Presence Of Oncogenic KRAS Research Articles

OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids invitro and metastasis invivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

High level of ANO1 promotes pancreatic cancer growth in concert with oncogenic KRAS.

Anoctamin-1 (ANO1) was identified as an unfavorable prognostic marker in pancreatic cancer. However, the exact implication of ANO1 in pancreatic cancer is still poorly understood. Here we investigated the effect of ANO1 in pancreatic cancer progression under the context of oncogenic KRAS, aiming at finding a new therapeutic target. Knockdown and overexpression of oncogenic KRAS as well as ANO1 in PDAC cell lines were performed by lentivirus infection. Cell proliferation and migration assay, RNA seq analysis were performed in PDAC cells bearing different status of ANO1 and KRAS. In vivo mice model was used to investigate the xenograft tumor growth with different status of KRAS and ANO1. Our results showed that ANO1 expression level is elevated in poorly differentiated cancer cells. Overexpression of ANO1 in PDAC cancer cells was found to promote cancer cell proliferation in vitro and in vivo, which synergized with the introduction of oncogenic KRAS. Consistently, knockdown of ANO1 expression was found to suppress cancer growth in vitro and in vivo. RNA seq analysis revealed that the observed synergistic cancer-promoting effect from ANO1 and oncogenic KRAS is likely due to concurrent activating key genes involved in lipid metabolism including HMGCS1. The outcome from our study suggests that ANO1 plays an important role in promoting pancreatic cancer development, especially at the presence of oncogenic KRAS. Considering the prevalence of KRAS mutation in pancreatic cancer patients, suppression ANO1 may represent a potential effective therapeutic measure in pancreatic cancer treatment.

Abstract A070: Comparison of pancreatic cancer mouse models identifies potential drivers of tumor initiation and progression

Abstract The five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is currently 11% [CP1] and by 2030 PDAC is expected to become the second leading cause of cancer related death. Understanding PDAC initiation and maintenance is important for the advancement of treatment. Mouse models allow us to study the mechanisms of PDAC initiation and progression. The most common genetically engineered mouse model (GEMM) of PDAC utilizes a knock-in of Cre recombinase [FB2] into the pancreatic transcription factor 1a (Ptf1a)locus to drive pancreas specific oncogenic KRAS expression. Alternative GEMMs express Cre recombinase from the Mist1/Bhlha15 locus (Mist1-CreER) or from elastase enzyme promoter-driven constructs (Ela-CreER). How these models differ in their ability to promote neoplasia is unknown. We hypothesized that differences in GEMMs may provide insight into mechanisms of PDAC initiation and progression. Here, we compare the tamoxifen-inducible Ptf1a-CreER, [FB3] Mist1-CreER and Ela-CreER models in their response to oncogenic KRAS expression. KRASG12D was induced with tamoxifen gavage 6-8 weeks postpartum and caerulein was used to induce pancreatitis. Histological analyses (H&E and IHC for amylase, cytokeratin 19, Fos and Jun) were performed on fixed tissue to assess associated phenotypic changes. Tissues from Ptf1a-CreER, Mist1-CreER and Ela-CreER mice were harvested 3 weeks after tamoxifen administration and processed for RNA sequencing. Phenotypically, in the presence of oncogenic KRAS and after pancreatitis induction, Ptf1a-CreER pancreata exhibited extensive tissue remodeling - acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) - with a dense fibroinflammatory response in contrast to both Mist1-CreER and Ela-CreER pancreata. Mist1-CreER and Ela-CreER pancreata had much more variable penetrance with tissues often showing little to no ADM and PanINs despite oncogenic KRAS expression and prior pancreatitis. At the gene expression level, Mist1-CreER pancreata showed fewer differentially expressed genes when compared to wild type tissue than Ptf1a-CreER under the same conditions. In total, less than 70 differentially expressed genes were common between the two models. Members of the AP-1 transcriptional complex (fos and c-jun) previously implicated in neoplastic progression were specifically increased only in Ptf1a-CreER mice.[FB4] Taken together, our histological, and transcriptional data indicates mice lacking one copy of Ptf1a (Ptf1a-CreER) undergo more dramatic histological changes after pancreatitis injury and oncogenic KRAS induction when compared to the GEMMs with both copies of Ptf1a intact. Additionally, mice in the Ptf1a-CreER group show a more marked molecular response to oncogenic KRAS induction, suggesting the absence of a single copy of the acinar fate transcription factor Ptf1a disrupted pancreatic homeostasis and poised it for transformation. Further comparison of PDAC GEMMs will yield clearer insight into mechanisms of PDAC initiation and progression. Citation Format: Joyce K. Thompson, Fatemeh Mousavi, Justine Lau, Emily Wu, Christopher Pin, Filip Bednar. Comparison of pancreatic cancer mouse models identifies potential drivers of tumor initiation and progression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A070.

Emerging Role of CREB in Epithelial to Mesenchymal Plasticity of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid malignancy with a high rate of metastasis and therapeutic resistance as its major hallmarks. Although a defining mutational event in pancreatic cancer initiation is the presence of oncogenic KRAS, more advanced PDAC lesions accumulate additional genomic alterations, including loss of tumor suppressor gene TP53. Co-occurrence of mutant KRAS and TP53 in PDAC promotes hyperactivation of cancer cell signaling pathways driving epithelial to mesenchymal plasticity (EMP). The cellular process of EMP influences the biological behavior of cancer cells by increasing their migratory and invasive properties, thus promoting metastasis. Our previous work has demonstrated that oncogenic KRAS-mediated activation of cyclic AMP response element-binding protein 1 (CREB) is one of the critical drivers of PDAC aggressiveness. The therapeutic approach of targeting this key transcription factor attenuates tumor burden in genetically engineered mouse models (GEMMs) of this disease. Herein, we discuss the significant role of CREB in perpetuating disease aggressiveness and therapeutic resistance through the EMP process. Furthermore, this review updates the therapeutic implications of targeting CREB, highlighting the challenges and emerging approaches in PDAC.

Abstract 788: Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment. Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1. Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation. Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 788.

A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer.

KRAS mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.

Abstract 2691: Cigarette smoke induced upregulation of endothelin axis in the initiation of pancreatic cancer

Abstract Background: Cigarette smoke is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Smoke induced inflammation accelerates the progression of PDAC in presence of constitutively active K-Ras mutation. Endothelin (ET) axis comprising of endothelin converting enzymes (ECE-1, ECE-2, and ECE-3), endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ETAR) and B (ETBR), is linked to pathobiology of pancreatitis and its components exhibit aberrant overexpression in PDAC. However, the expression of ET-axis in the oncogene-associated early Pancreatic Intraepithelial neoplastic (PanINs) lesions following exposure to cigarette smoke is unknown. We hypothesize that smoke induced alterations in the ET axis facilitate acinar to ductal metaplasia (ADM) in presence of oncogenic K-Ras. Thus, we characterized the impact of cigarette smoke exposure on ET axis components in the pancreas of mice harboring mutant K-Ras. Methods: Expression of ECE-1, ET-1, ETAR and ETBR was analyzed by IHC and RT-PCR first in the murine model of preneoplastic lesions [KC model: (Pdx1-Cre, KrasG12D)). To determine the changes in ET axis after smoke exposure, wild type (WT) and KC mutant mice were exposed to cigarette smoke for 20 weeks and expression was analyzed in the pancreas. The impact of smoking on ET-axis was also studied on murine acinar and tumor cell lines derived from KC mice (UN-KC6141) and KPC (Pdx1-Cre, p53 (R172H) KrasG12D) mice (UN-KPC-961) by western blot and RT-PCR analysis. Results: In contrast to the normal ducts, the expression of ECE-1, ET-1, ETAR and ETBR was upregulated in the early PaNIN lesions (20-30 weeks). In the advanced lesions (50 weeks), significant overexpression of all four molecules was noticeable in the tumor cells and stromal compartment. Smoke exposure resulted in significant increase in the transcripts of ET-1, ETAR and ETBR (p values = 0.04, 0.03 and 0.01 respectively) in the KC mice while the increase in WT mice was not significant. The changes in the expression were also corroborated by tissue IHC. Expression of ET axis components was predominantly seen in the islet cells in WT mice, while low immunoreactivity was observed in the acinar compartment. Smoke exposure of KC mice resulted in accelerated progression of PanIN lesions along with concomitant increase in the expression of ET axis components both in the ductal and stromal cells. In vitro exposure of cigarette smoke extract (CSE) to UN-KC6141 and UN-KPC-961 cells for 24 hours elicited a dose dependent upregulation of ECE 1 and ET-1. Conclusions: In presence of mutated KrasG12D, smoking-mediated inflammatory insult promotes PanIN progression and tumorigenesis. The sustained increase and activation of ET axis with increasing dysplasia in the ductal compartment during this progression in KC mice and its further upregulation following smoke exposure suggests its possible role in promoting inflammation-associated pancreatic tumor progression. Citation Format: Suprit Gupta, Satyanarayana Rachagani, Sushil Kumar, Surinder Kumar Batra, Maneesh Jain. Cigarette smoke induced upregulation of endothelin axis in the initiation of pancreatic cancer [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 2691. doi:10.1158/1538-7445.AM2017-2691

Abstract 1362: Argonaute 2 controls RAS activation in mouse embryonic fibroblasts

Abstract The RAS gene family is among the most commonly mutated genes within cancer, and while much research has elucidated the major downstream pathways, including MAPK and PI3K, little progress has been made in successfully targeting mutant RAS in cancer. We recently identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS. Furthermore, this interaction was found in all cell lines tested, expressing either wild-type (WT) or mutant KRAS. We found that stable knockdown of AGO2 in KRAS dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. Conversely, the overexpression of AGO2 in these cells lead to both an increase in KRAS expression and oncogenesis. In addition, this interaction inhibits the RNAi function of AGO2 by preventing microRNA unwinding in the presence of oncogenic KRAS compared to WT-KRAS. Despite a clear association between mutant KRAS and AGO2 mediating increased KRAS mediated oncogenesis, the precise function of this interaction remains unclear in normal physiology. In order to better assess the endogenous function of the KRAS-AGO2 interaction, we analyzed two mouse embryonic fibroblast cell lines (NIH 3T3 and MEF) with complete knockout of AGO2. Utilizing a Raf-1 RAS binding domain (RBD) pulldown method, we assessed activated WT-RAS levels in AGO2 null NIH 3T3 and MEF cells. We found that knockout of AGO2 lead to an increase in WT RAS-GTP activation compared to normal control cells. Immunoblot analysis also indicates that AGO2 null fibroblasts lead to increase in RAS downstream signaling through the MAPK/ERK and PI3K/AKT pathways. Furthermore, rescue of AGO2 knockout using full length mouse AGO2 decreased wild type RAS activation and its downstream signaling. Taken together, these observations suggest that the AGO2 interaction may suppress WT-KRAS activation, leading to maintenance of RAS-GDP levels. Using RNA-seq, proteome and microRNA analysis, we have begun to identify the pathways that may be involved in RAS activation in AGO2 null cells. Early analyses indicate that AGO2 controls WT-KRAS levels and activity through multiple mechanisms, laying the foundation for a better understanding of the RAS-AGO2 interaction in normal physiology. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Vijaya L. Dommeti, Malay Mody, Arul Chinnaiyan. Argonaute 2 controls RAS activation in mouse embryonic fibroblasts [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 1362. doi:10.1158/1538-7445.AM2017-1362

Isoform-Specific Effects of Wild-Type Ras Genes on Carcinogen-Induced Lung Tumorigenesis in Mice.

The gene KRAS is commonly mutated in lung cancer to encode a constitutively active and oncogenic protein that is well established to initiate and maintain lung tumorigenesis. However, the remaining wild-type KRAS protein, or the other family members HRAS and NRAS, can still be activated in the presence of oncogenic KRAS. Moreover, loss of any one of these three genes has been shown to increase the sensitivity of mice to the carcinogen urethane, which induces Kras mutation-positive early lung lesions. To determine the contribution of progressively disrupting Hras and Nras genes on urethane lung tumorigenesis, mice with different combinations of wild-type and null alleles of Hras and Nras were exposed with urethane and tumor burden was assessed. As previously reported, loss of one allele of Hras increased the sensitivity of mice to this carcinogen, and this effect was further exacerbated by the loss of the second Hras allele. However, loss of one or both alleles of Nras failed to alter tumor burden, either in the absence or presence of Hras, after exposure to urethane. Additionally, no obvious difference between lung lesions in mice with wild-type versus null alleles was detected, suggesting that wild-type Ras proteins may exert a tumor suppressive effects at the time of initiation, although other interpretations are certainly possible. In summary, these data suggest that in some genetic backgrounds inactivation of different wild-type Ras genes can have different effects on urethane-induced lung tumorigenesis.

Abstract 4191: Alterations in endothelin axis during pancreatic acinar to ductal metaplasia

Abstract Background: Acinar-ductal-metaplasia (ADM) is one the earliest recognizable alterations in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Inflammation-induced ADM, which is typically reversible upon removal of inflammatory insult, becomes irreversible in the presence of oncogenic K-Ras and progresses to Pancreatic Intraepithelial neoplastic lesions (PanINs) and subsequently PDAC. Endothelin (ET) axis comprising of endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ETAR) and B (ETBR), has been demonstrated to contribute to the pathobiology of pancreatitis and its components exhibit aberrant overexpression in pancreatic cancer PDAC. However, the expression patterns of ET axis in oncogene-associated early lesions remain unknown. We hypothesize that alterations in ET axis contribute to oncogene associated irreversible ADM. Thus, we studied expression pattern of ET axis in the pancreatic inflammation in the presence and absence of oncogenic KRas and in preneoplastic lesions. Methods: Expression of ET-1, ETAR and ETBR was analyzed in murine models of preneoplastic lesions [KC model: (Pdx1-Cre, KrasG12D)) by IHC. To determine the changes in ET axis during ADM, wild type (WT) and KC mutant mice were treated with cerulein to induce pancreatitis and tissues collected at day 0, 2, 7 and 21 days post treatment were analyzed for mRNA and Immunofluorescence analysis. Expression pattern of ET axis components was also determined in the pancreas of KC and WT animals following exposure to cigarette smoke. Results: A progressive increase in the expression of ET-1, ETAR and ETBR was observed in the PanIN lesions in KC mice. In mice with WT Kras, cerulein treatment resulted in a notable increase in the expression of ET-1 and ETAR at day 2, while the levels of ETBR increased marginally; however a recovery to basal levels was observed for all three molecules by day 7. In contrast, significant increase (p<0.005) in ET-1, ETAR and ETBR transcripts was observed following cerulein treatment and these levels continued to remain high even at 21 days post-trauma in KC mice. The changes in the expression ETAR and ETBR were corroborated by confocal microscopy. Cerulein treatment resulted in increased expression of ETAR and ETBR in acinar compartment in both WT and KC mice as indicated by their co-localization with amylase. In KC mice, cerulein induced acinar-to-ductal metaplasia and the resulting CK19 positive ductal components continued to express increased levels of both receptors with distinct magnitude and kinetics. Similarly, in the smoking models, ET-axis components exhibited a more robust and sustained overexpression in KC mice as compared to the WT. Conclusions: The expression of oncogenic KrasG12D results in an enhanced and sustained activation of ET-axis following inflammatory insults in the pancreatic tissues, suggesting its possible role in tumor initiation and progression. Citation Format: Suprit Gupta, Satyanarayana Rachagani, Sushil Kumar, Kavita Mallya, Surinder Kumar Batra, Maneesh Jain. Alterations in endothelin axis during pancreatic acinar to ductal metaplasia. [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 4191.

Cell hierarchies in colorectal cancer: focus on APC and BRAF.

The Wnt-APC-β-Catenin and RAS-RAF-MAPK signaling pathways serve to maintain homeostasis in the normal intestinal epithelium. This state is characterized by an equilibrium between stemness, proliferation, differentiation and cell death [1]. Recent studies by our group [2] and by Dow et al. [3] elucidate how oncogenes and tumor suppressors influence cell hierarchies during colorectal cancer (CRC) formation in the mouse and highlight the potential of this information for future approaches to cancer therapy. A majority of CRCs are initiated by APC mutations promoting Wnt/β-Catenin activity and stem cell fate [4]. As we described already in 2012, reversible transgenic expression of oncogenic β-Catenin can switch mouse intestinal epithelium between normal and adenomatous cell hierarchies [5]. For these experiments, we used organotypic cell cultures maintaining stem, proliferative and differentiated cells. β-Catenin promoted a universal capacity for self-renewal and expression of cancer stem cell markers, similar to cultures derived from APC−/− adenoma. Withdrawal of oncogenic β-Catenin re-established normal cell hierarchies driven by only few stem cells in the crypts. A new publication by Dow et al. [3] shows that reversible knockdown of APC in the intestine of mice establishes adenomatous growth in vivo, while restoration of APC results in reversion to normal cell hierarchies and tumor regression. When the authors combined reversible APC knockdown with conditional gain- and loss-of-function alleles for the KRAS oncogene and the p53 tumor suppressor, respectively, invasive colon carcinoma developed. Significantly, restoration of APC resulted in complete remission and reverted colon cancer tissue to normal intestinal epithelium even in the presence of oncogenic KRAS and absence of p53. These results demonstrate a surprising ability of intestinal epithelium to regain homeostasis. In contrast to Wnt/β-Catenin, the roles of MAPK activity in the control of intestinal cell hierarchies are not well established. EGFR-RAS-RAF-MAPK transduces crucial proliferative cues in the intestinal crypt. Oncogenic KRAS has a tumor-promoting role in conventional adenoma initiated by loss of APC, while oncogenic BRAF can initiate another type of CRC precursor, termed serrated adenoma, independently of APC [4]. We recently found that transgenic expression of oncogenic BRAF caused intestinal serration, but also abrogated maintenance of stem cells in the mouse intestine, which collectively converted to proliferative progenitors [2]. How can BRAF act as a CRC initiator despite its negative effect on stem cell support? The answer comes from a publication dissecting multi-step CRC development after oncogenic BRAF knock-in in the mouse [6]: activation of BRAF resulted in generalized intestinal hyperplasia with a reduced stem cell pool, and only progressive foci displayed activating mutations in the Wnt/β-Catenin pathway, nuclear β-Catenin and increased gene expression of Wnt targets and stem cell markers. This suggests that increased β-Catenin activity can supplement the stem cell pool after oncogenic activation of BRAF. Indeed, we found that activation of β-Catenin partially protected intestinal tissue from the deleterious effects of BRAF overexpression in vitro and in vivo [2]. Taken together, these results give insight into the mechanisms controlling cell hierarchies in CRC, and suggest that the Wnt/β-Catenin and MAPK signaling pathways must be activated in a coordinate manner to allow for CRC initiation and progression. Figure 1 Balancing of the stem cell pool in mouse models of intestinal cancer development How can constraints on cell hierarchies be exploited for novel cancer therapies? Dow et al. argue that cancer remission upon APC restoration provides a strong case for the Wnt signaling pathway as a therapeutic target [4]. Panels of organotypic cultures derived from human CRC have recently become available [7]. Thus, restoration of APC in human canceroids could provide further evidence for the validity of the concept proposed by Dow and colleagues. Therapies targeting the EGFR-MAPK axis are already in clinical use and in clinical trials, such as EGFR-inhibitors and combinatorial inhibition within the extended MAPK network [4]. Future strategies could take into account vulnerabilities of the stem cell pool towards MAPK signals in certain types of CRC.

Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway

KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

RON overexpression accelerates tumorigenesis and induces metastasis in a KRAS mutant mouse model of pancreatic cancer

Introduction The RON receptor is increasingly over-expressed during pancreatic cancer progression and has been implicated as a mediator of KRAS oncogene addiction. It remains unclear if RON promotes disease progression or simply represents an epiphenomenon. Methods To address this, we generated a transgenic mouse which over-expressed wt-RON in a pancreas-specific manner using Pdx-1, the identical promoter used to drive cre expression in the Kras-LSLGD12/Pdx-1-cre (KC) strain that develops pancreatic duct neoplasia. RON over-expression was verified via Western and IHC. Pdx-1/RON mice were crossed with LSL-KRASG12D mice to yield mice (RK), then bred to Pdx-1-Cre mice to combine RON over-expression in the presence of oncogenic KRAS (RCK). RCK mice were aged and sacrificed at various time points and histology compared to KC controls. Results Pdx-1-RON mice developed no pancreatic phenotype prior to 12 months. At 18 months, one of 4 animals developed primary pancreatic adenocarcinoma with lung metastasis. RON overexpression in KRAS mutant mice (RCK) led to accelerated PanIN progression compared to KC (p 1yr). Metastatic disease was visible in RCK mice at 9 months. Conclusions RON overexpression alone results in pancreatic cancer at long latency. In the presence of KRAS mutation, RON overexpression markedly accelerates PanIN progression to primary and metastatic pancreatic duct cancer. This data supports a role for RON in pancreatic carcinogenesis and this model may prove useful for investigations regarding the role of the RON receptor in pancreatic cancer biology.

Abstract 1200: Oncogenic KRAS sensitizes colorectal tumor cells to chemotherapy via p53-dependent induction of Noxa

Abstract Oxaliplatin and 5-fluorouracil currently form the backbone of conservative treatment in patients with metastatic colorectal cancer. Tumor responses to these agents are highly variable, but the underlying mechanisms are poorly understood. Our previous results have indicated that the presence of oncogenic KRAS in colorectal tumor cells sensitizes these cells to chemotherapy. In the present manuscript we show that the sensitization of colorectal tumor cells to oxaliplatin and 5-FU by oncogenic KRAS is critically dependent on the presence of wild type p53. KRAS status did not influence p53 stabilization, p21 induction, or cell cycle arrest. A screen for induction of apoptosis regulators revealed that KRAS selectively promoted expression of the pro-apoptotic p53 target gene Noxa. Suppression of Noxa by RNA interference strongly reduced KRAS/p53-dependent apoptosis following exposure to chemotherapy, both in vitro and in tumor xenografts in mice. Noxa knockdown did not affect p53-dependent p21 induction or cell cycle arrest. Our results identify oncogenic KRAS as an important determinant of the cellular response to p53 activation following exposure to oxaliplatin or 5-FU, by facilitating apoptosis induction through Noxa. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1200.

Phosphatome profiling reveals PTPN2, PTPRJ and PTEN as potent negative regulators of PKB/Akt activation in Ras-mutated cancer cells

Oncogenic Ras mutations render the protein constitutively active and promote tumorigenesis via chronic stimulation of effector pathways. In A549 lung adenocarcinoma approx. 50% of the total Ras population is constitutively active, yet these cells display only weak activation of the effectors: ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. In order to identify key negative regulators of oncogenic Ras signalling we performed a phosphatome RNAi (RNA interference) screen in A549 cells and ranked their effects on phosphorylation of Ser473 of Akt. As expected, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) emerged as a leading hit: knockdown elevated Akt activation to 70% of maximal generated by acute EGF (epidermal growth factor) stimulation. Importantly, we identified other phosphatases with similar potencies including PTPN2 (T-cell protein tyrosine phosphatase; also known as TC-PTP) and PTPRJ (protein tyrosine phosphatase receptor type J; also known as DEP-1/CD148). Potentiation of Akt phosphorylation by knockdown of PTEN or PTPRJ was contingent on the presence of oncogenic K-Ras. Our data reveal a synergy between oncogene function and the loss of a tumour suppressor within the same pathway that was necessary for full effector activation since each alone failed to elicit significant Akt phosphorylation. Taken together, these data reveal potent regulators of Akt signalling which contribute to ameliorating the consequences of oncogenic K-Ras activity.

TRAIL receptor upregulation and the implication of KRAS/BRAF mutations in human colon cancer tumors

TRAIL raises hopes as a promising anti-tumor agent due to its selectivity toward cancer cells. Higher expression of its pro-death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) attenuates higher sensitivity to TRAIL-induced apoptosis, and represents a marker for better cancer prognosis and treatment. Since receptor availability can be analogous to ligand efficacy, we performed RT-PCR analysis of DR4 and DR5 in 51 colon cancer biopsy specimens and respective normal mucosa, while 11 of these tumors were determined immunohistochemically for protein expression. Transcriptional analysis showed that DR4 and DR5 were significantly upregulated in 37 and 47% of the tumor samples respectively, while both DR4 and DR5 were coinstantaneously upregulated in 31% of the samples analyzed. Positive transcriptional regulation of DRs was recorded as early as Dukes' A stage. Furthermore, protein expression analysis yielded results comparable to DR4 and DR5 increased mRNA levels. Possible contributing events to DR upregulation involve presence of frequent oncogenic mutations in the MAPK pathway, and was investigated by direct sequencing in all 51 tumors. Samples (6/8) hosting either a KRAS(G12V) or BRAF(V600E) mutation, significantly amplified the upregulated expression of DR4 and DR5, showing strong inter-relation between overexpression and presence of oncogenic KRAS/ BRAF mutations. In the light of recent data concerning TRAIL receptor distribution, we contribute further by presenting DR5 as the most frequently upregulated DR in colon cancer. Furthermore, oncogenic mutations may directly or indirectly enhance DR expression, potentially sensitizing these tumors to TRAIL-based therapies.

Differential Notch and TGFβ Signaling in Primary Colorectal Tumors and Their Corresponding Metastases

Background: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFβ signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases. Methods: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (β-catenin), Notch (HES1) and TGFβ (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed. Results: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear β-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear β-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear β-catenin. Conclusions: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFβ signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFβ pathways is reduced in secondary colorectal tumors.

Paired-like homeoprotein ESXR1 acts as a sequence-specific transcriptional repressor of the human K-ras gene

Gain-of-function mutation of the K-ras gene is one of the most common genetic changes in human tumors. In tumors carrying K-ras mutation, the presence of oncogenic K-Ras is necessary for maintenance of the transformed phenotype. ESXR1 is a human paired-like homeodomain-containing protein expressed primarily in the testis. In cells, the 65-kDa full-length ESXR1 protein is proteolytically processed into an N-terminal 45-kDa fragment containing the homeodomain, which localizes exclusively within the nucleus, and a C-terminal 20-kDa fragment consisting of a proline-rich repeat region, which is located in the cytoplasm. In this work, we demonstrated that the N-terminal ESXR1 fragment specifically recognizes the TAATNNNATTA P3 consensus sequence for the paired-like homeodomain and functions as a sequence-specific transcriptional repressor. We also showed that the N-terminal ESXR1 fragment binds to the TAATGTTATTA sequence present within the first intron of the human K-ras gene and inhibits its expression at both mRNA and protein levels. Ectopic expression of the N-terminal ESXR1 fragment in human carcinoma cells that carry mutated K-ras reduces the level of K-Ras and thereby inhibits the tumor cell proliferation. Identification of ESXR1 as a transcriptional repressor of K-ras has an important implication for the development of cancer therapy that inhibits oncogenic K-Ras expression.

Ectopic expression of VAV1 reveals an unexpected role in pancreatic cancer tumorigenesis

Herein, we show that the hematopoietic-specific GEF VAV1 is ectopically expressed in primary pancreatic adenocarcinomas due to demethylation of the gene promoter. Interestingly, VAV1-positive tumors had a worse survival rate compared to VAV1-negative tumors. Surprisingly, even in the presence of oncogenic KRAS, VAV1 RNAi abrogates neoplastic cellular proliferation in vitro and in vivo, thus identifying Vav1 as a growth-stimulatory protein in this disease. Vav1 acts synergistically with the EGF receptor to stimulate pancreatic tumor cell proliferation. Mechanistically, the effects of Vav1 require its GEF activity and the activation of Rac1, PAK1, and NF-κB and involve cyclin D1 upregulation. Thus, the discovery of prooncogenic pathways regulated by Vav1 makes it an attractive target for therapeutic intervention.