Familial colorectal cancer: eleven years of data from a registry program in Switzerland

Deleterious germ-line variants involving the DNA mismatch repair (MMR) genes have been identified as the cause of the hereditary nonpolyposis colorectal cancer syndrome known as the Lynch syndrome, but in numerous familial clusters of colon cancer, the cause remains obscure. We analyzed data for 235 German-speaking Swiss families with nonpolyposis forms of colorectal cancer (one of the largest and most ethnically homogeneous cohorts of its kind) to identify the phenotypic features of forms that cannot be explained by MMR deficiency. Based on the results of microsatellite instability analysis and immunostaining of proband tumor samples, the kindreds were classified as MMR-proficient (n=134, 57%) or MMR-deficient (n=101, 43%). In 81 of the latter kindreds, deleterious germ-line MMR-gene variants have already been found (62 different variants, including 13 that have not been previously reported), confirming the diagnosis of Lynch syndrome. Compared with MMR-deficient kindreds, the 134 who were MMR proficient were less likely to meet the Amsterdam Criteria II regarding autosomal dominant transmission. They also had primary cancers with later onset and colon-segment distribution patterns resembling those of sporadic colorectal cancers, and they had lower frequencies of metachronous colorectal cancers and extracolonic cancers in general. Although the predisposition to colorectal cancer in these kindreds is probably etiologically heterogeneous, we were unable to identify distinct phenotypic subgroups solely on the basis of the clinical data collected in this study. Further insight, however, is expected to emerge from the molecular characterization of their tumor

A comprehensive look at transcription factor gene expression changes in colorectal adenomas

BACKGROUND: Biological processes are controlled by transcription networks. Expression changes of transcription factor (TF) genes in precancerous lesions are therefore crucial events in tumorigenesis. Our aim was to obtain a comprehensive picture of these changes in colorectal adenomas. METHODS: Using a 3-pronged selection procedure, we analyzed transcriptomic data on 34 human tissue samples (17 adenomas and paired samples of normal mucosa, all collected with ethics committee approval and written, informed patient consent) to identify TFs with highly significant tumor-associated gene expression changes whose potential roles in colorectal tumorigenesis have been under-researched. Microarray data were subjected to stringent statistical analysis of TF expression in tumor vs. normal tissues, MetaCore-mediated identification of TF networks displaying enrichment for genes that were differentially expressed in tumors, and a novel quantitative analysis of the publications examining the TF genes' roles in colorectal tumorigenesis. RESULTS: The 261 TF genes identified with this procedure included DACH1, which plays essential roles in the proper proliferation and differentiation of retinal and leg precursor cell populations in Drosophila melanogaster. Its possible roles in colorectal tumorigenesis are completely unknown, but it was found to be markedly overexpressed (mRNA and protein) in all colorectal adenomas and in most colorectal carcinomas. However, DACH1 expression was absent in some carcinomas, most of which were DNA mismatch-repair deficient. When networks were built using the set of TF genes identified by all three selection procedures, as well as the entire set of transcriptomic changes in adenomas, five hub genes (TGFB1, BIRC5, MYB, NR3C1, and TERT) where identified as putatively crucial components of the adenomatous transformation process. CONCLUSION: The transcription-regulating network of colorectal adenomas (compared with that of normal colorectal mucosa) is characterized by significantly altered expression of over 250 TF genes, many of which have never been investigated in relation to colorectal tumorigenesis

Preinvasive colorectal lesion transcriptomes correlate with endoscopic morphology (polypoid vs. nonpolypoid)

Improved colonoscopy is revealing precancerous lesions that were frequently missed in the past, and ∼30% of those detected today have nonpolypoid morphologies ranging from slightly raised to depressed. To characterize these lesions molecularly, we assessed transcription of 23,768 genes in 42 precancerous lesions (25 slightly elevated nonpolypoid and 17 pedunculated polypoid), each with corresponding samples of normal mucosa. Nonpolypoid versus polypoid morphology explained most gene expression variance among samples; histology, size, and degree of dysplasia were also linked to specific patterns. Expression changes in polypoid lesions frequently affected cell-cycling pathways, whereas cell-survival dysregulation predominated in nonpolypoid lesions. The latter also displayed fewer and less dramatic expression changes than polypoid lesions. Paradigmatic of this trend was progressive loss through the normal > nonpolypoid > polypoid > cancer sequence of TMIGD1 mRNA and protein. This finding, along with TMIGD1 protein expression patterns in tissues and cell lines, suggests that TMIGD1 might be associated with intestinal-cell differentiation. We conclude that molecular dysregulation in slightly elevated, nonpolypoid, precancerous colorectal lesions may be somewhat less severe than that observed in classic adenomatous polyps

Immunohistochemical analysis reveals high frequency of PMS2 defects in colorectal cancer

BACKGROUND & AIMS: Germline mutations in the DNA mismatch repair (MMR) genes MSH2, MSH6, or MLH1 predispose to colorectal cancer (CRC) with an autosomal dominant inheritance pattern. The protein encoded by PMS2 is also essential for MMR; however, alterations in this gene have been documented only in extremely rare cases. We addressed this unexpected finding by analyzing a large series of CRCs. METHODS: Expression of MSH2, MSH6, MLH1, and PMS2 was studied by immunohistochemistry in 1048 unselected, consecutive CRCs. Where absence of MMR proteins was detected, microsatellite instability and cytosine methylation of the respective gene promoter were analyzed. The DNA of patients presenting with PMS2-deficient cancers was examined for germline and somatic alterations in the PMS2 gene. RESULTS: An aberrant pattern of MMR protein expression was detected in 13.2% of CRCs. Loss of expression of MSH2, MSH6, or MLH1 was found in 1.4%, 0.5%, and 9.8%, respectively. PMS2 deficiency accompanied by microsatellite instability was found in 16 cases (1.5%) with a weak family history of cancer. The PMS2 promoter was not hypermethylated in these cases. Despite interference of the PMS2 pseudogenes, we identified several heterozygous germline mutations in the PMS2 gene. CONCLUSIONS: PMS2 defects account for a small but significant proportion of CRCs and for a substantial fraction of tumors with microsatellite instability. However, the penetrance of heterozygous germline mutations in PMS2 is considerably lower than that of mutations in other MMR genes. The possible underlying causes of this unorthodox inheritance pattern are discussed

KIAA1199 interactome and gene expression changes associated with KIAA1199 expression in SW480 cells.

<p><b>A.</b> The table shows cell lines and antibodies used in the three immunoprecipitation experiments performed to identify proteins that interact with KIAA1199. <b>B.</b> Representative Western blots showing the results of the immunoprecipitation experiments summarized in the table. Input: exp.1 and 3, KIAA1199-V5 extract; exp.2, KIAA1199-Cl.18 extract. <b>C.</b> Silver-stained gel showing unique bands present only in the KIAA1199 immunoprecipitate (black asterisk) following IP with antibodies against the V5 tag. (The immunoprecipitated KIAA1199 is indicated with a red asterisk.) <b>D.</b> Upper four strips: Western blot showing co-immunoprecipitation of ITPR3 with anti-KIAA1199 antibody and reciprocal co-immunoprecipitation of KIAA1199 with anti-ITPR3 antibody. Lower four strips: Western blot showing co-immunoprecipitation of EPHA2 with anti-KIAA1199 antibody (arrowheads) and reciprocal co-immunoprecipitation of KIAA1199 with anti-EPHA2 antibody. Input: KIAA1199-Cl.18 extract. The weak interactions of these two proteins with KIAA1199 might be related to the fact that IP was performed using extracts from cells in which these two proteins were endogenously expressed (instead of overexpressed). <b>E.</b> Top: Western blot showing KIAA1199 protein levels in SW480 Clone 13 total cell extracts and in conditioned medium before and after doxycycline induction. TUBB: loading control. Bottom: Principal component analysis (PCA) of log₂<i>KIAA1199</i> mRNA expression intensity values. The plot of PCA scores for the six samples 48 h after doxycycline induction shows clear separation of replicates with (n = 3) vs. without (n = 3) KIAA1199 expression. The first three principal components explain 72.9% of the total variance. <b>F.</b> Heatmap showing expression of the 490 differentially expressed genes (<i>y</i> axis) before and after doxycycline-induced KIAA1199 expression in three replicate experiments. (Green: upregulated; red: downregulated in KIAA1199-expressing cells; p value <0.025, and fold change >1.2).</p

Effect of ectopic constitutive KIAA1199 expression on SW480 cell morphology, proliferation, and invasiveness.

<p>SW480 KIAA1199-V5 cells were compared with SW480 Dest-V5 cells (empty vector controls). Each cell phenotype shown in this figure is based on the results of at least three independent experiments. Mean values ± SEM are reported. <b>A.</b> Morphology of control (left) and KIAA1199-V5-expressing cells (right) at low (top) and high (bottom) confluence. Control SW480 Dest cells, like the parental SW480 line <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069473#pone.0069473-Tomita1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069473#pone.0069473-Petrova1" target="_blank">[16]</a>, tended to be round (arrow), and confluent cultures were characterized by clusters of piled-up cells (ellipse). In contrast, KIAA1199-V5-expressing cells were flatter with a more epithelial-like aspect (arrowhead). Compared with controls, KIAA1199-V5-expressing cells also displayed: <b>B.</b> diminished proliferation rates; <b>C.</b> reduced colony formation; and <b>D.</b> a decrease in invasiveness of ∼45%.</p

Ectopic expression of KIAA1199 decreases cytoplasmic and nuclear levels of CTNNB1.

<p><b>A.</b> Immunocytochemical staining for total and active CTNNB1 in empty vector-transfected SW480 cells (Dest-V5) and SW480 cells expressing KIAA1199-V5 constitutively. The latter cells, presented decreased CTNNB1 levels in the nucleus and cytoplasm. <b>B.</b> Similar results were observed in SW480 KIAA1199-GFP cells (immunofluorescence experiments). Although only a fraction of these cells exhibited substantial KIAA1199-GFP expression upon doxycycline induction, the vast majority displayed CTNNB1 depletion (total and active forms) in both the nucleus and cytoplasm, which is probably a paracrine effect of secreted KIAA1199.</p

Gene ontologies (GO) and interactomes over-represented in the lists of putative KIAA1199 interactors and genes whose transcript levels were affected by KIAA1199 expression.

<p>Gene ontologies (GO) and interactomes over-represented in the lists of putative KIAA1199 interactors and genes whose transcript levels were affected by KIAA1199 expression.</p

KIAA1199 exerts negative feedback on canonical Wnt signaling.

<p><b>A.</b> Ectopic KIAA1199 expression in HEK293 cells inhibits the transcriptional activity elicited by wild-type CTNNB1 or by a constitutively active form of the protein, CTNNB1-T41A. Cells were transiently transfected with either a TOPflash or a FOPflash reporter vector, together with an empty vector or an expression vector for KIAA1199 or TCF4-DN. TOP/FOP ratios are the mean ± SEM of triplicate experiments. <b>B.</b> Total and active CTNNB1 levels in SW480 cells expressing KIAA1199 (V5) and in empty vector-transfected (Dest) controls. The table shows fold changes (vs. controls) in the expression of both CTNNB1 forms induced by KIAA1199 expression based on quantification of band intensity relative to that of CDH1 or TUBB in the same lane. TUBB was used as loading control for cytoplasmic and nuclear fractions since it was detectable in these fractions of the SW480-V5 and Dest cell extracts under the same extraction conditions. Furthermore, TUBB has been reported to shuttle to the nucleus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069473#pone.0069473-Akoumianaki1" target="_blank">[50]</a> and references herein).</p

Early insights into the function of KIAA1199, a markedly overexpressed protein in human colorectal tumors

We previously reported that the expression of KIAA1199 in human colorectal tumors (benign and malignant) is markedly higher than that in the normal colonic mucosa. In this study, we investigated the functions of the protein encoded by this gene, which are thus far unknown. Immunostaining studies were used to reveal its subcellular localization, and proteomic and gene expression experiments were conducted to identify proteins that might interact with KIAA1199 and molecular pathways in which it might play roles. Using colon cancer cell lines, we showed that both endogenous and ectopically expressed KIAA1199 is secreted into the extracellular environment. In the cells, it was found mainly in the perinuclear space (probably the ER) and cell membrane. Both cellular compartments were also over-represented in lists of proteins identified by mass spectrometry as putative KIAA1199 interactors and/or proteins encoded by genes whose transcription was significantly changed by KIAA1199 expression. These proteomic and transcriptomic datasets concordantly link KIAA1199 to several genes/proteins and molecular pathways, including ER processes like protein binding, transport, and folding; and Ca(2+), G-protein, ephrin, and Wnt signaling. Immunoprecipitation experiments confirmed KIAA1199's interaction with the cell-membrane receptor ephrin A2 and with the ER receptor ITPR3, a key player in Ca(2+) signaling. By modulating Ca(2+) signaling, KIAA1199 could affect different branches of the Wnt network. Our findings suggest it may negatively regulate the Wnt/CTNNB1 signaling, and its expression is associated with decreased cell proliferation and invasiveness