Activation Of K-ras Oncogene Research Articles

Cancer Stemness in Apc- vs. Apc/KRAS-Driven Intestinal Tumorigenesis

Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (<10−6) in the Apc–mutant adenomas. In contrast, the Lin−CD24hiCD29+ subpopulation of adenocarcinoma cells appear to be enriched in CSCs with increased levels of active β-catenin. Expression profiling analysis of the CSC-enriched subpopulation confirmed their enhanced Wnt activity and revealed additional differential expression of other signalling pathways, growth factor binding proteins, and extracellular matrix components. As expected, genes characteristic of the Paneth cell lineage (e.g. defensins) are co-expressed together with stem cell genes (e.g. Lgr5) within the CSC-enriched subpopulation. This is of interest as it may indicate a cancer stem cell niche role for tumor-derived Paneth-like cells, similar to their role in supporting Lgr5+ stem cells in the normal intestinal crypt. Overall, our results indicate that oncogenic KRAS activation in Apc-driven tumours results in the expansion of the CSCs compartment by increasing ®-catenin intracellular stabilization.

Oncogenesis and the Clinical Significance of K-ras in Pancreatic Adenocarcinoma

The RAS family genes encode small GTP-binding cytoplasmic proteins. Activated KRAS engages multiple effector pathways, notably the RAF-mitogen-activated protein kinase, phosphoinositide-3-kinase (PI3K) and RalGDS pathways. In the clinical field, K-ras oncogene activation is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. This mini-review aims to summarise information on Ras-induced oncogenesis and the clinical significance of K-ras.

Sa1681 Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer Cell Invasion and Metastasis.

Background: Kruppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highly expressed in the crypt cells of the intestinal epithelia and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted from either ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated at the transcriptional, translational, and post-translational level. Previous studies indicate that the protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradation of KLF5 protein via phosphorylation (GSK3β) and recognition (FBW7) of a phosphodegron sequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence (Sjoblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutation on KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant (S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter were transiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5 produced from the various constructs was investigated by cycloheximide chase and Western blot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferase reporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutants examined are more resistant to FBW7-mediated proteosomal degradation as compared to WT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5 but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experiments show that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7. Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation at Ser303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 and CDC2 promoter constructs demonstrate significantly elevated transcriptional activity of P301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, we show that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition of the phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels, and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutation in KLF5 is potentially oncogenic in colon cancer.

Abstract 205: SIAH2 modulates focal adhesion and cell junction proteins in response to oncogenic ERBB/K-RAS activation to promote cancer cell dissemination, invasion and metastasis.

Abstract Oncogenic EGFR/HER2/K-RAS activation is often associated with reduced focal adhesion, altered cell junctions, increased cell motility and enhanced cancer cell dissemination, aggressive tumor growth and metastasis, cancer relapse and drug resistance in human cancer. Deciphering novel mechanisms that promote invasion/metastasis in human cancer cells in response to oncogenic ERBB/K-RAS pathway activation and finding novel means to inhibit tumor invasion and cancer metastasis remain a paramount important goal in cancer biology. SIAH, which is the human homologue of a highly conserved family of RING-domain E3 ubiquitin ligases, is a key downstream signaling component required for proper ERBB/K-RAS signal transduction. We have shown that SIAH2-insufficiency impedes proper K-RAS signal transduction, thereby obstructing tumorigenesis and metastasis. While this suggests that SIAH2 E3 ligase is critical for K-RAS-dependent oncogenesis, the exact mechanism of SIAH2 function downstream of ERBB/K-RAS signaling pathway in driving cancer cell dissemination, tumor growth and metastasis has remained unknown. In this study, we affinity purified and isolated SIAH2-protein complex from human cancer cells with oncogenic K-RAS activation. Several focal adhesion and cell junction proteins were proteomically identified as SIAH-interacting proteins by Mass Spectrometry. Bi-directional co-immunoprecipitation (co-IP) and in vivo ubiquitination assays were performed to verify and validate that these newly identified SIAH-binding partners are indeed bona fide new SIAH substrates that play important roles in cancer cell dissemination, invasion and metastasis. Our immunofluorescent staining and confocal results showed that focal adhesion, cell junction and cell motility are severely compromised in the SIAH-deficient cancer cells while in contrast, the prominent focal adhesion sites are present in the SIAH-proficient cancer cells. Importantly, ectopic expression of these focal adhesion and cell junction proteins can successfully rescue SIAH2-deficient cancer cells from the pronounced defects in focal adhesion, adherens junction, cell motility, cell death and tumor growth. Hence, these focal adhesion and cell junction proteins may represent a new class of SIAH2 substrates. Their increased expression patterns have been confirmed and verified in human pancreatic cancer and breast tumor biospecimens. We may have uncovered a new molecular mechanism of SIAH enzymatic function in human cancer cells. The discovery that SIAH2 directly modulates focal adhesion, cell junction integrity and motility may provide a novel molecular mechanism to explain the well-documented changes in cancer cell dissemination, aggressive tumor invasion and systemic metastasis in response to oncogenic EGFR/HER2/K-RAS pathway activation in human cancer. Citation Format: Minglei Bian, Yang Liao, Rebecca L. Schmidt, Zandra E. Walton, Amy H. Tang. SIAH2 modulates focal adhesion and cell junction proteins in response to oncogenic ERBB/K-RAS activation to promote cancer cell dissemination, invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 205. doi:10.1158/1538-7445.AM2013-205

Intratumoral heterogeneity of KRAS mutation is rare in non-small-cell lung cancer

BackgroundSeveral lines of evidence indicate that mutational activation of KRAS is an early event in the carcinogenesis of non-small cell lung cancer (NSCLC). Nonetheless, previous studies report high frequencies of divergent KRAS mutational status between primary NSCLC and corresponding metastases. This suggests heterogeneity of the primary tumor in respect to its KRAS status. We therefore aimed to examine the frequency and the extent of such intratumoral heterogeneity. Methods40 NSCLC were examined for intratumoral heterogeneity of KRAS mutation (20 adenocarcinomas, 10 squamous cell carcinomas and 10 large cell carcinomas). Three to eight different tumor areas were analyzed for KRAS mutation and up to four corresponding lymph node metastases were included for analysis in nineteen cases. A combination of different methods for screening of heterogeneity and its validation were used including direct sequencing, laser-capture microdissection for tumor cell enrichment and the very sensitive ARMS/S method. ResultsMutations of KRAS were found in 13/30 adenocarcinomas and large cell carcinomas. No mutations were detected in 10 squamous cell carcinomas. Four cases showed heterogeneous KRAS results by direct sequencing. More sensitive methods for KRAS mutation analysis revealed false negative results due to admixture of non-neoplastic cells in all of these samples. Intratumoral heterogeneity of KRAS mutational status was therefore confirmed in none of the analyzed cases. In addition, identical KRAS mutations were present in the primary tumor and the corresponding lymph node metastases in 19 cases examined. ConclusionsIntratumoral heterogeneity of KRAS mutational status is rare in NSCLC but highly sensitive tools are required to reliably identify these mutations. This finding is in line with the hypothesis that oncogenic activation of KRAS is an early event and a bona fide “driver mutation” in NSCLC. Furthermore, future therapies targeting KRAS will not be limited by intratumoral heterogeneity.

Abstract 4169: The Wnt/β-catenin pathway is involved in KRAS-induced intestinal epithelial cell transformation

Abstract Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). The APC protein is an integral part of the destruction complex that controls cytoplasmic β-catenin levels by promoting ubiquitin-mediated degradation of β-catenin. Loss-of-function mutations in APC are commonly found in CRC, leading to constitutively high levels of β-catenin which then associates with TCF4, inducing expression of target genes that are important in cell growth. The KRAS/BRAF/MEK/ERK signaling pathway is also a major transforming growth regulatory signaling pathway. Indeed, gain-of-function mutations of KRAS and BRAF are detected in up to 60% of CRCs. Although both the Wnt/β-catenin and the ERK pathways are major pathways for tumorigenesis of intestinal epithelial cells (IECs), no significant interactions between these pathways have been identified. Methods. Retrovirus encoding the HA-tagged wild-type MEK1 (wtMEK) or activated MEK1 (MEK1S218D/S222D, caMEK) or oncogenic form of KRASG12V were used to infect and transform normal IECs in culture. The transcriptional activity of β-catenin/TCF4 complex was analyzed by using the luciferase reporter systems TOPFLASH and c-myc promoter (mutated or not for TCF4 binding elements). Expression levels of Wnt/β-catenin targets axin2, lef-1 and c-myc were evaluated by qPCR and protein localisation by immunofluorescence. A dominant-negative form of TCF4 (TCF4-DN) was used to verify the contribution of β-catenin/TCF4 complex in transformation induced by oncogenic activation of KRAS or MEK1. β-catenin transcriptional activity was analyzed in human CRC cells exhibiting APC mutation, treated or not with the MEK inhibitor U0126. Results. 1- Phase-contrast microscopy revealed that KRASG12V and caMEK-expressing IECs underwent a morphological change from an epithelial morphology to an elongated morphology. Cells clearly loose cell-to-cell contacts when compared to control cells. E-cadherin and β-catenin proteins disappeared from cell-cell junctions. 2- β-catenin/TCF4 transcriptional activity as well as mRNA levels of c-myc, axin2 and lef1 were markedly increased in IECs transformed by oncogenic MEK1 or KRAS. 3- Expression of the dominant-negative form of TCF4 severely attenuated IEC transformation induced by oncogenic MEK1 or KRAS. 4- Treatment of human CRC cell lines with U0126 significantly reduced axin2, lef-1 and c-myc mRNA levels, indicating that expression of these β-catenin target genes is likely dependent on MEK/ERK activity. Accordingly, β-catenin/TCF4 transcriptional activity was inhibited following MEK inhibition by U0126 or ERK dephosphorylation by DUSP4 expression. Conclusion. Our data indicate that the oncogenic activation of the KRAS/ERK signalling pathway in IECs up-regulates β-catenin/TCF4 transcriptional activity which contributes to the tumorigenic potential of this signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4169. doi:1538-7445.AM2012-4169

Abstract LB-205: Identification of tumorigenic cells in KrasG12D-induced lung adenocarcinoma

Abstract We established an inducible KrasG12D-driven lung adenocarcinoma in CCSP-rtTA/TetO-Cre/LSL-KrasG12D mice that enable pursuits of the cellular and molecular processes involved in Kras-induced tumorigenesis. To investigate the cellular origin of this cancer, we first report a strategy using FACS fractionation that could highly enrich bronchiolar Clara and alveolar type II cells, respectively. The EpCAM+MHCII− cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than EpCAM+MHCII+ cells (alveolar origin) in primary tumors that had been already initiated with oncogenic Kras activation. In addition, secondary tumors derived from EpCAM+MHCII− cells showed diversity of tumor locations compared to those derived from EpCAM+MHCII+ cells. In the alveolar region, secondary tumors from EpCAM+MHCII− cells expressed not only bronchiolar epithelial marker, panCK, but also differentiation marker, proSPC, consistent with the notion that cancer-initiating cells display not only the abilities for self-renewal but also the features of differentiation to generate heterogeneous tumors with phenotypic diversity. Furthermore, high level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM+MHCII− cells. Therefore, these results suggest that bronchiolar Clara cells are the origin of cells and tumorigenesis for KrasG12D-induced neoplasia in the lungs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-205. doi:1538-7445.AM2012-LB-205

Abstract 3291: Defining the role of the cellular polarity regulator Scrib in epithelial tumorigenesis

Abstract In recent years, loss of cell polarity and tissue architecture has been defined as a hallmark of epithelial cancer yet the molecular mechanisms underlying this predisposition are not fully understood. Two distinct polarity programs are required for the correct assembly and function of all mammalian epithelial tissues; apical-basal polarity and non-canonical Wnt/planar cell polarity (1). The Scribble complex functions to establish and maintain both aspects of epithelial polarity and has been shown to mediate cell adhesion, migration and asymmetric cell division (2). In human colorectal, breast, and cervical cancers, expression of the Scribble complex member SCRIB is often mislocalised and deregulated, suggesting that polarity regulators plays a role in maintaining homeostasis of epithelial tissues (1,3). Scrib null mice are neonatal lethal owing to a neural tube closure defect. To determine the tumor suppressive role of Scrib within epithelial tissues, we have employed tissue specific Cre-LoxP technology to deplete Scrib specifically within the prostate, mammary or lung. Scrib depletion in these epithelial tissues results in tumor initiation, supporting previous work in Drosophila (1). We and others have previously shown that SCRIB loss stimulates Ras/MAPK signaling in vitro, suggesting that SCRIB negatively regulates the Ras/MAPK cascade to suppress tumorigenesis (4,5). We now show elevated p-ERK1/2 expression is a common feature of Scrib deficient tumors in vivo. Accordingly, administration of the MEK inhibitor PD0325901 in Scrib+/− mice partially rescued the hyperplastic prostate phenotype. Together, these studies establish that depletion of the polarity regulator Scrib can initiate murine tumorigenesis in several epithelial tissues in a process involving deregulation of the Ras/MAPK cascade. Interestingly, tissue specific activation of oncogenic K-ras in combination with Scrib depletion accelerated tumor progression in both the prostate and lung. Thus, K-ras activation and Scrib deficiency synergise to facilitate tumor growth, demonstrating that Scrib depletion can contribute to tumor progression in the presence of an additional oncogenic mutation. The clinical relevance of this research is highlighted by our observation that SCRIB deregulation strongly correlated with PSA recurrence free survival in human prostate cancer (3). Together, our findings suggest that the polarity network may hold prognostic value or provide a novel route for therapeutic intervention. (1) Humbert PO et al (2008) Oncogene 27(55):6888-6907 (2) Kallay LM et al (2006) J Cell Biochem 99: 647-64 (3) Pearson HB et al (2011) J Clin Invest 121(11):4257-4267 (4) Dow LE et al (2008) Oncogene 27(46):5988-6001 (5) Nagasaka K et al (2010) Oncogene 29(38):5311-5321 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3291. doi:1538-7445.AM2012-3291

Oncogenic KRAS and BRAF activation of the MEK/ERK signaling pathway promotes expression of dual-specificity phosphatase 4 (DUSP4/MKP2) resulting in nuclear ERK1/2 inhibition

Gain-of-function mutations in KRAS and BRAF genes are found in up to 50% of colorectal cancers. These mutations result in the activation of the BRAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. Upon activation, ERK1/2 translocate from the cytoplasm to the nucleus. This process has been shown to be required for the induction of many cellular responses, although the molecular mechanisms regulating ERK nuclear function, especially under oncogenic stimulation, remain to be explored. Herein, we examined the spatiotemporal regulation of ERK1/2 activity upon oncogenic activation of KRAS(G12V) and BRAF(V600E) in normal intestinal epithelial crypt cells (IECs). Results demonstrate that expression of these oncogenes markedly stimulated ERK1/2 activities and morphologically transformed IECs. Importantly however, ERK phosphorylation was not observed in the nucleus, but restricted to the cytoplasm of KRAS(G12V)- and BRAF(V600E)-transformed IECs. The absence of nuclear ERK phosphorylation was due to a vanadate-sensitive phosphatase activity. Nuclear ERK dephosphorylation was found to be tightly correlated with the rapid expression of DUSP4 phosphatase induced in an MEK-dependent manner. In addition, MEK-dependent phosphorylation of T361, T363, S390 and S395 residues highly stabilized DUSP4 protein. Finally, in human colorectal cancer cells, ERK1/2 activities were also confined to the cytoplasm and treatment with pervanadate reactivated ERK1/2 in the nucleus. Accordingly, DUSP4 mRNAs were found to be highly expressed, in an MEK-dependent manner, in all colorectal cancer cells analyzed. These findings indicate that DUSP4 functions as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during intestinal tumorigenesis.

Tumor suppressor p16INK4a controls oncogenic K‐Ras function in human pancreatic cancer cells

Pancreatic cancer is characterized by oncogenic activation of K-Ras and inactivation of the cell cycle inhibitor p16(INK4a) . We previously demonstrated that reintroduction of p16(INK4a) reversed anoikis resistance and clonogenicity of human pancreatic cancer cells, properties commonly attributed to the transforming potential of oncogenic K-Ras. Therefore, we aimed to determine the role of Ras after p16(INK4a) re-expression. Here, we show that restitution of p16(INK4a) in pancreatic cancer cell lines elicits a profound suppression of K-Ras activity. A more detailed analysis in p16(INK4a) reconstituted Capan-1 cells indicated selective reduction of both K-Ras activity and protein stability. Re-expression of K-Ras in p16(INK4a) restituted Capan-1 cells reversed the anoikis-sensitive phenotype and increased colony formation, indicating that K-Ras suppression was required for p16(INK4a) -mediated reversion of the transformed phenotype. Inducible expression of p16(INK4a) in DanG cells confirmed inhibition of K-Ras activity as well as an increase in anoikis susceptibility. Thus, our results delineate a novel functional interaction with defined biological consequences for the two most frequent alterations observed in pancreatic cancer.

Identification of Tumorigenic Cells in KrasG12D-Induced Lung Adenocarcinoma

We established an inducible Kras(G12D)-driven lung adenocarcinoma in CCSP-rtTA/TetO-Cre/LSL-Kras(G12D) mice that enable pursuits of the cellular and molecular processes involved in Kras-induced tumorigenesis. To investigate the cellular origin of this cancer, we first report a strategy using fluorescence-activated cell sorting fractionation that could highly enrich bronchiolar Clara and alveolar type II cells, respectively. The EpCAM(+)MHCII(-) cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than EpCAM(+)MHCII(+) cells (alveolar origin) in primary tumors that had been already initiated with oncogenic Kras activation. In addition, secondary tumors derived from EpCAM(+)MHCII(-) cells showed diversity of tumor locations compared with those derived from EpCAM(+)MHCII(+) cells. In the alveolar region, secondary tumors from EpCAM(+)MHCII(-) cells expressed not only bronchiolar epithelial marker, panCK, but also differentiation marker, proSPC, consistent with the notion that cancer-initiating cells display not only the abilities for self-renewal but also the features of differentiation to generate heterogeneous tumors with phenotypic diversity. Furthermore, high level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM(+)MHCII(-) cells. Therefore, these results suggest that bronchiolar Clara cells are the origin of cells and tumorigenesis for Kras(G12D)-induced neoplasia in the lungs.

Diagnostic accuracy and clinical utility of K-ras mutation analysis on FNA aspirates of pancreatic masses under EUS guidance

Introduction/Objectives: Cytological analysis on specimen obtained by Endoscopic Ultrasound-Fine NeedleAspiration (EUS-FNA) is known as the most accurate technique for the diagnosis of pancreatic adenocarcinoma. Nonetheless, differenzial diagnosis between adenocarcinoma and other pseudotumoral lesions (such as chronic pancreatitis) can be challenging. Almost all pancreatic adenocarcinomas show mutational activation of K-ras oncogene. Analysis of mutations of K-ras on aspirates provided by EUS-FNA could be a helpful method to distinguish neoplasia from benign lesions.

Abstract B14: Attacking the Achilles' heel in the oncogenic K-RAS signaling pathway to block tumorigenesis and metastasis in human cancers

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive tumor growth and metastasis in human cancer. Currently, there are no effective ways to treat human cancers that have oncogenic K-RAS mutations that confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS is an urgent goal and the major challenge in cancer therapy and treatment. Instead of targeting an upstream signaling module such as EGFR/HER2/KRAS/B-RAF/MEK/MAPK/AKT/mT0R, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of seven-in-absentia (SINA), an evolutionarily conserved RING E3 ligase, an essential downstream signaling module, and a critical gatekeeper required for proper ERBB/RAS/RAF signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we conducted preclinical studies to dissect SIAH function in the context of ERBB/K-RAS-mediated tumorigenesis and metastasis in human cancer cells and tumor specimens. We found that: 1) SIAH is a novel biomarker for human cancer, i.e., SIAH specifically decorates proliferating tumor cells (not stromal or immune cells in the tumor microenvironment). SIAH expression is progressively and markedly upregulated as human cancer progresses to the advanced stages in human pancreatic, lung, breast, and prostate cancers; and 2) inhibiting SIAH function is highly effective to abolish tumorigenesis and metastasis in multiple of the most aggressive human cancer cell lines known in the literature that include pancreatic cancer cells (MiaPaCa and Panc-1), lung cancer cells (A549, H727, and UMC11), invasive breast cancer cells (MDA-MB-231, MDA-MB-468, and MDA-MB-435), colorectal cancer cells (SW620 and HCT116) and hormonal-refractory prostate cancer cells (PC-3 and DU-145) in our preclinical studies. These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS activation in human cancer cells. Using anti-SIAH molecules to block K-RAS signaling in human cancer is an excellent example of science going from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials). As a highly evolutionarily conserved E3 ligase and the most downstream gatekeeper of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is uniquely and logically positioned to become a next-generation anti-K-RAS drug target. Our preclinical studies have demonstrated that SIAH-dependent proteolysis is an Achilles' heel for human cancer cells. Albeit at an early preclinical stage, knowledge gained from our study has great promise that has generated many excitements with immediate translational values. Anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies. By attacking the oncogenic ERBB/K-RAS/B-RAF pathway using multipronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR), and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a great position to halt the genesis, progression, and metastasis of the most aggressive and the deadliest forms of human cancers. If successful, we hope to translate our findings to the cancer clinics to treat many cancer patients with metastatic diseases in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B14.

Prevention of K-Ras- and Pten-mediated intravaginal tumors by treatment with camptothecin-loaded PLGA nanoparticles.

Primary squamous cell carcinoma of the vagina is an uncommon disease that often exhibits few symptoms before reaching an advanced stage. Topical intravaginal therapies for resolving precancerous and cancerous vaginal lesions have the potential to be non-invasive and safer alternatives to existing treatment options. Two factors limit the testing of this approach: lack of a preclinical intravaginal tumor model and absence of safe and effective topical delivery systems. In this study, we present both an inducible genetic model of vaginal squamous cell carcinoma in mice and a novel topical delivery system. Tumors were generated via activation of oncogenic K-Ras and inactivation of tumor suppressor Pten in LSL-K-RasG12D/+PtenloxP/loxP mice. This was accomplished by exposing the vaginal epithelium to a recombinant adenoviral vector expressing Cre recombinase (AdCre). As early as 3 weeks after AdCre exposure exophytic masses protruding from the vagina were observed; these were confirmed to be squamous cell carcinoma by histology. We utilized this model to investigate an anticancer therapy based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with camptothecin (CPT); our earlier work has shown that PLGA nanoparticles can penetrate the vaginal epithelium and provide sustained CPT release. Particles were lavaged into the vaginal cavity of AdCre-infected mice. None of the mice receiving CPT nanoparticles developed tumors. These results demonstrate a novel topical strategy to resolve precancerous and cancerous lesions in the female reproductive tract.

Abstract 2335: Oncogenic K-RAS activation promotes cell motility, invasion, tumor growth and cancer metastasis via its most downstream signaling module, the SIAH2-dependent proteolytic machinery, in the highly aggressive and metastatic human cancer cells

Abstract Hyperactivation of EGFR/HER2/K-RAS is often associated with reduced cell/focal adhesion, altered cell junctions, increased cell motility, and enhanced invasion/metastasis in a large percentage of human cancers. Finding novel measures that inhibit activated ERBB/K-RAS signals and, thus, reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream component in the EGFR/HER2/K-RAS pathway, we have targeted the most downstream signaling module, called the SIAH-dependent proteolytic machinery. SIAH/SINA are evolutionarily conserved RING E3 ligases that act as an essential “gatekeeper” signaling module in the RAS signal transduction pathway. We have demonstrated that proper SIAH function is required for K-RAS signal transduction. SIAH is a novel and tumor-specific biomarker for proliferating cells in human cancer. Furthermore, we have shown that SIAH2 inhibition can successfully block both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. These observations highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. To elucidate the molecular mechanism of SIAH2 action in human cancer cells, a proteomic approach was taken to identify novel SIAH2-interacting proteins in three highly aggressive human cancer cell lines carrying K-RAS mutations (MiaPaCa, A549 and MDA-MB-435). From these studies, three focal adhesion LIM domain proteins (LPXN, TRIP6 and FHL2) were found to interact with SIAH2 endogenously in these human cancer cells. Here, we provide extensive biochemical evidence demonstrating that SIAH2 interacts with, ubiquitinates and degrades TRIP6/FHL2/LPXN in a dose-dependent fashion, both exogenously and endogenously. This study clearly demonstrates that LPXN, TRIP6 and FHL2 are bona fide SIAH2 substrates in vivo. Ectopic expression of LPXN/TRIP6/FHL2 successfully rescued the defects in cell motility and tumor growth observed in these SIAH2-deficient human cancer cells. These focal adhesion LIM domain proteins may, therefore, represent a novel class of SIAH2 substrates (logical RAS targets) that function downstream of SIAH2 and modulate focal adhesion, increased cell motility, invasion, tumorigenesis and metastasis in response to active ERBB/K-RAS signals. The identification of SIAH2 as an important regulator of focal adhesion and cell motility downstream of K-RAS pathway may help to explain the well-documented changes in cell adhesion, aggressive tumor growth, invasion and metastasis in response to K-RAS pathway activation in the most lethal forms of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2011-2335

Acute sensitivity of the oral mucosa to oncogenic K-ras.

Mouse models of cancer represent powerful tools for analysing the role of genetic alterations in carcinogenesis. Using a mouse model that allows tamoxifen-inducible somatic activation (by Cre-mediated recombination) of oncogenic K-rasG12D in a wide range of tissues, we observed hyperplasia of squamous epithelium located in moist or frequently abraded mucosa, with the most dramatic effects in the oral mucosa. This epithelium showed a sequence of squamous hyperplasia followed by squamous papilloma with dysplasia, in which some areas progressed to early invasive squamous cell carcinoma, within 14 days of widespread oncogenic K-ras activation. The marked proliferative response of the oral mucosa to K-rasG12D was most evident in the basal layers of the squamous epithelium of the outer lip with hair follicles and wet mucosal surface, with these cells staining positively for pAKT and cyclin D1, showing Ras/AKT pathway activation and increased proliferation with Ki-67 and EdU positivity. The stromal cells also showed gene activation by recombination and immunopositivity for pERK indicating K-Ras/ERK pathway activation, but without Ki-67 positivity or increase in stromal proliferation. The oral neoplasms showed changes in the expression pattern of cytokeratins (CK6 and CK13), similar to those observed in human oral tumours. Sporadic activation of the K-rasG12D allele (due to background spontaneous recombination in occasional cells) resulted in the development of benign oral squamous papillomas only showing a mild degree of dysplasia with no invasion. In summary, we show that oral mucosa is acutely sensitive to oncogenic K-ras, as widespread expression of activated K-ras in the murine oral mucosal squamous epithelium and underlying stroma can drive the oral squamous papilloma–carcinoma sequence. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models

KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function

Fine tuning of Ras activity is widely known as a mechanism to induce different cellular responses. Recently, we have shown that calmodulin (CaM) binds to K-Ras and that K-Ras phosphorylation inhibits its interaction with CaM. In this study we report that CaM inhibits K-Ras phosphorylation at Ser181 by protein kinase C (PKC) in vivo, and this is a mechanism to modulate K-Ras activity and signaling. Although CaM inhibition increased the activation of endogenous K-Ras, PKC inhibition decreased its activation status. We demonstrate that K-Ras phosphorylation decreased susceptibility to p120GAP activity. Accordingly, we also observed that non-phosphorylable K-Ras mutant exhibits a less sustained activation profile and do not efficiently activate AKT at low growth factor doses compared with wild-type K-Ras. It is interesting that the physiological responses induced by K-Ras are affected by this phosphorylation; when K-Ras cannot be phosphorylated it exhibits a remarkably decreased ability to stimulate proliferation in non-saturated serum conditions. Finally, we demonstrate that phosphorylation also regulates oncogenic K-Ras functions, as focus formation capacity, mobility and apoptosis resistance upon adriamycin treatment of cells expressing oncogenic K-Ras that cannot be phosphorylated are highly compromised. Moreover, at low serum concentration proliferation and survival is practically inhibited when cells cannot phosphorylate oncogenic K-Ras. In this condition, K-Ras phosphorylation is essential to ensure a proper activation of mitogen-activated protein kinase and PI3K/AKT pathways. In summary, our findings suggest that the interplay between CaM interaction and PKC phosphorylation is essential to regulate non-oncogenic and oncogenic K-Ras activity and functionality.

Nuclear β‐catenin expression and Wnt signalling: in defence of the dogma

Inactivation of the APC tumour suppressor gene represents the rate-limiting event in colorectal cancer. Loss of APC function leads to constitutive activation of the canonical Wnt-beta-catenin signalling pathway, thus resulting into a broad spectrum of cellular defects, ranging from stem cell self-renewal and differentiation, apoptosis, migration and proliferation. Recently, Phelps et al presented an alternative model where loss of APC does not primarily result in Wnt signalling activation but rather involves the transcriptional co-repressor CtBP1. According to this alternative scenario, oncogenic KRAS activation represents a conditio sine qua non for nuclear beta-catenin translocation and Wnt activation. In a recent issue of the Journal of Pathology, Obrador-Hevia and collaborators reaffirmed the broadly accepted textbook model by showing the presence of nuclear beta-catenin in both the presence and, more often, the absence of KRAS mutations.

Requirement of the NF-κB Subunit p65/RelA for K-Ras–Induced Lung Tumorigenesis

K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Because therapy directly targeting the activity of oncogenic Ras has been unsuccessful, a different approach for novel therapy design is to identify critical Ras downstream oncogenic targets. Given that oncogenic Ras proteins activate the transcription factor NF-kappaB, and the importance of NF-kappaB in oncogenesis, we hypothesized that NF-kappaB would be an important K-Ras target in lung cancer. To address this hypothesis, we generated a NF-kappaB-EGFP reporter mouse model of K-Ras-induced lung cancer and determined that K-Ras activates NF-kappaB in lung tumors in situ. Furthermore, a mouse model was generated where activation of oncogenic K-Ras in lung cells was coupled with inactivation of the NF-kappaB subunit p65/RelA. In this model, deletion of p65/RelA reduces the number of K-Ras-induced lung tumors both in the presence and in the absence of the tumor suppressor p53. Lung tumors with loss of p65/RelA have higher numbers of apoptotic cells, reduced spread, and lower grade. Using lung cell lines expressing oncogenic K-Ras, we show that NF-kappaB is activated in these cells in a K-Ras-dependent manner and that NF-kappaB activation by K-Ras requires inhibitor of kappaB kinase beta (IKKbeta) kinase activity. Taken together, these results show the importance of the NF-kappaB subunit p65/RelA in K-Ras-induced lung transformation and identify IKKbeta as a potential therapeutic target for K-Ras-induced lung cancer.