Marina Pasca di Magliano

Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis

Hedgehog signalling—an essential pathway during embryonic pancreatic development, the misregulation of which has been implicated in several forms of cancer—may also be an important mediator in human pancreatic carcinoma. Here we report that sonic hedgehog, a secreted hedgehog ligand, is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions: pancreatic intraepithelial neoplasia (PanIN). Pancreata of Pdx–Shh mice (in which Shh is misexpressed in the pancreatic endoderm) develop abnormal tubular structures, a phenocopy of human PanIN-1 and -2. Moreover, these PanIN-like lesions also contain mutations in K-ras and overexpress HER-2/neu, which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signalling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signalling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. These data suggest that this pathway may have an early and critical role in the genesis of this cancer, and that maintenance of hedgehog signalling is important for aberrant proliferation and tumorigenesis.

Hedgehog signalling in cancer formation and maintenance

The Hedgehog signalling pathway is essential for numerous processes during embryonic development. Members of this family of secreted proteins control cell proliferation, differentiation and tissue patterning in a dose-dependent manner. Although the overall activity of the pathway is diminished after embryogenesis, recent reports show that the pathway remains active in some adult tissues, including adult stem cells in the brain and skin. There is also evidence that uncontrolled activation of the pathway results in specific types of cancer.

Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice

Pancreatic cancer is almost invariably associated with mutations in the KRAS gene, most commonly KRASG12D, that result in a dominant-active form of the KRAS GTPase. However, how KRAS mutations promote pancreatic carcinogenesis is not fully understood, and whether oncogenic KRAS is required for the maintenance of pancreatic cancer has not been established. To address these questions, we generated two mouse models of pancreatic tumorigenesis: mice transgenic for inducible KrasG12D, which allows for inducible, pancreas-specific, and reversible expression of the oncogenic KrasG12D, with or without inactivation of one allele of the tumor suppressor gene p53. Here, we report that, early in tumorigenesis, induction of oncogenic KrasG12D reversibly altered normal epithelial differentiation following tissue damage, leading to precancerous lesions. Inactivation of KrasG12D in established precursor lesions and during progression to cancer led to regression of the lesions, indicating that KrasG12D was required for tumor cell survival. Strikingly, during all stages of carcinogenesis, KrasG12D upregulated Hedgehog signaling, inflammatory pathways, and several pathways known to mediate paracrine interactions between epithelial cells and their surrounding microenvironment, thus promoting formation and maintenance of the fibroinflammatory stroma that plays a pivotal role in pancreatic cancer. Our data establish that epithelial KrasG12D influences multiple cell types to drive pancreatic tumorigenesis and is essential for tumor maintenance. They also strongly support the notion that inhibiting KrasG12D, or its downstream effectors, could provide a new approach for the treatment of pancreatic cancer.

c-Met is a marker of pancreatic cancer stem cells and therapeutic target.

BACKGROUND & AIMS Growth of many different tumor types requires a population of self-renewing cancer stem cells (CSCs). c-Met is a marker of normal mouse pancreatic stem and progenitor cells; we investigated whether it is also a marker of human pancreatic CSCs that might be developed as a therapeutic target. METHODS We studied growth of primary human pancreatic adenocarcinoma in NOD SCID mice. The self-renewal capability of pancreatic cancer cells that expressed high levels of c-Met (c-Met(high)) was assessed using in vitro sphere assays and compared with those that were c-Met negative or expressed low levels of c-Met. The tumorigenicity of c-Met(high) pancreatic cancer cells was evaluated in NOD SCID mice. RESULTS c-Met(high) cells readily formed spheres, whereas c-Met-negative cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with small hairpin RNAs significantly inhibited tumor sphere formation. c-Met(high) cells had increased tumorigenic potential in mice; those that expressed c-Met and CD44 (0.5%-5% of the pancreatic cancer cells) had the capability for self-renewal and the highest tumorigenic potential of all cell populations studied. In pancreatic tumors established in NOD SCID mice, c-Met inhibitors slowed tumor growth and reduced the population of CSCs when given alone or in combination with gemcitabine. Administration of XL184 for 2 weeks after cardiac injection of cancer cells prevented the development of metastases. CONCLUSIONS c-Met is a new marker for pancreatic CSCs. It is required for growth and metastasis of pancreatic tumors in mice and is a therapeutic target for pancreatic cancer.

Common activation of canonical Wnt signaling in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

Interleukin-6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation and Oxidative Stress Resistance

Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the stepwise progression of the human disease. The inflammatory cytokine interleukin (IL)-6 is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL-6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL-6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL-6 synergizes with oncogenic Kras to activate the reactive oxygen species detoxification program downstream of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling cascade. In addition, IL-6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL-6 emerges as a key player at all stages of pancreatic carcinogenesis and a potential therapeutic target.

Metastatic Pancreatic Cancer Is Dependent on Oncogenic Kras in Mice

Pancreatic cancer is one of the deadliest human malignancies, and its prognosis has not improved over the past 40 years. Mouse models that spontaneously develop pancreatic adenocarcinoma and mimic the progression of the human disease are emerging as a new tool to investigate the basic biology of this disease and identify potential therapeutic targets. Here, we describe a new model of metastatic pancreatic adenocarcinoma based on pancreas-specific, inducible and reversible expression of an oncogenic form of Kras, together with pancreas-specific expression of a mutant form of the tumor suppressor p53. Using high-resolution magnetic resonance imaging to follow individual animals in longitudinal studies, we show that both primary and metastatic lesions depend on continuous Kras activity for their maintenance. However, re-activation of Kras* following prolonged inactivation leads to rapid tumor relapse, raising the concern that Kras*-resistance might eventually be acquired. Thus, our data identifies Kras* as a key oncogene in pancreatic cancer maintenance, but raises the possibility of acquired resistance should Kras inhibitors become available for use in pancreatic cancer.

Canonical Wnt Signaling Is Required for Pancreatic Carcinogenesis

Wnt ligand expression and activation of the Wnt/β-catenin pathway have been associated with pancreatic ductal adenocarcinoma, but whether Wnt activity is required for the development of pancreatic cancer has remained unclear. Here, we report the results of three different approaches to inhibit the Wnt/β-catenin pathway in a established transgenic mouse model of pancreatic cancer. First, we found that β-catenin null cells were incapable of undergoing acinar to ductal metaplasia, a process associated with development of premalignant pancreatic intraepithelial neoplasia lesions. Second, we addressed the specific role of ligand-mediated Wnt signaling through inducible expression of Dkk1, an endogenous secreted inhibitor of the canonical Wnt pathway. Finally, we targeted the Wnt pathway with OMP-18R5, a therapeutic antibody that interacts with multiple Frizzled receptors. Together, these approaches showed that ligand-mediated activation of the Wnt/β-catenin pathway is required to initiate pancreatic cancer. Moreover, they establish that Wnt signaling is also critical for progression of pancreatic cancer, a finding with potential therapeutic implications.

A Small‐Molecule Antagonist of the Hedgehog Signaling Pathway

The Hedgehog (Hh) signaling pathway plays an important role in embryonic pattern formation and adult tissue maintenance by directing cell differentiation and proliferation. In mammals, three Hh genes, Sonic (Shh), Indian (Ihh), and Desert (Dhh), have been identified. Binding of Hh protein to the membrane receptor Patched (Ptc) releases its inhibitory effect on Smoothened (Smo). Activated Smo further transduces downstream signals to activate the Gli family of transcription factors, which promote the expression of Hh signaling target genes. Hh signaling has recently attracted considerable interest based on the discovery that aberrant activation of Shh signaling leads to the formation of various tumors, which include pancreatic cancer, medulloblastoma, basal cell carcinoma, small cell lung cancer, and prostate cancer. 5] Cell-based phenotypic assays and, more recently, pathway screens of natural products and synthetic small molecules have provided useful chemical tools for modulating and/or studying complex cellular processes, both in vitro and in vivo. Several Hh antagonists, including cyclopamine, CUR61414, and SANT1-4 have been reported. Some of these antagonists exert antiproliferative effects by binding directly to Smo. However, cancer cells with mutations downstream of Smo are resistant to these antagonists. Therefore, Hh antagonists that interrupt downstream Hh signaling could lead to antiproliferative agents with a broader spectrum of activity. Here, we report the identification and characterization of the Hh signaling antagonist, JK184, and initial studies to characterize its biological mechanism of action. To screen small molecule libraries for compounds that antagonize Hh signaling, we developed a protocol using mesenchymal progenitor (C3H10T1/2) cells derived from the mouse embryonic mesoderm. These cells were stably transfected with a reporter construct that encoded luciferase, which was driven by Gli responsive elements, along with a neomycin resistanceconferring gene. Stably transfected C3H10T1/2 cells were plated into 384-well plates, and treated with a library of approximately 20000 heterocycles (2 mm, final concentration). After treatment with compound for 36 h in the presence of recombinant peptide that corresponded to the N terminus of Shh (100 ngmL ), luciferase activity was assayed, and a number of active 2,4-disubstituted thiazole compounds were identified. One of these compounds, JK184 (Figure 1A), inhibited Gli-dependent transcriptional activity in a dose-dependent manner with an IC50 value of 30 nm. This effect was fur-

Loss of the Transcription Factor GLI1 Identifies a Signaling Network in the Tumor Microenvironment Mediating KRAS Oncogene-induced Transformation

Background: KRAS is a known oncogene driving transformation in multiple tissues. Results: We demonstrate a role for the transcription factor GLI1 in KRAS-induced transformation through regulation of the IL-6/STAT3 axis in the tumor microenvironment. Conclusion: This study defines a novel oncogenic network downstream of KRAS modulating transformation. Significance: This knowledge will contribute to the understanding of the pathogenesis of tumors driven by KRAS. Although the biological role of KRAS is clearly established in carcinogenesis, the molecular mechanisms underlying this phenomenon are not completely understood. In this study, we provide evidence of a novel signaling network regulated by the transcription factor GLI1 mediating KRAS-induced carcinogenesis. Using pancreatic cancer (a disease with high prevalence of KRAS mutations) as a model, we show that loss of GLI1 blocks the progression of KRAS-induced pancreatic preneoplastic lesions in mice with pancreas-specific Cre-activated oncogenic mutant kras. Mice lacking GLI1 develop only low-grade lesions at low frequency, and in most cases, the pancreata are histologically normal. Further characterization of the phenotype showed a decrease in the activation of STAT3 in pancreatic preneoplastic lesions; STAT3 is a transcription factor required for the development of premalignant lesions and their progression into pancreatic cancer. Analysis of the mechanisms revealed a key role for GLI1 in maintaining the levels of activated STAT3 through the modulation of IL-6 signaling. GLI1 binds to the IL-6 mouse promoter and regulates the activity and expression of this cytokine. This newly identified GLI1/IL-6 axis is active in fibroblasts, a known source of IL-6 in the tumor microenvironment. Sonic hedgehog induces GLI1 binding to the IL-6 promoter and increases IL-6 expression in fibroblasts in a paracrine manner. Finally, we demonstrate that mutant KRAS initiates this cascade by inducing the expression of Sonic hedgehog in cancer cells. Collectively, these results define a novel role for GLI1 in carcinogenesis acting as a downstream effector of oncogenic KRAS in the tumor microenvironment.