krasV12 Expression Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Characterization of Aneuploid Populations with Trisomy 7 and 20 Derived from Diploid Human Colonic Epithelial Cells

Chromosomal instability leading to aneuploidy occurs in most sporadic colorectal cancers (CRCs) and is believed to be an early driving force in disease progression. Despite this observation, the cellular advantages conferred by these cytogenetic alterations are poorly understood. Here, we provide evidence that serum-free passage of originally diploid, immortalized human colonic epithelial cells (HCECs) gave rise to the acquisition of trisomy 7 (+7), an aneuploidy detected in more than 40% of colorectal adenomas. These cells remain diploid under long-term growth in 2% serum conditions. Analysis by GTG banding and fluorescent in situ hybridization detected no rare preexisting +7 cell in the original population, suggesting a conversion of diploid cells to an aneuploid state. The acquisition of +7 also precedes loss or truncation of the adenomatosis polyposis coli gene as both diploid and +7 cells express full-length, functional protein. Coculturing of fluorescent-labeled cells demonstrate that +7 HCECs have a growth advantage over diploid cells in serum-free conditions. Defects in cell migration and aberrant regulation of the epidermal growth factor receptor, located on chromosome 7p, are also detected in +7 HCECs. Interestingly, knockdown of TP53 and expression of K-RasV12 in +7 HCECs resulted in the emergence of trisomy 20, another nonrandom aneuploidy observed in ∼85% of CRC. In summary, we describe isogenic colonic epithelial cells that represent cytogenetic changes occurring frequently in sporadic CRC. The emergence and characterization of trisomy 7 and 20 demonstrate that these HCECs may serve as unique human cell-based models to examine the effects of chromosomal instability in CRC progression.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via phosphorylation of SRp30a

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non‐small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K‐RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage‐independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.

Alternative Splicing of Caspase 9 Is Modulated by the Phosphoinositide 3-Kinase/Akt Pathway via Phosphorylation of SRp30a

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four-exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathologic classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, epidermal growth factor receptor (EGFR) overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of phosphoinositide 3-kinase or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227, and 234. Taken together, our findings show that oncogenic factors activating the phosphoinositide 3-kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.

Rapid and Robust Transgenic High-Grade Glioma Mouse Models for Therapy Intervention Studies

To develop a transgenic mouse model of glioma that can be conveniently used for testing therapy intervention strategies. High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Typical for high-grade glioma is that glioma cells infiltrate extensively into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. High-grade gliomas were induced by stereotactic intracranial injection of lentiviral GFAP-Cre or CMV-Cre vectors into compound LoxP-conditional mice, resulting in K-Ras(v12) expression and loss of p16(Ink4a)/p19(Arf) with or without concomitant loss of p53 or Pten. Tumors reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. Especially, CMV-Cre injection into p53;Ink4a/Arf;K-Ras(v12) mice resulted in high-grade glioma with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up was accomplished by noninvasive bioluminescence imaging, and the practical utility for therapy intervention was shown in a study with temozolomide. We have developed a realistic high-grade glioma model that can be used with almost the same convenience as traditional xenograft models, thus allowing its implementation at the forefront of preclinical evaluation of new treatments.

Abstract 4187: A rapid and robust transgenic high-grade glioma mouse model for therapy-intervention studies

Abstract High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Glioma cells extensively infiltrate into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. We here present a novel transgenic mouse model of high-grade glioma that can be conveniently used for testing therapy-intervention strategies. High-grade gliomas were induced by stereotactic intracranial injection of self-deleting lentiviral GFAP-Cre or CMV-Cre vectors into adult immune-competent compound LoxP-conditional mice, resulting in localized expression of K-Rasv12 in conjunction with loss of p16Ink4a/p19Arf with or without concomitant loss of p53 or Pten. Tumors have been characterized by histology, immune-histochemistry and magnetic resonance imaging and reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. We are currently assessing which molecular alterations in cell signaling pathways have occurred. Especially CMV-Cre injection into p53;Ink4a/Arf;K-Rasv12 mice resulted in grade IV (glioblastoma multiforme) with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up of tumor growth can be accomplished conveniently by non-invasive bioluminescence imaging and the practical utility for therapy-intervention has been demonstrated in a study with temozolomide. Temozolomide delayed tumor progression modestly but significantly in line with what is observed in patients. Importantly, this novel model can be used with almost the same convenience as traditional xenograft models, thus allowing the implementation of realistic high grade glioma models at the forefront of preclinical evaluation. We expect that this model will be particularly useful to assess the potential clinical utility of agents that may augment temozolomide cytotoxicity, such as PARP-inhibitors or Wee1 kinase inhibitors, but also for agents targeting activated RAS and/or PI3K-Akt-mTor signaling pathways. 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 4187.

Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: The role of p53 and p21

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.

Ras Promotes Growth by Alternative Splicing-Mediated Inactivation of the KLF6 Tumor Suppressor in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown. In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2. In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation. Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.

Histone Deacetylase Inhibitors Induce Cell Death Selectively in Cells That Harbor Activated kRasV12: The Role of Signal Transducers and Activators of Transcription 1 and p21

Histone deacetylase (HDAC) inhibitors (HDACi) show potent and selective antitumor activity despite the fact that they induce histone hyperacetylation in both normal and tumor cells. In this study, we showed that the inducible expression of kRasV12 in nontransformed intestinal epithelial cells significantly lowered the mitochondrial membrane potential (MMP) and sensitized cells to HDACi-induced apoptosis. Consistent with our finding that colon cancer cell lines with mutant Ras have reduced expression of signal transducers and activators of transcription 1 (STAT1), we showed that inducible expression of mutant Ras markedly decreased both basal and inducible expression of STAT1, a transcription factor with tumor suppressor activity. To investigate whether reduced expression of STAT1 in cells that harbor mutant Ras contributes to their increased sensitivity to HDACi, we silenced the expression of STAT1 in HKe-3 cells with small interfering RNA. Despite the fact that silencing of STAT1 was not sufficient to alter the MMP, STAT1 deficiency, like Ras mutations, sensitized cells to apoptosis induced by HDACi. We showed that the induction of p21 by HDACi was significantly impaired in HKe-3 cells with silenced STAT1 expression and showed that the ability of butyrate to activate p21 transcription was diminished in STAT1-deficient HKe-3 cells. Finally, we used cells with targeted deletion of p21 to confirm that p21 protects cells from butyrate-induced apoptosis, strongly suggesting that in these cells STAT1 deficiency promotes butyrate-induced apoptosis through impaired induction of p21. Our data therefore establish that Ras mutations, and consequent reduction in the expression of STAT1, underlie the increased susceptibility of transformed cells to undergo apoptosis in response to treatment with inhibitors of HDAC activity.

P2-115: A Causal Relationship between Oncogenic K-Ras Expression and NF-kappaB Activation in Lung Cancer

In lung adenocarcinomas, the Ras signaling pathway is often constitutively activated, and K-Ras activating mutations are widespread. Given the failure in clinical trials of compounds that target the biological activity of the Ras proteins, the key molecular pathways downstream of K-Ras involved in promoting lung tumorigenesis need to be identified in order to provide better targets for lung cancer therapy. We hypothesize that a crucial target of oncogenic K-Ras in the lung is nuclear factor kappa B (NFχB). NFχB is a dimeric transcription factor that promotes proliferation, mediates resistance to apoptosis, and is abnormally activated in a large variety of human malignancies. In order to analyze the role of NFχB downstream of K-Ras in the airway epithelium, a tetracycline-inducible system was used to stably and inducibly express either the wild type form of K-Ras (K-RasWT) or the oncogenic form (K-RasV12) in the lung adenocarcinoma H1703 cell line. We also analyzed primary human bronchial cells that were either immortalized (AALEB) or immortalized and transformed with K-RasV12 (AALEB-K-Ras). NFχB activity in these cells was analyzed by western blotting, eletrophoretic mobility shift assays, luciferase assays and expression of NFχB target genes by real-time PCR. Expression of K-Ras-WT or K-RasV12 in H1703 cells after tetracycline induction was accompanied by both increased expression of NFχB target genes, and increased NFχB DNA binding activity. DNA binding was mediated primarily by p50 and p65 NFχB subunits. Although NFχB luciferase reporter activity was detectable prior to K-Ras expression, expression of K-RasV12, but not K-Ras-WT, enhanced NFχB activity. Interestingly, K-RasV12 does not activate NFχB by the canonical NFχB activation pathway; in both H1703 and AALEB cells expressing K-RasV12, there was no increase in IkappaBalpha (IχBα) phosphorylation/degradation. Nonetheless, activation seems to be dependent on IkappaB kinase (IKK) activity for the following reasons: (1) When compared to AALEB cells, AALEB K-Ras cells displayed increased IKK phosphorylation levels; and (2) specific inhibition of IKKβ activity in H1703 cells by the novel pyridine derivative compound 65-1942 (obtained under MTA agreement with Bayer) suppressed NFχB activity. Finally, as no increase in p65 phosphorylation at S536 was observed in either cell type expressing K-RasV12, the mechanism of NFχB activation does not involve direct phosphorylation of p65 at S536 by IKK. We have determined a causal link between K-RasV12 expression and NFχB activation in lung epithelial cells in vitro. NFχB in these cells is not activated either by the canonical pathway of IχBα phosphorylation/degradation or by p65 phosphorylation at S536. Although the specific mechanism of activation is being investigated both in H1703 and in primary AALEB cells, we have shown that it depends on IKK. IKKβ inhibitors, therefore, represent a potentially promising new therapeutic direction in lung cancer. Studies are underway to confirm their efficacy in a pre-clinical lung cancer mouse model.

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt