Narayan Shivapurkar

Tissue and Serum microRNAs in the KrasG12D Transgenic Animal Model and in Patients with Pancreatic Cancer

microRNAs (miRs) modulate the expression levels of mRNAs and proteins and can thus contribute to cancer initiation and progression. In addition to their intracelluar function, miRs are released from cells and shed into the circulation. We postulated that circulating miRs could provide insight into pathways altered during cancer progression and may indicate responses to treatment. Here we focus on pancreatic cancer malignant progression. We report that changes in miR expression patterns during progression of normal tissues to invasive pancreatic adenocarcinoma in the p48-Cre/LSL-KrasG12D mouse model mirrors the miR changes observed in human pancreatic cancer tissues. miR-148a/b and miR-375 expression were found decreased whereas miR-10, miR-21, miR-100 and miR-155 were increased when comparing normal tissues, premalignant lesions and invasive carcinoma in the mouse model. Predicted target mRNAs FGFR1 (miR-10) and MLH1 (miR-155) were found downregulated. Quantitation of nine microRNAs in plasma samples from patients distinguished pancreatic cancers from other cancers as well as non-cancerous pancreatic disease. Finally, gemcitabine treatment of control animals and p48-Cre/LSL-KrasG12D animals with pancreatic cancer caused distinct and up to 60-fold changes in circulating miRs that indicate differential drug effects on normal and cancer tissues. These findings support the significance of detecting miRs in the circulation and suggests that circulating miRs could serve as indicators of drug response.

Recurrence of Early Stage Colon Cancer Predicted by Expression Pattern of Circulating microRNAs

Systemic treatment of patients with early-stage cancers attempts to eradicate occult metastatic disease to prevent recurrence and increased morbidity. However, prediction of recurrence from an analysis of the primary tumor is limited because disseminated cancer cells only represent a small subset of the primary lesion. Here we analyze the expression of circulating microRNAs (miRs) in serum obtained pre-surgically from patients with early stage colorectal cancers. Groups of five patients with and without disease recurrence were used to identify an informative panel of circulating miRs using quantitative PCR of genome-wide miR expression as well as a set of published candidate miRs. A panel of six informative miRs (miR-15a, mir-103, miR-148a, miR-320a, miR-451, miR-596) was derived from this analysis and evaluated in a separate validation set of thirty patients. Hierarchical clustering of the expression levels of these six circulating miRs and Kaplan-Meier analysis showed that the risk of disease recurrence of early stage colon cancer can be predicted by this panel of miRs that are measurable in the circulation at the time of diagnosis (P = 0.0026; Hazard Ratio 5.4; 95% CI of 1.9 to 15).

βII-Spectrin (SPTBN1) suppresses progression of hepatocellular carcinoma and Wnt signaling by regulation of Wnt inhibitor kallistatin.

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)

Circulating microRNA profile predicts disease progression in patients receiving second-line treatment of lapatinib and capecitabine for metastatic pancreatic cancer

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.

Exercise – induced changes in cerebrospinal fluid miRNAs in Gulf War Illness, Chronic Fatigue Syndrome and sedentary control subjects

Gulf War Illness (GWI) and Chronic Fatigue Syndrome (CFS) have similar profiles of pain, fatigue, cognitive dysfunction and exertional exhaustion. Post-exertional malaise suggests exercise alters central nervous system functions. Lumbar punctures were performed in GWI, CFS and control subjects after (i) overnight rest (nonexercise) or (ii) submaximal bicycle exercise. Exercise induced postural tachycardia in one third of GWI subjects (Stress Test Activated Reversible Tachycardia, START). The remainder were Stress Test Originated Phantom Perception (STOPP) subjects. MicroRNAs (miRNA) in cerebrospinal fluid were amplified by quantitative PCR. Levels were equivalent between nonexercise GWI (n = 22), CFS (n = 43) and control (n = 22) groups. After exercise, START (n = 22) had significantly lower miR-22-3p than control (n = 15) and STOPP (n = 42), but higher miR-9-3p than STOPP. All post-exercise groups had significantly reduced miR-328 and miR-608 compared to nonexercise groups; these may be markers of exercise effects on the brain. Six miRNAs were significantly elevated and 12 diminished in post-exercise START, STOPP and control compared to nonexercise groups. CFS had 12 diminished miRNAs after exercise. Despite symptom overlap of CFS, GWI and other illnesses in their differential diagnosis, exercise-induced miRNA patterns in cerebrospinal fluid indicated distinct mechanisms for post-exertional malaise in CFS and START and STOPP phenotypes of GWI.

Author Correction: Exercise – induced changes in cerebrospinal fluid miRNAs in Gulf War Illness, Chronic Fatigue Syndrome and sedentary control subjects

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Cholecystokinin Receptor-Targeted Polyplex Nanoparticle Inhibits Growth and Metastasis of Pancreatic Cancer

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the cholecystokinin receptor that is overexpressed in this malignancy. Methods We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the cholecystokinin receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP. Results The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes. Conclusions Our polyplex nanoparticle platform establishes both a strong foundation for the development of receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.

Dietary Fat Stimulates Pancreatic Cancer Growth and Promotes Fibrosis of the Tumor Microenvironment through the Cholecystokinin Receptor

The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high-fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on the growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high-fat diet significantly increased growth and metastasis of pancreatic cancer compared with the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using clustered regularly interspaced short palindromic repeats technology and showed that without CCK-receptors, dietary fat was unable to stimulate cancer growth. We next demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor-associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK-receptor antagonist therapy because fibroblasts also have CCK receptors. The CCK-receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK-receptor pathway. NEW & NOTEWORTHY Diets high in long-chain saturated fats promote growth of pancreatic cancer independent of obesity. The mechanism through which dietary fat promotes cancer is mediated through the cholecystokinin (CCK) receptor pathway. Therapy with a CCK-receptor antagonist altered the tumor microenvironment by reducing fibrosis, increasing cluster of differentiation 8+ lymphocytes, increasing tumor suppressor genes, and thus decreasing metastases. Use of CCK-receptor antagonist therapy with standard chemotherapy for pancreatic cancer may improve response by altering the tumor microenvironment.

Abstract 3764: Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC β2SP(ELF) is one of adapter proteins for Smad3/Smad4 complex formation during TGF-β signal transduction. 40% of β2SP+/- mice spontaneously develop hepatocellular carcinoma (HCC) and most cases of human HCC, gastric cancer, and lung cancer demonstrate significant reductions in β2SP expression. In our study, we investigated the possible mechanism for loss of β2SP induced HCC tumorigenesis. Recent studies have highlighted the involvement of cancer stem cells during the formation and progression of various types of cancers, epithelial cells that have undergone EMT (epithelial mesenchymal transition) acquire stem-cell properties during cancer development. Giving the role of TGF-beta signaling in stem cell differentiation and EMT during embryonic development, we hypothesize loss of β2SP results in alteration of liver stem cell that may contribute to the development of HCC in β2SP+/− mice. It has been shown that EpCAM-positive adult liver cells has stem cell property and can differentiate into functional hepatocytes in xenograft model. We found that EpCAM-positive hepatocyte number doubled in β2SP+/− mouse compared to wild type by fluorescence-activated cell sorter (FACS). Furthermore EpCAM level in β2SP+/− mouse liver was three times higher than that in the liver of wild type mouse. To study the mechanism by which Epcam is regulated by β2SP, HCC cells were transfected by β2SP siRNA. Inhibition of β2SP increased the expression of Epcam, and EMT markers vimentin was increased and E-cadherin expression was decreased in PLC/PRF/5 and SNU449 cells, while TGF-β1 and Smad3 rescued the expression of Epcam and E-cadherin, but not vimentin. We also found that suppression of β2SP expression promotes adhesion, migration and cloning formation of PLC/PRF/5 and SNU449 cells. Our data suggests loss of β2SP enhances the stem-like traits and invasiveness of HCC cells. Citation Format: Xiuling Zhi, Ling Lin, Narayan Shivapurkar, Wilma Jogunoori, Lopa Mishra, Aiwu R He. Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells. [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 3764. doi:10.1158/1538-7445.AM2013-3764

Abstract 4667: Circulating microRNA profile predicts disease progression in patients receiving second-line treatment of lapatinib and capecitabine for metastatic pancreatic cancer.

Background: Serum microRNAs (miRNAs) have been reported as potential prognostic and/or predictive markers. We described an association between circulating miRNAs targeting the EGFR pathway and clinical outcomes in a phase II open-label, single arm study to evaluate the combination of lapatinib, a dual tyrosine kinase inhibitor of EGFR1 and HER-2, and capecitabine in treatment of metastatic pancreatic cancer. Methods: Seventeen patients with metastatic, gemcitabine-refractory pancreatic cancer received treatment of lapatinib 1,250 mg PO daily, and capecitabine 1,000 mg/m2 PO twice daily on days 1-14 of each 21-day cycle. The primary endpoint was median overall survival (OS). Serum samples were collected at baseline (before treatment) and every 3 weeks while on study. The expression profile of a panel of miRNAs that are focused on EGFR1 and HER-2 pathways (miR-16, miR-21, miR-210, miR-221, and miR-7) was analyzed for fold change in expression (compared to baseline). Results: Six of the 17 patients had disease progression after 2 cycles (nonresponders), 6 of 17 patients had stable disease and received more than 4 cycles (responders, range 4-22 cycles). The median PFS was 9 weeks (95% CI 7.1-18.9), median OS was 25 weeks (95% CI 11-34). 6-17% patients developed grade 3 adverse events including nausea, vomiting, diarrhea, limb edema and fatigue. Serum samples were collected serially from 8 patients (3 nonresponders and 5 responders). The mean survival from the time of enrollment was 6.5 months (median 6.5) in nonresponders v.s. 10.4 months (median 8.3) in responders. The miRNA analysis showed significant up-regulation of circulating miRNAs (target panel) at earlier time point (3-6 weeks) only in the nonresponders but not in the responders (fold change: 5.47 ± 1.47 v.s. 0.66 ± 0.08, p Citation Format: Zheng Wu, Narayan Shivapurkar, John L. Marshall, Jimmy Hwang, Michael J. Pishvaian, Tingting Zhuang, Anton Wellstein, Louis M. Weiner, Lisa Ley, Aiwu R. He. Circulating microRNA profile predicts disease progression in patients receiving second-line treatment of lapatinib and capecitabine for metastatic pancreatic cancer. [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 4667. doi:10.1158/1538-7445.AM2013-4667