NetrinG1+ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5ß1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5ß1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5ß1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.

Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

Fibroblasts Influence the Efficacy, Resistance, and Future Use of Vaccines and Immunotherapy in Cancer Treatment.

Tumors are composed of not only epithelial cells but also many other cell types that contribute to the tumor microenvironment (TME). Within this space, cancer-associated fibroblasts (CAFs) are a prominent cell type, and these cells are connected to an increase in tumor progression as well as alteration of the immune landscape present in and around the tumor. This is accomplished in part by their ability to alter the presence of both innate and adaptive immune cells as well as the release of various chemokines and cytokines, together leading to a more immunosuppressive TME. Furthermore, new research implicates CAFs as players in immunotherapy response in many different tumor types, typically by blunting their efficacy. Fibroblast activation protein (FAP) and transforming growth factor ß (TGF-ß), two major CAF proteins, are associated with the outcome of different immunotherapies and, additionally, have become new targets themselves for immune-based strategies directed at CAFs. This review will focus on CAFs and how they alter the immune landscape within tumors, how this affects response to current immunotherapy treatments, and how immune-based treatments are currently being harnessed to target the CAF population itself.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells.

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Anti-Cancer Tumor Cell Necrosis of Epithelial Ovarian Cancer Cell Lines Depends on High Expression of HDM-2 Protein in Their Membranes.

OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Does the Microbiome Affect the Outcome of Renal Transplantation?

The role of the human microbiome in health and disease is becoming increasingly apparent. Emerging evidence suggests that the microbiome is affected by solid organ transplantation. Kidney transplantation is the gold standard treatment for End-Stage Renal Disease (ESRD), the advanced stage of Chronic Kidney Disease (CKD). The question of how ESRD and transplantation affect the microbiome and vice versa includes how the microbiome is affected by increased concentrations of toxins such as urea and creatinine (which are elevated in ESRD), whether restoration of renal function following transplantation alters the composition of the microbiome, and the impact of lifelong administration of immunosuppressive drugs on the microbiome. Changes in microbiome composition and activity have been reported in ESRD and in therapeutic immunosuppression, but the effect on the outcome of transplantation is not well-understood. Here, we consider the current evidence that changes in kidney function and immunosuppression following transplantation influence the oral, gut, and urinary microbiomes in kidney transplant patients. The potential for changes in these microbiomes to lead to disease, systemic inflammation, or rejection of the organ itself is discussed, along with the possibility that restoration of kidney function might re-establish orthobiosis.

RASpecting the oncogene: New pathways to therapeutic advances.

RAS is the most commonly mutated driver of tumorigenesis, seen in about 30% of all cancer cases. There is a subset of tumors termed RAS-driven cancers in which RAS mutation or overactivation is evident, including as much as 95% in pancreatic and 50% in colon cancer. RAS is a family of small membrane bound GTPases that act as a signaling node to control both normal and cancer biology. Since the discovery of RAS' overall prominence in many tumor types and specifically in RAS-dependent cancers, it has been an obvious therapeutic target for drug development. However, RAS has proved a very elusive target, and after a few prominent RAS targeted drugs failed in clinical trials after decades of research, RAS was termed "undruggable" and research in this field was greatly hampered. An increase in knowledge about basic RAS biology has led to a resurgence in the generation of novel therapeutics targeting RAS signaling utilizing various and distinct approaches. These new drugs target RAS activation directly, block downstream signaling effectors and inhibit proper post-translational processing and trafficking/recycling of RAS. This review will cover how these new drugs were developed and how they have fared in preclinical and early phase clinical trials.

Inhibition of CX3CR1 reduces cell motility and viability in pancreatic adenocarcinoma epithelial cells.

Increased expression of the chemokine CX3CL1 and its sole receptor, CX3CR1 have been correlated with poor pancreatic cancer patient survival and time to recurrence, as well as with pancreatic perineural invasion. We have previously shown that metastasis of prostate and breast cancer is in part driven by CX3CL1, and have developed small molecule inhibitors against the CX3CR1 receptor that diminish metastatic burden. Here we ask if inhibition of this chemokine receptor affects the phenotype of PDAC tumor cells. Our findings demonstrate that motility, invasion, and contact-independent growth of PDAC cells all increase following CX3CL1 exposure, and that antagonism of CX3CR1 by the inhibitor JMS-17-2 reduces each of these phenotypes and correlates with a downregulation of AKT phosphorylation. These data suggest that PDAC tumor cell migration and growth, elements critical in metastatic progression, may susceptible to pharmacologic intervention.

CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype.

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(-/-)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(-/-)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes.Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696-707. ©2017 AACR.

Oncogenic Ras pushes (and pulls) cell cycle progression through ERK activation.

The Ras-Raf-MEK-ERK signaling cascade is capable of channeling a wide variety of extracellular signals into control of cell proliferation, differentiation, senescence, and death. Because aberrant regulation at all steps of this signaling axis is observed in cancer, it remains an area of great interest in the field of tumor biology. Here we present evidence of the intricate and delicate levels of control of this pathway as it pertains to cell cycle regulation and illustrate how this control is not simply a rheostat.

Analyzing Ras-associated cell proliferation signaling.

Ras-dependent signaling is an important regulator of cell cycle progression, proliferation, senescence, and apoptosis. Several of the downstream effectors of Ras play dual roles in each of these processes. Under one set of conditions, they promote cell cycle progression and proliferation; yet, in a different paradigm, they drive cell cycle arrest and apoptosis. Furthermore, there is cross talk between certain downstream effectors of Ras including the PI3K-AKT and Raf-MEK-ERK pathways. Here we describe a series of experiments used to dissect the effect of different Ras-dependent signaling pathways on cell cycle progression, proliferation, senescence, and apoptosis. Furthermore, we highlight the importance of consistent growth conditions of cells in culture when studying Ras-dependent signaling as we show that the activation of downstream effectors of Ras changes with the confluency at which the cells are grown.

Epicatechin-rich cocoa polyphenol inhibits Kras-activated pancreatic ductal carcinoma cell growth in vitro and in a mouse model.

Activated Kras gene coupled with activation of Akt and nuclear factor-kappa B (NF-κB) triggers the development of pancreatic intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used; however, there are not relevant cell-based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras-driven cell-based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA-grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa-based foods. The liquid chromatography (LC)-mass spectrometry (MS)/MS data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)-bound Ras protein, (iii) Akt phosphorylation and (iv) NF-κB transcriptional activity of premalignant and malignant Kras-activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras-PDE cell-originated tumors in a xenograft mouse model. LC-MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (i) Kras-driven cell-based model is an excellent model for testing intervention agents and (ii) CP is a promising chemopreventive agent for inhibiting PDAC development.

TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling.

BACKGROUND: TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallion's proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. RESULTS: To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. CONCLUSION: These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients.

K-Ras promotes angiogenesis mediated by immortalized human pancreatic epithelial cells through mitogen-activated protein kinase signaling pathways.

Activating point mutations in the K-Ras oncogene are among the most common genetic alterations in pancreatic cancer, occurring early in the progression of the disease. However, the function of mutant K-Ras activity in tumor angiogenesis remains poorly understood. Using human pancreatic duct epithelial (HPDE) and K-Ras4B(G12V)-transformed HPDE (HPDE-KRas) cells, we show that activated K-Ras significantly enhanced the production of angiogenic factors including CXC chemokines and vascular endothelial growth factor (VEGF). Western blot analysis revealed that K-Ras activation promoted the phosphorylation of Raf/mitogen-activated protein kinase kinase-1/2 (MEK1/2) and expression of c-Jun. MEK1/2 inhibitors, U0126 and PD98059, significantly inhibited the secretion of both CXC chemokines and VEGF, whereas the c-Jun NH(2)-terminal kinase inhibitor SP600125 abrogated only CXC chemokine production. To further elucidate the biological functions of oncogenic K-Ras in promoting angiogenesis, we did in vitro invasion and tube formation assays using human umbilical vein endothelial cells (HUVEC). HUVEC cocultured with HPDE-KRas showed significantly enhanced invasiveness and tube formation as compared with either control (without coculture) or coculture with HPDE. Moreover, SB225002 (a CXCR2 inhibitor) and 2C3 (an anti-VEGF monoclonal antibody) either alone or in a cooperative manner significantly reduced the degree of both Ras-dependent HUVEC invasiveness and tube formation. Similar results were obtained using another pair of immortalized human pancreatic duct-derived cells, E6/E7/st and its oncogenic K-Ras variant, E6/E7/Ras/st. Taken together, our results suggest that angiogenesis is initiated by paracrine epithelial secretion of CXC chemokines and VEGF downstream of activated oncogenic K-Ras, and that this vascular maturation is in part dependent on MEK1/2 and c-Jun signaling.

Ras-driven transformation of human nestin-positive pancreatic epithelial cells.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the means by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this signaling biology. This chapter describes the establishment and characterization of telomerase-immortalized human pancreatic duct-derived cells to study mechanisms of Ras growth transformation. An important strength of this model system is the ability of mutationally activated K-Ras to cause potent growth transformation in vitro and in vivo. We have utilized this cell system to evaluate the antitumor activity of small molecule inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade. This model will be useful for genetic and pharmacologic dissection of the contribution of downstream effector signaling in Ras-dependent growth transformation.

K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells.

BACKGROUND: The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. RESULTS: We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. CONCLUSIONS: RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

Genetic and pharmacologic dissection of Ras effector utilization in oncogenesis.

Ras proteins function as signaling nodes that are activated by diverse extracellular stimuli. Equally complex for this family of molecular switches is the multitude of downstream effectors and the pathways that they traverse to translate extracellular signals into a spectrum of cellular consequences. To better understand the individual and collective roles of these effector signaling networks, both genetic and pharmacological tools have been developed. By either stimulating or ablating specific components in a cascade downstream of Ras activation, one can gain insight into the specific signaling underlying a particular Ras phenotype, for example, malignant transformation. In this chapter, we describe the use of activating and dominant-negative mutations, both artificial and naturally occurring, of Ras and its effectors, as well as pharmacological inhibitors used to probe the effector pathways (Raf kinase, phosphoinositol 3-kinase, Tiam1, phospholipase C epsilon, and RalGEF) implicated in Ras-mediated oncogenesis.