Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, ...that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.
We have previously shown that rheumatoid arthritis (RA) risk alleles overlap between different ethnic groups. Here, we utilize a multiethnic approach to show that we can effectively discover RA risk ...alleles. Thirteen putatively associated SNPs that had not yet exceeded genome-wide significance (p < 5 × 10−8) in our previous RA genome-wide association study (GWAS) were analyzed in independent sample sets consisting of 4,366 cases and 17,765 controls of European, African American, and East Asian ancestry. Additionally, we conducted an overall association test across all 65,833 samples (a GWAS meta-analysis plus the replication samples). Of the 13 SNPs investigated, four were significantly below the study-wide Bonferroni corrected p value threshold (p < 0.0038) in the replication samples. Two SNPs (rs3890745 at the 1p36 locus p = 2.3 × 10−12 and rs2872507 at the 17q12 locus p = 1.7 × 10−9) surpassed genome-wide significance in all 16,659 RA cases and 49,174 controls combined. We used available GWAS data to fine map these two loci in Europeans and East Asians, and we found that the same allele conferred risk in both ethnic groups. A series of bioinformatic analyses identified TNFRSF14-MMEL1 at the 1p36 locus and IKZF3-ORMDL3-GSDMB at the 17q12 locus as the genes most likely associated with RA. These findings demonstrate empirically that a multiethnic approach is an effective strategy for discovering RA risk loci, and they suggest that combining GWASs across ethnic groups represents an efficient strategy for gaining statistical power.
Individuals and families carrying mutations in BRCA1 and BRCA2 ( BRCA1/2 ) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis ...targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients ( N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.
In mammals, large caliber axons are ensheathed by myelin, a glial specialization supporting axon integrity and conferring accelerated and energy‐efficient action potential conduction. Myelin basic ...protein (MBP) is required for normal myelin elaboration with maximal mbp transcription in oligodendrocytes requiring the upstream M3 enhancer. To further characterize the mechanism regulating mbp transcription, we defined M3 structure/function relationships by evaluating its evolutionary conservation, DNA footprints and the developmental programing conferred in mice by M3 derivatives. Multiple M3 regulatory element combinations were found to drive expression in oligodendrocytes and Schwann cells with a minimal 129 bp sequence conferring expression in oligodendrocytes throughout myelin elaboration, maintenance and repair. Unexpectedly, M3 derivatives conferred markedly different spatial and temporal expression programs thus illuminating striking transcriptional heterogeneity within post‐mitotic oligodendrocytes. Finally, one M3 derivative engaged only during primary myelination, not during adult remyelination, demonstrating that transcriptional regulation in the two states is not equivalent. GLIA 2016;64:175–194
Main Points
The mbp M3 enhancer drives transcription in oligodendrocytes and Schwann cells.
Different element combinations confer activity in the 2 cell types, in different CNS domains and during primary myelination and remyelination in the adult.
Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is ...relieved by occupancy of the NH2-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitrospecific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFκB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr688 or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH2-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr688 and the NH2-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr688in p85 leads to a novel mechanism of PI3K regulation.
Women with germ-line mutations in the mismatch repair genes (responsible for hereditary nonpolyposis colorectal cancer) face
an increased risk of colonic and endometrial cancer. However, these ...germ-line mutations are rare and are responsible for fewer
than 1% of endometrial cancers. Therefore, we examined whether or not common variants of the hereditary nonpolyposis colorectal
cancer–associated genes might also be associated with an increased risk of endometrial cancer. Three single-nucleotide polymorphisms
were selected in the MLH1 and MSH2 mismatch repair genes. All the various 672 women with endometrial cancer and 880 controls were genotyped. Each of these three
single-nucleotide polymorphisms was associated with an increased risk of endometrial cancer. Carriers of the MLH1 nt-93 A allele were at a 1.5-fold increased risk of developing endometrial cancer compared with controls 95% confidence interval
(95% CI), 1.2-2.0; P = 0.001. The risk was higher for homozygote carriers odds ratio (OR), 1.9; 95% CI, 1.2-3.2; P = 0.009. For carriers of the MSH2 rs2303428 C allele, the OR was 1.4 (95% CI, 1.0-1.9; P = 0.05), and for carriers of the MSH2 rs2059520 G allele, the OR was 1.3 (95% CI, 1.0-1.7; P = 0.03). More than 9% of endometrial cancer cases carried a variant allele in both MLH1 and MSH2 . For these women, the risk of endometrial cancer was particularly high (OR, 2.1; 95% CI, 1.2-3.6; P = 0.005). For patients younger than 50 years at diagnosis who carried both variants, the risk was even higher (OR, 3.4; 95%
CI, 1.7-6.6; P = 0.0005). In summary, two common variant alleles of the MLH1 and MSH2 genes make a substantial contribution to endometrial cancer incidence in Ontario. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1636–40)
Purpose: Increased levels of serum human kallikrein-2 (hK2) and an hK2 gene ( KLK2 ) variant are positively associated for prostate cancer, but the relationships between them remain unclear. We ...examined five
variants of the KLK2 gene to further define its relevance to prostate cancer susceptibility and hK2 levels.
Experimental Design: We genotyped 645 men with biopsy-proven prostate cancer (cases) and 606 males with biopsies negative for prostate cancer
(controls) for five additional single nucleotide polymorphisms (SNP) across the KLK2 gene and also tested for serum hK2 levels. These SNPs were identified from sequencing the KLK2 gene among 20 patients with aggressive prostate cancer. Odds ratios (OR) for prostate cancer detection and haplotype analysis
were done.
Results: Among the SNPs studied, the A allele of the KLK2-SNP1 (G > A, rs2664155) and the T allele of the KLK2-SNP5 (C > T, rs198977)
polymorphisms showed positive associations with prostate cancer, adjusted ORs for KLK2-SNP1 AG and AA genotypes being 1.4
95% confidence interval (95% CI), 1.2-1.8; P = 0.002 and for KLK2-SNP5 TT or CT genotypes being 1.3 (95% CI, 1.1-1.6; P = 0.05). Haplotype analyses also revealed a significant association between prostate cancer and the haplotype containing
both risk alleles (ACCTT), OR being 5.1 (95% CI, 1.6-6.5; P = 0.005). Analysis of serum hK2 revealed hK2 levels to be significantly increased in association with KLK2-SNP1 AA and AG
risk genotypes compared with the GG genotype ( P = 0.001) and also in association with the ACCTT risk haplotype compared with the most common non-risk haplotype ( P = 0.05).
Conclusions: These findings suggest a role for the KLK2 gene in prostate cancer susceptibility and imply that this role may be realized at least in part by the induction of increases
in hK2 production.
Genetic variation in specific G-protein coupled receptors (GPCRs) is associated with a spectrum of respiratory disease predispositions and drug response phenotypes. Although certain GPCR gene ...variants can be disease-causing through the expression of inactive, overactive, or constitutively active receptor proteins, many more GPCR gene variants confer risk for potentially deleterious endophenotypes. Endophenotypes are traits, such as bronchiole hyperactivity, atopy, and aspirin intolerant asthma, which have a strong genetic component and are risk factors for a variety of more complex outcomes that may include disease states. GPCR genes implicated in asthma endophenotypes include variants of the cysteinyl leukotriene receptors (CYSLTR1 and CYSLTR2), and prostaglandin D2 receptors (PTGDR and CRTH2), thromboxane A2 receptor (TBXA2R), beta2-adrenergic receptor (ADRB2), chemokine receptor 5 (CCR5), and the G protein-coupled receptor associated with asthma (GPRA). This review of the contribution of variability in these genes places the contribution of the cysteinyl leukotriene system to respiratory endophenotypes in perspective. The genetic variant(s) of receptors that are associated with endophenotypes are discussed in the context of the extent to which they contribute to a disease phenotype or altered drug efficacy.
Coligation of the Fc receptor on B cells, FcγRIIB1, with the B cell antigen receptor (BCR) leads to abortive BCR signaling. Here it was shown that the FcγRIIB1 recruits the phosphotyrosine ...phosphatase PTP1C after BCR coligation. This association is mediated by the binding of a 13-amino acid tyrosine-phosphorylated sequence to the carboxyl-terminal Src homology 2 domain of PTP1C and activates PTP1C. Inhibitory signaling and PTP1C recruitment are dependent on the presence of the tyrosine within the 13-amino acid sequence. Inhibitory signaling mediated by FcγRIIB1 is deficient in motheaten mice which do not express functional PTP1C. Thus, PTP1C is an effector of BCR-FcγRIIB1 negative signal cooperativity.
Abstract Objectives Inherited mutations in the MLH1 gene are associated with a proportion of families with the hereditary non-polyposis colon cancer syndrome (HNPCC). The cardinal features of the ...syndrome are a predisposition to colon, endometrial and ovarian cancers. Recently, it has been shown that a non-coding polymorphic variant in MLH1 (G>A nt-93) predisposes to colon and endometrial cancer, but with much reduced penetrance. We sought to establish whether or not this polymorphic variant also predisposes to ovarian cancer. Methods We genotyped 899 women with invasive ovarian cancer and 931 controls for the G>A nt-93 variant. Results The presence of the variant was associated with a modest, but highly significant risk of ovarian cancer (OR = 1.5; 95% CI 1.3–1.9; p = 0.00005). The association was present in cancers of all histologies except clear cell, and in all ethnic groups. Conclusions The G>A nt-93 variant of the MLH1 gene is associated with an increased risk of invasive ovarian cancer.
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