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Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial-mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1-Cre, and LSL-KrasG12D ) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E-Cadherin and upregulation of N-Cadherin, Snail, Slug, Zeb1, Nanog and BMI-1. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol-containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency-maintaining factors (cMyc, KLF4, SOX-2, and Oct-4), N-Cadherin, EMT-transcription factors (Snail, Slug, and Zeb1), and lower expression of E-cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of inflammatory cytokines (TNF-α, IL-6, and IL-8) and PTGS-2 (COX-2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.

Several studies have confirmed the involvement of cancer stem cells (CSC) in tumour progression, metastasis, drug resistance and cancer relapse. SATB2 (special AT‐rich binding protein‐2) acts as a transcriptional co‐factor and modulates chromatin architecture to regulate gene expression. The purpose of this review was to discuss the pathophysiological roles of SATB2 and assess whether it could be used as a therapeutic target for cancer. SATB2 modulated the expression of those genes which regulated pluripotency and self‐renewal. Overexpression of SATB2 gene in normal epithelial cells was shown to induce transformation, as a result transformed cells gained CSC’s characteristics by expressing stem cell markers and pluripotency maintaining factors, suggesting its role as an oncogene. In addition, SATB2 induced epithelial‐mesenchymal transition (EMT) and metastasis. Interestingly, the expression of SATB2 was positively correlated with the activation of β‐catenin/TCF‐LEF pathway. Furthermore, SATB2 silencing inhibited EMT and their positive regulators, and tumour growth, and suppressed the expression of stem cell markers, pluripotency maintaining factors, cell cycle and cell survival genes, and TCF/LEF targets. Based on the cancer genome atlas (TCGA) expression data and published papers, SATB2 alone or in combination with other proteins could be used a diagnostic biomarker for cancer. Although there is no pharmacological inhibitor of SATB2, studies using genetic approaches suggest that SATB2 could be a potential target for cancer treatment and prevention.

Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4810.

In the United States, Hepatocellular Carcinoma (HCC) incidence has tripled over the past two decades. The disease has disproportionately affected minority and disadvantaged populations. The purpose of this study was to examine the expression of SATB2 gene in HCC cells derived from African Americans (AA) and Caucasian Americans (CA) and assess its oncogenic potential by measuring cell viability, spheroid formation, epithelial‐mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in human primary hepatocytes, HCC cells derived from AA and CA, and HCC CSCs. Hepatocellular carcinoma cells derived from AA expressed the higher level of SATB2 than those from CA. By comparison, normal human hepatocytes did not express SATB2. Higher expression of SATB2 in HCC cells from AA was associated with greater growth rate, cell viability, colony formation and EMT characteristics than those from CA. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (c‐Myc, KLF4, SOX2 and OCT4), and EMT compared with non‐targeting control group. The expression of SATB2 was negatively correlated with miR34a. SATB2 rescued the miR‐34a‐mediated inhibition of CSC's viability. These data suggest that SATB2 is an oncogenic factor, and its higher expression may explain the disparity in HCC outcomes among AA.

3-Bromopyruvate (3 BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme of glycolysis pathway and ATP generation in cancer cells. In present investigation we have examined the effect of 3-BromoPyruvate employing panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. The clonogenic survival assay demonstrated dose dependent inhibition of cell growth, and we observed 95% cell death at 15 uM conc. Further, ATP production was severely inhibited in cancer cells treated with same concentration of 3-BP. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone. Electron microscopy revealed that 3-BromoPyruvate severely damaged mitochondrial integrity which might have severely affected ATP generation in cancer cells. We further tested 3-BromoPyruvate (2.5-10.0 mg/kg) in syngeneic panc-2 tumor mouse model and observed that 3-BP at10.0 mg/kg dose significantly reduced tumor growth (volume) about 35-40%. Immunohistochemistry (IHC) data showed that 3-BP inhibits hexokinase II (HK2) and TGFbeta1 and enhanced active caspase-3 expression in tumor tissues as compared to untreated control. The disruption of the interaction between VDAC1 and HK2 due to 3BP treatment was confirmed by CO-IP. Interestingly, 3 BP at higher dose induces caspase-3 activation in tumor derived organoid without showing any effect on normal pancreatic organoid. Altogether, the results in this study demonstrated that 3 BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit K. Roy, Tijana Dukic, Binny Bhandary, Aneesh Acharya, Kevin J. Tu, Young H. Ko, Hem D. Shukla. 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5243.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most lethal cancer in the United States with an estimated 60,430 new cases and 48,220 deaths in 2021. PDACs are characterized by extensive desmoplastic stroma and severe hypovascularity, resulting in an intra-tumoral hypoxic microenvironment. In response to hypoxic stress, cancer cells set off many adaptive responses including metabolism regulation, cell survival, and inflammation through the stabilization and activation of the hypoxia-inducible factor (HIF) family of transcription factors. In this study, we used the gene-editing tool CRISPR-cas9 to knock out HIF1A and investigate whether HIF1A affects the radioresistant and invasive characteristics of PDACs in a KPC cell line model. To first observe how HIF1A affects PDAC radioresistance, we performed a clonogenic survival assay with increasing doses of radiation on both wild-type and HIF1A knockout (KO) cells treated and untreated with CoCl2-induced hypoxia conditions (100 uM and 200 uM CoCl2). Our data showed that under hypoxia, KO cells exhibited significant cell death when treated with 6, 8, and 10 Gy of radiation as compared to wild-type KPC cells, emphasizing the role of HIF1A in radiation resistance. In addition, to understand the role of HIF1A in regulating the invasive behavior of PDACs, we performed a cell proliferation assay on wild-type and KO KPC cells. HIF1A KO cells treated with CoCl2 exhibited significantly reduced proliferation compared to wild-type cells also treated with CoCl2 (p0.05). Because western blot demonstrated increased HIF1A expression in wild-type cells following CoCl2 treatment, our results provided evidence for the role of HIF1A activation in promoting PDAC invasiveness. Through western blot, we also confirmed the association between HIF1A expression with p53 degradation in PDAC. We used COREMINE, a literature mining tool, to map a direct interaction between HIF1A with KRAS in PDACs (p=0.000016). We propose HIF1A as a switch to activate KRAS and degrade p53 under hypoxic conditions in PDAC proliferation. Thus, modulating the HIF1A switch may be an important mechanism to reduce the tumor-promoting microenvironment and inhibit cancer growth. Citation Format: Kevin J. Tu, Sanjit K. Roy, Binny Bhandary, Amit Sawant, Hem D. Shukla. Loss of HIF1A decreases resistance to radiation and invasiveness in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-126.

Radiation therapy plays a major role in the treatment of lung cancer patients. However, cancer cells develop resistance to radiation. Tumor radioresistance is a complex multifactorial mechanism which may be dependent on DNA damage and repair, hypoxic conditions inside tumor microenvironment, and the clonal selection of radioresistant cells from the heterogeneous tumor site, and it is a major cause of treatment failure in non–small cell lung cancer (NSCLC). In the present investigation caveolin-1 (CAV-1) has been observed to be highly expressed in radiation resistant A549 lung cancer cells. CRISPR-Cas9 knockout of CAV-1 reverted the cells to a radio sensitive phenotype. In addition, CAV-1 overexpression in parental A549 cells, led to radiation resistance. Further, gene expression analysis of A549 parental, radiation resistant, and caveolin-1 overexpressed cells, exhibited overexpression of DNA repair genes RAD51B, RAD18, SOX2 cancer stem cell marker, MMPs, mucins and cytoskeleton proteins in resistant and caveolin-1 over expressed A549 cells, as compared to parental A549 cells. Bioinformatic analysis shows upregulation of BRCA1, Nuclear Excision DNA repair, TGFB and JAK/STAT signaling pathways in radioresistant and caveolin-1 overexpressed cells, which may functionally mediate radiation resistance. Immunohistochemistry data demonstrated heterogeneous expression of CAV-1 gene in human lung cancer tissues, which was analogous to its enhanced expression in human lung cancer cell line model and mouse orthotopic xenograft lung cancer model. Also, TCGA PanCancer clinical studies have demonstrated amplification, deletions and missense mutation in CAV-1 gene in lung cancer patients, and that CAV-1 alteration has been linked to poor prognosis, and poor survival in lung cancer patients. Interestingly, we have also optimized ELISA assay to measure caveolin-1 protein in the blood of A549 radiation resistant human xenograft preclinical mouse model and discovered higher level of caveolin-1 (950 pg/ml) in tumor bearing animals treated with radiation, as compared to xenograft with radiosensitive lung cancer cells (450 pg/ml). Thus, we conclude that caveolin-1 is involved in radio-resistance and contributes to tumor aggression, and it has potential to be used as prognostic biomarker for radiation treatment response, and tumor progression for precision medicine in lung cancer patients.

In the US, hepatocellular carcinoma (HCC) incidence has tripled over the past two decades. This trend indicates that social determinants, genetic and environmental factors are driving the epidemic at the population level. Furthermore, the disease has disproportionately affected minority and disadvantaged populations. SATB2 (special AT-rich binding protein-2), a transcription factor and epigenetic regulator, influences gene expression both by modulating chromatin architecture and by functioning as a transcriptional co-factor. SATB2 is highly expressed in embryonic stem cells and progenitor cells, whereas its expression is low or absent in human normal liver tissues. We have recently demonstrated that overexpression of SATB2 can transform normal epithelial cells to cancer stem-like cells in pancreatic, colorectal and breast cancer models, and the expression of SATB2 was significantly higher in cancer tissues compared to normal tissues. The purpose of this study was to examine the expression of SATB2 gene in HCC from AA and CA patients and assess its oncogenic potential by measuring cell viability, epithelial-mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in primary hepatocytes, Hep3b (from AA), HepG2 (from CA) and HCC CSCs. Hep3b (higher), HepG2 (lower) and CSCs (highest) expressed SATB2 protein, whereas human primary normal hepatocytes did not express SATB2. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (cMyc, KLF4, SOX2 and OCT4), and EMT compared to NTC control group. The expression of SATB2 was significantly higher in HCC tissues than adjacent normal controls. Furthermore, the expression of SATB2 was significantly higher in HCC tissues derived from AA compared to those in CA. These data suggest that SATB2 is an oncogenic factor and its expression, which is higher in in HCC tissues derived from AA, may explain the disparity in HCC outcomes among AA. This abstract is also being presented as Poster B049. Citation Format: S.K. Roy, Y. Ma, D.M. Danos, S. Shankar, L. Miele, R.A. Scribner, R.K. Srivastava. Higher expression of SATB2 gene in hepatocellular carcinoma of African American patients determines aggressiveness phenotypes than those in Caucasian Americans [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr PR08.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-β- and retinoic acid-induced cell death, codes for a ≈16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

DAPK1, a ca(+2)/calmodulin regulated serine/threonine kinase, is a major tumor suppressor, whose expression is lost in multiple tumor types. However, the mechanisms contributing to it are unclear. We have recently shown that CCAAT/Enhancer binding protein-beta (C/EBP-beta) is required for the basal and interferon gamma (IFN-gamma)-induced expression of dapk1 in many cell types. C/EBP-beta interacts with the transcriptional Mediator, a multisubunit complex that couples enhancer bound transcription factors to the basal transcriptional machinery in an IFN-gamma dependent manner for regulating dapk1 expression. Specifically, the Med1 (TRAP220/PBP/DRIP220/CRSP220) subunit associates with the enhancer bound C/EBP-beta at the CRE/ATF site of dapk1 in an IFN-gamma dependent manner for stimulating gene expression. Therefore, we investigated if the mechanism responsible for the loss of dapk1 expression in human cancers involves a failure to recruit C/EBP-beta and/or Med1 to the dapk1 promoter. We compared the relative occupancy of these factors at the dapk1 promoter at CRE/ATF sites in normal and cancer cell lines. A significantly lower binding of these factors to the CRE/ATF site of dapk1 promoter occurred in human cancer cell lines than in normal cells. We show that loss of Med1 expression correlates with a corresponding loss of dapk1 expression in a number of primary human lung carcinomas. Med1 levels were significantly lower in cancer cell lines than in normal controls. Importantly, we show that restoration of Med1 induces the expression of dapk1 in these cancer cells and also attenuates their metastatic potential in vivo. Our studies reveal a critical parameter limiting dapk1 expression in cancer cell lines.

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2Rgammanull mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs. Citation Format: Junsheng Fu, Mariana Rodova, Sanjit K. Roy, Sharmila Shankar, Rakesh K. Srivastava. GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4012. doi:10.1158/1538-7445.AM2013-4012

Our previous studies showed that B cell receptor (BCR) induced apoptosis in lymphoma was preceded by suppression of PI-3K [Carey GB, Scott DW (2001) J. Immunol, 166: 1618-1626]. PI-3K stimulates mTOR, a suppressor of autophagy and mitophagy. In addition, Bcl-XL is known to inhibit BCR-driven apoptosis in lymphoma, although the underlying mechanism is not clear. Therefore the present study examined autophagy in BCR induced apoptosis in lymphoma cells expressing high or low levels of Bcl-XL. WEHI-231 and WEHI-231 cells expressing Bcl-XL (WEHI-231/Bcl-XL) were treated with anti-IgM to cross-link the BCR and to determine the cellular processes leading to apoptosis. The results confirmed dramatic mitochondrial dysfunction followed by apoptosis in parental WEHI-231 compared to WEHI-231/BclXL. BCR-crosslinking resulted in a massive and rapid induction of the autophagic response in parental WEHI-231 compared to those expressing Bcl-XL. Furthermore, treatment with STF-62247, a small molecule inducer of autophagy, resulted in apoptosis in WEHI-231 but not WEHI-231/BclXL cells. These data suggest that BclXL prevents BCR-driven apoptosis in lymphoma via blocking autophagic and mitophagic responses. Hence, combinations of small molecule autophagy inducers and Bcl-XL antagonists may prove to be effective in addressing lymphoma. Supported by NCI Grant UH2CA158689 to G.B.C. Citation Format: Gregory B. Carey, Sanjit K. Roy, Alphius Sesay. Bcl-XL overexpression prevents B Cell receptor driven autophagy in IgM+ lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2013-1728

s / Pancreatology 13 (2013) e1–e94 e11 mTOR signaling in PaSC. Further, conditioned medium from PanIN cells isolated from KRAS mice enhanced fibrogenic responses in PaSC. Histological analysis of tissue microarrays of patient-matched normal human pancreas parenchyma and PanIN lesions showed profuse active PaSC surrounding both low-grade and advanced PanINs, and acini in adjacent normal parenchymal sections. Conclusions: Our data support a key role for Akt/mTOR signaling in PaSC fibrogenic responses, and indicate that interplay between PanINs and PaSC provide the mechanistic underpinnings of environmental risk factor promotion of tumor progression.

BACKGROUND: The forkhead transcription factors of the O class (FOXO) play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1). Resveratrol induced cell cycle arrest by up-regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes. CONCLUSIONS: These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO transcription factors which are involved in resveratrol-mediated pancreatic tumor growth suppression.