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// Jheelam Banerjee 1 , Arokya M.S. Papu John 1 , Mohammed H. Al-Wadei 1 , Hildegard M. Schuller 1 1 Experimental Oncology Laboratory, Department of Biomedical & Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA Correspondence to: Hildegard M. Schuller, email: hmsch@utk.edu Keywords: pancreatic cancer, prevention, γ-aminobutyric acid, cyclic adenosine monophosphate, cancer stem cell signaling Received: December 2, 2015 Accepted: May 22, 2016 Published: June 02, 2016 ABSTRACT Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). We have previously shown that these cancers overexpressed stress neurotransmitters and cyclic adenosine monophosphate (cAMP) while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using a hamster model, the current study has tested the hypothesis that cAMP decrease 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 cAMP and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers and phosphorylated signaling proteins, which stimulate cell proliferation, and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP decrease by GABA supplementation inhibits multiple cancer stimulating pathways in 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.

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

The understanding of signaling cascades involved in the induction, promotion, and progression of cancer, although advanced in recent years, is still incomplete. Tracing the imbalance of the impaired, physiologically-essential cellular signaling that drives the neoplastic process is a complex issue. This review discusses the role of the regulator of the fight or flight response, the beta-adrenergic signaling cascade, as a mediator of cancer growth and progression in in vitro and in vivo cancer models. We review a series of experiments from our own laboratory and those of others examining the contribution of this signaling network to lung and other human malignancies and thereby identifying potential targets for chemotherapeutic interventions. The stimulation of the β-adrenergic receptor by lifestyle and environmental factors, as well as a preexisting risk for neoplasm, activates downstream effector molecules (adenylyl cyclase/cAMP/PKA/CREB) concomitant to the transactivation of related pathways (EGFR) that lead to pro-oncogenic signaling; this β-adrenergic pathway thereby encourages cancer growth by evasion of apoptosis, invasion, angiogenesis, and metastasis. GABAergic signaling acts as an antagonist to the β-adrenergic cascade by intercepting adenylyl cyclase activation, and thereby neutralizing the pro-oncogenic effects of β-adrenergic stimulation. The regulation of cancer cell growth by neurobiological signals expands the possibilities for pharmacological interventions in cancer therapy.

GABA is a bioactive constituent of fruits, vegetables, cereals and is believed to play a role in defense against stress in plants. In animals, it acts as an inhibitory neurotransmitter in brain while also expressed in non-neuronal cells. Studies have implicated the regulator of fight or flight stress responses, β-AR signaling cascade, as mediators of cancer growth and progression in in vitro and in vivo models of pancreatic malignancies. Pancreatic cancer is the fourth leading cause of cancer mortality in western countries. This malignancy is generally unresponsive to conventional radio- and chemotherapy, resulting in mortality rate near 100% within 6 months of diagnosis. We review a series of experiments from our laboratory and those of others examining the contribution of this signaling network to pancreatic and other human malignancies. Stimulation of the β-adrenergic receptor by lifestyle and environmental factors, as well as a pre-existing risk of neoplasm, activates downstream effector molecules that lead to pro-oncogenic signaling and thereby aid cancer growth. GABAergic signaling mediated by the serpentine receptor GABA(B) acts as an antagonist to β-adrenergic cascade by intercepting adenylyl cyclase. These evidences enhance the pharmacological value of human diets rich in GABA for use as an adjuvant to standard therapies.

Small airway epithelial cell-derived adenocarcinoma is the most common human lung cancer and is particularly prevalent in women. We have previously reported that the proliferation of immortalized human small airway epithelial cells HPL1D is stimulated by a single dose of the tobacco carcinogen NNK via cAMP signaling downstream of the beta-1-adrenergic receptor (beta1-AR) and that estrogen enhances this response. In the current study we show that gamma-aminobutyric acid (GABA) blocks this cooperative signaling of NNK and estrogen in HPL1D cells. NNK additionally stimulated the production of noradrenaline, an effect mediated by the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), while reducing GABA production via desensitization of the alpha4nAChR. Chronic exposure to NNK, estrogen or the combination of both upregulated and sensitized the alpha7nAChR, resulting in an enhanced noradrenergic response to agonist. At the same time, chronic NNK and estrogen suppressed the production of GABA by desensitizing its regulatory alpha4beta2nAChR. This selective imbalance in stimulatory and inhibitory signaling may contribute to the development and progression of small airway-derived adenocarcinoma in women who smoke.

Background 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. Methods Using mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1. Results Our data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were 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. Conclusions These findings suggest GABA as a promising single agent for the therapy of pancreatic cancer and to overcome nicotine-induced gemcitabine resistance whereas treatment with baclofen may increase gemcitabine resistance.

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.

Pancreatic cancer has a high mortality rate and alcoholism is a risk factor independent of smoking. We have shown that nicotinic acetylcholine receptors (nAChR) regulate pancreatic ductal epithelia and pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production of the stress neurotransmitters noradrenaline and adrenaline that signal through β-adrenergic receptors (β-AR). Our current study has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by γ-amino butyric acid (GABA). Using MTT assays, cell migration assays, Western blotting, immunoassays, and gene knockdown of individual nAChRs in two PDAC cell lines and in immortalized human pancreatic duct epithelial cells, our data show that treatment for seven days with ethanol induced the protein expression and sensitivity of nAChRs α3, α5, and α7 resulting in increased production of noradrenaline and adrenaline, which drive proliferation and migration via cyclic AMP (cAMP)-dependent signaling downstream of β-ARs. Treatment with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration below base levels in untreated cells. Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling pathways by upregulating the protein expression and sensitivity of nAChRs that regulate stress neurotransmitter production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in individuals at risk due to chronic alcohol consumption. Cancer Prev Res; 6(2); 139–48. ©2012 AACR.

Pancreatic ductal adenocarcinoma (PDAC) has a mortality rate near 100%. Smoking is a documented risk factor. However, the mechanisms of smoking-associated pancreatic carcinogenesis are poorly understood. We have shown that binding of nicotine to nicotinic acetylcholine receptors (nAChRs) expressing subunits α7, α3 and α5 in PDAC and pancreatic duct epithelial cells in vitro triggered the production of the neurotransmitters noradrenaline and adrenaline by these cells. In turn, this autocrine catecholamine loop significantly stimulated cell proliferation via cyclic adenosine 3',5'-monophosphate-dependent signaling downstream of beta-adrenergic receptors. However, the observed responses only represent acute cellular reactions to single doses of nicotine whereas nicotine exposure in smokers is chronic. Using the PDAC cell lines BxPC-3 and Panc-1 and immortalized pancreatic duct epithelial cell line HPDE6-C7, our current experiments reveal a significant sensitization of the nAChR-driven autocrine catecholamine regulatory loop in cells pre-exposed to nicotine for 7 days. The resulting increase in catecholamine production was associated with significant inductions in the phosphorylation of signaling proteins ERK, CREB, Src and AKT, upregulated protein expression of nAChR subunits α3, α4, α5 and α7 and increased responsiveness to nicotine in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and cell migration assays. All three cell lines produced the inhibitory neurotransmitter γ-aminobutyric acid, an activity inhibited by gene knockdown of the α4β2nAChR and suppressed by chronic nicotine via receptor desensitization. All of the observed adverse effects of chronic nicotine were reversed by treatment of the cells with γ-aminobutyric acid, suggesting the potential usefulness of this agent for the improvement of PDAC intervention strategies in smokers.

Pancreatic cancer is the fourth leading cause of cancer deaths in developed countries. Smoking is an established risk factor for this malignancy but the underlying mechanisms are poorly understood. Previous reports have provided evidence that nicotinic acetylcholine receptors (nAChR) and beta adrenergic receptors (β-AR) stimulate the growth and migration of pancreatic cancer cells. However, a potential cooperation of these two receptor families in the regulation of pancreatic cancer has not been studied to date. Using two pancreatic cancer cell lines and immortalized pancreatic duct epithelia in vitro, our current data show that all three cell lines synthesized and released the catecholamine neurotransmitters noradrenaline and adrenaline upon exposure to nicotine and that this activity was regulated by α3, α5, and α7-nAChRs. In accordance with the established function of these catecholamines as β-AR agonists, nicotine-induced cell proliferation was blocked by the β-AR antagonist propranolol. Nicotine-induced proliferation was also abolished by the α7-nAChR antagonist α-bungarotoxin, whereas catecholamine production in response to nicotine was blocked by gene knockdown of the α3, α5, and α7-nAChRs. The nicotinic agonists acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK induced the phosphorylation of CREB, ERK, Src, and AKT and these responses were inhibited by propranolol. Our findings identify this hitherto unknown autocrine catecholamine loop as an important regulatory cascade in pancreatic cancer that may prove a promising new target for cancer intervention. Mol Cancer Res; 10(2); 239–49. ©2011 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer deaths and is unresponsive to existing therapy. Smoking and alcohol-induced pancreatitis are among the risk factors for PDAC. We have previously reported that beta-adrenergic receptors (beta-ARs) stimulate the proliferation and migration of human PDAC cells in vitro by cAMP-dependent signaling and that the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates this pathway directly in vitro while additionally stimulating the release of noradrenaline/adrenaline by binding to alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) in hamsters. In this study, we have tested the hypothesis that the beta-AR antagonist propranolol prevents the development of PDAC induced in hamsters with ethanol-induced pancreatitis by NNK. We found that propranolol had strong cancer preventive effects in this animal model. Western blots of pancreatic duct cells and PDAC cells harvested by laser capture microscopy showed significant upregulation of the alpha7 nAChR associated with significant inductions of p-CREB, p-ERK1/2, and increases in epidermal growth factor and vascular endothelial growth factor in PDAC cells of hamsters not treated with propranolol. These effects were reversed by treatment with propranolol. Our data suggest that propranolol may prevent the development of PDAC by blocking cAMP-dependent intracellular signaling, cAMP-dependent release of epidermal growth factor, and PKA-dependent release of vascular endothelial growth factor while additionally downregulating the alpha7 nAChR by inhibiting cAMP-mediated subunit assembly. We conclude that increased cAMP signaling is an important factor that drives the development and progression of PDAC and that the inhibition of cAMP formation is a promising new target for the prevention and adjuvant therapy of PDAC.

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

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 has a high mortality rate and alcoholism is a risk factor independent of smoking. We have shown that nicotinic acetylcholine receptors (nAChRs) regulate pancreatic ductal epithelia and pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production of the stress neurotransmitters noradrenaline and adrenaline that signal through beta-adrenergic receptors (β-ARs). Our current study has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by γ-amino butyric acid (GABA). Treatment for seven days with ethanol at a concentration equivalent to the legal blood alcohol limit in the United States induced protein expression and sensitivity of nAChRs α3, α5 and α7 resulting in increased production of noradrenaline and adrenaline which drive proliferation and migration via cAMP-dependent signaling downstream of β-ARs. Treatment with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration below base levels in untreated cells. Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling pathways by up-regulating the protein expression and sensitivity of nAChRs that regulate stress neurotransmitters production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in individuals at risk due to chronic alcohol consumption. Supported by R01CA042829 and R01CA130888 with the National Cancer Institute Citation Format: Mohammed H. Al-Wadei, Hussein A.N. Al-Wadei, Hildegard M. Schuller. GABA counteracts ethanol induced stimulation of pancreatic cancer cells and normal pancreatic duct epithelia . [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 3593. doi:10.1158/1538-7445.AM2013-3593

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is associated with high levels of psychological distress. We have shown that beta-adrenergic receptors (β-ARs), which are activated by stress neurotransmitters, regulate PDAC cells via cyclic AMP (cAMP)-dependent signaling in vitro, that social stress promotes PDAC progression in mouse xenografts and that γ-aminobutyric acid (GABA) inhibits these responses in vitro and in vivo. The targeted inhibition of stress-induced regulatory pathways may abolish the potentially negative impact of psychological stress on clinical outcomes. Our current data show that chronic exposure of PDAC cell lines Panc-1 (activating point mutations in K-ras) and BXPC-3 (no mutations in K-ras) in vitro to the stress neurotransmitter epinephrine at the concentration (15 nM) previously measured in the serum of mice exposed to social stress significantly increased proliferation and migration. These responses were inhibited in a concentration-dependent manner by celecoxib. The effects of celecoxib alone and in combination with γ-aminobutyric acid (GABA) on the progression of subcutaneous mouse xenografts from the cell line (BXPC-3) most responsive to epinephrine were then investigated in the presence and absence of social stress. Cancer-stimulating factors (VEGF & prostaglandin E(2) [PGE(2)]) and levels of cAMP were measured by immunoassays in blood and xenograft tissue. Phosphorylation of the signaling proteins ERK, CREB, Src, and AKT was assessed by ELISA assays and Western blotting. Expression of COX-2, 5-lipoxygenase, and p-5-LOX were determined by semi-quantitative Western blotting. Celecoxib alone significantly inhibited xenograft progression and decreased systemic and tumor VEGF, PGE2, and cAMP as well as phosphorylated signaling proteins in stress-exposed and stress-free mice. These responses were significantly enhanced by co-treatment with GABA. The celecoxib-induced downregulation of COX-2 protein and p-5-LOX was also significantly enhanced by GABA under both experimental conditions. Our findings identify the targeted inhibition of stress-induced pathways as a promising area for more effective cancer intervention in pancreatic cancer.

Pancreatic cancer has a poor prognosis and is associated with high levels of psychological distress. We have earlier shown that beta-adrenergic receptors, which are activated by stress neurotransmitters, regulate pancreatic cancer cells via cAMP-dependent signaling in vitro and that psychological distress promotes pancreatic cancer growth in vivo. The observation that psychological distress acts as an ancillary factor in growth promotion of pancreatic cancer have implications for cancer prevention strategies where stress initiated growth stimulus may retard the efficacy of cancer preventive regimens. Therefore this study tested the hypothesis that psychological stress inhibits the cancer preventive effects of celecoxib on pancreatic cancer progression whereas α-aminobutyric acid (GABA) which neutralizes the stress neurotransmitter stimulated cAMP signaling may potentiate the chemopreventive outcome of anti-cancer drugs. The effects of psychological distress in the absence and presence of treatment (with COX-2 inhibitor celecoxib alone / celecoxib with GABA co-adminstration) was tested in mice carrying xenograft from human pancreatic cancer cell line BxPC-3. Neuroendocrine responses (noradrenaline, adrenaline, cortisol & GABA), cancer stimulating factors (VEGF & prostaglandin PGE2) and levels of cAMP were measured by immunoassays in serum and xenograft tissue. Phosphorylation of nicotinic receptors (≥4 and α7), beta-adrenergic receptors, celecoxib targets (COX-2 & 5-lipoxygenase) and signaling proteins downstream of α-ARs were determined by semi-quantitative Western blots. The results show a significant inhibition of cancer preventive effects of celecoxib by psychological stress. By contrast, GABA significantly potentiated the preventive effects of celecoxib in this tumor model. The effector molecules of stress signaling via the cAMP pathway were downregulated by GABA and celecoxib additionally inhibited COX-2 and 5-lipoxygenase lead to significant inhibition of tumor growth in our xenografts model. Our findings identify the interruption of stress-induced c-AMP-dependent neuroendocrine stimulation as central regulator of pancreatic cancer that negatively impacts cancer prevention. In addition, our data identify the anti-stress GABA signaling as an effective adjuvant to standard interventions which in turn shows induce in the COX-2 phosphorylation and increase PGE2 level in the social stress groups which are inhibited by GABA or celecoxib or combination treatment. Better understanding in the above areas may help to develop new therapeutic for pancreatic cancer development. Grant Support: RC1CA144640 and R01CA042829 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 3696. doi:1538-7445.AM2012-3696

Pancreatic cancer is the fourth leading cause of cancer deaths in developed countries. Smoking is an established risk factor for this malignancy but the underlying mechanisms are poorly understood. Previous reports have provided evidence that nicotinic acetylcholine receptors (nAChRs) and beta-adrenergic receptors (α-ARs) stimulate the growth and migration of pancreatic cancer cells. But a potential cooperation of these two receptor families in the regulation of pancreatic cancer has not been studied to date. Using two pancreatic cancer cell lines and immortalized pancreatic duct epithelia in vitro, our current data show, that all three cell lines synthesized and released the catecholamine neurotransmitters noradrenaline and adrenaline upon exposure to nicotine and that this activity was regulated by the α3, 5 & 7-nAChRs. In accord with the established function of these catecholamines as α-AR agonists, nicotine-induced cell proliferation was blocked by the α-AR antagonist propranolol. Nicotine-induced proliferation was also abolished by the α7-nAChR antagonist α-bungarotoxin and catecholamine production in response to nicotine was blocked by α3, 5 or 7-nAChR gene knockdown. Moreover, our results show that the nicotinic agonists acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK induced the phosphorylation of CREB, ERK, Src and AKT and that those responses were inhibited by the beta-blocker, propranolol. Our findings identify this hitherto unknown autocrine catecholamine loop as an important regulatory cascade in pancreatic cancer that may prove a promising new target for cancer intervention. Supported by R01CA042829 and R01CA130888 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 4432. doi:1538-7445.AM2012-4432

Lung cancer is the leading cause of cancer death; 80-85% of lung cancer cases are non-small cell lung cancer (NSCLC). Smoking is a documented risk factor for the development of this cancer. Although nicotine does not have the ability to initiate carcinogenic events, recent studies have implicated nicotine in growth stimulation of NSCLC. Using three NSCLC cell lines (NCI-H322, NCI-H441 and NCI-H1299), we identified the cooperation of nicotinic acetylcholine receptors (nAChRs) and β-adrenergic receptors (β-ARs) as principal regulators of these effects. Proliferation was measured by thymidine incorporation and MTT assays, and Western blots were used to monitor the upregulation of the nAChRs and activation of signaling molecules. Noradrenaline and GABA were measured by immunoassays. Nicotine-treated NSCLC cells showed significant induction of the α7nAChR and α4nAChR, along with significant inductions of p-CREB and p-ERK1/2 accompanied by increases in the stress neurotransmitter noradrenaline, which in turn led to the observed increase in DNA synthesis and cell proliferation. Effects on cell proliferation and signaling proteins were reversed by the α7nAChR antagonist α-BTX or the β-blocker propranolol. Nicotine treatment also down-regulated expression of the GABA synthesizing enzyme GAD 65 and the level of endogenous GABA, while treatment of NSCLC cells with GABA inhibited cell proliferation. Interestingly, GABA acts by reducing β-adrenergic activated cAMP signaling. Our findings suggest that nicotine-induced activation of this autocrine noradrenaline-initiated signaling cascade and concomitant deficiency in inhibitory GABA, similar to modulation of these neurotransmitters in the nicotine-addicted brain, may contribute to the development of NSCLC in smokers. Our data suggest that exposure to nicotine either by tobacco smoke or nicotine supplements facilitates growth and progression of NSCLC and that pharmacological intervention by β blocker may lower the risk for NSCLC development among smokers and could be used to enhance the clinical outcome of standard cancer therapy.

Pancreatic cancer is the leading cause of cancer mortality in both men and women in western countries due to its unresponsiveness to available therapeutics. It is well documented that smoking, diabetes and pancreatitis from chronic alcohol consumption are risk factors for this type of cancer. More than 95% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDACs) that arise from pancreatic ductal epithelia. Our lab has previously shown that nicotine stimulates the proliferation and migration of human small airway epithelial cells and the adenocarcinomas derived from them in vitro due to nicotine induced changes in the sensitivity of the nicotinic acetylcholine receptors. Such changes caused an increase in the production and secretion of excitatory neurotransmitters, but decreased the production and secretion of inhibitory neurotransmitter γ-aminobutyric acid (GABA). In analogy to these findings in lung cells, both nicotine and ethanol stimulated cell proliferation in PDAC cell lines Panc-1 and BXPC-3 and in immortalized pancreatic duct epithelial cells HPDE6-C7. Acute and chronic treatment of all cell lines with nicotine or ethanol alone and in combination significantly increased the production of noradrenaline and adrenaline by the cells while significantly reducing GABA production. Western blots are currently underway to assess the potential modulation of nicotinic receptor protein expression as well signaling pathways. Our findings suggest a potential role for these pathways contributing to the development of smoking and alcohol related pancreatic carcinogenesis and may result in the identification of novel targets for the prevention and therapy of this malignancy. Supported by R01CAo42829 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 1949. doi:10.1158/1538-7445.AM2011-1949

Human lung adenocarcinoma (AC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common types of human cancer and have a poor prognosis in all populations investigated. Smoking is a risk factor for both malignancies. Nicotine addiction is characterized by changes of nicotinic acetylcholine receptors (nAChRs) in the brain to increase the production of excitatory neurotransmitters while reducing inhibitory neurotransmitters. The current study has tested the hypothesis that chronic nicotine stimulates the growth of PAC and PDAC xenografts in nude mice via similar mechanisms. Groups of nude mice carrying xenografts of AC cell line NCI-H322 or PDAC cell line Panc-1 remained untreated, were given nicotine in the drinking water for 30 days in the presence and absence of daily injections with GABA or were treated with GABA alone. The diameters of xenografts were measured. Levels of noradrenaline and adrenaline, the stress hormone cortisol, the neurotransmitter γ-aminobutyric acid (GABA) in serum and xenograft tissues and levels of cAMP in the cellular fraction of blood and in xenograft tissue were assessed by immunoassays. The levels of GAD65, GAD67, nAChRs and signaling proteins downstream of β-ARs in the tumor tissue were determined by Western blotting. Our data show a significant stimulation of xenografts from both cell lines by chronic nicotine that was reversed by treatment with GABA. Nicotine also significantly increased the systemic levels of adrenaline, noradrenaline and cAMP while decreasing GABA. The protein levels of nicotinic acetylcholine receptors α4 and α7, p-CREB and p-ERK1/2 in the tumor tissue was significantly increased by nicotine and reversed by GABA. Nicotine additionally reduced the protein levels of both GAD isozymes in tumor tissue. In summary, our findings suggest that nicotine-induced predominance of stimulatory noradrenaline and deficiency in inhibitory GABA similar to nicotine-addiction in the brain may contribute to the development of lung adenocarcinoma in smokers and that GABA may be useful for marker-guided prevention and adjuvant therapy for PAC and PDAC. Supported by R01 CA042829 and R01 CA130888 with National Institutes of Health. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A6.

Small airway epithelial cell-derived adenocarcinoma is the most common human lung cancer and is particularly prevalent in women. We have previously reported that the proliferation of immortalized human small airway epithelial cells HPL1D is stimulated by a single dose of the tobacco carcinogen NNK via cAMP signaling activated by binding of NNK to the beta-1-adrenergic receptor (β1-AR) and that estrogen enhances this response. In the current study we show that γ-aminobutyric acid (GABA) blocks this cooperative signaling of NNK and estrogen in HPL1D cells by inhibiting the activation of adenylyl cyclase. In addition, HPL1D cells produced the stress neurotransmitters noradrenaline and adrenaline in response to binding of a single dose of NNK to the α7 nicotinic acetylcholine receptor (α7nAChR). Since both stress neurotransmitters are beta-adrenergic agonists, this response further enhanced cAMP-dependent stimulatory signaling. On the other hand, GABA production by these cells was reduced by a single dose of NNK via desensitization of the α4β2 nAChR. Exposure to NNK, estrogen or the combination of both agents for 7 days upregulated and sensitized the α7nAChR, resulting in an enhanced noradrenergic response to agonist. At the same time, chronic NNK and estrogen suppressed the production of GABA by desensitizing its regulatory α4β2nAChR. The resulting predominance of stimulatory cAMP signaling over inhibitory GABA signaling may contribute to the development and progression of small airway-derived adenocarcinoma in women who smoke and offer a novel target for the marker-guided prevention of this cancer. Supported by a grant from the National Lung Cancer Partnership and LUNGevity Foundation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4124.