Sabita Rakshit

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.

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.

Generation and expression of a Hoxa11eGFP targeted allele in mice.

Hox genes are crucial for body axis specification during embryonic development. Hoxa11 plays a role in anteroposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximodistal patterning of the limbs. Hoxa11 expression is also observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock‐in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in‐frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis. Developmental Dynamics 237:3410–3416, 2008.

The role of Pax2 in mouse prostate development

Loss‐of‐function of Pax2 results in severe defects of the male reproductive system, and Pax2 expression is detected in mouse prostate lobes and human prostatic cancers. However, the role for Pax2 in prostate development remains poorly understood.

Abstract B66: MAPK signaling mediates Kras-driven PanIN formation and maintenance.

Pancreatic ductal adenocarcinoma (PDA) is almost uniformly associated to the presence of a constitutive active mutation of the Kras gene, most often K-rasG12D (hereby Kras*). Several studies using mouse models have shown that mutant Kras* drives the formation of pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesions for pancreatic cancer. Moreover, our group has recently shown that Kras* is required for maintenance of PanINs and of invasive PDA. However, the lack of inhibitors targeting Kras prevents clinical applications of our findings. Therefore, we have set out to explore which downstream effector(s) of Kras mediate its effects during pancreatic carcinogenesis, with the goal to identify potential therapeutic targets. The MAPK/ERK pathway is activated by Kras, and its activation accompanies PanIN formation and is retained in invasive tumors. Inhibitors for the key pathway component MEK are currently in clinical trials for several malignancies. Here, we have explored the effect of inhibiting the MAPK/ERK pathway during pancreatic carcinogenesis. We have utilized two mouse models of pancreatic cancer, the KC mouse, based on tissue-specific expression of Kras*, and the iKras* mouse that allows inducible, and reversible, expression of Kras* in the pancreas. In a first set of experiments, we investigated the role of MAPK/ERK signaling during early tumorigenesis. Inhibition of MAPK/ERK signaling in vivo, prevents as well as PanIN formation in both iKras* and KC mice. To monitor individual acini over time to address whether MAPK/ERK signaling is required for acinar-ductal metaplasia (ADM), believed to precede PanINs, we utilized a 3-D culture system. Acini from both KC and iKras* models exhibit ADM over a period of three to five days in culture. Blocking the MAPK/ERK pathway inhibited ADM, thus confirming our in vivo results. In a second set of experiments, we set out to study the role of MAPK signaling in the maintenance of PanIN lesions. Treatment of PanIN-bearing iKras* mice with a MEK inhibitor resulted in regression of the PanIN lesions and regeneration of acinar tissue. In addition, we observed reversion of the fibrosis and inflammatory response that accompanies PanIN lesions, recapitulating the effect of Kras* inactivation. Taken together, our data indicate that activation of the MAPK/ERK pathway during tumorigenesis is required for both the formation and maintenance PanIN lesions. Our future directions include long-term inhibition of MAPK/ERK signaling in PanINs. Moreover, we will investigate the effect of pathway inhibition on invasive tumors, both in spontaneous and transplantation models. Finally, we will study the effect of MAPK/ERK inhibition in primary human pancreatic cancer cells. Citation Format: Meredith A. Collins, Yaqing Zhang, Sabita Rakshit, Judith S. Sebolt-Leopold, Marina Pasca di Magliano. MAPK signaling mediates Kras-driven PanIN formation and maintenance. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B66.

Abstract A104: IL6 signaling is required for pancreatitis-induced pancreatic intra-epithelial neoplasia progression and maintenance in mice.

Background: Epidemiological and experimental data suggest a causal link between chronic pancreatitis and pancreatic cancer. The cytokine interleukin 6 (IL6) is upregulated during the course of pancreatitis; moreover, recent studies have shown that the cytokine interleukin 6 (IL6) plays a role in pancreatic cancer initiation and progression. Our goal was to investigate whether IL6 upregulation provides a causal link in pancreatitis-induced carcinogenesis in mice. Methods: We previously described the iKras* mouse as a new model of pancreatic tumorigenesis based on pancreas-specific expression of the oncogenic form of Kras, KrasG12D. By crossing iKras* mice with IL6-deficient mice we have generated iKras*; IL6-/-mice, as well as iKras*; IL6+/-. To induce pancreatitis, we injected the colecistokinin agonis Caerulein administered by intraperitoneal injection. The pancreatic tissue was collected at several time-points for histopathological analysis. In addition, the immune cells infiltrated were analyzed by flow cytometry. Results: 1] IL6 is required for pancreatitis induced PanIN maintenance. PanIN lesions arose from both iKras* and iKras*;IL6-/- mice 3 weeks post-pancreatitis. By 17 weeks after pancreatitis the iKras* pancreata were totally replaced by PanINs; however, iKras*; IL6-/- pancreata appeared atrophic, devoid of PanINs and largely populated by acini. Our data further suggest that, in absence of IL6, epithelial cell survival is affected in PanIN lesions. 2] IL6 is a key regulator of inflammation during carcinogenesis. Flow cytometry analysis revealed that fewer macrophage and myeloid-derived suppressor cells (MDSCs) were recruited to iKras*; IL6-/- pancreata 3 weeks post-pancreatitis. MDSCs have been involved in maintaining an immune-suppressive microenvironment in tumors. 3] Taking advantage of the reversible expression of KrasG12D in iKras* mice, we also show that deletion of IL6 signaling accelerates PanIN reprogramming (ductal to acinar redifferentiation) following Kras* inactivation. 4] Finally, in a set of in vitro 3-dimensional culture experiments, we show that IL6 promotes acinar-ductal metaplasia (ADM)/PanIN formation and progression in acinar clusters embedded in Matrigel. Conclusion: Previous studies had shown that IL6 signaling is required for the development of PanIN lesions in mice. Here, we show that IL6 is essential for PanIN maintenance following induction of acute pancreatitis. Thus, IL6 potentially represents a therapeutic target in pancreatic cancer. Citation Format: Yaqing Zhang, Filip Bednar, Wei Yan, Meredith Anne Collins, Sabita Rakshit, Marina Pasca Di Magliano. IL6 signaling is required for pancreatitis-induced pancreatic intra-epithelial neoplasia progression and maintenance in mice. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A104.