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Supplementary Figure 4 from Role for Stromal Heterogeneity in Prostate Tumorigenesis

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Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). Using a hamster model of pancreatitis-associated PDAC induced by treatment with ethanol in the drinking water and the nicotine-derived nitrosamine 4-methylnitrosamino-(3-pyridyl)-1-butanone (NNK) by subcutaneous injection as well as immunohistochemistry of human PDAC tissue microarrays, we have previously shown that these cancers overexpressed stress neurotransmitters and cAMP while the inhibitory neurotransmitter ã-aminobutyric acid (GABA) was suppressed. Using our hamster model, the current study has tested the hypothesis that cAMP control by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cyclic adenosine monophosphate (cAMP) and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers, phosphorylated signaling proteins associated with cell proliferation and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP control by GABA supplementation inhibits multiple cancer supporting pathways at the level of cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism. Citation Format: Jheelam Banerjee, Arokya M. Papu John, Mohammed H. Al-Wadei, Hildegard M. Schuller. Prevention of pancreatic cancer by cAMP control. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 823.

Non small-cell lung cancer (NSCLC) is the leading type of lung cancer with a poor prognosis. Smoking is a risk factor but NSCLC develops in a significant number of nonsmokers. We have reported that social stress significantly promoted the growth of NSCLC xenografts, a response mediated by multiple cAMP-driven signaling cascades downstream of Gαi-coupled beta-adrenergic receptors activated by the stress neurotransmitters norepinephrine and epinephrine and reversed by γ-aminobutyric acid (GABA) via Gαi-coupled GABA-B-receptor signaling. In addition, improved clinical outcomes in NSCLC patients with incidental beta-blocker therapy have been reported. These findings suggest that psychological stress with the associated increase in systemic stress neurotransmitter levels and suppression of physiological agonists for Gαi-coupled receptors may significantly contribute to the high incidence and poor therapeutic response of NSCLC. On the other hand, these findings suggest that stress reduction may have significant inhibitory effects on NSCLC. To test this hypothesis, we achieved stress reduction in athymic nude mice by maintaining the animals in larger cages and by providing them with several enrichment items, resulting in reduced systemic levels of corticosterone, norepinephrine and epinephrine while the levels of GABA and the physiological agonists for Gαi-coupled opioid receptors, dynorphin A and B and met-enkephalin increased. We found that stress reduction significantly inhibited the establishment of xenografts and significantly reduced xenograft sizes. Xenografts in the stress reduction group expressed lower levels of cAMP, VEGF and sonic hedgehog (SHH) accompanied by reduced protein expression of p-ERK, p-AKT, p-CREB, p-SRc and Gli1 while cleaved caspase-3 and p53 proteins were induced. Based on the observed reduction of the cancer stem cell markers SHH and Gli1 in xenograft tissues, we conducted additional mechanistic experiments with cancer stem cells enriched from three NSCLC cell lines under selective sphere formation conditions over 21 days with subcultures every seven days. We found that epinephrine significantly increased stem cell self renewal associated with increased intracellular cAMP, increased levels of the stem cell markers SHH and aldehyde dehydrogenase-1 and increased expression of Gli1. Simultaneous treatment of the enriched cancer stem cells with GABA or dynorphin B completely reversed all of these effects. We conclude that stress reduction can act as a powerful inhibitor of NSCLC by restoring cAMP homeostasis and should be incorporated as important component into existing NSCLC prevention and therapy protocols to improve clinical outcomes. Supported in part by 5RC1CA144640 and State of Tennessee Center of Excellence for Human and Livestock Disease Citation Format: Jheelam Banerjee, Arokya M. S Papu John, Hildegard M. Schuller. Inhibition of non small-cell lung cancer by stress reduction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3729. doi:10.1158/1538-7445.AM2015-3729

Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer. Using pancreatic cancer cell lines BXPC-3 and PANC-1, our data show that GABA significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. This effect of GABA was accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of cAMP formation after single dose exposures to stimulation of cAMP formation in cells pretreated for seven days. These findings suggest GABA as a promising agent to overcome nicotine-induced gemcitabine resistance in pancreatic cancer whereas treatment of alcoholism by baclofen may increase gemcitabine resistance. Supported by grants RO1CA130888 and RO1CA042829 with the National Cancer Institute. Citation Format: Jheelam Banerjee, Hussein A. N Al-Wadei, Mohammed H. Al-Wadei, Koami Dagnon, Hildegard M. Schuller. Nicotine-induced gemcitabine resistance is reversed by gamma-aminobutyric acid but enhanced by baclofen in pancreatic cancer xenografts and in pancreatic cancer cells in vitro. [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 1703. doi:10.1158/1538-7445.AM2014-1703

Urothelial bladder cancer (UBC) is the 7th most common cancer in men and the 17th most common cancer in women. Smoking is an established risk factor for UBC. However, the mechanisms how smoking induces bladder cancer are poorly understood. Using two established human urothelial bladder cancer cell lines, our data show that nicotine significantly increased the proliferation of both cell lines. Both cell lines produced the stress neurotransmitters norepinephrine and epinephrine and nicotine further enhanced this activity. The broad-spectrum beta-blocker propranolol strongly inhibited base level and nicotine-induced proliferation in both cell lines, identifying beta-adrenergic receptors as mediators. Proliferation in response to exogenous addition of epinephrine, norepinephrine or the selective β-adrenergic agonist isoproterenol and complete blockage of these responses by propranolol support this interpretation. Treatment with the inhibitory neurotransmitter γ-aminobutyric acid (GABA), that inhibits beta-adrenergic receptor-mediated proliferation of other cancer types via inhibition of cAMP formation, was less effective. These findings suggest that urothelial bladder cancer cells are stimulated in their growth via beta-adrenergic receptor signaling independent of cAMP by nicotine or psychological stress. Additional investigations are underway to further dissect the signal transduction pathways involved. Supported by the State of Tennessee Center of Excellence in Livestock Diseases and Human Health. Citation Format: Arokya M. Papu John, Jheelam Banerjee, Hildegard M. Schuller. Regulation of urothelial bladder cancer by nicotine and stress neurotransmitters. [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 5298. doi:10.1158/1538-7445.AM2014-5298

Pancreatic ductal adenocarinoma (PDAC) is a leading cause of cancer deaths in developed countries. The nucleoside analog Gemcitabine, which induces apoptosis, is widely used for the therapy of pancreatic cancer. Smoking and alcoholism are risk factors for pancreatic cancer. Nicotine replacement therapy often accompanies chemotherapy while the GABA-B receptor (GABA-B-R) agonist Baclofen has recently been suggested as an effective agent for the treatment of alcohol dependence. Our laboratory has shown that the proliferation and migration of PDAC and pancreatic duct epithelial cells in vitro is regulated by the nicotinic receptor-mediated synthesis and release of stress neurotransmitters that bind to beta-adrenoreceptors (beta-ARs). We have additionally shown that nicotine in the drinking water at a high dose (432 μmole/L) comparable to nicotine exposure in heavy smokers significantly stimulated the growth of PDAC xenografts whereas identical exposure of mice to low dose nicotine (1 μmole/L) reduced gemcitabine-induced apoptosis, thus significantly increasing resistance to gemcitabine. In the current study, we have investigated the potential prevention of nicotine-induced gemcitabine resistance by γ-aminobutyric acid (GABA) and Baclofen in PDAC xenografts. We found that GABA significantly reduced nicotine-induce drug resistance. By contrast, Baclofen failed to reduce nicotine-induced resistance to gemcitabine while even slightly increasing xenograft growth in mice not exposed to nicotine. Investigation of xenograft tissues for the expression levels of the GABA-B-R, intracellular cAMP and signaling proteins associated with cell proliferation, apoptosis and metastasis by immunoassays and western blots revealed effective inhibition of cAMP-dependent signaling in xenografts of mice treated with GABA. By contrast, Baclofen did not inhibit cAMP-dependent signaling and decreased the protein expression of the GABA-R, suggesting downregulation of the receptor. Our findings identify GABA as a promising agent for the prevention of nicotine-induced resistance to gemcitabine in PDAC. On the other hand, our data suggest that treatment of alcohol dependence by Baclofen should be avoided in PDAC patients. Supported by grants RO1CA130888 and RO1CA042829 with the National Cancer Institute. Citation Format: Jheelam Banerjee, Mohammed H. Al-Wadei, Hussein A. N. Al-Wadei, Hildegard M. Schuller. GABA but not baclofen prevents gemcitabine resistance induced by low dose nicotine in pancreatic cancer xenografts. [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 2191. doi:10.1158/1538-7445.AM2013-2191

Pancreatic cancer is a leading cause of cancer deaths in developed countries. The nucleoside analog Gemcitabine, which induces apoptosis, is widely used for the therapy of pancreatic cancer. Smoking is an established risk factor for pancreatic cancer and nicotine replacement therapy often accompanies chemotherapy. Using the two human pancreatic cancer cell lines PANC-1 and BXPC-3 in vitro, our data show that low concentrations of nicotine significantly reduces the growth inhibiting effects of gemcitabine while also significantly inhibiting gemcitabine-induced apoptosis as indicated by determination of cleaved caspase-3 in immunoassays. Analysis of other signaling proteins is currently in progress. In a mouse xenograft model with BXPC-3 cells, a 1 micromolar concentration of nicotine in the drinking water significantly reduced the therapeutic response to gemcitabine while additionally reducing the induction of pro-apoptotic proteins and the inhibition of growth stimulating proteins by gemcitabine. Our data suggest that nicotine replacement therapy and possibly also the exposure to second hand smoke may negatively impact therapeutic outcomes of gemcitabine in pancreatic cancer patients. Nicotine addiction should be treated with non nicotine agents in such patients and exposure to tobacco smoke in any form should be avoided. Supported by grants RO1CA130888 and RO1CA042829 with the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 152. doi:1538-7445.AM2012-152

Smoking is an established risk factor for pancreatic cancer. More than 95% of all pancreatic cancers are pancreatic ductal adenocarcinomas (PDACs). Our data has earlier established that nicotine contributes to the progression of smoking associated PDAC by increasing the systemic levels of the stress neurotransmitters adrenaline and noradrenaline in pancreatic cancer xenografts. These findings suggested that development and progression of PDAC is subject to significant modulation by stimulatory stress neurotransmitters. Here we demonstrate that nicotine abrogates the apoptotic activity of gemcitabine, gefitinib, and meloxicam, which are standard therapy for PDAC, in a variety of human PDAC cell lines. We first evaluated the effect of gemcitabine, gefitinib and meloxicam on cell proliferation using a varied range of concentrations and timepoints on pancreatic cancer cell lines PANC-1 and BXPC-3. The results demonstrated that all the three anti-cancer drugs significantly inhibited the proliferation of pancreatic cancer cells PANC-1 and BXPC-3. The IC50 values were determined for optimal concentration and timepoint. It was next examined whether chronic nicotine could confer protection against apoptosis induced by gemcitabine, gefitinib, and meloxicam, which are widely used to treat PDAC. Cells were stimulated with 1 µM nicotine (pre-determined concentration) in the presence or absence of the IC50 concentrations of the indicated drugs. Our results indicated that nicotine suppresses apoptosis induced by gemcitabine, gefitinib, and meloxicam in pancreatic cancer cells, as measured by TUNEL assays, viability assays and western blotting of apoptosis associated proteins. In conclusion, the antiapoptotic effects of nicotine, negatively impacts the chemosensitivity of PDAC cells against anticancer drugs. These findings suggest that continued exposure to nicotine during cancer therapy may significantly reduce the responsiveness to anti-cancer agents. This work is supported by grants RO1CA130888 and RO1CA042829 with the National Cancer Institute. 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 1010. doi:10.1158/1538-7445.AM2011-1010

DNA damage of stromal fibroblastic cells is known to promote tumorigenesis in number of tissues including the breast and pancreas. 69% of prostate cancer patients lose expression of the Tgfbr2 in the stromal compartment. We previously described, transforming growth factor-beta (TGF-ß) responsiveness of the prostate stromal fibroblastic cells can promote tumorigenesis in transgenic mice. It is known TGF-ß promotes DNA stability, however the mechanism is not known. We found that human prostate cancer stroma has significant number of cells with DNA double stranded breaks, as determined by phosphorylated Ser139-histone H2 (γH2AX) immunolocalization. Similarly, prostate stromal cells from mice with a conditional Tgfbr2-knockout were positive for γH2AX staining. Further promoter methylation array analysis of prostate stromal cells from Tgfbr2-knockout and Tgfbr2-flox (control) indicated 17 DNA damage repair genes to be methylated in Tgfbr2-knockout cells versus the control. Thus, we hypothesized that the loss of stromal TGF-ß responsiveness facilitates stromal DNA damage accumulation through epigenetic regulation. We sought to test the role of stromal TGF-ß signaling in epigenetic regulation. Fourteen of 17 genes were RT-PCR verified to be silenced in Tgfbr2-KO cells and re-expressed following 5-azaDC (de-methylating agent) treatment. Homologous genes were found to be epigenetically silenced in human prostate CAF (carcinoma associated fibroblasts) cultured from primary tumors compared to NAF (normal prostate fibroblasts) from 10 prostate cancer patients. A downstream mediator of TGF-ß signaling includes Smad3 activation. Thus, a similar methylation array analysis of Smad3-knockout prostate stromal cells was performed. We found none of the DNA damage repair genes to be silenced in Smad3-KO cells. Next, we measured DNA methyltransferase-1 (DNMT1) mRNA, protein and activity in Tgfbr2-KO and Tgfbr2-flox cultured prostate stromal cells. Interestingly, protein expression and activity of DNMT1 was 3-fold greater in Tgfbr2-KO stroma over Tgfbr2-flox cells, in the absence of DNMT1 mRNA expression differences. DNMT3b differed little between the two cell types. The use of proteasome inhibitor, MG-132, suggested DNMT1 to be down regulated by TGF-ß in a post-translational manner. Finally, tissue recombinant experiments of Tgfbr2-KO prostate stromal cells with LNCaP, human prostate cancer epithelia, developed large tumors in orthotopic grafts. Similar grafts with wild type and Smad3-KO stromal cells with LNCaP epithelia developed small tumors. Strikingly Tgfbr2-KO stromal cells treated with 5-azaDC and subsequently grafted with LNCaP cells developed smaller tumors. These results for the first time support the role of TGF-ß signaling on stromal DNA stability in tumorigenesis and support a mechanism for TGF-ß responsiveness results in epigenetic silencing of DNA damage repair genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1429.

Introduction: A previously described mouse model, Tgfbr2-fspKO, with a conditional stromal knockout of the TGF-beta type II receptor (Tgfbr2), developed prostatic intra-epithelial neoplasia (PIN) lesions and subsequently progressed to prostate adenocarcinoma. Tissue recombination of wild-type (WT) prostatic organoids with 100% Tgfbr2-floxE2/floxE2 prostatic stroma resulted in grafts with normal prostatic glandular architecture, whereas recombination with 100% Tgfbr2-fspKO stroma resulted in grafts with PIN lesions. Interestingly, only grafts made with a mixture of WT and KO stroma resulted in prostatic adenocarcinoma. Furthermore, loss of stromal Tgfbr2 expression occurs in a proportion of human prostate cancer tissues. The objective of the study was to elucidate the mechanism(s) contributing to the cancer progression in the context of a mixed heterogeneous stroma. Method: Prostate stromal-epithelial interactions were described by a hybrid computational model involving a hypothesized 2-step mechanism (initial transformation followed by invasion), where these steps are controlled by stromal-derived paracrine morphogenic factors. Biological parameters for the candidate morphogens (Wnt3a and SDF-1/CXCL12) were assessed by qPCR and ELISA from stromal mixing co-culture experiments. Results: Modeling indicated that the expression of the initiating morphogen increases as a function of the % of Tgfbr2-KO stromal cells. The incidence of invasion promoted by the pro-invasive morphogen was biphasic with a 50/50 mixture of WT and Tgfbr2-KO stromal cells resulting in maximal cancer progression. Wnt3a is produced at 4-fold higher levels in Tgfbr2-KO vs WT stroma and antibody mediated depletion of Wnt3a from stromal conditioned media reduced the growth rate of LNCaP cells. SDF-1 is produced at nearly 4-fold higher levels in WT vs. Tgfbr2-KO stroma and SDF-1 mRNA expression is synergistically increased when WT and Tgfbr2-KO stroma are co-cultured. Additionally, CD90 expression is heterogeneously expressed by stromal tissues associated with primary human prostate cancer as compared to benign tissue as demonstrated by immunofluorescence staining. Conclusions: The model and data support the importance of stromal heterogeneity in the development of prostatic adenocarcinoma in two independent tumorigenic steps. Each step is facilitated by a different stromal component, so that stromal heterogeneity results in the maximal tumorigenesis. Tgfbr2-KO stromal cells produce an altered cytokine profile that drives the proliferation of the epithelial compartment and likely primes the WT stromal cells to produce additional cytokines, like SDF-1, that promote epithelial invasion and cancer progression. While most studies tend to focus on the altered stroma, this study suggests that the WT stroma cells play a necessary and important role required as a rate-limiting step for prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1428.

Stromal cells promote prostate cancer initiation and progression. Metastasized prostate cancers form osteoblastic bone lesions. The prostate cancer associated stroma expresses alpha-smooth muscle actin (alpha-SMA). Additionally, 69% of human prostate cancer tissues lost stromal TGFbeta type II receptor (Tgfbr2) expression. Conditional stromal knockout of the Tgfbr2 mouse model developed prostate adenocarcinoma. In parallel to the primary site, we observed human prostate cancer associated stroma in bone metastasis gained the expression of androgen receptor and alpha-SMA, which were negative in the normal bone marrow. Tgfbr2 expression was also lost in prostate cancer associated stroma in bone marrow, compared to its high expression in the normal bone marrow. This led us to question whether the stromal cells from the primary site or the bone marrow contribute the prostate cancer osteoblastic bone lesion development. In vitro, the conditioned media from Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells (PSC) or bone marrow stromal cells (BMSC) were incubated on the C42B prostate cancer epithelia. Expressions of PTHrP, RANKL osteolytic factors and ET1, RUNX2, BMP2 osteoblastic factors by C42B cells were analyzed by qRT-PCR. The Tgfbr2-flox PSC conditioned media significantly increased the C42B cell expression of all the osteolytic and osteoblastic factors examined compared to C42B cells without conditioned media. However, the Tgfbr2-KO PSC conditioned media decreased the expression of PTHrP and RANKL, while further increased the expression of osteoblastic factors, ET1, RUNX2 and BMP2 by C42B cells by at least 2-fold. Conditioned media of Tgfbr2-flox and Tgfbr2-KO BMSC increased the expression of C42B cells on osteolytic factors (10-fold PTHrP and 2-fold RANKL), but neither has significant effect on expression of osteoblastic factors. Importantly, Tgfbr2-KO PSC and BMSC conditioned media increased C42B proliferation similarly by 2-fold over that of respective Tgfbr2-Tgfbr2-flox conditioned media. We found that the reduced PTHrP and RANKL expression was associated with respective promoter methylation, when C42B cells were incubated with Tgfbr2-KO PSC, but not BMSC. Epigenetic silencing of such osteolytic genes, would suggest for the first time, that the prostate stroma could influence osteoblastic tumor at a distant site. So, GFP labeled C42B cells, following pre-incubation with PSC or BMSC conditioned media, were injected into the tibia of SCID mice. X-rays of the bone lesions and fluorescence imaging of GFP-expressing tumor growth were measured. The data suggested, that the stromal cells from primary prostates, but not from the second metastasis site of bone marrow, promotes prostate cancer growth in the bone and osteoblastic bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1418.