Sarita Prabhakaran

Bortezomib Stabilizes Mitotic Cyclins and Prevents Cell Cycle Progression via Inhibition of UBE2C in Colorectal Carcinoma

Substantial evidence implicates the ubiquitin-conjugating enzyme E2C (UBE2C) gene, in several human cancers, including colorectal carcinoma (CRC). We therefore investigated the prognostic value of UBE2C alterations in CRC and UBE2C signaling in CRC cell lines. UBE2C protein expression and UBE2C gene copy number were evaluated on clinical samples by immunohistochemistry and fluorescence in situ hybridization in a TMA format. The effect of the proteasome inhibitor bortezomib and small-interfering RNA knockdown was assessed by apoptotic assays and immunoblotting. UBE2C dysregulation was associated with proliferative marker Ki-67, accumulation of cyclin A and B1, and a poor overall survival. UBE2C expression was an independent prognostic marker in early-stage (I and II) CRC. UBE2C depletion resulted in suppression of cellular growth and accumulation of cyclin A and B1. In vitro, bortezomib treatment of CRC cells caused inhibition of cell viability via down-regulation of UBE2C. UBE2C knockdown by bortezomib or transfection with specific small-interfering RNA against UBE2C also caused cells to be arrested at the G2/M level, leading to accumulation of cyclin A and cyclin B1. In vivo, a significant reduction in tumor volume and weight was noted in mice treated with a combination of subtoxic doses of oxaliplatin and bortezomib compared with treatment with oxaliplatin or bortezomib alone. Altogether, our results suggest that UBE2C and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention in CRC.

Prognostic significance of TRAIL death receptors in Middle Eastern colorectal carcinomas and their correlation to oncogenic KRAS alterations

BackgroundTumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor cytokine family that induces apoptosis upon binding to its death domain containing receptors, TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). Expression of TRAIL receptors is higher in colorectal carcinoma (CRC) as compared to normal colorectal mucosa and targeted therapy with TRAIL leads to preferential killing of tumor cells sparing normal cells.MethodsWe investigated the expression of TRAIL and its receptors in a tissue microarray cohort of 448 Middle Eastern CRC. We also studied the correlation between TRAIL receptors and various clinico-pathological features including key molecular alterations and overall survival.ResultsCRC subset with TRAIL-R1 expression was associated with a less aggressive phenotype characterized by early stage (p = 0.0251) and a histology subtype of adenocarcinomas (p = 0.0355). Similarly CRC subset with TRAIL-R2 expression was associated with a well-differentiated tumors (p < 0.0001), histology subtype of adenocarcinomas (p = 0.0010) and tumors in left colon (p = 0.0009). Over expression of pro apoptotic markers: p27KIP1 and KRAS4A isoforms was significantly higher in CRC subset with TRAIL-R1 and TRAIL-R2 expression; TRAIL-R1 expression was also associated with cleaved caspase-3(p = 0.0011). Interestingly, TRAIL-R2 expression was associated with a microsatellite stable (MS--S/L) phenotype (p = 0.0003) and with absence of KRAS mutations (p = 0.0481).ConclusionTRAIL-R1 expression was an independent prognostic marker for better survival in all CRC samples and even in the CRC group that received adjuvant therapy. The biological effects of TRAIL in CRC models, its enhancement of chemosensitivity towards standard chemotherapeutic agents and the effect of endogenous TRAIL receptor levels on survival make TRAIL an extremely attractive therapeutic target.

A very low incidence of BRAF mutations in Middle Eastern colorectal carcinoma.

BackgroundRecent studies emphasize the role of BRAF as a genetic marker for prediction, prognosis and risk stratification in colorectal cancer. Earlier studies have reported the incidence of BRAF mutations in the range of 5-20% in colorectal carcinomas (CRC) and are predominantly seen in the serrated adenoma-carcinoma pathway characterized by microsatellite instability (MSI-H) and hypermethylation of the MLH1 gene in the setting of the CpG island methylator phenotype (CIMP). Due to the lack of data on the true incidence of BRAF mutations in Saudi Arabia, we sought to analyze the incidence of BRAF mutations in this ethnic group.Methods770 CRC cases were analyzed for BRAF and KRAS mutations by direct DNA sequencing.ResultsBRAF gene mutations were seen in 2.5% (19/757) CRC analyzed and BRAF V600E somatic mutation constituted 90% (17/19) of all BRAF mutations. BRAF mutations were significantly associated with right sided tumors (p = 0.0019), MSI-H status (p = 0.0144), CIMP (p = 0.0017) and a high proliferative index of Ki67 expression (p = 0.0162). Incidence of KRAS mutations was 28.6% (216/755) and a mutual exclusivity was noted with BRAF mutations (p = 0.0518; a trend was seen).ConclusionOur results highlight the low incidence of BRAF mutations and CIMP in CRC from Saudi Arabia. This could be attributed to ethnic differences and warrant further investigation to elucidate the effect of other environmental and genetic factors. These findings indirectly suggest the possibility of a higher incidence of familial hereditary colorectal cancers especially Hereditary non polyposis colorectal cancer (HNPCC) syndrome /Lynch Syndrome (LS) in Saudi Arabia.

ALK alteration is a frequent event in aggressive breast cancers

IntroductionBreast cancer is the most common female malignancy worldwide and, despite improvements in treatment modalities, there are increased chances of recurrence and metastasis in a substantial number of cases and it remains one of the major causes of mortality among female cancer patients. Anaplastic lymphoma kinase (ALK) gene has been found to be altered in several solid and hematologic tumors. We aimed to comprehensively study the prevalence of ALK expression, and changes in copy number and translocation in a large cohort of breast cancer cases in a Middle Eastern population.MethodsALK protein expression was investigated by immunohistochemistry and numerical and structural variations of the ALK gene were analyzed by fluorescence in situ hybridization (FISH) in a tissue microarray format in a cohort of more than 1000 Middle Eastern breast cancers. The data were correlated with clinicopathologic parameters and other important molecular biomarkers.ResultsImmunohistochemical analysis showed ALK overexpression in 36.0 % of the breast cancer patients and gene amplification was present in 13.3 % of cases, seen by FISH analyses. ALK overexpression was significantly associated with ALK gene amplification (p = 0.0031). ALK-overexpressing tumors showed significant association with high-grade tumors (p = 0.0039), ductal histologic subtype (p = 0.0076), triple-negative phenotype (p = 0.0034), and high Ki-67 (p = 0.0001) and p-AKT (p <0.0001).ConclusionsImmunohistochemical analysis showed ALK is overexpressed in a substantial proportion of breast cancers and possibly plays a significant role in the aggressive behavior of this cancer. Gene amplification is hypothesized to be a possible cause for a significant proportion of this overexpression. Based on these findings, a potential role for an ALK inhibitor, as a therapeutic agent targeting aggressive subtypes of breast cancer, merits further investigation.

Abstract 4412: Prognostic significance of NF-κB in Middle Eastern diffuse large B cell lymphoma and efficacy of NF-κB inhibition as a viable therapeutic target

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL NF-kB is frequently over expressed in variety of NHL and has been implicated in lymphomagenesis; however, its role in diffuse large B-cell lymphoma (DLBCL) as a prognostic biomarker has not been fully elucidated. Therefore, we investigated the role of NF-kB and its association with clinico-pathological features in a tissue microarray cohort of 230 DLBCL patient samples. We then elucidated the role of NF-kB inhibition on cell viability and apoptosis in-vitro using DLBCL cell lines. Using immunohistochemistry, NF-kB was detected in 25.6% (52/203) DLBCL tumors, was associated with activated B cell (ABC) phenotype (p=0.0054) and overexpression of anti apoptotic marker, XIAP (p=0.0013). DLBCL with nuclear expression of NF-kB showed a significantly poor overall survival as compared to those with no NF-kB expression (p=0.0236). In the multivariate analysis using Cox Proportional Hazard model for IPI and NF-kB expression, the relative risk was 2.97 for high NF-kB expression (95% CI 1.27-6.94; p=0.0113) and 7.55 for high IPI group (95% CI 3.34-18.35; p<0.0001). In-vitro, Bay11-7085 inhibited constitutively active NF-kB expression in a dose dependent manner. Inhibition of NF-kB also down-regulated expression of down-stream target gene products, such as Bcl-2, Bcl-Xl, XIAP and Survivin leading to apoptosis via the mitochondrial apoptotic pathway. NF-kB overexpression was found to be an independent prognostic marker for poor survival in DLBCL. Inhibition of NF-kB caused apoptosis via activation of the mitochondrial apoptotic pathway. Altogether, these results suggest that NF-kB may be a useful prognostic biomarker and a potential target for therapeutic intervention in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4412. doi:10.1158/1538-7445.AM2011-4412

The role of HER2 overexpression in Middle Eastern papillary thyroid cancer

Background: HER2 oncogene is involved in many cancers and serves as a prognostic marker and therapeutic target in breast cancer. The purpose of this study was to learn more on the prevalence and clinical significance of HER2 overexpression and its association with clinical parameters in Middle Eastern papillary thyroid carcinoma (PTC). Methods: A tissue microarray (TMA) containing >1,000 PTC cases with follow-up data was used. TMA sections were analyzed on protein and DNA level using immunohistochemistry and fluorescence in situ hybridization (FISH). FISH analyses were performed to look for the gain or amplifications in HER2 gene. Results: Immunohistochemical analysis showed HER2 overexpression in 19.7% of our cases. Elevated expression was almost exclusively 2+ (194 tumors) and only rarely 3+ (1 tumor). No amplification was seen by FISH. Only 3% of our cases showed mildly elevated HER2 gene copy numbers not reaching the threshold for amplification. HER2 overexpression and copy number gains were unrelated to tumor stage, metastasis, patient survival and other clinical and pathological parameters. Conclusions: Our results demonstrated that HER2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. Additionally, expression of HER2 seems to hold no clinical value as prognostic factor in PTC.

Abstract 2727: Molecular markers and pathway analysis of Middle Eastern colorectal carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Colorectal cancer (CRC) is one of the common cancers in the world. In Saudi Arabia CRC is the most common cancer in males and third most common in females. A newly proposed integrated pathways comprising traditional, alternate and serrated by genetic and epigenetic factors was defined recently and hypothesized to role in the pathogenesis of CRC; however there is a paucity of information about these proposed molecular pathways in different ethnic group. Hence we analyzed a large cohort of Saudi colorectal tumors to understand the role of these molecular pathways. 770 archival CRCs specimen were evaluated for Microsatellite Instability (MSI), BRAF, KRAS and 500 cases for CpG Island Methylator Phenotype (CIMP). Traditional pathway constituted 33.4% of CRC cases; the alternate pathway comprised 11.6% of cases. Strikingly, the serrated molecular pathway accounted for only 0.8% of Middle Eastern CRC that is extremely below compared to publish data. 54.2% CRC cases did not qualify to fit into any pathway and thus were designated an unassigned group. Molecular pathways were significantly associated with tumor site and grade. In our attempt to further classify unassigned group a subset of the uncategorized pathway showed a significant survival difference (p = 0.0079). The Unassigned group that accounted for the majority of our cases reflects the heterogeneity of colorectal cancers and warrants the need to unravel the molecular genetic basis of this disease to further subcategorize these CRC cases. It also identifies the need to do further studies on different populations for better understanding of their exact role and incidence. Keywords: colorectal; molecular pathways; serrated Citation Format: Shaham Beg, Sarita Prabhakaran, Rong Bu, Mohammed Al-Assiri, Rami Sairafi, Fouad Al-Dayel, Nasser Al-Sanea, Abdul K. Siraj, Shahab Uddin, Khawla Al-Kuraya. Molecular markers and pathway analysis of Middle Eastern colorectal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2727. doi:10.1158/1538-7445.AM2015-2727

Abstract 4323: ALK overexpression is associated with activation of PI3K/AKT signaling pathway in PTC

Aberrant activation of anaplastic lymphoma kinase (ALK) gene caused by mutation, rearrangement or overexpression plays a fundamental role in tumorigenesis in several tumors and represents an important therapeutic target. Currently, only limited data is available on thyroid cancer with respect to the genetic alteration of ALK. Therefore we investigate the ALK overexpression and amplification, and its correlation with various clinicopathological features and molecular biomarkers in a large cohort of 1040 Middle Eastern PTC. In our study, ALK protein expression was determined by immunohistochemistry. ALK gene amplification and rearrangement were analyzed by in situ hybridization (FISH). We also correlated the data with clinicopathological characteristics and other molecular markers. Our data showed that ALK is overexpressed in 136 of the 997 (13.6%) PTC samples. ALK amplification and rearrangement were not observed by FISH. ALK overexpression was significantly associated with surgical margin, but had no effect on the survival of PTC patients. Interestingly, ALK overexpression was found to be significantly correlated with the higher expression of pAKT and PIK3CA in PTC samples, suggesting that ALK overexpression is associated with activation of PI3K/AKT signaling pathway in PTC. In addition, ALK overexpression is also correlated with overexpression of anti-apopototic marker XIAP and BCL-XL and cell cycle regulatory marker SKP2. Altogether, our results suggest that ALK overexpression presents in a small subset of samples and is not associated with survival in PTC. However ALK overexpression is significantly correlated with PI3K/AKT signaling pathway and might provide a potential therapeutic target for PTC patients with ALK overexpression. Citation Format: Rong Bu, Sarita Prabhakaran, Shaham Beg, Zeenath Jehan, Abdul K. Siraj, Maqbool Ahmed, Azhar Hussain, Saif A. Alsobhi, Fouad Al-Dayel, Shahab Uddin, Khawla S. Al-Kuraya. ALK overexpression is associated with activation of PI3K/AKT signaling pathway in PTC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2015-4323

Abstract 1213: Clinicopathological significance of ALK alterations in colorectal carcinoma.

ALK has been shown to be a putative oncogene in lung cancer and neuroblastoma that can be potentially targeted with selective inhibitors. Ongoing clinical trials using ALK inhibitors such as Crizotinib have shown promising results. ALK gene copy gain number in colorectal cancer (CRC) patients have been shown to be associated with tumors that have a more aggressive phenotype. We investigated the alterations in ALK gene by FISH analysis, immunohistochemistry and screened for ALK mutations by sequencing the ALK gene in 737 CRC. We screened for mutations in ALK by sequencing the tyrosine kinase domain that covers exons 20 -28. ALK alterations were correlated with tumor pathological features including TNM staging, MSI status, KRAS and BRAF mutations, as well as clinical outcome. Overall survival was analyzed using Kaplan Meyer plots. On FISH analysis in colorectal cancers, ALK amplifications were observed in 2.6%. Amplifications and gain with increase in gene copy number(4 to 6 copies) were seen in 3.4%. No translocations were seen in our study. There were no associations with nodal metastasis and other clinical or molecular parameters. However in Stage III & IV, ALK amplification was associated with KRAS mutations. CRC subgroup with ALK amplifications showed a poor overall survival overall survival and ALK gene amplifications was an independent prognostic marker in multivariate Cox proportional Hazards model. IHC showed cytoplasmic overexpression in 14.7% CRCs and no associations with any clinical or molecular parameters. We are screening for ALK mutations in 100 CRC samples and results are awaited. The insights gained from our current study and future clinical trials are likely to directly benefit subgroups of CRC patients whose tumors are driven by ALK and will pave the way to more targeted approaches to cancer treatment. Citation Format: Zeenath Jehan, Sarita Esther Prabhakaran, Rong Bu, Nasser Al-Sanea, Alaa Abduljabbar, Luai Ashari, Samar Al-Homoud, Fouad Al Dayel, Shahab Uddin, Prashant Bavi, Khawla Sami Al-Kuraya. Clinicopathological significance of ALK alterations in colorectal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2013-1213

Abstract 1069: Clinicopathological significance of CARD10 alterations in Middle Eastern colorectal cancer

The incidence of Colorectal Carcinoma (CRC) in Saudi Arabia is rising and CRC is among the top 3 Saudi cancers in both men and women. NF-κB gene plays a role in tumorgenesis through the transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, tumor promotion, inflammation and suppression of apoptosis. NF-κB protein activation, defined as nuclear expression of NF-kB by immunohistochemistry was seen in 43.2% (164/380) CRC and was an independent prognostic marker for poor survival. CARD10 and A20 genes are known positive and negative regulators of the NF-kB activation pathway and can activate the NF-kB activity. CARD10 is an enzyme that is encoded by the CARD10 gene and it associates with IKK complex resulting in NF-κB activation. CARD11 which shares a high degree of structure, domain and functional homology to CARD10 is oncogenic in diffuse large B cell lymphoma and results in activation of NF-κB. Considering the potential therapeutic utility of targeting NF-kB and its key modulators, we studied CARD10 genetic alterations (mutations and amplifications) and immunohistochemical expression of CARD10 in over 300 CRC cases and 13 colon cell lines. We detected missense mutations in 5.2 % (15/288) and 2 colon cell lines. Our results showed that CARD10 protein is widely expressed in CRC (67.7%) and expression levels of CARD10 progressively increase from normal colorectal mucosa to pre-malignant colorectal adenomas to CRC. CARD10 protein expression associated significantly with NF-KB activation (P = 0.0313). Interestingly, there was a mutual exclusivity between CARD10 protein overexpression and A20 loss of expression which might suggest that NFKB can be activated by alteration in one of these genes in CRC. Furthermore, CARD10 amplification (15.3%) was significantly associated with CARD10 protein expression and presence of distance metastasis (p=0.0487) Interestingly CARD10 mutations were detected in 21.4% of MSI CRC and only 78.6% of MSS CRC (p=0.0251), suggesting that CARD10 mutations play a role in both subsets of CRC but might represent more important oncogenic event for the development/progression of MSI CRC than MSS CRC. Our study highlights for the first time the role of CARD10 protein overexpression as a novel mechanism for NF-kB activation and demonstrates clinico-pathological significance of CARD10 alterations in Middle Eastern CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2011-1069