Jason Castellanos

emerging targets in pancreatic cancer: epithelial- mesenchymal transition and cancer stem cells

Pancreatic ductal adenocarcinoma is one of the most aggressive solid malignancies and is characterized by poor response to current therapy and a dismal survival rate. Recent insights regarding the role of cancer stem cells (CSCs) and epithelial–mesenchymal transition (EMT) in tumorigenesis have brought further understanding to the field and have highlighted new therapeutic targets. CSCs are a distinct subset of cancer cells, with the ability to differentiate into other cell types and self-renew in order to fuel the maintenance of tumor amplification. Transition of a cancer cell from an EMT leads to increased migratory and invasive properties, and thus facilitates initiation of metastasis. EMT is regulated by a complex network of factors that includes cytokines, growth factors, aberrant signaling pathways, transcription factors, and the tumor microenvironment. There is emerging evidence that the EMT process may give rise to CSCs, or at least cells with stem cell-like properties. We review the key pathways involved in both of these processes, the biomarkers used to identify CSCs, and new therapeutic approaches targeting CSCs and EMT in pancreatic ductal adenocarcinoma.

Optical Imaging of Drug-Induced Metabolism Changes in Murine and Human Pancreatic Cancer Organoids Reveals Heterogeneous Drug Response.

Objectives Three-dimensional organoids derived from primary pancreatic ductal adenocarcinomas are an attractive platform for testing potential anticancer drugs on patient-specific tissue. Optical metabolic imaging (OMI) is a novel tool used to assess drug-induced changes in cellular metabolism, and its quantitative end point, the OMI index, is evaluated as a biomarker of drug response in pancreatic cancer organoids. Methods Optical metabolic imaging is used to assess both malignant cell and fibroblast drug response within primary murine and human pancreatic cancer organoids. Results Anticancer drugs induce significant reductions in the OMI index of murine and human pancreatic cancer organoids. Subpopulation analysis of OMI data revealed heterogeneous drug response and elucidated responding and nonresponding cell populations for a 7-day time course. Optical metabolic imaging index significantly correlates with immunofluorescence detection of cell proliferation and cell death. Conclusions Optical metabolic imaging of primary pancreatic ductal adenocarcinoma organoids is highly sensitive to drug-induced metabolic changes, provides a nondestructive method for monitoring dynamic drug response, and presents a novel platform for patient-specific drug testing and drug development.

Adiponectin receptor agonists inhibit leptin induced pSTAT3 and in vivo pancreatic tumor growth

Obesity is a significant risk factor for pancreatic cancer, harboring a chronic inflammatory condition characterized by dysregulation of the adipokines, leptin and adiponectin, that in turn alter oncogenic signaling pathways. We and others have shown that leptin promotes the proliferation and an invasive potential of pancreatic cancer cells through STAT3 mediated signaling. However, the role of adiponectin on the tumorigenicity of pancreatic cancer has not been elucidated. Adiponectin represents an important negative regulator of cytokines, which acts through two receptors, ADIPOR1 and ADIPOR2, to elicit pro-apoptotic, anti-inflammatory, and anti-angiogenic responses. We show that the level and expression of both adiponectin receptors are decreased in pancreatic tumors relative to normal pancreatic tissue. In vitro stimulation with adiponectin or a small molecule adiponectin receptor agonist, AdipoRon, increases apoptosis while inhibiting pancreatic cancer cell proliferation, colony formation, and anchorage independent growth. In addition, adiponectin receptor agonism inhibits leptin mediated STAT3 activation. In vivo, treatment of mice with AdipoRon inhibits orthotopic pancreatic tumor growth. These results demonstrate that adiponectin receptor activation is a key regulator of pancreatic cancer growth and AdipoRon provides a rational agent for the development of novel therapeutic strategies for pancreatic cancer.

Abstract 5259: Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related mortality in the United States. Most patients present with an advanced disease and the majority die within five years, many surviving less than six months. Cytotoxic chemotherapy including Gemcitabine (Gem), FOLFIRINOX, nab-paclitaxel offer modest improvement in survival, albeit at the cost of increased side effects and unwanted toxicities. Therefore, developing novel chemotherapeutic agents for PDAC treatment is critical to improve survival. Ellagic acid/ellatitannins are abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin A (UA). Oral administration of UA has shown to be highly bioavailable and non-toxic. UA inhibits multiple kinases that are known to be involved in PDAC progression and metastasis. Therefore, we hypothesized that UA would elicit potent anti-cancer therapeutic potential in PDAC. The effect of UA on kinase activity was assessed . Inhibition of AKT (downstream of PI3K/PDK1), p70 S6 Kinase (PS6K) and STAT3 activation was quantified in PDAC cells treated with UA in dose-dependent manner. The mechanism of action was validated for UA’s activity on PI3K/PDK1, PS6K and STAT3 activation using immunoblot analysis. MiaPaCa2 cells were treated with specific inhibitors for either AKT (MK2206) or STAT3 (AZD1480) and analyzed for tumorigenicity. UA treated PDAC cells were analyzed for cell proliferation, cell invasion and colony formation. Cell cycle analysis and cell apoptosis were measured by flow cytometry. To test the efficacy of UA in vivo, cells were implanted subcutaneously in athymic nude mice. The animals received UA daily and tumor volume was measured for 5 weeks. Next, we assessed tumor growth and overall survival (OS) in PKT (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) mice, an aggressive genetically engineered PDAC mouse model, in response to UA and/or Gem treatment. Tissues from the xenografts and PKT mice treated with vehicle or UA were analyzed for cell proliferation (Ki67) and apoptosis (cleaved Caspase 3) by immunohistochemistry. High expression levels of activated STAT3 or AKT correlate with decreased survival in PDAC. UA treated MiaPaCa2 cells showed significant dose-dependent increase in apoptosis and decrease in anchorage-independent growth. UA inhibited AKT, PS6K and STAT3 signaling. As a single agent, UA effectively reduced in vivo PDAC tumor growth. Immunohistochemistry of UA treated tissues from tumor xenografts and PKT mice showed inhibition of Ki67 positive tumor cells and increased cleaved caspase 3 staining. PKT mice treated with UA showed a decrease in tumor size and an increased OS when compared to vehicle or Gem treated mice alone. These findings show that UA is a novel inhibitor/modulator/regulator for multiple signaling pathways in PDAC. These results suggest UA has potential for pre-clinical development in pancreatic cancer. Citation Format: Supriya Srinivasan, Venkatakrishna Jala, Kumar Honnenahally, Jason Castellanos, Praveen Kumar Vermula, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli. Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 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 5259. doi:10.1158/1538-7445.AM2017-5259

Abstract A46: AdipoRon suppresses ERK and STAT3 to inhibit pancreatic cancer growth

Introduction: The increasing incidence of pancreatic cancer is associated with a rising prevalence of obesity, a documented risk factor for the disease. Obesity harbors a systemic chronic inflammatory disorder characterized by increased production and secretion of pro-inflammatory adipokines leptin, TNF-α, and IL-6; while exhibiting a decrease in the anti-inflammatory adipokine; adiponectin. Dysregulation of these factors is thought to be a key mechanism of obesity associated cancers, contributing to increased activation of mitogenic pathways including PI3K and MAPK. Adiponectin represents an important negative regulator of leptin, TNF-α and IL-6. We previously demonstrated that adiponectin inhibits pancreatic cancer proliferation and tumor growth, however, the molecular mechanisms by which adiponectin regulates these processes are unknown. We hypothesize that Adiponectin Receptor (AdipoR) agonists elicit anti-tumor effects through suppression of RAS-MAPK mediated pathways and its downstream signaling components in pancreatic cancer progression. Experimental Procedures: The anti-tumor effects of AdipoRon, a novel small molecule agonist of the AdipoR, were assessed in vitro on human (MiaPaca-2 and Panc-1) and murine (P-4313 and K8484) pancreatic cancer cell lines. Cells were treated with AdipoRon in a dose-dependent manner and then assayed for cellular proliferation, apoptosis, colony formation and anchorage-independent growth. The effect of AdipoRon on activation of key RAS-MAPK signaling regulators was investigated by immunoblot analysis. To determine whether AdipoRon could inhibit the effects of obesity associated pro-tumorigenic cytokines, human and mouse pancreatic cancer cells were exposed to plasma collected from obese mice or specifically with recombinant cytokines. To determine whether AdipoRon could inhibit tumor growth in vivo, mice were orthotopically injected in the pancreas with the murine KrasG12D mutant P-4313 cell line. Tumors were allowed to establish for two weeks and treated with either vehicle or AdipoRon. Tumor size and number of Ki67 positive cells were assessed. Results: Compared to vehicle treatment, in vitro assessment confirmed that AdipoRon was highly effective at inhibiting cell proliferation, increasing apoptosis and preventing colony formation for all pancreatic cell lines tested. Anchorage independent growth was drastically reduced for both Panc1 (3.8 fold) and MiaPaca-2 (5.1 fold) cell lines in the presence of AdipoRon. Treatment of both murine and human pancreatic cancer cell lines with AdipoRon caused a significant dose dependent decrease in pSTAT3, pERK1, and pERK2 with a simultaneous increase in pAMPK. Importantly, AdipoRon completely antagonized the stimulatory effects of obese plasma or recombinant IL-6 on the activation of pSTAT3. Administration of AdipoRon to P-4313 orthotopic pancreatic tumor bearing mice resulted in four fold decrease in tumor size and a 50% reduction in tumor cell proliferation. Conclusions: AdipoRon, an adiponectin receptor agonist, suppresses KRAS signaling mediators ERK and STAT3 while simultaneously increasing AMPK resulting in inhibition of pancreatic cancer proliferation and tumor growth. Targeting of adiponectin receptors can provide a viable therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Fanuel Messaggio, Alisha M. Mendonsa, Jason A. Castellanos, Casey Roberts, Nagaraj S. Nagathihalli, Nipun B. Merchant, Lee D. Gorden, Michael N. VanSaun.{Authors}. AdipoRon suppresses ERK and STAT3 to inhibit pancreatic cancer growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A46.

Abstract B71: Resistance to MEK inhibition in pancreatic cancer is associated with amphiregulin mediated EGFR-STAT3 activation

Introduction: Mutations in the KRAS oncogene occur in the majority of pancreatic ductal adenocarcinomas (PDAC), resulting in aberrant activation of the MAPK (RAS-RAF-MEK-ERK) pathway, driving malignant progression. Targeting KRAS has remained an elusive goal. Therefore, efforts have focused on targeting downstream effectors of RAS. The clinical efficacy of MEK inhibitors in other malignancies confirms that targeting the MAPK pathway has therapeutic potential. Unfortunately, clinical trials of MAPK-directed therapies have been unsuccessful in PDAC. Here, we report a novel mechanism of resistance to MAPK-directed therapies, which is associated with amphiregulin (AREG)-mediated activation of EGFR-STAT3 signaling. Experimental procedure: The effects of MEK inhibition on the phosphorylation of multiple signaling proteins and EGF family ligands was assessed. Activation of MAPK and STAT3 was quantified in human pancreatic tissues, and then correlated with overall survival (OS). Cell lysates from mouse PanIN lines derived from the LSL-KrasG12D/+;Pdx1Cre/+ and PDA line from LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ mouse models of PDAC were immunoblotted for activated MAPK and STAT3 expression. AREG release was measured in the conditioned media of PDAC cells treated with MEK, EGFR, STAT3, and/or TACE inhibitors and TACE siRNA knock down cells treated with MEK, EGFR, or STAT3 inhibitors. Cell invasion, colony formation, spheroid formation, metabolic activity, cell cycle analysis, and apoptosis assays were performed with human PDAC cells treated with inhibitors for TACE, MEK, EGFR and STAT3 in combinations. PDAC xenografts and patient derived xenografts (PDXs) were treated either with vehicle, the MEK inhibitor AZD6244, the STAT3 inhibitor AZD1480 or both drugs and analyzed for spheroid growth. PKT mice (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) were treated with AZD6244 and AZD1480 and assessed for OS. Tissues from the xenografts and PKT mice were analyzed for cell proliferation and apoptosis markers. Plasma from the PKT mice treated with the drugs and their combinations was analyzed released AREG by ELISA. Results: Targeting the MAPK pathway is a novel approach to inhibit KRAS mutant tumor progression and improve therapeutic response in PDAC. However, our results show that MAPK inhibition leads to activation of TACE and EGFR and subsequent activation of STAT3 signaling. Combined inhibition of MEK/STAT3 or MEK/EGFR resulted in sustained blockade of MEK, EGFR and STAT3 signaling, decreased cell invasion, colony formation, reduced spheroid size, and metabolic activity in vitro. Growth of flank PDAC xenografts and human PDX tumors in vivo were significantly decreased with combined MEK and STAT3 inhibition when compared to vehicle or monotherapy treatment. OS in PKT mice was extended to a median of 85 days with combined MEK and STAT3 inhibition vs. 52 days for vehicle treated mice (p Conclusions: Our study provides insights into the molecular mechanisms that help explain the heterogeneous response and therapeutic resistance of PDAC to MAPK pathway inhibition and provide a strong rationale that AREG mediated EGFR-STAT3 pathway activation is a major resistance mechanism that impairs the efficacy of MEK inhibitors. Citation Format: Nagaraj Nagathihalli, Jason Castellanos, Chanjuan Shi, Casey Roberts, Michael VanSaun, Nipun Merchant.{Authors}. Resistance to MEK inhibition in pancreatic cancer is associated with amphiregulin mediated EGFR-STAT3 activation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B71.

Abstract 2618: Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in pancreatic cancer

Introduction: Cyclic AMP (cAMP) response element binding (CREB) overexpression in pancreatic ductal adenocarcinoma (PDAC) is associated with poor outcome, however, the mechanism(s) driving CREB overexpression in PDAC are not fully understood. We investigated the association of CREB activation with oncogenic KRAS, MEK-ERK and AKT signaling pathways. Experimental procedure: Mouse lines derived from the Ptf1aCre/+; LSL-KrasG12D/+ (K518), Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT), LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA) mouse models of PDAC, human immortalized pancreatic ductal epithelial lines (HPDE6-E6E7 or H6c7, HPNE E6/E7 and HPNE E6/E7/KRAS) were immunoblotted for phospho-CREB (pCREB) and total CREB expression. KRAS mutant PDAC cell lines and CREB shRNA flank xenografts (PKT GEMM and nude mice) were treated with CREB (ICG-001), MEK (AZD6244) and AKT (MK-2206) inhibitors. The effects on downstream signaling targets were interrogated with cell cycle, apoptosis, survival and anchorage-independent growth analysis. Human CREB shRNA cells were treated with AZD6244 and MK-2206 to confirm the molecular mechanism of the CREB inhibition together with MEK and AKT. Results: The expression of pCREB was higher in cells with KRAS mutation. MEK inhibition resulted in activation of AKT, while combined inhibition of CREB and MEK prevented AKT reactivation. Treatment with the combination of MEK and CREB inhibitors significantly decreased tumorigenic potential and increased cell apoptosis. Combined MEK, AKT and CREB inhibition synergistically enhanced these effects further, more so in KRAS mutant cell lines. To explore the relationship of CREB, MEK and AKT signaling in vivo, PKT mice were treated with their respective inhibitors individually and in combination. Either CREB/MEK or CREB/AKT two drug combinations significantly extended the median survival compared with individual agents. Lysates from MEK and AKT inhibited tumors showed decreased phosphorylation of CREB, confirming that CREB is activated through both, MEK and AKT signaling in vivo. Conclusions: Our study demonstrates that oncogenic KRAS activation enhances the expression of CREB through MEK and AKT signaling. CREB inhibition results in increased sensitivity to MEK and AKT targeted therapy in KRAS mutant PDACs. Citation Format: Jason Castellanos, Supriya Srinivasan, Kumar Honnenahally, Chanjuan Shi, Michael VanSaun, David Robbins, Nipun Merchant, Nagaraj Nagathihalli. Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in 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 2618.

Abstract 924: GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Nicotine and nitrosamine exposure from smoking causes pancreatic cell injury and contributes to a cascade of oncogenic events that may be contributing to the rising rate of pancreatic cancer (PDAC). Cytokines activate kinases and transcription factors including cyclic AMP response element binding (CREB) protein. CREB activation through phosphorylation regulates diverse cellular responses. We studied whether granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent phosphorylated CREB plays a role in smoking-induced pathogenesis of PDAC. Experimental procedure: Human tissue microarray analysis was performed to determine the significance of pCREB expression amongst smokers and non-smokers. Total RNA extracted from immortalized human pancreatic ductal cell (H6c7) and pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) were treated with tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and RNA sequencing (RNA-seq) was performed. Network, pathway and functional analyses of the transcriptome were conducted. To analyze the signaling pathway involved in NNK-induced tumorigenicity, we performed a phospho-kinase antibody array and cytokine antibody array on NNK treated H6c7 and PanIN cells or conditioned media (CM) protein. Western blot and ELISA were used to validate the array data findings. NNK-induced activation of GM-CSF, G-CSF and IL-6 were blocked using monoclonal antibodies. PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice and nude mice with MiaPaCa2 flank xenografts were treated with MEK inhibition (AZD6244) with or without NNK/in vivo smoking. Tumors from these mice were immunoblotted for phosphorylation of MEK1/2, MSK1/2, RSK and CREB. PDAC cells with CREB siRNA or human rGM-CSF were treated with NNK and analyzed for in vitro functional assays and EMT characteristics. Results: Expression of pCREB was significantly higher (p<0.001) in smokers when compared to non-smokers. Overall survival of smokers with high pCREB expression in their primary tumor was associated with significantly decreased median survival when compared to non-smokers with low pCREB expression. Exposure of cells to NNK resulted in phosphorylation of CREB, c-Jun and β-catenin, and release of GM-CSF, G-CSF and IL-6. RNA-seq analysis confirmed activation of MEK/ERK signaling. Studies were performed to elucidate the possible regulatory mechanism behind NNK mediated induction of GM-CSF and its downstream signaling. Importantly, blocking GM-CSF inhibited NNK-induced phosphorylation of CREB and it was mediated through MEK signaling. The in vivo and in vitro tumorigenicity assays showed an increase in the tumorigenic potential and EMT of NNK treated PDAC cells. Conclusions: Our study demonstrates that NNK induces pancreatic tumorigenesis through GM-CSF mediated activation of CREB. Citation Format: Jason Castellanos, Kumaraswamy Honnenahally, Chanjuan Shi, Nipun Merchant, Nagaraj Nagathihalli. GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis. [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 924. doi:10.1158/1538-7445.AM2015-924