Mutational analysis of the tyrosine kinome in colorectal cancers.
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Amino Acid Sequence↗
Biomedical subjects
Sanford Markowitz
Publications and source records attributed to Sanford Markowitz.
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.
Alternative Splicing↗
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.
Active Transport, Cell Nucleus↗
DEC1 is a downstream target of TGF-beta with sequence-specific transcriptional repressor activities.
To identify genes that mediate transforming growth factor-beta (TGF-beta) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-beta. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-beta in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-beta.
Animals↗
Prevalence of somatic alterations in the colorectal cancer cell genome.
Although a small fraction of human cancers have increased rates of somatic mutation because of known deficiencies in DNA repair, little is known about the prevalence of somatic alterations in the vast majority of human cancers. To systematically assess nonsynonymous somatic alterations in colorectal neoplasia, we used DNA sequencing to analyze approximately 3.2 Mb of coding tumor DNA comprising 1,811 exons from 470 genes. In total, we identified only three distinct somatic mutations, comprising two missense changes and one 14-bp deletion, each in a different gene. The accumulation of approximately one nonsynonymous somatic change per Mb of tumor DNA is consistent with a rate of mutation in tumor cells that is similar to that of normal cells. These data suggest that most sporadic colorectal cancers do not display a mutator phenotype at the nucleotide level. They also have significant implications for the interpretation of somatic mutations in candidate tumor-suppressor genes.
Colorectal Neoplasms↗
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.
Adaptor Proteins, Signal Transducing↗
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.
Colonic Neoplasms↗
Small changes in expression affect predisposition to tumorigenesis.
We have used quantitative measures of gene expression to show that constitutional 50% decreases in expression of one adenomatous polyposis coli tumor suppressor gene (APC) allele can lead to the development of familial adenomatous polyposis.
Adenoma↗