Nirish Shah

Muscarinic receptors and ligands in cancer

Emerging evidence indicates that muscarinic receptors and ligands play key roles in regulating cellular proliferation and cancer progression. Both neuronal and nonneuronal acetylcholine production results in neurocrine, paracrine, and autocrine promotion of cell proliferation, apoptosis, migration, and other features critical for cancer cell survival and spread. The present review comprises a focused critical analysis of evidence supporting the role of muscarinic receptors and ligands in cancer. Criteria are proposed to validate the biological importance of muscarinic receptor expression, activation, and postreceptor signaling. Likewise, criteria are proposed to validate the role of nonneuronal acetylcholine production in cancer. Dissecting cellular mechanisms necessary for muscarinic receptor activation as well as those needed for acetylcholine production and release will identify multiple novel targets for cancer therapy.

Genetic Ablation of M3 Muscarinic Receptors Attenuates Murine Colon Epithelial Cell Proliferation and Neoplasia

Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline [10 wild-type (WT) mice] or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.

Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice.

M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apc(min/+) mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apc(min/+) mice and compared intestinal morphology and tumor number in 12-week-old Apc(min/+)Chrm3(-/-) and Apc(min/+)Chrm3(+/+) control mice. Compared with Apc(min/+)Chrm3(+/+) mice, Apc(min/+)Chrm3(-/-) mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, β-catenin nuclear staining was reduced in Apc(min/+)Chrm3(-/-) compared with Apc(min/+)Chrm3(+/+) mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apc(min/+)Chrm3(-/-) mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apc(min/+) mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and β-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.

Scopolamine Treatment and Muscarinic Receptor Subtype 3 Gene Ablation Augment Azoxymethane-Induced Murine Liver Injury

Previous work suggests that vagus nerve disruption reduces hepatocyte and oval cell expansion after liver injury. The role of postneuronal receptor activation in response to liver injury has not been ascertained. We investigated the actions of scopolamine, a nonselective muscarinic receptor antagonist, and specific genetic ablation of a key cholinergic receptor, muscarinic subtype-3 (Chrm3), on azoxymethane (AOM)-induced liver injury in mice. Animal weights and survival were measured as was liver injury using both gross and microscopic examination. To assess hepatocyte proliferation and apoptosis, ductular hyperplasia, and oval cell expansion, we used morphometric analysis of 5-bromo-2′-deoxyuridine-, activated caspase-3-, hematoxylin and eosin-, cytokeratin-19-, and epithelial cell adhesion molecule-stained liver sections. Sirius red staining was used as a measure of collagen deposition and its association with oval cell reaction. In AOM-treated mice, both muscarinic receptor blockade with scopolamine and Chrm3 ablation attenuated hepatocyte proliferation and augmented gross liver nodularity, apoptosis, and fibrosis. Compared with control, scopolamine-treated and Chrm3(−/−) AOM-treated mice had augmented oval cell reaction with increased ductular hyperplasia and oval cell expansion. Oval cell reaction correlated robustly with liver fibrosis. No liver injury was observed in scopolamine-treated and Chrm3(−/−) mice that were not treated with AOM. Only AOM-treated Chrm3(−/−) mice developed ascites and had reduced survival compared with AOM-treated wild-type controls. In AOM-induced liver injury, inhibiting postneuronal cholinergic muscarinic receptor activation with either scopolamine treatment or Chrm3 gene ablation results in prominent oval cell reaction. We conclude that Chrm3 plays a critical role in the liver injury response by modulating hepatocyte proliferation and apoptosis.

Liver Failure Requiring Transplantation After Orlistat Use

Orlistat is a United States Food and Drug Administration (FDA)‐approved drug indicated for the management of obesity. The FDA has issued a warning of rare, but severe, reports of liver injury after orlistat use. We describe a 40‐year‐old woman with no significant medical history who experienced fulminant liver failure after orlistat use. Two weeks after taking the drug at a low dosage of 60 mg/day for 4 days, she developed significant fatigue, nausea, right upper abdominal discomfort, and icterus, and her liver enzyme panel showed significant abnormalities. Five weeks later, after further deterioration in her clinical status, she was transferred to our medical center with severe cholestasis and coagulopathy Liver ultrasonography, serologies for viral hepatitis, and autoimmune markers were unremarkable. Early cessation of the drug was not sufficient to stop the progression of liver injury, and she required an orthotopic liver transplant. Two weeks after transplantation, she was discharged in good condition. Published reports of liver injury associated with orlistat use describe a presentation similar to that of our patient. Although use of the Naranjo adverse reaction probability scale indicated a possible relationship (score of 3) between the patients development of fulminant liver failure and orlistat, we believe this was a drug‐induced case and is consistent with previous reports. To our knowledge, this is the first published report of orlistat‐induced liver failure in the United States. Although orlistat may be a useful drug for weight loss, clinicians should be aware that its use can rarely cause idiosyncratic hepatotoxicty characterized by subacute hepatitis, which may progress to serious injury.

M1 Muscarinic Receptor Deficiency Attenuates Azoxymethane-Induced Chronic Liver Injury in Mice.

Cholinergic nervous system regulates liver injury. However, the role of M1 muscarinic receptors (M1R) in modulating chronic liver injury is uncertain. To address this gap in knowledge we treated M1R-deficient and WT mice with azoxymethane (AOM) for six weeks and assessed liver injury responses 14 weeks after the last dose of AOM. Compared to AOM-treated WT mice, M1R-deficient mice had attenuated liver nodularity, fibrosis and ductular proliferation, α-SMA staining, and expression of α1 collagen, Tgfβ-R, Pdgf-R, Mmp-2, Timp-1 and Timp-2. In hepatocytes, these findings were associated with reductions of cleaved caspase-3 staining and Tnf-α expression. In response to AOM treatment, M1R-deficient mice mounted a vigorous anti-oxidant response by upregulating Gclc and Nqo1 expression, and attenuating peroxynitrite generation. M1R-deficient mouse livers had increased expression of Trail-R2, a promotor of stellate cell apoptosis; dual staining for TUNNEL and α-SMA revealed increased stellate cells apoptosis in livers from M1R-deficient mice compared to those from WT. Finally, pharmacological inhibition of M1R reduced H2O2-induced hepatocyte apoptosis in vitro. These results indicate that following liver injury, anti-oxidant response in M1R-deficient mice attenuates hepatocyte apoptosis and reduces stellate cell activation, thereby diminishing fibrosis. Therefore, targeting M1R expression and activation in chronic liver injury may provide therapeutic benefit.

674 Concomitant Knockout of M1 Muscarinic Receptors (CHRM1) in Azoxymethane-Treated Chrm3−/− Mice Nullifies Reductions in Colon Tumor Number and Size

G A A b st ra ct s (HP). Animals were analyzed 3 and 12 months after inoculation. Morphology of the fundus was examined in sections stained with H&E. Expression of MIP-2, TNF-alfa, INF-gamma, DCAMKL1, villin, activation-induced cytidine deaminase (AID), TFF2 and COX-2 genes were measured by QRT-PCR. Distribution of parietaland DCAMKL1-positive cells was analyzed immunohistochemistry. Western blots were used to assess the phosphorylation of Smad1-5-8 and STAT3. RESULTS: The mucosa of the TG-mice exhibited decreased phosphorylation of Smad1-5-8, confirming inhibition of BMP signaling. Histological analysis showed increased height, dilated glands, decreased parietal cell number and amodest increase in inflammatory cells. The TG-mice also exhibited a 2to 3-fold increase in the expression of the inflammatory genes MIP-2, INF-gamma TNF-alfa, and COX-2. Inhibition of BMP signaling led to increased expression of the gastric progenitor cell (GPC) markers DCAMKL1 and villin, and of AID and TFF2, molecules associated with gastric carcinogenesis. Infection of the TG-mice with HP, led to a dramatic increase in inflammation, characterized by enhanced cytokine gene expression and by a robust influx of inflammatory cells. HP-infected non-TG mice exhibited inflammatory changes that were less severe then those seen in the infected TG-animals. Intraepithelial neoplasia was seen in TG-mice infected for 12 months but not in age matched, non-infected TG-mice, or HP-infected non-TG-mice. HP infection also led to enhanced expression of AID, TFF2, villin and DCAMKL1 and to the phosphorylation and activation of the oncogenic molecule STAT3. CONCLUSIONS: Loss of gastric BMP signaling leads to a pro-inflammatory state, resulting in extreme responses and accelerated development of intraepithelial neoplasia with HP infection, suggesting that BMPs normally temper gastric inflammatory responses. Low BMP signaling and heightened inflammation stimulate GPCs, including increased marker expression and cell number. Thus BMPs play an important role in modulating inflammatory responses in the development of gastric cancer.