levels by FACS (Fig D).Lastly, we wanted to validate our findings in primary patients samples. We quantified FAS expression in FL biopsies with known ARID1A mutation status by nCounter gene expression profiling (GEP; N=51, 12 mut vs 39 WT) and quantitative multispectral imaging (QMI; N=44, 10 mut vs 34 WT) (Fig E). Both approaches showed significantly reduced FAS expression in ARID1A mutant FL (P<0.05 for GEP, P<0.0001 for QMI; Fig E).In summary, we show that ARID1A loss is directly linked to reduced FAS expression via a novel RUNX3/ETS1 transcriptional network, potentially opening avenues for therapeutic targeting of this clinically relevant perturbation. [Display omitted] DisclosuresSubklewe: Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment; Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding.

SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth.

Trimethylation of histone 3 lysine 9 (H3K9me3) at gene promoters is a major epigenetic mechanism that silences gene expression. We have developed a small molecule inhibitor for the H3K9me3-specific histone methyltransferase SUV39H1. We report here that FAS expression is significantly down-regulated and SUV39H1 expression is significantly up-regulated in human colorectal carcinoma (CRC) as compared to normal colon. SUV39H1-selective inhibitor F5446 decreased H3K9me3 deposition at the FAS promoter, increased Fas expression, and increased CRC cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, inhibition of SUV39H1 altered the expression of genes with known functions in DNA replication and cell cycle in the metastatic colon carcinoma cells, which is associated with cell cycle arrest at S phase in the metastatic human colon carcinoma cells, resulting in tumor cell apoptosis and growth inhibition in a concentration-dependent manner in vitro. Moreover, F5446 increased 5-FU-resistant human CRC sensitivity to both 5-FU- and FasL-induced apoptosis and inhibited tumor cell growth in vitro. More importantly, F5446 suppressed human colon tumor xenograft growth in vivo. Our data indicate that pharmacological inhibition of SUV39H1 is an effective approach to suppress human CRC.

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

BackgroundFibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood.MethodsPrimary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young’s moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP.ResultsFas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1.ConclusionsSoluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Fas promoter -670 polymorphism and the risk of cervical cancer: A case-control study in multi-ethnic Malaysia

Background/Objective: Single nucleotide polymorphism (SNP) is a commonly occurring DNA sequence variation within the human population. The prevalence of these sequence variations differs in various populations and may lead to the suboptimal regulation of genes, including those of the apoptosis pathway. The regulation of cellular death is partially controlled by the interaction between tumor necrosis factor receptor family gene, Fas and its ligand, FasL. A base substitution in Fas -670 A&gt;G (rs1800682) has been found to alter the binding affinity of the Fas protein to its activators, potentially altering its apoptotic potential. The lack of proper apoptosis regulation can lead to a wide plethora of human conditions involving uncontrolled cellular growth such as cancer. The purpose of the study was to investigate whether there was any association between the Fas -670 polymorphism and risk of cervical cancer in multi-ethnic Malaysian women.Methods: Using restriction fragment length polymorphism (RFLP-PCR), Chi-square and logistic regression analysis, this study investigated the Fas -670 SNP and its associations with cervical cancer in the multi-ethnic population of Malaysia.Results: No significant associations were found between Fas -670 A&gt;G SNP and risk of cervical cancer using the genotype model, dominant model and allele frequency model analysis, even after stratification into the Malay, Chinese and Indian ethnic subgroups; and cancer types.Conclusions: Our results showed that the A&gt;G SNP of Fas -670 does not affect the risk of cervical carcinogenesis in Malaysian females.

Abstract 1028: Genetic disruption of Kras sensitizes lung cancer cells to Fas-mediated apoptosis

Abstract Genetic lesions that activate KRAS account for ~30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras. Here we show that tumor cells can survive CRISPR-mediated knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically upregulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas-receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone-deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles’ heel in tumors initiated by oncogenic Kras. Citation Format: Haiwei Mou, Jill Moore, Sunil Malonia, Yingxiang Li, Deniz Ozata, Soren Hough, Chunqing Song, Jordan Smith, Has Yin, Andrew Fisher, Daniel Anderson, Shipping Weng, Michael Green, Wen Xue. Genetic disruption of Kras sensitizes lung cancer cells to Fas-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1028. doi:10.1158/1538-7445.AM2017-1028

Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis

Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

Promoter hypermethylation and downregulation of the FAS gene may be involved in colorectal carcinogenesis.

Aberrant DNA methylation has been investigated in carcinogenesis and as biomarker for the early detection of colorectal cancer (CRC). The present study aimed to define the methylation status in the regulatory elements of two proapoptotic genes, Fas cell surface death receptor (FAS) and BCL2-associated X protein (BAX). DNA methylation analysis was performed in tumor and adjacent normal tissue using HpaII/MspI restriction digestion and methylation-specific polymerase chain reaction (PCR). The results observed downregulation of the FAS and BAX genes in the CRC tissues compared with the adjacent normal samples. Furthermore, demethylation using 5-aza-2'-deoxycytidine treatment followed by reverse-transcription quantitative PCR were performed on the HT-29 cell line to measure BAX and FAS mRNA expression following demethylation. The 5-aza-2'-deoxycytidine treatment resulted in significant FAS gene upregulation in the HT-29 cell line, but no significant difference in BAX expression. Furthermore, analysis of CpG islands in the FAS gene promoter revealed that the FAS promoter was significantly hypermethylated in 53.3% of tumor tissues compared with adjacent normal samples. Taken together, the results indicate that decreased expression of the FAS gene due to hypermethylation of its promoter may lead to apoptotic resistance, and acts as an important step during colorectal carcinogenesis.

H3K9 Trimethylation Silences Fas Expression To Confer Colon Carcinoma Immune Escape and 5-Fluorouracil Chemoresistance.

The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL(+) and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

P0751 : Polymorphisms in the FAS promoter region are not associated with fibrosis progression in HCV-1 and -4 patients

P0751 : Polymorphisms in the FAS promoter region are not associated with fibrosis progression in HCV-1 and -4 patients

APO-1/FAS Promoter (-670A/G) Polymorphisms and Risk of Lupus Nephritis in SLE Egyptian Female Patients

Background: Self-immunization in systemic lupus is driven by defective in apoptosis. Fas, is an apoptosis-promoting cell surface receptor. The present study evaluate the possible association between APO-1/FAS Promoter (-670A/G) Polymorphism and sFAS level with susceptibility to lupus nephritis in SLE patients. Design and Methods: This study was performed on 88 female patients with SLE (mean age, 39.82 ± 10.16 years). 82 patients with lupus nephritis (mean age, 42.50 ± 6.65 years). 150 age and sex-matched person served as controls. All participants were genotyped for the APO-1/FAS Promoter (-670A/G) Polymorphism, manifestations and serum sFAS were correlated with the genotypes. Results: Serum sFAS was significantly higher in patients with -670 AA genotype compared to others. (-670A/G) AA genotype frequencies were significantly higher in the lupus nephritis and SLE patients groups compared with the controls and were associated with increased risk for lupus nephritis and SLE development (odds ratio, 4.08 and 1.91 respectively). Conclusions: The APO-1/FAS Promoter (-670A/G) A allele can be used as a genetic marker for lupus nephritis susceptibility in SLE and was associated with high sFAS level.

Abstract 2498: Detection of S-nitrosylated heat shock protein 90 in renal cell cancer

Abstract Purpose: Nitric oxide (NO) is a versatile signaling molecule. Its roles in a variety of physiological functions in mammals are beginning to be understood. It has been demonstrated that NO up-regulates the expression of the Fas receptor on human tumor cells via specific inactivation of transcription repressor YY1 DNA binding activity to the silencer region of the Fas promoter (Hermes G., et al. 2001). We also showed that inactivation of YY1 via protein S-nitrosylated by NO in prostatic cancer could be identified (Hongo F., et al. 2005). This post-translational modification, known as S-nitrosylation, has emerged as a highly conserved and spatiotemporally specific signaling mechanism. The objective of our study was to examine the effect of NO donor treatment on renal cell cancer. Methods: We examined the effect of the NO donor DETA-NONOate (100-500uM) on the following renal cell cancer cell lines; CAKI-1, NC65, and ACHN. Anti-S-nitroso-Cysteine antibody in the rabbit (SIGMA-ALDRICH) was applied as a primary antibody for immunohistochemistry. The biotin-switch technique was applied to detect specific S-nitrosylated proteins. Whole cell lysates were extracted from ACHN cells employed with DETA-NONOate. Using an S-nitrosylated detection kit (Cayman Chemical Company), S-NO was replaced by Biotin. Cell lysates were incubated at 4 oC with 30 μL of Dynabeads M-280 Streptavidin (Invitrogen) for 3 hrs. After five washes with wash buffer (S-Nitrosylation Wash buffer), the protein complex was eluted from anti-biotin dynabeads by incubation with 1x volume of elution buffer (0.1M glycine (pH2.0)) twice for 15 min. Each sample was added to an SDS-polyacrylamide gel for mass spectrometry. Results: Significant up-regulation of S-nitrosylated proteins employed with DETA-NONOate was observed by immunohistochemistry. Specific S-nitrosylated proteins in ACHN cells were detected by the biotin-switch technique and included HSP90 (heat shock protein 90), GRP78 (HSP70 family), HSP70, and pyruvate kinase M2. In ACNH cells, Significant up regulation of S-nitrosylated HSP90 by western blot. Conclusions: Our data showed that NO treatment significantly increased S-nitrosylation of several proteins. S-nitrosylation should be considered as one of the mechanisms by which NO acts on cancer cells. Citation Format: Fumiya Hongo, Takashi Ueda, Saya Ito-Ueda, Masakatsu Oishi, Terukazu Nakamura, Yoshio Naya, Tsuneharu Miki. Detection of S-nitrosylated heat shock protein 90 in renal cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2014-2498

Epigenetic Control of NF-κB-Dependent FAS Gene Transcription during Progression of Myelodysplastic Syndromes

The death domain containing TNF receptor 6 (CD95/Fas) is a direct target for the NF-κB transcription factor and is repressed in solid tumors such as colon carcinomas. Previously, we reported that the Fas death receptor, while overexpressed in low-risk myelodysplastic syndromes (MDS), becomes undetectable on CD34(+) progenitors when the disease progresses to secondary acute myeloid leukemia (AML). This study determined the interplay between NF-κB and Fas during MDS progression. We first observed that Fas was induced by TNF-α in the HL60 cell line. In these cells, p65 (RELA) was associated with the FAS promoter, and inhibition of the NF-κB pathway by an IKKα inhibitor (BAY11-7082) or lentiviral expression of a nondegradable mutant of IκBα (IκSR) blocked Fas expression. In contrast, TNF-α failed to induce Fas expression in the colon carcinoma cell line SW480, due to hypermethylation of the FAS promoter. Azacitidine rescued p65 binding on FAS promoter in vitro, and subsequently Fas expression in SW480 cells. Furthermore, inhibition of the NF-κB pathway decreased the expression of Fas in MDS CD45(lo)CD34(+) bone marrow cells. However, despite the nuclear expression of p65, Fas was often low on CD45(lo)CD34(+) AML cells. TNF-α failed to stimulate its expression, while azacitidine efficiently rescued p65 binding and Fas reexpression. Overall, these data suggest that DNA methylation at NF-κB sites is responsible for FAS gene silencing.

AB0132 Genetic polymorphisms of fas and fasl apoptotic proteins as susceptibility factors to systemic lupus erythematosus in brazilian patients

BackgroundSystemic Lupus Erythematosus (SLE) is an autoimmune chronic inflammatory disease that affects multiple organs and systems, presenting a wide range of clinical manifestations. Immunological, environmental, hormonal and genetic factors may...