In around 30% of families with colorectal adenomatous polyposis, no germline mutation in the previously-implicated genes APC, MUTYH, POLE, POLD1, or NTHL1 can be identified, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis was performed. We identified two unrelated individuals with differing compound-heterozygous loss-of-function germline mutations in the mismatch repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1g>a, c.2760delC, c.3001-2a>c) was indicated on RNA and protein level. Analysis of the diseased individuals’ tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST) and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the loss-of-function effect and causal relevance of the mutations. The pedigrees, genotypes, and the frequency of MSH3 mutations in the general population are consistent with an autosomal recessive mode of inheritance. Both index persons had an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing Constitutional Mismatch Repair Deficiency Syndrome (CMMRD). Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study we describe biallelic germline mutations of MSH3 in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis.

Overexpression of the epidermal growth factor receptor (EGFR) and its association with the tyrosine kinase, c-Src, is correlated with increased cellular proliferation and tumorigenesis. Previous studies have shown that EGFR and c-Src co-overexpression and association leads to the c-Src-mediated phosphorylation of tyrosine 845 of the EGFR and that mutation of Tyr(845) ablates epidermal growth factor (EGF)-induced DNA synthesis. Here, we investigate the contribution of the signal transducers and activators of transcription (STAT5b) in the signaling pathways regulated by EGFR and c-Src overexpression in human breast tumor cell lines as well as in a mouse fibroblast model (C3H10T1/2). We demonstrate that 1) activation of STAT5b by EGF requires overexpression of the EGFR, 2) co-overexpression of c-Src alone does not result in EGF-induced activation of STAT5b but enhances that seen in EGFR-overexpressing cells, and 3) EGF-induced tyrosine phosphorylation of STAT5b requires Tyr(845) of the EGFR. Furthermore, the stable overexpression of a kinase-defective c-Src in the context of EGFR overexpression results in a decrease in the tyrosine phosphorylation of STAT5b in response to EGF and a more dramatic decrease in EGF-induced transcriptional activation of STAT5b, suggesting an integral role for c-Src in the physiological actions of STAT5b. Using a dominant negative STAT5b, we provide evidence that one such physiological action is to mediate EGF-induced DNA-synthesis. Finally, the use of site-specific tyrosine mutants demonstrates that EGF-induced phosphorylation of STAT5b involves not only tyrosine 699 of STAT5b, which is required for its transcriptional activation, but also three previously identified tyrosines in the C terminus of STAT5b (Tyr(725)/Tyr(740)/Tyr(743)).

Our analysis further confirms inherited inactivating ARMC5 mutations as a cause of familial PMAH and suggests an additional role for the development of concomitant intracranial meningiomas.

Of the seven signal transducers and activators of transcription that have been identified, STATs 1, 3, and 5a/5b can be activated not only by a multitude of cytokines but also by some growth factors. The data presented here demonstrate that, in contrast to activation by the cytokine, growth hormone (GH), the activation of STAT5b by the growth factor, epidermal growth factor (EGF), requires overexpression of the EGF receptor (EGFR The signal transducers and activators of transcription (STATs) 1 transmit signals from cytokine receptors at the membrane of the cell to the nucleus. Seven STAT proteins have been cloned and four of these (STATs 1, 3, 5a, and 5b) can be activated by a number of different cytokines (1-3). Similar to growth factor receptors, cytokine receptors are single transmembrane receptors that are activated by ligand-induced dimerization. However, the cytokine receptors do not contain a tyrosine kinase activity; rather they recruit one or more of a family of intracellular tyrosine kinases known as JAK kinases (JAKs 1, 2, 3, and tyk2) to the cytokine receptor complex. Activation of these kinases results in the tyrosine phosphorylation of one or more of the recruited STAT proteins (2). Some growth factor receptors have also been shown to activate the tyrosine phosphorylation of STAT proteins (4). Platelet-derived growth factor treatment results in the activation of STAT1 and STAT3, and EGF can activate STATs 1, 3, and 5 (5-8). Although JAK kinases are activated after growth factor stimulation, they are not required for growth factorstimulated tyrosine phosphorylation of STAT1 or STAT3 (4). In response to cytokine treatment, STAT proteins are activated by the phosphorylation of a single tyrosine residue at the C terminus of the molecule. Tyrosine phosphorylation of the STATs results in STAT dimerization through phosphotyrosine-SH2 domain interactions. Dimerization leads to nuclear translocation and binding of STAT dimers to consensus elements upstream of regulated genes. There is evidence that STATs 1, 3, and 5 are also serine-phosphorylated in their C-terminal transactivation domain resulting in maximal transcriptional activation (9). The importance of this C-terminal domain is evidenced by the fact that its truncation results in dominant negative STAT proteins that are able to inhibit the transcriptional activity of the wild type STAT proteins (2). Therefore, these C-terminal truncated proteins provide a direct means of inhibiting the biological function of wild type STATs at the cellular level. The biological actions of STAT proteins are diverse and involve a number of cellular processes, including proliferation, differentiation, and apoptosis (10).In our previous work on growth hormone (GH) receptor signaling, a member of the cytokine receptor superfamily, we identified and cloned the human forms of STAT5a and STAT5b, encoded by two separate genes that result in two highly homologous proteins (94% homology at the amino acid level) (11). Cytokine activation results in the phosphorylation of tyrosi...

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours.