Narayan Shivapurkar

Pancreatic cancer is characterized by an extensive fibrotic stroma largely driven by activated pancreatic stellate cells (PSCs)/fibroblasts, which also function to support tumor growth and metastasis. Cholecystokinin-B receptors (CCK-BRs) are expressed on pancreatic stellate cells (PSCs) and have emerged as a key regulator of PSC activation and tumor-stromal interactions. We hypothesized that disrupting CCK-BR function shifts PSCs to a more quiescent phenotype and reduces their pro-fibrotic and tumor-supportive activity to decrease growth of pancreatic cancer. Murine PSCs were genetically engineered with CRISPR-Cas9 to knockout the CCK-BR. In a series of experiments, the role of the CCK-BR expression was evaluated on cell migration, proliferation, differentially expressed genes, molecular signaling pathways, and in co-culture with murine pancreatic cancer epithelial cells. Next, primary human pancreatic stellate cells were treated with proglumide, a CCK-BR antagonist, to study the effects of pharmacologic blockade of the CCK-BR on cellular signaling and proliferative pathways by RNA sequencing. Knockout of the CCK-BR led to significant decreases in PSC activation and the ability to stimulate growth of pancreatic cancer cells in co-culture. Both genetic knockdown and pharmacologic blockade of the CCK-BR downregulated genes implicated in fibrosis, proliferation, fibroblast activation, and tumorigenesis, while genes implicated in apoptosis and tumor suppression were upregulated. Flow cytometry showed increased apoptosis markers in CCK-BR-knockout cells compared to controls. These experiments combine transcriptomic profiling with functional validation to provide a comprehensive analysis of how targeting CCK-BR interrupts the cross-communication between cancer cells and fibroblasts. Blockade or downregulation of the CCK-BR on pancreatic fibroblasts may provide a strategy to disrupt oncogenic signaling pathways and reprogram the tumor microenvironment.

Chronic pancreatitis (CP) is a rare but debilitating condition with an 8-fold increased risk of developing pancreatic cancer. In addition to the symptoms that come from the loss of endocrine and exocrine function in CP, the management of chronic pain is problematic. We previously showed that the CCK-receptor antagonist called proglumide could decrease inflammation, acinar-ductal metaplasia, and fibrosis in murine models of CP. We hypothesized that proglumide would be safe and diminish pain caused by CP. A Phase 1 open-labeled safety study was performed in subjects with clinical and radiographic evidence of CP with moderate to severe pain. After a 4-week observation period, the subjects were treated with proglumide in 400 mg capsules three times daily (1200 mg per day) by mouth for 12 weeks, and then subjects returned for a safety visit 4 weeks after the discontinuation of the study medication. The results of three pain surveys (Numeric Rating Scale, COMPAT-SF, and NIH PROMIS) showed that the patients had significantly less pain after 12 weeks of proglumide compared to the pre-treatment observation phase. Of the eight subjects in this study, two experienced nausea and diarrhea with proglumide. These side effects resolved in one subject with doses reduced to 800 mg per day. No abnormalities were noted in the blood chemistries. A blood microRNA blood biomarker panel that corresponded to pancreatic inflammation and fibrosis showed significant improvement. We conclude that proglumide is safe and well tolerated in most subjects with CP at a dose of 1200 mg per day. Furthermore, proglumide therapy may have a beneficial effect by decreasing pain associated with CP.

CCK receptors are expressed on pancreatic cancer epithelial cells, and blockade with receptor antagonists decreases tumor growth. Activated pancreatic stellate cells or myofibroblasts have also been described to express CCK receptors, but the contribution of this novel pathway in fibrosis of the pancreatic cancer microenvironment has not been studied. We examined the effects of the nonselective CCK receptor antagonist proglumide on the activation, proliferation, collagen deposition, differential expression of genes, and migration in both murine and human PSCs. CCK receptor expression was examined using western blot analysis. Collagen production using activated PSCs was analyzed by mass spectroscopy and western blot. Migration of activated PSCs was prevented in vitro by proglumide and the CCK-B receptor antagonist, L365,260, but not by the CCK-A receptor antagonist L365,718. Proglumide effectively decreased the expression of extracellular matrix-associated genes and collagen-associated proteins in both mouse and human PSCs. Components of fibrosis, including hydroxyproline and proline levels, were significantly reduced in PSC treated with proglumide compared to controls. CCK peptide stimulated mouse and human PSC proliferation, and this effect was blocked by proglumide. These investigations demonstrate that targeting the CCK-B receptor signaling pathway with proglumide may alter the plasticity of PSC, rendering them more quiescent and leading to a decrease in fibrosis in the pancreatic cancer microenvironment.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15–20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.

Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRAS^G12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-Kras^G12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide’s interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

Pancreatic cancer is resistant to chemotherapy in part due to the dense desmoplastic fibrosis surrounding the tumor, the immunosuppressive cells in the tumor microenvironment (TME), and the early rate of metastases. In this study, we examined the effects of a CCK receptor antagonist, proglumide, alone and in combination with gemcitabine in murine models of pancreatic cancer. Tumor growth rate, metastases, and survival were assessed in mice bearing syngeneic murine or human pancreatic tumors treated with PBS (control), gemcitabine, proglumide, or the combination of gemcitabine and proglumide. Excised tumors were evaluated histologically for fibrosis, immune cells, molecular markers, and uptake of chemotherapy by mass spectroscopy. Peripheral blood was analyzed with a microRNAs biomarker panel associated with fibrosis and oncogenesis. Differentially expressed genes between tumors of mice treated with gemcitabine monotherapy and combination therapy were compared by RNAseq. When given in combination the two compounds exhibited inhibitory effects by decreasing tumor growth rate by 70%, metastases, and prolonging survival. Proglumide monotherapy altered the TME by decreasing fibrosis, increasing intratumoral CD8⁺ T-cells, and decreasing arginase-positive cells, thus rendering the tumor sensitive to chemotherapy. Proglumide altered the expression of genes involved in fibrosis, epithelial–mesenchymal transition, and invasion. CCK-receptor antagonism with proglumide renders pancreatic cancer susceptible to chemotherapy.

Background: Serial analysis of biomarkers in the circulation of patients undergoing treatment (“liquid biopsies”) can provide new insights into drug effects. In particular the analysis of cell‐free, circulating nucleic acids such as microRNAs (miRs) can reveal altered expression patterns indicative of mechanism of drug action, cancer growth, and tumor–stroma interactions.Results: Here we analyzed plasma miRs in patients with hormone receptor positive, metastatic breast cancer with prior disease progression during aromatase inhibitor therapy (n = 8) in a phase I/II trial with the multiple tyrosine kinase inhibitor dovitinib (TKI258). Plasma miR levels were measured by quantitative RT‐qPCR before and after treatment with dovitinib. A candidate miR signature of drug response was established from a 379 miR screen for detectable plasma miRs as well as from the published literature. Changes in miR expression patterns and tumor sizes were compared. In this analysis we identified miR‐21‐5p, miR‐100‐5p, miR‐125b‐5p, miR‐126‐3p, miR‐375 and miR‐424‐5p as potential indicators of a response to dovitinib. The altered expression patterns observed for the six circulating miRs separated patients with resistant disease from those with drug responsive disease. There was no relationship between adverse effects of dovitinib treatment and identifiable changes in miR patterns.Conclusion: We conclude that changes in the expression patterns of circulating miRs can be indicators of drug responses that merit prospective studies for validation.

Patients exhibiting pancreatic cancer possess poor rates of survival. Therefore, the identification of a biomarker that can be measured non-invasively and be used to predict patient outcomes is required for the successful treatment of pancreatic cancer. The present study evaluated serum microRNA (miRNA/miR) profiles in patients exhibiting pancreatic cancer, who were treated with lapatinib and capecitabine in a phase II trial. Serum samples were collected for the measurement of a panel of miRNAs (miR‑21, miR‑210, miR‑221 and miR‑7) associated with the epidermal growth factor receptor (EGFR)1 and human epidermal growth factor receptor (HER)2 pathways. Preclinically, human pancreatic cancer PANC‑1, MIA PaCa‑2 and BXCP‑3 cell lines were utilized for miRNA and drug resistance studies. In total, 6/17 patients treated experienced disease progression following 2 cycles of treatment [non‑responders (NRS)], while another 6/17 patients exhibited a stable disease state and received >4 cycles of treatment [responders (RS); range, 4‑22 cycles]. Five patients withdrew from the study due to severe toxicity or mortality. The mean overall survival time was 6.5 vs. 10.4 months for NRS and RS, respectively. Significant upregulation of serum miRNAs at earlier time points (3‑6 weeks) was observed in NRS. miRNA levels increased with cancer progression, and lapatinib and 5‑fluorouracil (5‑FU; the active form of capecitabine) treatment increased the miRNA levels (specifically miR‑210 and miR‑221) in the treatment‑resistant pancreatic cancer PANC‑1 and MIA PaCa‑2 cell lines. However, lapatinib and 5‑FU treatment did not increase the miRNA levels in the treatment‑sensitive BXPC‑3 cell line. Inhibition of miR‑221 increased the sensitivity of the PANC‑1 cells to treatment. In conclusion, an increase in specific serum miRNAs was associated with resistance to lapatinib and capecitabine treatment. Additional investigation is required with regard to the application of the miRNA panel investigated in the present study as a potential predictor of patient responses to anti-EGFR/HER2 treatment.

βII‐Spectrin (SPTBN1) is an adapter protein for Smad3/Smad4 complex formation during transforming growth factor beta (TGF‐β) signal transduction. Forty percent of SPTBN1^+/− mice spontaneously develop hepatocellular carcinoma (HCC), and most cases of human HCC have significant reductions in SPTBN1 expression. In this study, we investigated the possible mechanisms by which loss of SPTBN1 may contribute to tumorigenesis. Livers of SPTBN1^+/− mice, compared to wild‐type mouse livers, display a significant increase in epithelial cell adhesion molecule‐positive (EpCAM⁺) cells and overall EpCAM expression. Inhibition of SPTBN1 in human HCC cell lines increased the expression of stem cell markers EpCAM, Claudin7, and Oct4, as well as decreased E‐cadherin expression and increased expression of vimentin and c‐Myc, suggesting reversion of these cells to a less differentiated state. HCC cells with decreased SPTBN1 also demonstrate increased sphere formation, xenograft tumor development, and invasion. Here we investigate possible mechanisms by which SPTBN1 may influence the stem cell traits and aggressive behavior of HCC cell lines. We found that HCC cells with decreased SPTBN1 express much less of the Wnt inhibitor kallistatin and exhibit decreased β‐catenin phosphorylation and increased β‐catenin nuclear localization, indicating Wnt signaling activation. Restoration of kallistatin expression in these cells reversed the observed Wnt activation. Conclusion: SPTBN1 expression in human HCC tissues is positively correlated with E‐cadherin and kallistatin levels, and decreased SPTBN1 and kallistatin gene expression is associated with decreased relapse‐free survival. Our data suggest that loss of SPTBN1 activates Wnt signaling, which promotes acquisition of stem cell‐like features, and ultimately contributes to malignant tumor progression. (Hepatology 2015;61:598‐612)