Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival.

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.

TISSUE REGENERATION. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration.

Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

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.

Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells.

Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently up-regulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low-glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low-glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired KRAS mutations not present in their parents. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.

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.

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.

Ugene, a newly identified protein that is commonly overexpressed in cancer and binds uracil DNA glycosylase.

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA glycosylase 2 (UNG2), an important enzyme in the base excision repair (BER) pathway, was identified as a partner protein that binds to Ugene. Coimmunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH(2) terminus. We suggest that Ugene induction in cancer may contribute to the cancer phenotype by interacting with the BER pathway.

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.

The genomic landscapes of human breast and colorectal cancers.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene "mountains" and a much larger number of gene "hills" that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

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.

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.

The consensus coding sequences of human breast and colorectal cancers.

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of approximately 90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

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.

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.

Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.

BACKGROUND: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. METHODS: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. RESULTS: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval [CI] = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). CONCLUSIONS: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.

Reorganization of ErbB family and cell survival signaling after Knock-down of ErbB2 in colon cancer cells.

The role of the ErbB family in supporting the malignant phenotype was characterized by stable transfection of a single chain antibody (ScFv5R) against ErbB2 containing a KDEL endoplasmic reticulum retention sequence into GEO human colon carcinoma cells. The antibody traps ErbB2 in the endoplasmic reticulum, thereby down-regulating cell surface ErbB2. The transfected cells showed inactivation of ErbB2 tyrosine phosphorylation and reduced heterodimerization of ErbB2 and ErbB3. This resulted in greater sensitivity to apoptosis induced by growth deprivation and delayed tumorigenicity in vivo. Furthermore, decreased heterodimerization of ErbB2 and ErbB3 led to a reorganization in ErbB function in transfected cells as heterodimerization between epidermal growth factor receptor (EGFR) and ErbB3 increased, whereas ErbB3 activation remained almost the same. Importantly, elimination of ErbB2 signaling resulted in an increase in EGFR expression and activation in transfected cells. Increased EGFR activation contributed to the sustained cell survival in transfected cells.

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.

15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-beta-induced suppressor of human gastrointestinal cancers.

Marked increased expression of cyclooxygenase 2 (COX-2), a prostaglandin-synthesizing enzyme that is pharmacologically inhibited by nonsteroid anti-inflammatory-type drugs, is a major early oncogenic event in the genesis of human colon neoplasia. We report that, in addition to inducing expression of COX-2, colon cancers further target the prostaglandin biogenesis pathway by ubiquitously abrogating expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme that physiologically antagonizes COX-2. We find that 15-PGDH transcript and protein are both highly expressed by normal colonic epithelia but are nearly undetectable in colon cancers. Using gene transfection to restore 15-PGDH expression in colon cancer cells strongly inhibits the ability of these cells to form tumors in immune-deficient mice and demonstrates 15-PGDH to have functional colon cancer tumor suppressor activity. In interrogating the mechanism for 15-PGDH expression loss in colon cancer, we determined that colonic 15-PGDH expression is directly controlled and strongly induced by activation of the TGF-beta tumor suppressor pathway. These findings thus delineate an enzymatic pathway that induces colon cancer suppression, a pathway that is activated by TGF-beta and mediated by 15-PGDH.