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Pancreatic ductal adenocarcinoma (PDAC) manifests aggressive tumor growth and early metastasis. Crucial steps in tumor growth and metastasis are survival, angiogenesis, invasion, and immunosuppression. Our prior research showed that chemokine CXC- receptor-2 (CXCR2) is expressed on endothelial cells, innate immune cells, and fibroblasts, and regulates angiogenesis and immune responses. Here, we examined whether tumor angiogenesis, growth, and metastasis of CXCR2 ligands expressing PDAC cells are regulated in vivo by a host CXCR2-dependent mechanism. C57BL6 Cxcr2(−/−) mice were generated following crosses between Cxcr2(−/+) female and Cxcr2(−/−) male. Cxcr2 ligands expressing Kirsten rat sarcoma (KRAS-PDAC) cells were orthotopically implanted in the pancreas of wild-type or Cxcr2(−/−) C57BL6 mice. No significant difference in PDAC tumor growth was observed. Host Cxcr2 loss led to an inhibition in microvessel density in PDAC tumors. Interestingly, an enhanced spontaneous and experimental liver metastasis was observed in Cxcr2(−/−) mice compared with wild-type mice. Increased metastasis in Cxcr2(−/−) mice was associated with an increase in extramedullary hematopoiesis and expansion of neutrophils and immature myeloid precursor cells in the spleen of tumor-bearing mice. These data suggest a dynamic role of host CXCR2 axis in regulating tumor immune suppression, tumor growth, and metastasis.

The discovery of early detection markers of pancreatic cancer (PC) disease is highly warranted. We analyzed the expression profile of different CXC-receptor-2 (CXCR2) ligands in PC cases for the potential of biomarker candidates. Analysis of different PDAC microarray datasets with matched normal and pancreatic tumor samples and next-generation sequenced transcriptomics data using an online portal showed significantly high expression of CXCL-1, 3, 5, 6, 8 in the tumors of PC patients. High CXCL5 expression was correlated to poor PC patient survival. Interestingly, mRNA and protein expression analysis of human PC cell lines showed higher CXCL2, 3, and 5 expressions in cell lines derived from metastatic sites than primary tumors. Furthermore, we utilized immunohistochemistry (IHC) to evaluate the expression of CXCR2 ligands in the human PC tumors and observed positive staining for CXCL1, 3, and 8 with a higher average IHC composite score of CXCL3 in the PC tissue specimens than the normal pancreas. We also observed an increase in the expression of mouse CXCL1, 3, and 5 in the pre-cancerous lesions of tumors and metastasis tissues derived from the PDX-cre-LSL-Kras(G12D) mouse model. Together, our data suggest that different CXCR2 ligands show the potential of being utilized as a diagnostic biomarker in PC patients.

Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils' contribution to early tumor dissemination.

Semaphorin-5A (SEMA5A) has differential cell surface expression between normal and cancer cells and represents an attractive target for therapeutic intervention in pancreatic cancer (PC). In this study, we delineated the pathological expression and significance of SEMA5A during PC progression and metastasis. We utilized human tissue microarrays and different PC mouse models (Pdx1-cre; LSL- Kras(G12D), Pdx1-Cre; LSL-Kras(G12D); LSL-p53(R172H) and RIP1-Tag2) to analyze SEMA5A expression during PC progression. Using human patients and different mouse models, we demonstrated that SEMA5A expression was highest in liver metastases, followed by primary pancreatic tumors, and the lowest expression was found in the normal pancreas. SEMA5A expression was localized on tumor cells with no staining in the surrounding stroma. To understand the functional significance of SEMA5A, we treated PC cell lines with recombinant SEMA5A. We observed an increase in migration, chemotaxis, and scattering of PC cells. To delineate the signaling axis of SEMA5A, we generated SEMA5A receptor-Plexin-B3 knockdown in T3M-4 and CD18/HPAF PC cell lines and observed that the effect of SEMA5A treatment was absent in the Plexin-B3 knockdown counterparts of T3M-4 and CD18/HPAF cells. SEMA5A treatment leads to phosphorylation of cMET in Plexin-B3 dependent manner. Our data demonstrate that there is an increase in SEMA5A expression during PC progression and the elevation of this expression takes place at metastatic sites especially the liver in both exocrine and endocrine tumors. SEMA5A can elicit a migratory response in cells by activating cMET through the Plexin-B3 receptor. In conclusion, SEMA5A signaling represents a potential molecule for targeting metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PDAC. The tumor microenvironment contributes significantly to tumor progression and spread. Cancer-associated fibroblasts (CAFs) facilitate therapy resistance and metastasis. Recent reports emphasized the concurrence of multiple subtypes of CAFs with diverse roles, fibrogenic, and secretory. C-X-C motif chemokine receptor 2 (CXCR2) is a chemokine receptor known for its role during inflammation and its adverse role in PDAC. Oncogenic Kras upregulates CXCR2 and its ligands and, thus, contribute to tumor proliferation and immunosuppression. CXCR2 deletion in a PDAC syngeneic mouse model produced increased fibrosis revealing a potential undescribed role of CXCR2 in CAFs. In this study, we demonstrate that the oncogenic Kras-CXCR2 axis regulates the CAFs function in PDAC and contributes to CAFs heterogeneity. We observed that oncogenic Kras and CXCR2 signaling alter CAFs, producing a secretory CAF phenotype with low fibrogenic features; and increased secretion of pro-tumor cytokines and CXCR2 ligands, utilizing the NF-κB activity. Finally, using syngeneic mouse models, we demonstrate that oncogenic Kras is associated with secretory CAFs and that CXCR2 inhibition promotes activation of fibrotic cells (myofibroblasts) and impact tumors in a mutation-dependent manner.

Amyloid precursor protein (APP) and its family members amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are type 1 transmembrane glycoproteins that are highly conserved across species. The transcriptional regulation of APP and APLP2 is similar but not identical, and the cleavage of both proteins is regulated by phosphorylation. APP has been implicated in Alzheimer's disease causation, and in addition to its importance in neurology, APP is deregulated in cancer cells. APLP2 is likewise overexpressed in cancer cells, and APLP2 and APP are linked to increased tumor cell proliferation, migration, and invasion. In this present review, we discuss the unfolding account of these APP family members’ roles in cancer progression and metastasis.

// Abhilasha Purohit 1 , Michelle Varney 1 , Satyanarayana Rachagani 2 , Michel M. Ouellette 3 , Surinder K. Batra 1, 2, 3 , Rakesh K. Singh 1 1 Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE, USA 2 Department of Biochemistry and Molecular Biology, 985870 Nebraska Medical Center, Omaha, NE, USA 3 Eppley Institute, 985950 Nebraska Medical Center, Omaha, NE, USA Correspondence to: Rakesh K. Singh, e-mail: rsingh@unmc.edu Keywords: KRAS (G12D) , PDAC, CXCR2, autocrine growth, ERK Received: August 05, 2015 Accepted: November 25, 2015 Published: January 13, 2016 ABSTRACT Pharmacological inhibition of RAS , the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS (G12D) -induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL- Kras (G12D) mice. Knocking-down CXCR2 in KRAS (G12D) -bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS (G12D) -bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS (G12D) -bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS (G12D) -induced growth transformation and progression in PDAC.

The major complexity of pancreatic cancer (PC) cases is late clinical presentation and metastasis at the time of detection. Achievement of significant clinical advancement in the treatment of PC disease needs extensive research efforts to discover early detection markers of this disease. In this study, we wanted to analyze the expression profile of different CXC-receptor 2 (CXCR2) ligands, well documented in the literature for their role in inflammation, in pancreatic ductal adenocarcinoma (PDAC) cases for the potential of biomarker candidates. With this perspective, we utilized different online portals to evaluate mRNA expression of different CXCR2 ligands CXCL1-3 and 5-8 in PDAC tumors in comparison with the normal pancreas. Analysis of GEO PDAC microarray dataset, GSE15471 containing 39 pancreatic tumors with corresponding matched normal samples, showed significantly higher expression of CXCL-3,5,6,8 in pancreatic tumors in comparison with their matched normal tissue. Similarly, analysis of next-generation sequenced transcriptomics data on MiPanda portal showed higher expression of CXCL-1,3,5,6,8 in pancreatic tumors of 417 PC patients in comparison with the normal pancreas. Interestingly, only CXCL5 ligand showed association with patient survival data based on the mRNA, miRNA, or lncRNA expression on Oncolnc-TCGA portal. PC patients with lower CXCL5 expression showed significantly higher survival in comparison with patients with higher CXCL5 expression. Next, we evaluated the expression pattern of CXCR2 ligands CXCL1, 3, and 8 in the human PC tissue specimens and using pancreatic tissues derived from disease progression mouse model of PDX-cre-LSL-KRASG12D. Immunohistochemical (IHC) analysis revealed positive staining for CXCL1, and 3 in both in the ducts and the stroma of the human PC tumors and normal pancreas specimens. However, in normal pancreas, the expression was localized only to the acinar cell compartment and pancreatic ducts were negative for expression. Overall the average composite score of CXCL3 IHC was higher in the PC tissue specimens versus the normal pancreas. Furthermore, we observed an increase in the expression of mCXCL1, 3, and 5 in the pre-cancerous lesions of tumors and metastasis derived from PDX-cre-LSL-KrasG12D. However, unlike the human tissues the normal murine pancreas was negative for the expression of mCXCL1 and 5. Lastly, mRNA expression analysis of nine different human PC cell lines showed higher CXCL2, 3, and 5 in cell lines derived from metastatic sites in comparison with cell lines derived from primary tumors. Our observations resonated with CXCL5 protein levels in these cell lines. In conclusion, our data suggest that among different CXCR2 ligands, CXCL5 shows the potential of being used as a biomarker in PC patients. Citation Format: Sugandha Saxena, Abhilasha Purohit, Evie Ehrhorn, Pranita Atri, Caitlin Molczyk, Michelle Varney, Rakesh K. Singh. Evaluation of the differential expression profile of CXC-receptor-2 ligands for potential biomarker candidates in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6194.

Background Pancreatic cancer (PC) is a highly aggressive disease, and the lethality of this disease stems from early metastatic dissemination where surgical removal cannot provide a cure. Improvement of the therapeutic outcome and overall survival of PC patients requires to understand the fundamental processes that lead to metastasis such as the gain of cellular migration ability. One such family of proteins, which are essential players of cellular migration, is Semaphorin. Previously, we have identified one of the Semaphorin family member, Semaphorin-5A (SEMA5A) to be involved in organ-specific homing during PC metastasis. We have also demonstrated that SEMA5A has a constitutive expression in PC cell lines derived from metastatic sites in comparison with low endogenous expression in the primary tumor-derived cell line. In this study, we examined whether constitutive SEMA5A expression in metastatic PC cells regulates tumor growth and metastatic potential. Methods We generated SEMA5A knockdown in T3M-4 and CD18/HPAF cells and assessed their phenotypes on in vitro motility, tumor growth, and metastatic progression. Results In contrary to our initial expectations, orthotopic injection of SEMA5A knockdown cells into nude mice resulted in a significant increase in both tumor burden and liver metastases in comparison with the Control cells. Similarly, we observed higher in vitro migratory potential with pronounced morphological changes associated with epithelial-mesenchymal transition (EMT), a decrease in the expression of epithelial marker E-cadherin (E-Cad), increase in the expression of mesenchymal markers N-cadherin (N-Cad) and Snail and the activation of the Wnt-signaling pathway in SEMA5A knockdown cells. Furthermore, re-establishing SEMA5A expression with a knockdown resistant mouse Sema5A in SEMA5A knockdown cells resulted in a reversion to the epithelial state (mesenchymal-epithelial transition; MET), as indicated by the rescue of E-Cad expression and a decrease in N-Cad and Snail expression. Conclusions Collectively, our data suggest that SEMA5A expression maintains epithelial phenotype in the metastatic microenvironment. Electronic supplementary material The online version of this article (10.1186/s12885-018-5204-x) contains supplementary material, which is available to authorized users.

Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the US and remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PC. In addition to the autonomous aggressiveness feature of PC malignant cells, host tumor microenvironment contribute greatly to tumor progression and spread. Cancer-associated fibroblasts (CAFs), represent the major component of the tumor microenvironment in PC, are implicated in facilitating therapy resistance and metastasis. Recent reports emphasize the concurrence of multiple subtypes of CAFs that carry out different functions. CXCR2 is a chemokine receptor that is known for its role during inflammation by recruiting innate immune cells and the induction of angiogenesis. In PC, oncogenic Kras upregulates CXCR2 and its ligands (CXCL1-3 and 5-8), which are linked to inducing tumor proliferation and immunosuppression as well as induction of malignant cells stemness contributing to therapy resistance. Deletion of CXCR2 in a syngeneic mouse model (Cxcr2-/-) of PC increased the abundance of fibrosis revealing a potential undescribed role of CXCR2 in regulating CAFs. We hypothesize that CXCR2 regulates CAFs function in PC and contribute to CAFs heterogeneity. Co-culture of PC tumor cells in the conditioned media of CAFs and co-culturing CAFs in conditioned media of tumor cells revealed an oncogenic Kras-dependent interaction. CAFs enhanced the growth of PC cells with oncogenic Kras; whereas, conditioned media of the PC cells with oncogenic Kras has inhibited CAFs growth. Paracrine factors derived from tumor cells decreased the expression of the fibrotic CAFs-associated markers including αSMA and collagen I, and increased the expression of immunosuppressive cytokines and tumor-promoting chemokines including IL-4, IL-10, IL-13 and CXCL7. Utilizing recombinant CXCR2 chemokines and CXCR2 inhibitors, we confirmed the involvement of CXCR2 in this secretory phenotype. CXCL1 and CXCL8 exhibited a similar effect on CAFs by downregulating αSMA and upregulating IL-4, IL-13 and CXCL7. CXCR2 inhibitors reverted some of the observed effects. Examining downstream signaling pathways revealed that this effect is mediated through activation of NFκB. We further confirmed these findings in-vivo. Immunohistochemistry revealed higher αSMA expression in the tumors implanted in Cxcr2-/- compared to wildtype mice. Altogether, we demonstrate that sustained signaling through CXCR2 activates NFκB and induces a secretory phenotype of CAFs in PC that upregulates pro-tumor immunosuppression growth factors. Citation Format: Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Lingyun Wu, Sugandha Saxena, Sushil Kumar, Surinder K. Batra, Rakesh K. Singh. Sustained CXCR2 signaling promotes secretory phenotype in cancer-associated fibroblasts of pancreatic cancer via activation of NFkB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1982.

Objective: The present study was designed to examine the anti-hypothyroid activity of Withania somnifera (Ashwagandha)’s leaf extract on 6-n-propyl- 2-thio-uracil (PTU) induced hypothyroid rats.Method: Ethanolic extracts of W. somnifera leaf and Eltroxin were administrated to PTU induced hypothyroid rats. The animals were divided into control, PTU treatment, W. somnifera leaf extract treatment, and Eltroxin treatment groups for 60 days. The serum T3 and T4 were estimated, and biochemical and hematological parameters of the blood serum were also evaluated.Results: PTU induction caused a significant decrease (p≤0.001) in T3 and T4 level when compared with the control group. Adverse effects of PTU were also observed in blood sugar, cholesterol, alkaline phosphate, protein, albumin, globulin, liver function test, and renal function test parameters. Non-significant changes in LFT,RFT and Other parameters were observed. W.Somnifera’s leaf extract and Eltroxin treatment group recovered both of the thyroid hormone secretion as compare to control.

Pancreatic cancer (PC) remains one of the deadliest types of solid malignancies. Making progress in dealing with PC starts by understanding the complex interaction of cellular components in the tumor microenvironment and identifying the crucial checkpoints for tumor progression and metastasis. Desmoplasia and inflammation are two major hallmarks of PC. Cancer-associated fibroblasts (CAFs) are known for inducing fibrosis in several pathologies of the pancreas including PC. Recent reports have described that CAFs can assume the fibrosis-inducing phenotype, when close or have a direct contact with tumor cells, or a tumor-promoting inflammatory phenotype, through distant paracrine signaling. Known for their role in recruiting granulocytes and inducing angiogenesis, CXC chemokines (1-3 and 5-8) contain a conserved motif of Glutamic acid-Leucine-Arginine (ELR) and signal through CXCR1/2 chemokine receptors. In PC, ELR+ chemokines produced by malignant cells and stromal cells contribute to tumor growth, angiogenesis, and tumor-supporting immunosuppression. The deletion of CXCR2 in the genetically engineered PC mouse model (KC) promoted fibrosis and tumor metastasis. Taken together, we hypothesize that ELR+ chemokines signaling through CXCR2 may trigger the inflammatory phenotype of CAFs in PC that promotes tumor growth and progression. Utilizing unidirectional coculture studies, incubating CAFs in conditioned media of PC cells inhibited their growth, decreased the expression of the fibrotic CAFs-associated markers such as αSMA and collagen I, and increased the expression of immunosuppressive cytokines and tumor-promoting chemokines including IL-4, IL-10, IL-13, and CXCL7. Treating CAFs with exogenous ELR+ chemokines (CXCL1 and CXCL8) exhibited a similar phenotype. Furthermore, treating CAFs with exogenous ELR+ chemokines or PC cells conditioned media in the presence of pharmacological inhibitors of CXCR2 rescued this phenotype. In summary, we believe that ELR+ chemokine signaling through CXCR2 induces the inflammatory phenotype of CAFs that promotes immunosuppression, tumor growth, and tumor progression of PC. Citation Format: Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Surinder Batra, Rakesh Singh. CXCR2 signaling in pancreatic cancer induces a tumor supporting inflammatory phenotype of the cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5085.

Pancreatic cancer (PC) is a grotesque disease featured by an inflamed complex tumor microenvironment (TME) that contribute to advancing tumor progressing. Oncogenic K-ras, the most common mutation in PC, amplifies inflammation within TME through overexpression of multiple immune factors such as CXCR2 and its ligands. K-ras induced overexpression of CXCR2 axis, made CXCR2 seem intuitively a good target for PC treatment. In KC, a spontaneous PC mouse model with oncogenic K-ras, genetic ablation of CXCR2 caused anti-tumor events such as increased apoptosis and decreased angiogenesis; and also presented pro-tumorigenic events; most notably was the increased fibrosis and metastasis to the liver. These observations have shed light on the possible role the CXCR2 on the other TME component especially pancreatic stellate cells (PSCs). The aim of this study is to determine the differential effect of K-ras status and CXCR2 chemokines expression in the PC cells on their interaction with PSCs. Unidirectional segregated co-culture studies were conducted using conditioned media (CM). CM collected from PSCs were used to grow multiple PC cell lines, and CM obtained from different PC cell lines were used to culture PSCs. Cellular proliferation and gene expression were analyzed. Furthermore, PSCs proliferation potentials were assessed as those cells treated with exogenous CXCL1 and CXCL8, or PC cell lines derived-CM in the presence or absence of CXCR2 antagonists. Co-culture studies revealed that PSC-derived CM had increased proliferation of PC cell lines positive to oncogenic K-ras as well as increasing their expression of multiple chemokines such as CXCL1, CXCL5, and CCL2. On the other hand, PC cell lines with a wildtype K-ras were inhibited by PSC-derived CM treatment. PSCs proliferation potentials were decreased with CM derived from oncogenic K-ras positive PC cell lines and increased with CM derived from PC cell lines with wildtype K-ras. Furthermore, CM from oncogenic K-ras PC cell lines has increased the expression of multiple pro-tumorigenic genes in the PSCs including cytokines such as IL-10, IL-4 and IL-13, and chemokines such as CXCL2 and CXCL7. Also, treating PSCs with exogenous CXCL1 and CXCL8 exhibited a similar proliferation inhibition to that observed with oncogenic K-ras PC derived CM treatment. CXCR2 antagonists have shown rescued inhibition or enhanced proliferation of PSCs when incorporated with CXCR2 chemokine or PC cell line derived CM treatment. These observations suggest that K-ras status of PC dictates their interaction with PSCs within the TME. We have demonstrated that oncogenic K-ras through increased production of CXCR2 chemokine would dampen PSCs proliferation and further orient them to support tumor progression by increased expression of pro-tumorigenic genes. Besides, we observed that CXCR2 inhibition in PSCs would increase their proliferation which may further their fibrogenic activity. Citation Format: Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Surinder K. Batra, Rakesh K. Singh. CXCR2 acts as a checkpoint regulator of the pancreatic stellate cells activity within pancreatic cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 942. doi:10.1158/1538-7445.AM2017-942

Pancreatic cancer (PC) is one of the deadliest forms of cancers. In spite of recent advances, minimal progress has been made in our understanding of PC progression, metastasis, and treatment of PC patients with advanced disease. Striking similarity between the process of cancer metastasis and guidance of neuronal cells to their target sites has generated interest in pharmacologically targeting guidance cue molecules for treatment of metastasis. Among the guidance cue family members, Semaphorin5A (SEMA5A), was found to be involved in organ-specific homing during PC metastasis. With an interest to delineate the function of SEMA5A in PC, we generated SEMA5A knockdown in metastatic PC cell lines. Knock down of SEMA5A expression resulted in loss of cellular differentiation and epithelial phenotype with higher motility as compared to vector control cells. This observation is in accordance with our previous finding that SEMA5A expression was higher in well-differentiated tumors in comparison with undifferentiated pancreatic tumors. Loss of SEMA5A increased TGF-β2 production, decreased expression of E-cadherin, and nuclear translocation of β-catenin. Furthermore, we observed higher Wnt activity and increased expression of the transcription factor SNAIL in SEMA5A knockdown cells in comparison with vector control cells. Higher Wnt signaling along with increased TGF-β2 production explains the loss of differentiation and epithelial markers. Moreover, we observed that non canonical TGF-β2/AKT mediated inhibition of GSK3-β is responsible for increased stability of β-catenin and SNAIL, thereby activating Wnt signaling in SEMA5A knockdown cells. Our observations demonstrate that SEMA5A has a potential role in maintaining the epithelial phenotype in PC cells by keeping cross-talk between Wnt and TGF-β signaling under check. Citation Format: Sugandha Saxena, Abhilasha Purohit, Surinder K. Batra, Rakesh K. Singh. Semaphorin 5A preserves epithelial phenotype in pancreatic cancer cells by regulating cross-talk between Wnt and TGF-β signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5797. doi:10.1158/1538-7445.AM2017-5797

The ultraprecise wiring of neurons banks on the instructions provided by guidance cue proteins that steer them to their appropriate target tissue during neuronal development. Semaphorins are one such family of proteins. Semaphorins are known to play major physiological roles during the development of various organs including the nervous, cardiovascular, and immune systems. Their role in different pathologies including cancer remains an intense area of investigation. This review focuses on a novel member of this family of proteins, semaphorin 5A, which is much less explored in comparison to its other affiliates. Recent reports suggest that semaphorins play important roles in the pathology of cancer by affecting angiogenesis, tumor growth and metastasis. We will firstly give a general overview of the semaphorin family and its receptors. Next, we discuss their roles in cellular movements and how that makes them a connecting link between the nervous system and cancer. Finally, we focus our discussion on semaphorin 5A to summarize the prevailing knowledge for this molecule in developmental biology and carcinogenesis.

Pancreatic cancer (PC) is a highly aggressive disease and in general has metastasized to distant organs by the time of its clinical diagnosis. Poor five-year survival rate of 7% with an average survival time of 5-8 months highlight the gravity of the situation and necessitate a better understanding of the molecules and mechanisms that lead to metastatic dissemination in PC. One of the key processes that lead to metastasis is the gain of cellular migration ability. Previous reports from our laboratory have identified semaphorin5A (SEMA5A), an axon guidance cue molecule regulating cellular migration, as a putative cell adhesion molecule which is involved in organ-specific homing during PC metastasis. Also, differential expression of SEMA5A was observed in cells derived from metastatic sites in comparison to those arising from primary tumors. On the basis of the these findings, we hypothesize that SEMA5A regulates epithelial to mesenchymal changes necessary to drive cellular migration and further leads to establishment of these cells at secondary sites. We utilized various cellular models such as the L3.3 and L3.6pl cell lines with different metastatic potential, cell lines derived from metastatic sites (Capan-1 and CD18/HPAF) with stable knock down of SEMA5A, and cell lines derived from primary tumors (Panc1) with overexpression of SEMA5A. We evaluated epithelial and mesenchymal markers, in vitro cellular migration and the metastatic potential of these cells. We observed down regulation of E-cadherin expression and re-localization of beta-catenin from the membrane to the nucleus in SEMA5A knockdown cells. Similarly, overexpression of SEMA5A in Panc1 cells resulted in higher E-cadherin expression. We also observed higher SEMA5A and E-cadherin expression in the highly metastatic L3.6pl cells in comparison with L3.3 cells. Furthermore, knock-down SEMA5A in Capan-1 cells resulted in enhanced in vitro cellular migration and higher metastasis when control or SEMA5A knockdown cells were orthotopically implanted in nude mice. Our observations suggest that SEMA5A plays a bi-functional role in mediating epithelial to mesenchymal transition at primary site while regulating mesenchymal to epithelial transition and establishment at secondary sites. Citation Format: Sugandha Saxena, Abhilasha Purohit, Michelle Varney, Surinder K. Batra, Rakesh K. Singh. Dual role of semaphorin5A in maintaining epithelial and mesenchymal phenotype in metastatic pancreatic cancer. [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 1683.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor survival. Somatic KRAS mutations are present in almost 95% of PDAC patients and are known to a play pivotal role in PDAC genesis and progression. The G12D substitution is the predominant form detected in PDAC. By constitutively activating diverse downstream signaling pathways (like ERK/MEK) KRAS mutations are known to drive autocrine growth transformation in cancer cells. However, the lack of therapeutics for direct targeting of mutant KRAS necessitate a proper understanding of its downstream mechanisms, which may aid in the development of specific therapeutics. CXCR2 is a receptor for the ELR-positive CXC subfamily of chemokines, which are established regulators of leukocyte chemotaxis during inflammation. Aberrant expression of CXCR2 and its ligands has been reported in various cancers including PDAC. Both autocrine and paracrine events mediated by CXCR2 signaling lead to the manifestation of tumor growth, metastasis and angiogenesis. RAS is a known regulator of cytokine production including CXC chemokines. Whilst much of the work done formerly has indicated a paracrine role for KRAS-induced production of CXC chemokines in the biology of PDAC the precise autocrine effects of KRAS induced CXCR2 signaling remain uncertain. We examined the tumors derived from PDX-cre-LSL-kras(G12D) mice and observed a specific expression of Cxcr2 in the cytokeratin positive duct cells. Based on this preliminary observation the objective of our current study is to evaluate the autocrine role of CXCR2 signaling in the KRAS(G12D) -induced PDAC development. Two human cell line models having exogenously expressed KRAS(G12D): hTERT-HPNE-/KRAS(G12D) and hTERT-HPNE-E6-E7-st/KRAS(G12D) were used for our in vitro studies. CXCR2 inhibition achieved using antagonists (SCH-527123 and SCH-479833) significantly (P ≤ 0.05) suppressed the in vitro proliferation, anchorage independent growth and migration potential of KRAS(G12D) bearing cells in a dose dependent manner. We generated stable clones of hTERT-HPNE-E6-E7-st/KRAS(G12D) cells having knock-down for CXCR2. Complementing our observations from CXCR2 inhibition the hTERT-HPNE-E6-E7-st/KRAS(G12D)-shCXCR2 cells demonstrated a significant (P ≤ 0.05) decrease in in vitro cell growth, colony formation and migration compared to the control cells. Additionally, both hTERT-HPNE-E6-E7-st/KRAS(G12D)-shCXCR2 cells and hTERT-HPNE-E6-E7-st/KRAS(G12D) cells treated with SCH-527123 demonstrated inhibited phosphorylation of Erk downstream of KRAS. We next extended our investigation in vivo. The hTERT-HPNE-E6-E7-st/KRAS(G12D)-shCXCR2 cells showed reduced tumor growth compared to the control cells in subcutaneous implants. To conclude, our data provides evidence for the role of the CXCR2 signaling axis in regulation of KRAS(G12D) induced autocrine cellular transformation in PDAC. Citation Format: Abhilasha Purohit, Michelle Varney, Satya Rachagani, Pavan Myneni, Michel Ouellette, Surinder K. Batra, Rakesh K. Singh. Targeting CXCR2 signaling inhibits KRAS(G12D) induced autocrine growth transformation by suppressing ERK activation in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2015-3178

Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the United States. The high mortality can be accredited to late clinical presentation and early metastasis. Somatic mutations in oncogene KRAS(G12D) are the most common and earliest events during development of PC, suggesting their crucial role in tumor initiation; however, the precise functional molecular mechanism(s) of action remains unclear. CXCR2, a CXC chemokine receptor, and its ligands have also been found to be important in regulating inflammation, angiogenesis, and metastasis in various cancers. The purpose of the current study was to test the functional role of CXCR2 signaling in KRAS(G12D)-induced PC development. Immunohistochemical analysis of tumors derived from PDX-cre-LSL-kras(G12D) mice demonstrated significantly higher expression of Cxcr2 and its ligands Cxcl1 and -3. Expression of Cxcr2 and its ligands Cxcl1 and -3 was observed as early as 10 weeks and increased consistently as the lesions advanced. We demonstrated the direct link between KRAS(G12D) mutation and CXCR2 signaling using cell line models hTERT-HPNE and hTERT-HPNE -E6-E7-st with or without exogenously expressed KRAS(G12D). Real time PCR, ELISA and western blot analysis of these cells revealed that KRAS (G12D) up-regulates the expression of CXCR2 and its ligands at both the transcriptional and protein levels. In order to delineate the significance of CXCR2 in KRAS(G12D) induced autocrine growth, we treated the cell line models with various concentrations of SCH-527123 (CXCR2 antagonist) or CXCR2 neutralizing antibody. Our data shows that KRAS(G12D) bearing cells show significantly (P ≤ 0.05) greater percent inhibition in cell growth in comparison to their control counterparts. Next to evaluate the role of CXCR2 autocrine signaling in KRAS(G12D) induced cell migration, we performed a scratch assay by treating the hTERT-HPNE -E6/E7-st- KRAS(G12D) cells with SCH-527123 for 24 hours. Our data demonstrated a significant inhibition of cell migration in CXCR2 antagonist treated cultures. Together, our data demonstrate that KRAS(G12D) mutation up regulates CXCR2 and its ligands, which, in turn, play key roles in the autocrine regulation of cellular growth and migration in PC. Note: This abstract was not presented at the meeting. Citation Format: Abhilasha Purohit, Michelle Varney, Satya Rachagani, Michel Ouellette, Surinder K. Batra, Rakesh K. Singh. CXCR2 signaling axis- a helping hand to KRAS(G12D) mutation in pancreatic cancer initiation. [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 4874. doi:10.1158/1538-7445.AM2014-4874

Pancreatic cancer (PC) is considered the fourth leading cause of cancer-related deaths in the United States. The high mortality-to-incidence ratio for the disease can be attributed to late clinical presentation. Thus, the detection of early markers of this disease remains a major challenge in the field of PC research. Somatic mutations in oncogene KRAS are the most common and earliest events during development of PC, suggesting their crucial role in tumor initiation; however, the precise functional molecular mechanism of action remains poorly understood. CXCR2, a CXC chemokine receptor, and its ligands have also been found to be important in regulating angiogenesis and metastasis. Their enhanced expression is observed as early as the precursor lesions of PC. The objective of this study was to investigate the role of CXCR2 signaling in the KRAS(G12D)-induced PC development. To validate that mutant KRAS(G12D) is essential for this increased secretome, studies were performed using two cell line models having both exogenous and endogenous expression of mutant KRAS(G12D): Htert-HPNE-E6-E7-st/KRAS(G12D), which is an immortalized pancreatic duct-derived cell line having exogenous expression of mutant KRAS (G12D), and CD18/HPAF KRAS (G12D) -scram/Sh, which is a tumor-derived cell line. The endogenous expression of mutant KRAS (G12D) was silenced by stable expression of Sh-RNA specific to the KRAS(G12D) allele. Real time PCR, ELISA and western blot analysis of these cells revealed that mutant KRAS (G12D) regulates the expression of CXCR2 and its ligands at both transcription and protein levels. We observed an enhanced expression of the CXCR2 and its ligands in Htert-HPNE-E6-E7-st-KRAS(G12D) cells. Interestingly, stable knock-down of KRAS in CD18/HPAF-shKRAS(G12D) cells upregulated CXCR2 expression, but inhibited the expression of its ligands. In addition, immunohistochemical analysis of PC lesions from Pdx-cre-LSL-KRAS (G12D) mice showed a higher expression of both receptor and its ligands as the lesions progress to advanced stages. Taken together, these results suggest that KRAS effector pathways are essential for modulating the expression of CXCR2 and its ligand, which, in turn, can mediate cellular- responses in PC growth and progression. Citation Format: Abhilasha Purohit, Michelle Varney, Satya Rachagani, Michelle Ouellete, Surinder K. Batra, Rakesh K. Singh. Upregulation of CXCR2 and its ligands in KRAS oncogene-induced initiation of 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 1431. doi:10.1158/1538-7445.AM2013-1431