Manish Gala

Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells.

Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1α knockdown DLD-1 colon cancer cells (DLD-1HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1α (DLD-1HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1HIF-kd but not DLD-1HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-κB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1HIF-kd but not DLD-1HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted.

Hypoxic Regulation of Vascular Endothelial Growth Factor through the Induction of Phosphatidylinositol 3-Kinase/Rho/ROCK and c-Myc

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1α was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between –418 and –223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between –300 and –251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at –271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1α but instead requires the activation of PI3K/Rho/ROCK and c-Myc.

Hereditary Colon Cancer Syndromes

Colon cancer is associated with a family history in up to 25% of cases. As many as 5% are associated with an established hereditary syndrome, demonstrating the profound influence of inheritable genetic mechanisms in the development of this disease. These syndromes confer a diverse spectrum of risk, age of presentation, endoscopic and histological findings, extracolonic manifestations, and modes of inheritance. As the molecular characteristics of these disorders become better described, enhanced genotype-phenotype correlations may offer a more targeted approach to diagnosis, screening, and surveillance. While the strategies for diagnosis and management of familial adenomatous polyposis (FAP) and Lynch syndrome are more established, the approach to newly recognized syndromes such as MUTYH-associated polyposis (MAP) and hyperplastic polyposis syndromes continues to evolve. Effective cancer prevention in affected individuals and at-risk family members first requires timely recognition of these hereditary colon cancer syndromes followed by integration of genetic testing and clinical examinations.

Genome-wide association study of colorectal cancer identifies six new susceptibility loci

Genetic susceptibility to colorectal cancer is caused by rare pathogenic mutations and common genetic variants that contribute to familial risk. Here we report the results of a two-stage association study with 18,299 cases of colorectal cancer and 19,656 controls, with follow-up of the most statistically significant genetic loci in 4,725 cases and 9,969 controls from two Asian consortia. We describe six new susceptibility loci reaching a genome-wide threshold of P<5.0E-08. These findings provide additional insight into the underlying biological mechanisms of colorectal cancer and demonstrate the scientific value of large consortia-based genetic epidemiology studies.

Molecular Pathways: Aspirin and Wnt Signaling-A Molecularly Targeted Approach to Cancer Prevention and Treatment

The anti-inflammatory properties of aspirin have resulted in its widespread use as an analgesic, antipyretic, and cardioprotective agent. Beyond these applications, multiple observational studies and randomized controlled trials have demonstrated a chemopreventative role for aspirin, particularly in the development of colorectal neoplasia. Given the critical importance of Wnt dysregulation in colorectal carcinogenesis, the interplay between aspirin and canonical Wnt signaling has become a focus of investigation. These studies have illuminated our understanding of the anticancer mechanisms of aspirin, yielding the identification of potential biomarkers for which aspirins chemopreventative efficacy can be safely optimized into routine clinical practice and providing leads into the discovery of novel preventive and therapeutic targets. In this review, we summarize key experimental and clinical studies of this interaction, as well as highlighting future strategies to advance their clinical translation. Clin Cancer Res; 21(7); 1543–8. ©2014 AACR.

Identification of a common variant with potential pleiotropic effect on risk of inflammatory bowel disease and colorectal cancer

Although genome-wide association studies (GWAS) have separately identified many genetic susceptibility loci for ulcerative colitis (UC), Crohns disease (CD) and colorectal cancer (CRC), there has been no large-scale examination for pleiotropy, or shared genetic susceptibility, for these conditions. We used logistic regression modeling to examine the associations of 181 UC and CD susceptibility variants previously identified by GWAS with risk of CRC using data from the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. We also examined associations of significant variants with clinical and molecular characteristics in a subset of the studies. Among 11794 CRC cases and 14190 controls, rs11676348, the susceptibility single nucleotide polymorphism (SNP) for UC, was significantly associated with reduced risk of CRC (P = 7E-05). The multivariate-adjusted odds ratio of CRC with each copy of the T allele was 0.93 (95% CI 0.89-0.96). The association of the SNP with risk of CRC differed according to mucinous histological features (P heterogeneity = 0.008). In addition, the (T) allele was associated with lower risk of tumors with Crohns-like reaction but not tumors without such immune infiltrate (P heterogeneity = 0.02) and microsatellite instability-high (MSI-high) but not microsatellite stable or MSI-low tumors (P heterogeneity = 0.03). The minor allele (T) in SNP rs11676348, located downstream from CXCR2 that has been implicated in CRC progression, is associated with a lower risk of CRC, particularly tumors with a mucinous component, Crohns-like reaction and MSI-high. Our findings offer the promise of risk stratification of inflammatory bowel disease patients for complications such as CRC.

Inhibition of cell transformation by sulindac sulfide is confined to specific oncogenic pathways

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce the risk of colorectal cancer (CRC). They are also known to induce the regression of colorectal adenomas, which are precursors to CRC. Despite these evidences, the exact mechanism by which NSAIDs exerts its anti-oncogenic effect is not completely understood. Using a focus formation assay, here we show that sulindac sulfide, a NSAID, specifically inhibits cell transformation mediated by oncogenic Ha-Ras, but not by other established oncogene products such as v-Src, Galpha12, and Galpha13. Our results suggest that the ability of sulindac sulfide to suppress transformation is confined to specific oncogenic pathways. Further studies of the sulindac-resistant oncogenic pathways may lead to identification of novel therapeutic agents that are effective in the prevention or treatment of CRC.

Adrenomedullin is a therapeutic target in colorectal cancer

The KRAS oncogene influences angiogenesis, metastasis and chemoresistance in colorectal cancers (CRCs), and these processes are all enhanced in hypoxic conditions. To define functional activities of mutant KRAS in a hypoxic microenvironment, we first performed cDNA microarray experiments in isogenic DKs5 and DKO3 colon cancer cell lines that differ only by their expression of mutant KRAS (K‐rasD13). Adrenomedullin (ADM) was identified as one of the most significantly upregulated genes in DKs5 cells that express the KRAS oncogene in hypoxia (3.2‐fold, p = 1.47 × 10−5). Ectopic expression of mutant KRAS (K‐rasV12) in Caco‐2 cells (K‐rasWT) induced ADM, whereas selective knockdown of mutant KRAS alleles (K‐rasD13 or K‐rasV12) in HCT116, DLD1 and SW480 colon cancer cells suppressed the expression of ADM in hypoxia. Knockdown of ADM in colon tumor xenografts blocked angiogenesis and stimulated apoptosis, resulting in tumor suppression. Furthermore, ADM also regulated colon cancer cell invasion in vitro. Among 56 patients with CRC, significantly higher expression levels of ADM were observed in samples harboring a KRAS mutation. Collectively, ADM is a new target of oncogenic KRAS in the setting of hypoxia. This observation suggests that therapeutic targets may differ depending upon the specific tumor microenvironment.

Common genetic variation and survival after colorectal cancer diagnosis: a genome-wide analysis

Genome-wide association studies have identified several germline single nucleotide polymorphisms (SNPs) significantly associated with colorectal cancer (CRC) incidence. Common germline genetic variation may also be related to CRC survival. We used a discovery-based approach to identify SNPs related to survival outcomes after CRC diagnosis. Genome-wide genotyping arrays were conducted for 3494 individuals with invasive CRC enrolled in six prospective cohort studies (median study-specific follow-up = 4.2-8.1 years). In pooled analyses, we used Cox regression to assess SNP-specific associations with CRC-specific and overall survival, with additional analyses stratified by stage at diagnosis. Top findings were followed-up in independent studies. A P value threshold of P < 5×10(-8) in analyses combining discovery and follow-up studies was required for genome-wide significance. Among individuals with distant-metastatic CRC, several SNPs at 6p12.1, nearest the ELOVL5 gene, were statistically significantly associated with poorer survival, with the strongest associations noted for rs209489 [hazard ratio (HR) = 1.8, P = 7.6×10(-10) and HR = 1.8, P = 3.7×10(-9) for CRC-specific and overall survival, respectively). No SNPs were statistically significantly associated with survival among all cases combined or in cases without distant-metastases. SNPs in 6p12.1/ELOVL5 were associated with survival outcomes in individuals with distant-metastatic CRC, and merit further follow-up for functional significance. Findings from this genome-wide association study highlight the potential importance of genetic variation in CRC prognosis and provide clues to genomic regions of potential interest.

Genetics of the Serrated Pathway to Colorectal Cancer

Accounting for ~15% of all colorectal cancers (CRCs), the serrated pathway represents an alternate mechanism of colorectal carcinogenesis that yields microsatellite stable (MSS) tumors and the overwhelming fraction of “sporadic” microsatellite instability high (MSI-H) tumors. Moreover, the MSI-H tumors derived from the serrated pathway are more common of the two, and frequently display excessive CpG island promoter hypermethylation (CIMP-high; Table 1). This promoter hypermethylation results in epigenetic silencing of a large number of tumor-suppressor genes, including MLH1, which causes the associated MSI-H phenotype. Somatic mutations typically include activating mutations in BRAF (V600E), and less commonly KRAS or aberrant EGFR activation, which occur during the early stages of serrated polyp development. In contrast to tubular adenomas, bilallelic inactivation of APC is not an initiating event in this pathway (Figure 1). Despite this molecular understanding and the development of novel drugs to target them, additional treatments are still needed given the inferior outcomes observed in BRAF-mutated colon cancers within the context of their microsatellite status.1 Figure 1 Serrated Pathway to Colorectal Cancer. Schematic comparing the mutational changes involved from normal mucosa to colorectal cancer. The top half represents the conventional pathway to colorectal cancer, with the most frequent mutational events described. ... Table 1 Important acronyms and definitions Discovery of high-risk genetic variants for this pathway represents a promising strategy to identify additional therapeutic targets for this subset of colorectal cancers. Epidemiologic data from families of serrated polyposis patients strongly suggest a heritable predisposition exists toward serrated colorectal carcinogenesis. First-degree relatives of serrated polyposis patients are at significantly higher elevated risk of developing serrated polyps themselves. In addition, elevated pleiotropic cancer risks are present in these families. An Australian cohort demonstrated that relatives are at an increased risk of pancreatic cancer.2 First-degree relatives of those with MSI-H CRC who do not have Lynch syndrome or serrated polyposis are at increased risk (standardized incidence ratios) of developing stomach, ovarian, and liver cancers.3 Through exome sequencing of individuals who develop multiple sessile serrated polyps and/or serrated polyposis, we recently identified novel high-risk variants for the serrated pathway.4 We demonstrated that such individuals are approximately fourfold enriched for rare, germline loss-of-function (LoF) mutations (defined as nonsense and splice-site mutations) in genes responsible for oncogene-induced senescence (OIS) mechanisms. OIS is a tumor-suppressive mechanism that is activated by the replicative and metabolic stress caused by oncogenic transformation. This hypothesis was based on the observations from genetic mouse models of serrated neoplasia in which the BRAF V600E mutation or activating KRAS mutation was alone sufficient to induce serrated neoplasia in the long-term; however, in the short-term, OIS barriers prevented rapid tumorigenesis.5, 6, 7 Concurrent inactivation of these OIS mechanisms with activating BRAF/KRAS mutations greatly expedited serrated neoplasia formation. In humans, activation of these critical OIS pathways (ATM–ATR DNA damage pathway and p16-RB pathway) has been previously demonstrated to be relevant in colonic precursor lesions in addition to lesions in other tissue types. Several of the OIS genes identified in serrated polyposis patients in this study (ATM, RBL1, and XAF1) have been previously implicated in human or animal studies of colorectal carcinogenesis. We next demonstrated that the remaining patients with serrated polyposis (who do not have an obvious loss-of-function mutation) actually harbor deleterious variants in genes previously unassociated, but critical to these OIS mechanisms. Cross-referencing all rare LoF mutations found in patients with an orthogonal database (not dependent on senescence characteristics) of all genes implicated in cancer by genome-wide association studies, we discovered two unrelated individuals with identical nonsense mutations in RNF43, a gene frequently mutated in mucinous neoplasms of the pancreas and stomach that encodes for a negative regulator of Wnt signaling through Wnt receptor endocytosis. This enrichment in cases of serrated polyposis was significant over controls with sizeable effect sizes (odds ratio 460, P=6.8 × 10−5). Analysis of publicly available microarrays of sporadic serrated polyps and tubular adenomas, we found RNF43 to be significantly downregulated in the serrated pathway. Through functional experiments in pancreatic duct cells harboring the KRAS G12D oncogene, we demonstrated that silencing of RNF43 impaired ATR–ATM DNA damage signaling in response to UV radiation, as evident by impaired phosphorylation of Chk1 and p53. Subsequently, another group has further generalized the importance of RNF43 to the development of sporadic MSI-H colorectal cancers.8 Performing whole-exome sequencing on 185 formalin-fixed, paraffin-embedded colon cancers from two Harvard cohorts, Giannakis et al. discovered deleterious somatic mutations in RNF43 to be present in 18.9% of these cancers, in addition to being frequently mutated in endometrial cancers. Interestingly, 50% of the deleterious mutations discovered were frameshift mutations occurring at microsatellite loci within the gene. Validation of a small subset of tumors by next-generation sequencing or Sanger sequencing demonstrated an overall accuracy of 97% for the RNF43 mutation calls made by software. To replicate these results, the authors reanalyzed 222 colorectal cancer cases from The Cancer Genome Atlas.9 RNF43 mutations were present reliably in 17.6% of cases, and 49 cases were from the initial publication. The discrepancy between these results and previously published analyses of the TCGA data set may be attributable to newer algorithms in the detection of significant insertion and deletion events, which continue to be an ongoing challenge in their accuracy compared with single-nucleotide polymorphisms. Under prior methodologies, many true mutations at microsatellite sites were falsely discarded as errors due to their resemblance to sequencing artifacts caused by polymerase slippage during the exome enrichment step. Consistent with the importance of this RNF43 in the serrated pathway, these mutations were particularly enriched in those tumors with MSI-H status, occurring in ~80% of this subset of colorectal cancers (P<2.2 × 10−16, Fishers exact test). Complementary to our initial microarray comparisons of sporadic polyps from the serrated and conventional pathway of colorectal carcinogenesis, somatic RNF43 mutations appeared mutually exclusive with deleterious APC mutations. The results from these studies have clinical consequences for epidemiology, genetic testing, and treatment strategies. First, these mutations provide firm genetic support for the multiple cancer risks observed in families and first-degree relatives of those afflicted with serrated polyposis. Many of the genes found in the primary mechanisms of OIS contain tumor-suppressor genes with already established pleiotropic effects. The discoveries of germline and somatic RNF43 mutations in serrated lesions provide additional genetic evidence of such pleiotropy as the gene is found frequently mutated in gastric, pancreatic, ovarian, and endometrial cancers. The germline mechanisms disrupted in serrated polyposis patients and the somatic mutations found in sporadic MSI-H tumors should promote clinical trials of newly developed chemotherapeutics for sporadic serrated colorectal tumors. Clinical trials of poly(ADP-ribose) polymerase inhibitors have demonstrated promise in individuals with germline mutations in upstream DNA damage repair pathways, notably those with BRCA1 or BRCA2 mutations, for individuals with breast or ovarian cancers.10, 11 Such agents push tumor cells with deficiencies in DNA repair pathways into mitotic catastrophe with the accumulation of double-stranded DNA breaks. The presence of germline and somatic RNF43 mutations also confers the possibility of using an additional class of therapeutic agents. Recently, porcupine inhibitors, a new class of drugs that impair Wnt secretion, have demonstrated efficacy in slowing the tumor growth of pancreatic cancer cell lines that harbor deleterious somatic RNF43 mutations.12 Despite the initial focus upon epigenetics due to the observed CIMP-high phenotype, the genetics of serrated neoplasia have a critical role in determining disease risk and therapeutic strategies. Additional experiments with larger cohorts of serrated polyposis will likely reveal additional high-risk genes given the broad genetic heterogeneity evident, leading to discovery of additional novel targets. In contrast to other tumor types with hypermethylation, the search for the inciting somatic mechanisms that trigger the widespread epigenetic changes observed in these cancers remains uncertain. These efforts over the upcoming years have the potential to further change preventative and therapeutic approaches toward colon cancer.