Prashant Bavi

Homozygous Mutations in ADAMTS10 and ADAMTS17 Cause Lenticular Myopia, Ectopia Lentis, Glaucoma, Spherophakia, and Short Stature

Weill-Marchesani syndrome (WMS) is a well-characterized disorder in which patients develop eye and skeletal abnormalities. Autosomal-recessive and autosomal-dominant forms of WMS are caused by mutations in ADAMTS10 and FBN1 genes, respectively. Here we report on 13 patients from seven unrelated families from the Arabian Peninsula. These patients have a constellation of features that fall within the WMS spectrum and follow an autosomal-recessive mode of inheritance. Individuals who came from two families and met the diagnostic criteria for WMS were each found to have a different homozygous missense mutation in ADAMTS10. Linkage analysis and direct sequencing of candidate genes in another two families and a sporadic case with phenotypes best described as WMS-like led to the identification of three homozygous mutations in the closely related ADAMTS17 gene. Our clinical and genetic findings suggest that ADAMTS17 plays a role in crystalline lens zonules and connective tissue formation and that mutations in ADAMTS17 are sufficient to produce some of the main features typically described in WMS.

Genome wide expression analysis of Middle Eastern colorectal cancer reveals FoxM1 as a novel target for cancer therapy

To identify genes potentially playing an important role in the progression of colorectal carcinoma (CRC), we screened global gene expression using cDNA expression array on 41 CRC tissue samples and 25 noncancerous colorectal tissue samples. Among the up-regulated genes, forkhead box M1 (FOXM1) has been shown to play a critical role in pathogenesis of various malignancies. Using immunohistochemistry on 448 Saudi CRC samples in tissue microarray format, FoxM1 protein overexpression was seen in 66% of CRC tissues and was significantly associated with poorly differentiated and highly proliferative tumors (P = 0.0200 and 0.0018, respectively). FoxM1 expression was also significantly associated with MMP-9 protein expression (P = 0.0002). In vitro data using CRC cell lines showed that inhibition of FoxM1 by thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. Overexpression of FoxM1 potentiated cell proliferation, cell transformation, and migration/invasion of CRC cells via up-regulation of FoxM1 target genes MMP2 and MMP9 and protected these cells from thiostrepton-mediated antiproliferative effects. Finally, in vivo, overexpression of FoxM1 promoted growth of CRC-cell line xenograft tumors in nude mice. Altogether, our data indicate that FoxM1 signaling contributes to aggressiveness in a subset of CRC and that the FOXM1 gene may serve as a useful molecular biomarker and potential therapeutic target.

Overexpression of leptin receptor predicts an unfavorable outcome in Middle Eastern ovarian cancer

BackgroundRecent epidemiological studies have suggested that obesity is associated with ovarian cancer. Obesity hormone leptin and its receptor (Ob-R) contribute to tumor development by enhancing cell growth and survival. This study was design to investigate the prevalence of leptin and Ob-R in Middle Eastern epithelial ovarian cancer (EOC) and to analyze the role of leptin and the mechanisms under its action in EOC tissue sample and cell lines.MethodsThe expression of leptin and Ob-R was examined by immunohistochemistry in a tissue microarray of 156 EOC samples. Proliferation of EOC cells in response to leptin was assessed by MTT assays, and its anti-apoptotic effects were determined by flow cytometry. Effect of leptin on PI3K/AKT signaling pathway was further determined by western blotting.ResultsIn clinical samples, Ob-R overexpression was seen in 59.2% EOCs and was significantly associated with poor progression free survival (p = 0.0032). Furthermore, Ob-R expression was associated with anti apoptotic proteins Bcl-XL (p = 0.0035) and XIAP (p = 0.0001). In vitro analysis using EOC cell lines showed that leptin stimulated cell proliferation and inhibits apoptosis via activation of PI3K/AKT signaling pathway. Inhibition of PI3K activity by LY294002, a specific inhibitor of PI3-kinase abrogated leptin mediated PI3K/AKT signaling. Gene silencing of Ob-R with Ob-R siRNA in EOC cells resulted in down regulation of phospho-AKT and its down stream targets.ConclusionOur findings have potential clinical implication for EOC development and progression.

Mutations in C2orf37, encoding a nucleolar protein, cause hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome.

Hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome (also referenced as Woodhouse-Sakati syndrome) is a rare autosomal recessive multisystemic disorder. We have identified a founder mutation consisting of a single base-pair deletion in C2orf37 in eight families of Saudi origin. Three other loss-of-function mutations were subsequently discovered in patients of different ethnicities. The gene encodes a nucleolar protein of unknown function, and the cellular phenotype observed in patient lymphoblasts implicates a role for the nucleolus in the pathogenesis of this disease. Our findings expand the list of human disorders linked to the nucleolus and further highlight the developmental and/or maintenance functions of this organelle.

FREM1 Mutations Cause Bifid Nose, Renal Agenesis, and Anorectal Malformations Syndrome

An autosomal-recessive syndrome of bifid nose and anorectal and renal anomalies (BNAR) was previously reported in a consanguineous Egyptian sibship. Here, we report the results of linkage analysis, on this family and on two other families with a similar phenotype, which identified a shared region of homozygosity on chromosome 9p22.2-p23. Candidate-gene analysis revealed homozygous frameshift and missense mutations in FREM1, which encodes an extracellular matrix component of basement membranes. In situ hybridization experiments demonstrated gene expression of Frem1 in the midline of E11.5 mouse embryos, in agreement with the observed cleft nose phenotype of our patients. FREM1 is part of a ternary complex that includes FRAS1 and FREM2, and mutations of the latter two genes have been reported to cause Fraser syndrome in mice and humans. The phenotypic variability previously reported for different Frem1 mouse mutants suggests that the apparently distinct phenotype of BNAR in humans may represent a previously unrecognized variant of Fraser syndrome.

Cyclooxygenase‐2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer

Cyclooxygenase‐2 (COX‐2) expression contributes to tumor growth and invasion in epithelial ovarian cancer (EOC). COX‐2 inhibitors exhibit important anticarcinogenic potential against EOC, but the molecular mechanisms underlying this effect and relation with PI3‐kinase/AKT signaling remain the subject of intense investigations. Therefore, the role of COX‐2 in EOC and its cross talk with PI3‐kinase/AKT pathway were investigated using a large series of EOC tissues in a tissue micro array (TMA) format followed by in vitro and in vivo studies using EOC cell lines and NUDE mice. Clinically, COX‐2 was overexpressed in 60.3% of EOC and was significantly associated with activated AKT (p < 0.0001). Cox‐1 expression was seen in 59.9% but did not associate with AKT. Our in vitro data using EOC cell line showed that inhibition of COX‐2 by aspirin, selective inhibitor NS398 and gene silencing by COX‐2 specific siRNA impaired phosphorylation of AKT resulting decreased downstream signaling leading to cell growth inhibition and induction of apoptosis. Finally, treatment of MDAH2774 cell line xenografts with aspirin resulted in growth inhibition of tumors in NUDE mice via down‐regulation of COX‐2 and AKT activity. These data identify COX‐2 as a potential biomarker and therapeutic target in distinct molecular subtypes of ovarian cancer.

Bortezomib (Velcade) Induces p27Kip1 Expression through S-Phase Kinase Protein 2 Degradation in Colorectal Cancer

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell cycle regulators including cycle-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancers. We investigated the role of SKP2 and its ubiquitin-proteasome pathway in colorectal carcinoma using a panel of cell lines, clinical samples, and the NUDE mouse model. Using immunohistochemical analysis on a large tissue microarray of 448 samples, an inverse association of SKP2 expression with p27Kip1 protein levels was seen. A colorectal cancer (CRC) subset with high level of SKP2 and low level of p27Kip1 showed a decreased overall survival (P = 0.0057). Treatment of CRC cell lines with bortezomib or expression of small interfering RNA of SKP2 causes down-regulation of SKP2 and accumulation of p27Kip1. Furthermore, treatment of CRC cells with bortezomib causes apoptosis by involving the mitochondrial pathway and activation of caspases. In addition, treatment of CRC cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Finally, treatment of CRC cell line xenografts with bortezomib resulted in growth inhibition of tumors in NUDE mice via down-regulation of SKP2 and accumulation of p27Kip1. Altogether, our results suggest that SKP2 and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention for treatment of CRC.

Prognostic significance of alterations in KRAS isoforms KRAS-4A/4B and KRAS mutations in colorectal carcinoma

Somatic KRAS mutation is an early well‐known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico‐pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real‐time PCR assay, to study the protein expression of KRAS4A and ‐4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0.0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5‐year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0.0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0.0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0.0234) and inversely correlated with p27kip1 protein (p = 0.0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC. Copyright

Leptin receptor expression in Middle Eastern colorectal cancer and its potential clinical implication

We investigated the role of leptin receptor (Ob-R) and its relationship with phosphatidylinositol 3-kinase (PI3K)/AKT activation in colorectal carcinomas (CRCs) tissues followed by in vitro studies using a panel of CRC cell lines. Obesity serves an important risk factor of several cancers including CRC that ranks as the second most common cancer in Saudi Arabia. High levels of adipokine leptin (Ob) and its Ob-R are seen in obesity and also in various carcinomas including CRC. We investigated the proliferative and antiapoptotic effect of Ob on human CRC cell lines Caco-2, HT-29 and SW-840 and the role of PI3K/AKT-signaling pathway in mediating these actions. Then the expression of Ob-R and its relationship with clinicopathological features was analyzed in 448 CRC, 229 normal colon mucosa and 24 colorectal adenomas using tissue microarray technology. Treatment with Ob resulted in increased proliferation of CRC cell lines and involved activation of PI3K/AKT-signaling pathway. Pretreatment with Ob-R small interfering RNA or PI3K inhibitor inhibited these responses. Ob-R was significantly overexpressed in primary CRC relative to adenomas and normal colonic mucosa. In primary CRC, Ob-R significantly correlated with Ob expression, early stage and well-differentiated tumors. Intriguingly, patient with Ob-R positive tumors showed significantly better overall survival (P = 0.0098). Ob plays a critical role in CRC carcinogenesis through PI3K/AKT pathway via Ob-R. Ob-R is a prognostic marker associated with better survival.

Prognostic significance of XIAP expression in DLBCL and effect of its inhibition on AKT signalling

The inhibitor of apoptosis protein (IAP) family member X‐linked inhibitor of apoptosis protein (XIAP) is essential for cell survival in lymphoma. However, the role of XIAP overexpression in diffuse large B‐cell lymphoma (DLBCL) is not fully elucidated. Therefore, we analysed the expression of XIAP protein and its clinicopathological correlation in a large cohort of DLBCLs by immunohistochemistry in a tissue micro‐array format. XIAP was found to be overexpressed in 55% of DLBCLs and significantly associated with poor clinical outcome (p = 0.0421). To further elucidate the role of XIAP in DLBCL and the inter‐relationship with PI3‐kinase/AKT signalling, we conducted several in vitro studies using a panel of DLBCL cell lines. We found that pharmacological inhibition of XIAP led to caspase‐dependent apoptosis in DLBCL cells. We also detected an inter‐relationship between XIAP expression and activated AKT in DLBCL cells that may explain cellular resistance to PI3‐kinase/AKT inhibition‐mediated apoptosis. Finally, this anti‐apoptotic effect was overcome by simultaneous pharmacological inhibition of XIAP and PI3‐kinase/AKT signalling leading to a more potent synergistically induced apoptosis. In summary, our data suggest that XIAP expression is a poor prognostic factor in DLBCL and the XIAP‐AKT relationship should be explored further as a potential therapeutic target in DLBCL. Copyright