GPR37 is processed in the N-terminal ectodomain by ADAM10 and furin.

GPR37 is an orphan G protein-coupled receptor (GPCR) implicated in several neurological diseases and important physiological pathways in the brain. We previously reported that its long N-terminal ectodomain undergoes constitutive metalloprotease-mediated cleavage and shedding, which have been rarely described for class A GPCRs. Here, we demonstrate that the protease that cleaves GPR37 at Glu167↓Gln168 is a disintegrin and metalloprotease 10 (ADAM10). This was achieved by employing selective inhibition, RNAi-mediated downregulation, and genetic depletion of ADAM10 in cultured cells as well as in vitro cleavage of the purified receptor with recombinant ADAM10. In addition, the cleavage was restored in ADAM10 knockout cells by overexpression of the wild type but not the inactive mutant ADAM10. Finally, postnatal conditional depletion of ADAM10 in mouse neuronal cells was found to reduce cleavage of the endogenous receptor in the brain cortex and hippocampus, confirming the physiological relevance of ADAM10 as a GPR37 sheddase. Additionally, we discovered that the receptor is subject to another cleavage step in cultured cells. Using site-directed mutagenesis, the site (Arg54↓Asp55) was localized to a highly conserved region at the distal end of the ectodomain that contains a recognition site for the proprotein convertase furin. The cleavage by furin was confirmed by using furin-deficient human colon carcinoma LoVo cells and proprotein convertase inhibitors. GPR37 is thus the first multispanning membrane protein that has been validated as an ADAM10 substrate and the first GPCR that is processed by both furin and ADAM10. The unconventional N-terminal processing may represent an important regulatory element for GPR37.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates ß1-Adrenergic Receptor N-terminal Cleavage.

The ß1-adrenergic receptor (ß1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ß-adrenergic antagonists, such as ß-blockers, relatively little is yet known about its regulation. We have shown previously that ß1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ß1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ß1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ßAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ß1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

Altered O-glycosylation of ß1-adrenergic receptor N-terminal single-nucleotide variants modulates receptor processing and functional activity.

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human ß1-adrenergic receptor (ß1AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P52↓L53 and R31↓L32), respectively, or abolished the major site at R31↓L32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer ß-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a ß1AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

Matriptase-2 gene (TMPRSS6) variants associate with breast cancer survival, and reduced expression is related to triple-negative breast cancer.

Matriptase-2 (TMPRSS6) has been identified as a breast cancer risk factor. Here, we examined relationships between TMPRSS6 genetic variations and breast cancer risk and survival, and determined the gene and protein expressions in breast tumors and assessed their clinical importance. Thirteen TMPRSS6 polymorphisms were genotyped in 462 invasive breast cancer cases and 458 controls. Gene expression was analyzed from 83 tumors and protein expression from 370 tumors. We then assessed the statistical significance of associations among genotypes, clinicopathological characteristics and survival. The TMPRSS6 variant rs2543519 was associated with breast cancer risk (p = 0.032). Multivariate analysis showed that four variants had effects on survival-rs2543519 (p = 0.017), rs2235324 (p = 0.038), rs14213212 (p = 0.044) and rs733655 (p = 0.021)-which were used to create a group variable that was associated with poorer prognosis correlating with more alleles related to reduced survival (p = 0.006; risk ratio, 2.375; 95% confidence interval, 1.287-4.382). Low gene expression was related to triple-negative breast cancer (p = 0.0001), and lower protein expression was detected in undifferentiated (p = 0.019), large (p = 0.014) and ductal or lobular tumors (p = 0.036). These results confirm the association of TMRRSS6 variants with breast cancer risk and survival. Matriptase-2 levels decrease with tumor progression, and lower gene expression is seen in poor-prognosis-related triple-negative breast cancers. Our study is the first to show that matriptase-2 gene variants are related to breast cancer prognosis, supporting matriptase-2 involvement in tumor development.

Transcription factors zeb1, twist and snai1 in breast carcinoma.

BACKGROUND: Epitheliomesenchymal transition (EMT) is the process where cancer cells attain fibroblastic features and are thus able to invade neighboring tissues. Transcriptional factors zeb1, snai1 and twist regulate EMT. METHODS: We used immunohistochemistry to investigate the expression of zeb1, twist and snai1 in tumor and stromal compartments by in a large set of breast carcinomas. The results were compared with estrogen and progesterone receptor status, HER2 amplification, grade, histology, TNM status and survival of the patients. RESULTS: Nuclear expression for twist was seen in the epithelial tumor cell compartment in 3.6% and for snai1 in 3.1% of the cases while zeb1 was not detected at all in these areas. In contrast, the tumor stromal compartment showed nuclear zeb1 and twist expression in 75% and 52.4% of the cases, respectively. Although rare, nuclear expression of twist in the epithelial tumor cell compartment was associated with a poor outcome of the patients (p = 0.054 log rank, p = 0.013, Breslow, p = 0.025 Tarone-Ware). Expression of snai1, or expression of zeb1 or twist in the stromal compartment did not have any prognostic significance. Furthermore, none of these factors associated with the size of the tumors, nor with the presence of axillary or distant metastases. Expression of zeb1 and twist in the stromal compartment was positively associated with a positive estrogen or progesterone receptor status of the tumors. Stromal zeb1 expression was significantly lower in ductal in situ carcinomas than in invasive carcinomas (p = 0.020). Medullary carcinomas (p = 0.017) and mucinous carcinomas (p = 0.009) had a lower stromal expression of zeb1 than ductal carcinomas. Stromal twist expression was also lower in mucinous (p = 0.017) than in ductal carcinomas. CONCLUSIONS: Expression of transcriptional factors zeb1 and twist mainly occur in the stromal compartment of breast carcinomas, possibly representing two populations of cells; EMT transformed neoplastic cells and stromal fibroblastic cells undergoing activation of zeb1 and twist due to growth factors produced by the tumor. However, epithelial expression of twist was associated with a poor prognosis, hinting at its importance in the spread of breast carcinoma.

Twist and snai1 expression in pharyngeal squamous cell carcinoma stroma is related to cancer progression.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial process in tumorigenesis since tumor cells attain fibroblast-like features enabling them to invade to surrounding tissue. Two transcription factors, TWIST and SNAI1, are fundamental in regulating EMT. METHODS: Immunohistochemistry was used to study the expression of TWIST and SNAI1 in 109 pharyngeal squamous cell carcinomas. RESULTS: Tumors with intense stromal staining of TWIST relapsed more frequently (p = 0.04). Tumors with both positive TWIST and SNAI1 immunoreactivity in the stroma were at least Stage II (p = 0.05) and located more often in hypopharynx (p = 0.035). Tumors with negative immunostaining of TWIST and SNAI1 in the stromal compartment were smaller (T1-2) (p = 0.008), less advanced (SI-II) (p = 0.031) and located more often in the oropharynx (p = 0.007). Patients with negative SNAI1 and TWIST immunostaining in tumor stroma had a better 5-year disease-specific and overall survival (p = 0.037 and p = 0.014 respectively). CONCLUSION: TWIST and SNAI1 expression in stromal cells is associated with clinical and histopathological characteristics that indicate progressive disease. Negative expression of these EMT-promoting transcription factors predicts a better outcome.

Nuclear expression of Snail1 in borderline and malignant epithelial ovarian tumours is associated with tumour progression.

BACKGROUND: Transcription factor Snail1 has a central role in induction of epithelial-mesenchymal transition (EMT). The aim of the present study was to elucidate the expression of Snail1 protein during epithelial ovarian tumourigenesis and to study the association of Snail1 expression with clinicopathological factors and prognosis. METHODS: Epithelial and stromal fibroblast-like fusiform cells of 14 normal ovarian samples, 21 benign, 24 borderline and 74 malignant epithelial ovarian tumours were studied for Snail1 protein using immunohistochemistry. RESULTS: Nuclei of surface peritoneal cells of normal ovaries (n = 14) were regarded as negative for Snail1. Nuclear expression of Snail1 protein in epithelial ovarian tumours was increased during tumour progression from precursor lesions into carcinomas both in epithelial (p = 0.006) and stromal cells (p = 0.007). Nuclei of benign tumours (n = 21) were negative for Snail1. In borderline tumours (n = 24) occasional positive epithelial cells were found in 2 (8%) samples and in 3 (13%) samples stromal cells were focally positive for Snail1. In carcinomas (n = 74) focal Snail1 staining in epithelial cells was present in 17 (23%) tumours, and in stromal cells in 18 (24%) tumours. Nuclear expression of Snail1 in epithelial or stromal cells was not associated with clinicopathological factors or prognosis. CONCLUSION: Nuclear Snail1 expression seems to be related to tumour progression, and expression in borderline tumours indicates a role for Snail1 in early epithelial ovarian tumour development. Snail1 also appears to function more generally in tissue remodelling as positive staining was demonstrated in stromal cells.

Refinement of the 22q12-q13 breast cancer--associated region: evidence of TMPRSS6 as a candidate gene in an eastern Finnish population.

Although many risk factors for breast cancer are known, most of the genetic background and molecular mechanisms still remain to be elucidated. We have previously published an autosome-wide microsatellite scan for breast cancer association and here we report a follow-up study for one of the detected regions. Ten single nucleotide polymorphisms (SNP) were genotyped in an Eastern Finnish population sample of 497 breast cancer cases and 458 controls to refine the 550-kb region on 22q12-q13 and identify the breast cancer-associated gene(s) in this region. We also studied 22q12-q13 for allelic imbalance for the detection of a possible tumor suppressor gene and to see whether the breast cancer association and allelic imbalance in this region could be connected. A SNP (rs733655) in matriptase-2 gene (TMPRSS6) was detected to associate with breast cancer risk. The genotype frequencies of rs733655 differed significantly between cases and controls in the entire sample and in the geographically and genetically more homogeneous subsample with P = 0.044 and P = 0.0003, respectively. The heterozygous genotype TC was observed to be the risk genotype in both samples (odds ratios, 1.39; 95% confidence intervals, 1.06-1.83 and odds ratios, 2.11; 95% confidence intervals, 1.46-3.05). An associated two-marker haplotype involving SNP rs733655 (empirical P = 0.041) provides further evidence for breast cancer risk factor locating on 22q12-q13, possibly being TMPRSS6. Our results suggest that matriptase-2 gene is associated with breast cancer risk in the Eastern Finnish population.

An autosome-wide scan for linkage disequilibrium-based association in sporadic breast cancer cases in eastern Finland: three candidate regions found.

Breast cancer is the most common of cancers among women in industrialized countries. Many of breast cancer risk factors are known, but the majority of the genetic background is still unknown. Linkage disequilibrium-based association is a powerful tool for mapping disease genes and is suitable for mapping complex traits in founder populations. We report the results of a two-stage, autosome-wide scan for LD with breast cancer. Our aim was to identify genetic risk factors for sporadic breast cancer in an eastern Finnish population. Our case-control set is from the province of northern Savo in the late-settlement area of eastern Finland. This population is relatively young and genetically homogeneous. We used 435 autosomal microsatellite markers spaced by an average of 10 cM in a set of 49 breast cancer cases and 50 controls. In the first-stage scan, we found 21 markers in LD with breast cancer (Ps = 0.003-0.046, Fisher's exact test). In the second-stage scan with markers flanking 21 positive loci, four significant markers were found (Ps = 0.013-0.046, Fisher's exact test). Haplotype analysis using global score method with two, three, or four markers also revealed four positive marker combinations (simulated P for global score = 0.003-0.021). Our results suggest breast cancer-associated regions on 3p26, 11q23, and 22q13.1 in an eastern Finnish population.

Transcription factor snail1 expression and poor survival in pharyngeal squamous cell carcinoma.

Snail1, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in tumour progression. Previous studies of snail1 have mainly focused on the epithelial tumour cells. The objective of this study was to evaluate the expression of snail1 protein in endothelial cells, stromal myofibroblasts and malignant epithelial cells of pharyngeal squamous cell carcinomas (PSCC), as well as its relation to clinicopathological features and survival. One hundred and ten tissue microarray samples were analyzed for snail1 expression using immunohistochemistry. In endothelial cells snail1 expression was observed in 51 (48%) of 107 cases and it predicted reduced disease specific survival (DSS) (p=0.009). In 49 (46%) tumour samples snail1 immunostaining was detected in stromal myofibroblasts and there was a tendency to poorer DSS in that group (p=0.067). Snail1 expression in endothelial cells and stromal myofibroblasts is also associated with hypopharyngeal tumours (p=0.01 and p=0.038 respectively), increasing T category (T3-4) (p=0.005, p=0.037 respectively) and poorer general condition of the patient (Karnofsky performance status score <70; p=0.029, p=0.039 respectively). Moreover endothelial expression correlated with advanced stage (III-IV) (p=0.005) and poorer differentiation (grade 2-3; p=0.012). In malignant epithelial cells snail1 immunostaining was detected in 75 of 110 cases (68%). Expression of the protein was more common in hypopharyngeal tumours (p=0.044). Snail1 positive tumours associated with a lower Karnofsky performance status score (p=0.039) and regional failure (p=0.042). Our findings indicate that snail1 protein expression in endothelial cells and to some extent also in tumour stromal myofibroblasts seems to be a predictor of poor survival in PSCC. The presence of snail1 protein in tumour microenvironment rather than in malignant epithelial tumour cells may induce tissue remodelling and tumour progression.

Frequent gene dosage alterations in stromal cells of epithelial ovarian carcinomas.

Stromal cells are an active and integral part of epithelial neoplasms. We have previously observed allelic imbalance on chromosome 3p21 in both stromal and epithelial cells of ovarian tumors. This study was designed to explore gene dosage alterations throughout human chromosomes from stromal and epithelial cells of epithelial ovarian carcinomas. Thirteen stromal and 24 epithelial samples, microdissected from epithelial ovarian carcinomas, were analyzed using multiplex ligation-dependent probe amplification technique. Analysis covered 110 cancer related genes. Frequent genetic alterations were detected both in the stroma and epithelium of ovarian carcinomas. The mean number of altered genes per tumor was 10.8 in stroma and 23.6 in epithelium. In the stroma, the mean number of gains was 6.6 and of losses 4.2 and in the epithelium 13.7 and 9.9. The high number of changes associated with advanced tumor stage (p = 0.035) and death due to ovarian cancer (p = 0.032). The most frequent alteration was the deletion of the deleted in colorectal carcinoma (DCC) on chromosome 18q21.3 in 62% of samples. Loss of DCC was related to endometrioid subtype (p = 0.033). Large chromosomal aberrations were detected on the basis of alterations in adjacent genes. Most importantly, 38 genes showed similar genetic alterations (gain-gain or loss-loss) in stromal and epithelial compartments of 11 tumor pairs. Thus, frequent genetic alterations in stromal cells of epithelial ovarian carcinomas resembled those of malignant epithelial cells and may indicate a common precursor cell type. Epithelial-mesenchymal transition may generate transformed cancer cells and modify the tumor microenvironment with distinct properties.

Marked allelic imbalance on chromosome 5q31 does not explain alpha-catenin expression in epithelial ovarian cancer.

OBJECTIVE: Human alpha-catenin gene (CTNNA1) on chromosome 5q31 is aberrantly expressed in various types of cancer including epithelial ovarian tumors. Allelic imbalance on this region has also been described in several malignant diseases. In the present work, the role of CTNNA1 as a candidate tumor suppressor gene was studied by comparing protein expression with allelic imbalance in human epithelial ovarian tumors. METHODS: Alpha-catenin protein expression was determined from two areas of 41 tumors, and tissues from these areas were microdissected. After DNA extraction, AI analysis was carried out with eight microsatellite markers. RESULTS: Altogether, 93% of the tumors (38 of 41) showed allelic imbalance at one or more loci. Two distinct common regions of allelic imbalance were identified, one comprising markers D5S2002 and D5S1995 and the other markers D5S393 and D5S476. Loss of the CTNNA1 gene did not appear to be involved in down-regulation of alpha-catenin in ovarian tumors, since allelic imbalance with a variety of markers, including CTNNA1 associated marker D5S476, was found in tumor samples independently of alpha-catenin expression. Furthermore, allelic imbalance analyses of two different samples from the same tumor revealed genetic heterogeneity. CONCLUSIONS: High allelic imbalance frequency indicates that chromosomal region 5q31 is functionally important in epithelial ovarian cancer. Allelic imbalance occurs at two distinct regions of which one includes the CTNNA1 gene. However, this gene is likely to be inactivated by mechanisms other than allelic imbalance. In addition, genetic heterogeneity observed in these tumors demonstrates the multiclonal nature of epithelial ovarian tumors.

Genetic alterations in the peritumoral stromal cells of malignant and borderline epithelial ovarian tumors as indicated by allelic imbalance on chromosome 3p.

Stromal accumulation of hyaluronan in epithelial ovarian cancers is an independent predictor of tumor spreading and unfavorable outcome of the disease. We started to screen for chromosomal causes of this accumulation by studying deletions in 3p21.3, a region harboring 3 hyaluronidase genes (HYAL1-3) among other potentially important tumor suppressors. Using 6 microsatellite markers from this region, allelic imbalance was found in 60-87% of the informative tumor cells microdissected from histologic sections of 58 patients with epithelial ovarian cancer. However, adjacent stromal cells originally intended as controls showed allelic imbalance at a frequency almost as high as the tumor cells (52-80%). A further laser capture microdissection on 10 borderline tumors also showed a high rate of allelic imbalance, both in the epithelial and stromal cells, but with a pattern slightly different from cancers. Allelic imbalance in the tumor epithelium or stroma was not correlated with the accumulation of hyaluronan or clinicopathologic parameters, including tumor stage and grade. The results suggest that factors other than inactivation of the HYAL1-3 genes are responsible for hyaluronan accumulation in epithelial ovarian tumors. Moreover, the results indicate that the stromal cells of the epithelial ovarian cancers not only respond to the signals from malignant epithelium but also have themselves undergone genetic alterations in markers partly identical to those in the cancer epithelial cells and may actively contribute to the development of the tumor from its early stages to the late determinants of patient mortality.