Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.

Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.

Receptor tyrosine kinases (RTKs) such as the fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor are overexpressed in a variety of cancers. In addition to overexpression, the FGFRs are found mutated in some cancers. The Src homology 2 domaincontaining phosphotyrosine phosphatase (SHP2) is a critical mediator of RTK signaling, but its role in oncogenic RTK-induced cell transformation and cancer development is largely unknown. In the current report, we demonstrate that constitutively activated FGFR3 (K/E-FR3) transforms NIH-3T3 cells, and that SHP2 is a critical mediator of this transformation. Infection of K/E-FR3-transformed 3T3 cells with a retrovirus carrying a dominant-negative mutant of SHP2 (C/S-SHP2) retarded cell growth, reversed the transformation phenotype and inhibited focus-forming ability. Furthermore, treatment of K/E-FR3-transformed NIH-3T3 cells with PD98059 or LY294002, specific inhibitors of MEK and PI3K, respectively, inhibited focus formation. Biochemical analysis showed that K/E-FR3 activates the Ras-ERK and the PI3K signaling pathways, and that the C/S SHP2 mutant suppressed this effect via competitive displacement of interaction of the endogenous SHP2 with FRS2. However, the C/S SHP2 protein did not show any effect on receptor autophosphorylation, FRS2 tyrosine phosphorylation or interaction of Grb2 with K/E-FR3 or FRS2. Together, the results show that K/E-FR3 is transforming and that the Ras-ERK and the PI3K-Akt signaling pathways, which are positively regulated by SHP2, are important for K/E-FR3-induced transformation.

Pancreatic cancer is one of the deadliest human malignancies. A striking feature of pancreatic cancer is that activating Kras mutations are found in ∼90% of cases. However, apart from a restricted population of cells expressing pancreatic and duodenal homeobox 1 (PDX1), most pancreatic cells are refractory to Krasdriven transformation. In the present study, we sought to determine which subsets of PDX1+ cells may be responsible for tumor growth. Using the Lox-Stop-Lox-KrasG12D genetic mouse model of pancreatic carcinogenesis, we isolated a population of KrasG12D-expressing PDX1+ cells with an inherent capacity to metastasize. This population of cells bears the surface phenotype of EpCAM+CD24+CD44+CD133-SCA1− and is closer in its properties to stem-like cells than to more mature cell types. We further demonstrate that the tumorigenic capacity of PDX1+ cells is limited, becoming progressively lost as the cells acquire a mature phenotype. These data are consistent with the hypothesis that the adult pancreas harbors a dormant progenitor cell population that is capable of initiating tumor growth under conditions of oncogenic stimulation. We present evidence that constitutive activation of the mitogen-activated protein kinase (MAPK/ERK) signaling and stabilization of the MYC protein are the two main driving forces behind the development of pancreatic cancer cells with stem-cell-like properties and high metastatic potential. Our results suggest that pancreatic cells bearing Kras mutation can be induced to differentiate into quasi-normal cells with suppressed tumorigenicity by selective inhibition of the MAPK/ERK/MYC signaling cascade.pancreatic ductal adenocarcinoma | cell of origin P ancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, exceptional resistance to all forms of anticancer treatment, and a high propensity for metastatic spread. Paradoxically, despite growing understanding of the genetic causes of PDAC, the mechanism and timing of cancer metastases, the main cause of deaths in pancreatic cancer patients, remain relatively unexplored. A striking feature of pancreatic cancer is that mutations in the Kras gene are found in ∼90% of cases. Genetic studies demonstrated that oncogenic Kras activity is required for the initiation and maintenance of both primary and metastatic pancreatic lesions (1-5). However, apart from a restricted population of pancreatic and duodenal homeobox 1 (PDX1)-expressing cells or subsets thereof, adult pancreatic cells are refractory to transformation by oncogenic Kras even in the context of loss of p53 or INK4A (CDKN2A) tumor suppressors (2-4). PDX1 is a transcription factor expressed in early pancreatic precursor cells, subsequently becoming restricted to insulin-producing islet cells (6). Some exocrine cells express PDX1, albeit at low levels (6). Unlike PDAC, islet cell tumors are relatively rare, and oncogenic Kras mutations are extremely rare in pancreatic islet cell neoplasms (7). Targeting expression of mutant Kras to acinar...

The recepteur d’origne nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, the macrophage stimulating protein, when expressed in epithelial cells. In the current study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON is mediated in part by Src-family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.

V-SEA is the transforming component of S13 Avian Erythroblastosis Retrovirus that causes erythroblastosis and anemia in chicken. Like all members in the family (MET, RON, SEA), its cytosolic domain possesses two tyrosine autophosphorylation sites in the tandemly arranged bidentate motif that serve as docking sites for SH2 domain-containing proteins. Here, we investigated phosphotyrosine-dependent activation of signaling pathways and their signi®cance in V-SEA-induced transformation and/or proliferation. We demonstrated that V-SEA activates the PI3K-Akt signaling pathway primarily in Y557-and secondarily in Y564-dependent manner. V-SEA was also shown to induce the tyrosine phosphorylation of the Gab2 protein, leading to PI3K association and thus providing an alternative route for PI3K activation. On the other hand, activation of the Ras-ERK pathway is primarily via Y564 and secondarily via Y557. A dominant-negative form of Ras inhibited V-SEA-induced ERK phosphorylation in concentration dependent manner suggesting the importance of the Grb2-Ras signaling axis in V-SEA-induced ERK activation. The biological signi®cance of activation of the PI3K-Akt and the Ras-ERK pathways in V-SEAinduced transformation was analysed in the V-SEA-RAT1 and V-SEA-3T3 cell lines by employing speci®c inhibitors, LY294002 and PD98059 compounds. Both the PD and LY compounds inhibited cell growth, but only the PD compound caused reversion of the transformed phenotype. In addition, both compounds inhibited focal colony formation by the transformants in soft agar. Thus, transformation by the V-SEA oncogene is a function of the concomitant activation of, at least, the PI3K-Akt and Ras-ERK signaling pathways that regulate cell growth and morphology.

No abstract