Novel recurrently mutated genes in African American colon cancers.

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers.

Aberrant glycosylation is a pathological alteration that is widespread in colon cancer, and usually accompanies the onset and progression of the disease. To date, the molecular mechanisms underlying aberrant glycosylation remain largely unknown. In this study, we identify somatic and germ-line mutations in the gene encoding for polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) in individuals with colon cancer. Biochemical analyses demonstrate that each of the 8 GALNT12 mutations identified inactivates the normal function of the GALNT enzyme in initiating mucin type O-linked protein glycosylation. Two of these inactivating GALNT12 mutations were identified as acquired somatic mutations in a set of 30 microsatellite stable colon tumors. Relative to background gene mutation rates, finding these somatic GALNT12 mutations was statistically significant at P < 0.001. Six additional inactivating GALNT12 mutations were detected as germ-line changes carried by patients with colon cancer; however, no inactivating variants were detected among cancer-free controls (P = 0.005). Notably, in 3 of the 6 individuals harboring inactivating germ-line GALNT12 mutations, both a colon cancer and a second independent epithelial cancer had developed. These findings suggest that genetic defects in the O-glycosylation pathway in part underlie aberrant glycosylation in colon cancers, and they contribute to the development of a subset of these malignancies.

Colorectal cancer: mutations in a signalling pathway.

Protein kinases are enzymes that are important for controlling cellular growth and invasion, and their malfunction is implicated in the development of some tumours. We analysed human colorectal cancers for genetic mutations in 340 serine/threonine kinases and found mutations in eight genes, including in three members of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for therapeutic intervention.

Integrated analysis of homozygous deletions, focal amplifications, and sequence alterations in breast and colorectal cancers.

We have performed a genome-wide analysis of copy number changes in breast and colorectal tumors using approaches that can reliably detect homozygous deletions and amplifications. We found that the number of genes altered by major copy number changes, deletion of all copies or amplification to at least 12 copies per cell, averaged 17 per tumor. We have integrated these data with previous mutation analyses of the Reference Sequence genes in these same tumor types and have identified genes and cellular pathways affected by both copy number changes and point alterations. Pathways enriched for genetic alterations included those controlling cell adhesion, intracellular signaling, DNA topological change, and cell cycle control. These analyses provide an integrated view of copy number and sequencing alterations on a genome-wide scale and identify genes and pathways that could prove useful for cancer diagnosis and therapy.

The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancers.

MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR-126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR-126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR-126 gene targets revealed p85beta, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal, as one of the potential substrates. Restoration of miR-126 in cancer cells induced a > or =3-fold reduction in p85beta protein levels, with no concomitant change in p85alpha, a gene that is functionally related to p85beta but not a supposed target of miR-126. Additionally, using reporter constructs, we show that the p85beta-3' untranslated region is directly targeted by miR-126. Furthermore, this miR-126 mediated reduction of p85beta was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR-126 down-regulation together with an increase in the p85beta protein level. Taken together, we propose that miR-126 regulates PI3K signaling partly by targeting p85beta, and that the loss of miR-126 may provide a selective growth advantage during colon carcinogenesis.

Mutational inactivation of TGFBR2 in microsatellite unstable colon cancer arises from the cooperation of genomic instability and the clonal outgrowth of transforming growth factor beta resistant cells.

The mutational inactivation of transforming growth factor beta receptor type II (TGFBR2) occurs in approximately 30% of colon cancers and promotes the formation of colon cancer by inhibiting the tumor suppressor activity of the TGFB signaling pathway. TGFBR2 mutations occur in >90% of microsatellite unstable (MSI) colon cancers and affect a polyadenine tract in exon 3 of TGFBR2, called BAT-RII, which is vulnerable to mutation in the setting of DNA mismatch repair (MMR) system deficiency. In light of the vulnerable nature of the BAT-RII tract in the setting of MMR inactivation and the favorable effects of TGFBR2 inactivation in colon cancer, analysis of TGFBR2 inactivation provides an opportunity to assess the roles of genomic instability vs. clonal selection in cells acquiring TGFBR2 BAT-RII tract mutations in MSI colon cancer formation. The contribution of genomic instability and/or clonal evolution to the mutational inactivation of TGBFR2 in MSI colon cancers has not been studied in a systematic way that would allow a determination of the relative contribution of these two mechanisms in the formation of MSI colon cancer. It has not been demonstrated whether the BAT-RII tract mutations are strictly a consequence of the BAT-RII region being hypermutable in the setting of MMR deficiency or if the mutations are rather a consequence of clonal selection pressure against the TGFB receptor. Through the use of defined cell line systems, we show that both genomic instability and clonal selection of TGFB resistant cells contribute to the high frequency of TGFBR2 mutations in MSI colon cancer.

A novel mechanism of resistance to epidermal growth factor receptor antagonism in vivo.

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.

Blockade of EGFR and ErbB2 by the novel dual EGFR and ErbB2 tyrosine kinase inhibitor GW572016 sensitizes human colon carcinoma GEO cells to apoptosis.

Coexpression of the epidermal growth factor receptor (EGFR) family receptors is found in a subset of colon cancers, which may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. Recently, efforts have been made to develop novel 4-anilinoquinazoline and pyridopyrimidine derivatives to inhibit EGFR and ErbB2 kinases simultaneously. In this study, we tested the efficacy of a novel reversible dual inhibitor GW572016 compared with the selective EGFR and ErbB2 tyrosine kinase inhibitors (TKI) AG1478 and AG879 and their combination, using the human colon adenocarcinoma GEO mode. GEO cells depend on multiple ErbB receptors for aberrant growth. A synergistic effect on inhibition of cell proliferation associated with induction of apoptosis was observed from the combination of AG1478 and AG879. Compared with AG1478 or AG879, the single TKI compound GW572016 was a more potent inhibitor of GEO cell proliferation and was able to induce apoptosis at lower concentrations. Western blot analysis revealed that AG1478 and AG879 were unable to suppress both EGFR and ErbB2 activation as well as the downstream mitogen-activated protein kinase (MAPK) and AKT pathways as single agents. In contrast, GW572016 suppressed the activation of EGFR, ErbB2, MAPK, and AKT in a concentration-dependent manner. Finally, in vivo studies showed that GW572016 treatment efficiently blocked GEO xenograft growth at a dose range of 30 to 200 mg/kg with a twice-daily schedule. In summary, our study indicates that targeting both EGFR and ErbB2 simultaneously could enhance therapy over that of single agents directed at EGFR or ErbB2 in cancers that can be identified as being primarily heterodimer-dependent.

Colon carcinoma cells harboring PIK3CA mutations display resistance to growth factor deprivation induced apoptosis.

PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival.

Colon cancer secreted protein-2 (CCSP-2), a novel candidate serological marker of colon neoplasia.

Cancers of the colon and rectum are the second leading cause of cancer death among adult Americans. When detected at early stages, colon cancer is highly curable. Colonoscopy, an effective but invasive screening test, has been limited in its public acceptance. The goal of this study was to identify novel serum markers of colon cancers and precancerous colon adenomas as potential candidates for noninvasive detection of early colon neoplasms. Employing expression microarrays, we identified colon cancer secreted protein-2 (CCSP-2) as a novel transcript whose expression is generally absent in normal colon and other normal body tissues, but that is induced an average of 78-fold in Stage II, III, and IV colon cancers, as well as in colon adenomas and colon cancer cell lines. These findings were validated by real-time PCR analysis in an independent panel of colon cancer cases. Moreover, CCSP-2 was shown to encode a secreted protein that circulates stably and is detectable in the blood of mice bearing human cancer xenografts transfected with epitope-tagged CCSP-2. As a novel secreted protein that is markedly induced in colon adenomas and cancers, CCSP-2 is a novel candidate for development as a diagnostic serum marker of early stage colon cancer.

Silence of chromosomal amplifications in colon cancer.

Oncogene activation by gene amplification is a major pathogenetic mechanism in human cancer. Using comparative genomic hybridization, we determined that metastatic human colon cancers commonly acquire numerous extra copies of chromosome arms 7p, 8q, 13q, and 20q. We then examined the consequence of these amplifications on gene expression using DNA microarrays. Of 55,000 transcripts profiled, 2,146 were determined to map to one of the four common colon cancer amplicons and to also be expressed in normal or malignant colon tissues. Of these, only 81 transcripts (3.8%) demonstrated a 2-fold increase over normal expression among cancers bearing the corresponding chromosomal amplification. Chromosomal amplifications are common in colon cancer metastasis, but increased expression of genes within these amplicons is rare.

PMEPA1, a transforming growth factor-beta-induced marker of terminal colonocyte differentiation whose expression is maintained in primary and metastatic colon cancer.

To identify potential effectors of transforming growth factor (TGF)-beta-mediated suppression of colon cancer, we used GeneChip expression microarrays to identify TGF-beta-induced genes in VACO 330, a nontransformed TGF-beta-sensitive cell line derived from a human adenomatous colon polyp. PMEPA1 was identified as a gene highly up-regulated by TGF-beta treatment of VACO 330. Northern blot analysis confirmed TGF-beta induction of PMEPA1 in VACO 330, as well as a panel of three other TGF-beta-sensitive colon cell lines. PMEPA1 induction could be detected as early as 2 h after TGF-beta treatment and was not inhibited by pretreatment of cells with cycloheximide, suggesting that PMEPA1 is a direct target of TGF-beta signaling. Wild-type PMEPA1 and an alternative splice variant lacking the putative transmembrane domain were encoded by the PMEPA1 locus and were shown by epitope tagging to encode proteins with differing subcellular localization. Both variants were found to be expressed in normal colonic epithelium, and both were shown to be induced by TGF-beta. Consistent with TGF-beta playing a role in terminal differentiation of colonocytes, in situ hybridization of normal colonic epithelium localized PMEPA1 expression to nonproliferating, terminally differentiated epithelium located at the top of colonic crypts. Intriguingly, in situ hybridization and Northern blot analysis showed that the expression of PMEPA1 was well maintained both in colon cancer primary tumors and in colon cancer liver metastases. PMEPA1 is thus a novel TGF-beta-induced marker of a differentiated crypt cell population. Moreover, as PMEPA1 expression is maintained, presumptively in a TGF-beta-independent manner after malignant transformation and metastasis, it demonstrates that even late colon cancers retain a strong capacity to execute many steps of the normal colonic differentiation program.

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.

Evidence of selection for clones having genetic inactivation of the activin A type II receptor (ACVR2) gene in gastrointestinal cancers.

The activin signaling pathway parallels the transforming growth factor (TGF)-beta pathway. Both use extracellular ligands and cell surface receptors that are structurally and functionally related, as well as the same intracellular mediators (SMADs 2-4) to transmit these signals. Members of both pathways have been characterized previously as tumor suppressor genes on the demonstration of inactivating mutations in human neoplasms, e.g., genetic inactivation of the activin type I receptor was reported recently in pancreatic cancer. Here, we present evidence of selection for mutations of the activin A type II receptor (ACVR2) gene during human gastrointestinal carcinogenesis. Two 8-bp polyadenine tracts of the ACVR2 gene are targets for inactivating frameshift mutations in gastrointestinal neoplasms having microsatellite instability (MSI). These mutations are similar to those of the 10-bp polyadenine tract within the TGF-beta type II receptor (TGFBR2), a well-characterized target of frameshift mutations in the same neoplasms. We identified biallelic mutations of ACVR2 in 25 of 28 MSI colorectal and pancreatic cancers. In addition, a mutation in the ACVR2 gene combined with loss of the wild-type allele was found in a non-MSI pancreatic cancer. This evidence is compatible with a high degree of selection for inactivation of the ACVR2 gene in tumorigenesis, supporting ACVR2 as a candidate tumor suppressor gene in gastrointestinal cancers.

Three classes of genes mutated in colorectal cancers with chromosomal instability.

Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined. To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers. We found 19 somatic mutations in five genes representing three distinct instability pathways. Seven mutations were found in MRE11, whose product is involved in double-strand break repair. Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation. Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction. This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.

TGF-beta-induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines.

Smad4 is a tumor suppressor gene that is commonly lost or mutated in colorectal and pancreatic cancers. The activated transforming growth factor-beta (TGF-beta) receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus. Smad4 mutations when detected as present in some human cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe a colon cancer cell line, VACO-9M, that is Smad4 null when analysed by multiple assays. To study the role of Smad4 in TGF-beta-induced translocation of the receptor-activated Smads to the nucleus, we analysed by immunofluorescence the cellular localization of endogenous Smad2 and Smad3 after TGF-beta treatment of VACO-9M, plus four additional Smad4 null cell lines of breast (MDA-MB-468), or pancreatic (BxPC3, Hs766T, CFPAC-1) origin. In each cell line, TGF-beta treatment resulted in both Smad2 and Smad3 moving to the nucleus in a Smad4-independent fashion. Nuclear translocation of Smad2 and Smad3 was, however, not sufficient to activate reporters for TGF-beta-induced transcriptional responses, which were however restored by transient transfection of wild-type Smad4. We conclude that Smad4 is not required for nuclear translocation of Smad2 and Smad3, but is needed for activation of at least certain transcriptional responses.

Autocrine heregulin generates growth factor independence and blocks apoptosis in colon cancer cells.

The aim of this study was to determine whether constitutive ErbB2 activation controls growth and apoptosis in colon cancer cells. Growth arrested GEO cells showed constitutive activation of ErbB2 in the absence of exogenous growth factors or serum supplementation. Higher levels of heregulin and ErbB2 activation were observed in the growth-arrested state and cell cycle re-entry was independent of exogenous growth factors. Blockade of ErbB2 activation by heregulin neutralizing antibodies and by AG879 resulted in prevention of cell cycle re-entry. This indicated that autocrine heregulin activity was responsible for growth factor independence and for cell cycle re-entry. Activation of ErbB2 was the result of heregulin mediated interaction with ErbB3 and generated downstream activation of the ERK and the PI3K/AKT pathways. Heregulin neutralizing antibody treatment of growth arrested GEO cells also generated apoptosis as reflected by PARP cleavage and DNA fragmentation indicating a cell survival signal was also induced by the constitutively activated ErbB2. The activation of AKT but not the MAPK pathway was responsible for cell survival in these cells.

Chromosomal autonomy of hMLH1 methylation in colon cancer.

Silencing of hMLH1 expression by aberrant hMLH1 promoter methylation accounts for the majority of sporadic colon cancers with microsatellite instability. We have previously shown hMLH1 silencing is biallelic and actively maintained. To study the mechanism of aberrant hMLH1 methylation, we assayed whether an hMLH1 methylated cell could transfer methylation and silencing to an exogenous hMLH1 promoter in somatic cell hybrids between hMLH1 methylated-silenced and hMLH1 unmethylated-expressing colon cancer cells. Conversely, we assayed whether these hybrids could reactivate expression of initially methylated and silenced hMLH1 alleles. Compellingly, within the hybrids each hMLH1 allele remained unchanged, retaining the expression status of its parental cell of origin. This chromosomal autonomy may not be simply determined by DNA methylation, as it is reasserted after experimentally forced demethylation of all hMLH1 alleles in the hybrids. Confirming findings included hMLH1 methylated cells being unable to methylate single transferred exogenous hMLH1 expressing chromosomes or transfected hMLH1 reporter constructs. hMLH1 silencing does not conform to either a dominant or recessive model, and is not determined by trans-acting factors differing between hMLH1 expressing or silenced genomes. We posit that hMLH1 methylation is dependent on and maintained by cis chromosomal marks, whose nature remains to be elucidated.