Satyanarayana Rachagani

MicroRNA in pancreatic cancer: Pathological, diagnostic and therapeutic implications

MicroRNAs (miRNAs) are a group of small non-coding RNA molecules of 17-25 nucleotides (nt) in length, predicted to control the activity of about 30% of all protein-coding genes in mammals. Altered expressions of miRNAs are reported in various cancers and may associate with cancer pathogenesis, apoptosis, and cell growth, thereby functioning as either tumor suppressors or oncogenes. Recent reports showed that deregulation of miRNA contribute to tumor development and progression and hence, have diagnostic and prognostic value in several human malignancies. This review discusses the current status of miRNA in pancreatic cancer development, progression, diagnosis, and therapy.

Current status of mucins in the diagnosis and therapy of cancer

Mucins are the most abundant high molecular weight glycoproteins in mucus. Their nature and glycosylation content dictates the biochemical and biophysical properties of viscoelastic secretions, pointing out an important role in diverse biological functions, such as differentiation, cell adhesions, immune responses, and cell signaling. Mucins are expressed in tubular organs by specialized epithelial cells in the body. Their aberrant expression is well documented in a variety of inflammatory or malignant diseases. From a prognosis point of view, their expression and alterations in glycosylation are associated with the development and progression of malignant diseases. Therefore, mucins can be used as valuable markers to distinguish between normal and disease conditions. Indeed, this alteration in glycosylation patterns generates several epitopes in the oligosaccharide side chains that can be used as diagnostic and/or prognostic markers. Furthermore, these characteristic tumor‐associated epitopes are extensively used as appropriate immunotargets of malignant epithelial cells. Therefore, in an effort to detect and treat cancer at the earliest stage possible, mucins are analyzed as potential markers of disease for diagnosis, progression, and for therapeutic purposes. In this review, we focused on the current status of the distribution of mucins in normal and pathologic conditions and their clinical use both in cancer diagnosis and therapeutics treatments.

Overexpression of macrophage inhibitory cytokine-1 induces metastasis of human prostate cancer cells through the FAK-RhoA signaling pathway.

An elevated level of macrophage inhibitory cytokine-1 (MIC-1) is reported in the sera of patients with metastatic prostate cancer compared with that of benign diseases and healthy adults. We investigated the mechanistic role of MIC-1 overexpression in the metastasis of prostate cancer cells. Our study showed a progressive increase in secretory MIC-1 production correlated with the increase in the metastatic potential of PC-3 and LNPCa prostate cancer metastatic variants. Further, the in vitro studies using ‘loss-’ and ‘gain’-of-function approaches showed that ectopic overexpression of MIC-1 (PC-3-MIC-1) and forced downregulation of MIC-1(PC-3M-siMIC-1) enhanced and reduced the motility and invasiveness of these cells, respectively. Supporting our in vitro observations, all the mice orthotopically implanted with PC-3-MIC-1 cells developed metastasis compared with none in the PC-3-vector group. Our results showed that MIC-1 overexpression was associated with apparent changes in actin organization. In addition, an enhanced phosphorylation of focal adhesion kinase (FAK) and guanosine-5′-triphosphate (GTP)-bound RhoA was also seen; however, no significant change was observed in total FAK and RhoA levels in the PC-3-MIC-1 cells. Altogether, our findings show that MIC-1 has a role in prostate cancer metastasis, in part, by promoting the motility of these cells. Activation of the FAK–RhoA signaling pathway is involved in MIC-1-mediated actin reorganization, and thus, leads to an increase in the motility of prostate cancer cells.

Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m3 total suspended particulate (TSP)), P=0.014; high dose (247 mg/m3 TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88±35.96 ng/ml; high dose, 216.25±29.95 ng/ml) and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS upregulates the MUC4 mucin in PC via the α7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of PC.

Graviola: A novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism

Pancreatic tumors are resistant to conventional chemotherapies. The present study was aimed at evaluating the potential of a novel plant-derived product as a therapeutic agent for pancreatic cancer (PC). The effects of an extract from the tropical tree Annona Muricata, commonly known as Graviola, was evaluated for cytotoxicity, cell metabolism, cancer-associated protein/gene expression, tumorigenicity, and metastatic properties of PC cells. Our experiments revealed that Graviola induced necrosis of PC cells by inhibiting cellular metabolism. The expression of molecules related to hypoxia and glycolysis in PC cells (i.e. HIF-1α, NF-κB, GLUT1, GLUT4, HKII, and LDHA) were downregulated in the presence of the extract. In vitro functional assays further confirmed the inhibition of tumorigenic properties of PC cells. Overall, the compounds that are naturally present in a Graviola extract inhibited multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties in PC cells. Collectively, alterations in these parameters led to a decrease in tumorigenicity and metastasis of orthotopically implanted pancreatic tumors, indicating promising characteristics of the natural product against this lethal disease.

Pathobiological Implications of MUC16 Expression in Pancreatic Cancer

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.

Activated Kras G12D is associated with invasion and metastasis of pancreatic cancer cells through inhibition of E-cadherin

Background:Pancreatic cancer (PC) harbours an activated point mutation (KrasG12D) in the Kras proto-oncogene that has been demonstrated to promote the development of PC.Methods:This study was designed to investigate the effect of the oncogenic KrasG12D allele on aggressiveness and metastatic potential of PC cells. We silenced the oncogenic KrasG12D allele expression in CD18/HPAF and ASPC1 cell lines by stable expression of shRNA specific to the KrasG12Dallele.Results:The KrasG12D knockdown cells exhibited a significant decrease in motility (P<0.0001), invasion (P<0.0001), anchorage-dependent (P<0.0001) and anchorage-independent growth (P<0.0001), proliferation (P<0.005) and an increase in cell doubling time (P<0.005) in vitro and a decrease in the incidence of metastases upon orthotopic implantation into nude mice. The knockdown of the KrasG12D allele led to a significant increase in the expression of E-cadherin (mRNA and protein) both in vitro and in vivo. This was associated with a decrease in the expression of phoshpo-ERK-1/2, NF-κB and MMP-9, and transcription factors such as δEF1, Snail and ETV4. Furthermore, the expression of several proteins involved in cell survival, invasion and metastasis was decreased in the KrasG12D knockdown cells.Conclusions:The results of this study suggest that the KrasG12D allele promotes metastasis in PC cells partly through the downregulation of E-cadherin.

MUC4 potentiates invasion and metastasis of pancreatic cancer cells through stabilization of fibroblast growth factor receptor 1

MUC4 is a type-1 transmembrane mucin differentially expressed in multiple cancers and has previously been shown to potentiate progression and metastasis of pancreatic cancer. In this study, we investigated the molecular mechanisms associated with the MUC4-induced invasion and metastasis in pancreatic cancer. Stable silencing of MUC4 in multiple pancreatic cancer cells resulted in the downregulation of N-cadherin and its interacting partner fibroblast growth factor receptor 1 (FGFR1) through downregulation of partly by pFAK, pMKK7, pJNK and pc-Jun pathway and partly through PI-3K/Akt pathway. The downregulation of FGFR1 in turn led to downregulation of pAkt, pERK1/2, pNF-κB, pIkBα, uPA, MMP-9, vimentin, N-cadherin, Twist, Slug and Zeb1 and upregulation of E-cadherin, Occludin, Cytokeratin-18 and Caspase-9 in MUC4 knockdown BXPC3 and Capan1 cells compared with scramble vector transfected cells. Further, downregulation of FGFR1 was associated with a significant change in morphology and reorganization of the actin-cytoskeleton, leading to a significant decrease in motility (P < 0.00001) and invasion (P < 0.0001) in vitro and decreased tumorigenicity and incidence of metastasis in vivo upon orthotopic implantation in the athymic mice. Taken together, the results of the present study suggest that MUC4 promotes invasion and metastasis by FGFR1 stabilization through the N-cadherin upregulation.

Guggulsterone decreases proliferation and metastatic behavior of pancreatic cancer cells by modulating JAK/STAT and Src/FAK signaling.

Inadequate efficacy, high toxicity and drug resistance associated with existing chemotherapeutic agents mandate a need for novel therapeutic strategies for highly aggressive Pancreatic Cancer (PC). Guggulsterone (GS) exhibits potent anti-proliferative effects against various cancer cells and has emerged as an attractive candidate for use in complementary or preventive cancer therapies. However, the knowledge regarding the therapeutic potential of GS in PC is still limited and needs to be explored. We studied the effect of GS on PC cell growth, motility and invasion and elucidated the molecular mechanisms associated with its anti-tumor effects. Treatment of Capan1 and CD18/HPAF PC cells with GS resulted in dose- and time-dependent growth inhibition and decreased colony formation. Further, GS treatment induced apoptosis and cell cycle arrest as assessed by Annexin-V assay and FACS analysis. Increased apoptosis following GS treatment was accompanied with Bad dephosphorylation and its translocation to the mitochondria, increased Caspase-3 activation, decreased Cyclin D1, Bcl-2 and xIAP expression. Additionally, GS treatment decreased motility and invasion of PC cells by disrupting cytoskeletal organization, inhibiting activation of FAK and Src signaling and decreased MMP9 expression. More importantly, GS treatment decreased mucin MUC4 expression in Capan1 and CD18/HPAF cells through transcriptional regulation by inhibiting Jak/STAT pathway. In conclusion, our results support the utility of GS as a potential therapeutic agent for lethal PC.

Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

BackgroundPancreatic cancer (PC) is a lethal malignancy primarily driven by activated Kras mutations and characterized by the deregulation of several genes including mucins. Previous studies on mucins have identified their significant role in both benign and malignant human diseases including PC progression and metastasis. However, the initiation of MUC expression during PC remains unknown because of lack of early stage tumor tissues from PC patients.MethodsIn the present study, we have evaluated stage specific expression patterns of mucins during mouse PC progression in (KrasG12D;Pdx1-Cre (KC)) murine PC model from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and quantitative real-time PCR.ResultsIn agreement with previous studies on human PC, we observed a progressive increase in the expression of mucins particularly Muc1, Muc4 and Muc5AC in the pancreas of KC (as early as PanIN I) mice with advancement of PanIN lesions and PDAC both at mRNA and protein levels. Additionally, mucin expression correlated with the increased expression of inflammatory cytokines IFN-γ (p < 0.0062), CXCL1 (p < 0.00014) and CXCL2 (p < 0.08) in the pancreas of KC mice, which are known to induce mucin expression. Further, we also observed progressive increase in inflammation in pancreas of KC mice from 10 to 50 weeks of age as indicated by the increase in the macrophage infiltration. Overall, this study corroborates with previous human studies that indicated the aberrant overexpression of MUC1, MUC4 and MUC5AC mucins during the progression of PC.ConclusionsOur study reinforces the potential utility of the KC murine model for determining the functional role of mucins in PC pathogenesis by crossing KC mice with corresponding mucin knockout mice and evaluating mucin based diagnostic and therapeutic approaches for lethal PC.