Zeeshan Qadri

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.

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

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis

BackgroundBreast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored.MethodsWe examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors.ResultsXIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo.ConclusionThese data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

FoxM1 is an independent poor prognostic marker and therapeutic target for advanced Middle Eastern breast cancer

Breast cancer (BC) is the most common cause of cancer-related death in females in Saudi Arabia. BC in Saudi women tend to behave more aggressively than breast cancer in the West. Therefore, identification of new molecular targets and treatment strategies are highly warranted to improve patient outcome. FoxM1 has been shown to play a critical role in pathogenesis of various malignancies. In this study, we explored the prevalence and clinical implication of FoxM1 overexpression in Saudi breast cancer. FoxM1 protein overexpression was seen in 79% (770/975) of BC tissues and was associated with aggressive clinical parameters such as younger age (< 30 yrs) (p = 0.0172), high grade (p < 0.0001), mucinous histology (p < 0.0001) and triple negative phenotype (p < 0.0001). Overexpression of FoxM1 was significantly associated with activated AKT (p < 0.0001), Ki67 expression (p < 0.0001), VEGF (p < 0.0001), MMP-9 (p < 0.0001), XIAP (p < 0.0001) and Bcl-xL (p = 0.0300). Importantly, FoxM1 overexpression is found to be an independent prognostic marker in multivariate analysis in advanced stage (Stage III and IV) breast cancer (p = 0.0298). In vitro data using BC cell lines showed that down-regulation of FoxM1 using specific inhibitor, thiostrepton or siRNA inhibited cell migration, invasion and angiogenesis. In addition, treatment of BC cell lines with thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. In vivo, thiostrepton treatment regressed MDA-MB-231 cells generated xenografts via down-regulation of FoxM1 and its downstream targets. Our results suggest that FoxM1 may be a potential therapeutic target for the treatment of aggressive breast cancers.

Abstract 3560: Bcl-Xl over-expression is a poor prognostic marker in papillary thyroid cancer and can be therapeutically targeted to induce apoptosis and autophagy

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Bcl-Xl is a member of the Bcl-2 family of proteins that are divided into either pro-apoptotic proteins or anti-apoptotic proteins on the basis of their functionality. All the members of the Bcl-2 family share common domains known as Bcl-2 Homology (BH1-4) domain. The pro-apoptotic family members include proteins that only contain the BH-3 domain and include Bax, Bak, Bid, PUMA, NOXA, Bim and Bad while the anti-apoptotic proteins include members such as Bcl-2, Bcl-Xl and Mcl-1. In normal conditions, there is a balance between the pro- and anti-apoptotic members of the Bcl-2 family. Because of their important role in cell survival, there has been lot of interest in utilizing dysregulation of Bcl-2 family members to counter cancer growth and induce apoptosis. Bcl-Xl is an anti-apoptotic protein that has found to be over-expressed in various cancers including lung cancer, DLBCL and breast cancer. However, the role of Bcl-Xl in papillary thyroid cancer (PTC) from Middle Eastern region has not been fully illustrated. In order to investigate the role of Bcl-Xl in PTC, we examined the expression of Bcl-Xl in a cohort of 1022 PTC clinical cases by immunohistochemistry in a tissue microarray format and found that Bcl-Xl was over-expressed in 51.7% of PTC cases. Bcl-Xl over-expression was significantly associated with aggressive high proliferative markers such as older age (p = 0.0009), extra-thyroidal extension (p<0.0001), tumor size (p = 0.0081), nodal involvement (p = 0.0067), metastasis (p = 0.0013) and showed a poor overall 5 year survival (0.0438). Bcl-Xl over-expression was was also found to be significantly associated with p-AKT (p<0.0001), XIAP (p<0.0001) and proliferative marker Ki67 (p = 0.0041). In vitro, targeting Bcl-Xl expression in PTC cell lines using a small molecular inhibitor of Bcl-Xl; AB141657 (Z36) showed a dose dependent inhibition of cell viability and induction of apoptosis after 48 hours treatment. Using 5 and 10μM Z36, we found that PTC cells not only underwent apoptosis detected by flow cytometry, inactivation of AKT and activation of mitochondrial apoptotic pathway but also autophagy as detected by up-regulation of LC1-3, inactivation of p-mTOR1 and p-mTOR2 and their downstream targets. These data clearly indicate a role of Bcl-Xl in the pathogenesis of PTC as well as the importance of targeting Bcl-Xl using Z36 to induce both; apoptosis and autophagy in Bcl-Xl over-expressing PTC cells. Citation Format: Azhar R. Hussain, Maqbool Ahmed, Shaham Beg, Rong Bu, Roxanne Melosantos, Zeeshan Qadri, Saif Al-Sobhi, Fouad Al-Dayel, Abdul Khalid Siraj, Khawla S. Al-Kuraya. Bcl-Xl over-expression is a poor prognostic marker in papillary thyroid cancer and can be therapeutically targeted to induce apoptosis and autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3560.

Abstract 3117: Pleiotrophin is a marker of poor prognosis in Middle Eastern papillary thyroid carcinoma

Pleiotrophin (PTN) is a heparin binding growth factor known to have role in neuronal development. It is highly expressed in embryo but has a very limited expression in adult tissues. PTN is considered a proto-oncogene and has been hypothesized to play role in oncogenesis as its expression is found to be increased in many different cancer subtypes. Its role in cell transformation, cell growth, survival, migration and angiogenesis has also been shown in various different types of cancers. The function of PTN is hypothesized to be carried out by its interaction with cell surface proteoglycans or binding to its selective cell surface receptor, protein tyrosine phosphatase receptor Z1 (PTPRZ1). The significance of role of PTN in pathogenesis of thyroid cancer has not been explored especially with the fact that papillary thyroid carcinoma (PTC) originating in this ethnic population is the second most common female malignancy, after breast. So in search for novel druggable molecular target we sought for PTN expression in a large cohort of Saudi PTC. We analysed PTN alteration in more than 1000 primary papillary thyroid carcinoma in a tissue microarray format with clinical follow up data. We found that PTN was overexpressed in 65.5% (658/1006) of PTC and was significantly associated with aggressive clinical parameters such as tall cell variant histological subtype (p = 0.0333), extrathyroidal extension (p = 0.0292), lymphovascular invasion (p = 0.0182) and large tumour size (p = 0.0160). Important significant molecular association was seen with PTPRZ-1 (p = 0.0316), Midkine (p = 0.0008) and pSTAT-3 (p Keywords: Papillary Thyroid Cancer; Pleiotrophin; Pleiotrophin Receptor Citation Format: Shaham Beg, Maqbool Ahmed, Azhar Hussein, Rong Bu, Zeeshan Qadri, Saif AlSobhi, Fouad Al Dayel, Abdul K. Siraj, Khawla S. Al-Kuraya. Pleiotrophin is a marker of poor prognosis in Middle Eastern papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3117.

Abstract 2980: Clinico-pathological correlation of UBE2C alterations in colorectal carcinoma and efficacy of UBE2C inhibition by proteosome inhibitor as a viable therapeutic target

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Substantial evidence implicates UBE2C in several human cancers including colorectal carcinoma. We sought to determine the prognostic value of the UBE2C alterations in colorectal cancer (CRC), UBE2C signaling in CRC cell lines and effect of protesome inhibitors in modulating UBE2C expression in vitro and in vivo nude mice xenografts. We examined UBE2C expression by immunohistochemistry and UBE2C gene copy number by FISH in CRC.The oncogenic role of UBE2C in CRC cell lines was explored and potential therapeutic value of Bortezomib in treating CRC by inhibiting UBE2C was also investigated using in vivo and in vitro analysis. UBE2C dysregulation was seen in a large percentage of colorectal carcinomas: 45.9% at the protein level; 30.7 % at the gene copy number and UBE2C alteration in 61.5 % of the CRC. UBE2C dysregulation occurs early in the progression from adenoma to carcinoma sequence and was associated with proliferative marker Ki-67(p=0.0023), accumulation of cyclin A (p=0.0175) and B1 (p=0.0026). UBE2C expression was associated with a poor overall survival (p=0.0267) in all CRC patients and interestingly, in the early stage CRC subgroup, UBE2C expression was an independent (RR 3.67 95% C.I 1.15-14.03) prognostic marker.Thus, the above findings show the potential of targeting UBE2C enzyme in early stage CRC as well as in pre neoplastic adenomatous lesions. In CRC cell lines, UBE2C depletion resulted in suppression of cellular growth and accumulation of cyclin A and B1, whereas over overexpression of UBE2C promoted cell proliferation. Bortezomib treatment of CRC cells and xenograft downregulated the expression of UBE2C at mRNA as well as protein level, inhibited cell proliferation resulted in accumulation of cyclin A and cyclin B1 and reduced tumor growth. Altogether, our results establish UBE2C as a key molecule in colorectal carcinogenesis and identify a subgroup of CRC patients with high UBE2C alterations showing a poor overall survival. We have also shown for the first time the efficacy of Bortezomib in inhibiting UBE2C expression at the protein as well as the mRNA level and stabilization of cyclin A and B1. These studies may have important implications for future preclinical and clinical studies in CRC aimed at determining the usefulness of a novel strategy for treating CRC with inhibitors of proteasome pathways, either alone or in combination with other agents. Further work is ongoing to check if treatment with the UBE2C siRNA sensitize the cells to Bortezomib treatment or other commonly used colorectal cancer therapies and if Bortezomib-induced tumor regression comparable to conventional chemotherapeutics like oxaliplatin or 5FU treatment. 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 2980. doi:10.1158/1538-7445.AM2011-2980

Abstract 2711: JC virus T-Antigen (SV40) is an independent prognostic marker for poor disease free survival in epithelial ovarian carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Epithelial ovarian cancer is the leading cause of death from gynecologic cancers and because of the pelvic location of the ovaries, the patients present at an advanced stage that is generally incurable. SV40 is a type of polyoma virus has been shown to induce tumors in rodents, transform cells in tissue culture and promote tumor transformation in transgenic mice. Many studies have reported the presence of simian virus 40 (SV40) deoxyribonucleic acid (DNA) or protein in human brain tumors, bone cancers, non-Hodgkins lymphomas, malignant mesothelioma and other solid epithelial tumors including epithelial ovarian carcinomas. We studied the expression of SV40 in a tissue microarray cohort of 156 epithelial ovarian cancers by immunohistochemistry. The incidence of SV40 expression was 44.3% (58/131) and poorly differentiated carcinomas showed a trend towards higher expression of SV40 as compared to moderately and well differentiated tumors (p=0.0898). Validation of SV40 IHC expression was done by polymerase chain reaction (PCR) assay in selected clinical samples and ovarian cancer cell lines. A good concordance was observed between immunohistochemical expression of SV40 and amplification by PCR. Earlier studies have reported various molecular targets of SV40: up-regulation of c-MET oncogene and IGF-1; inhibition of tumor suppressor genes like p53, RASS1FA and protein phosphotase PP2A. To elucidate the link between these targets and SV40 we assessed the immunohistochemical expression of p53, RASS1FA, PP2A, c-MET and IGF-1 the cohort of epithelial ovarian carcinomas. In addition we also analyzed the association of SV40 with known oncogenic mutations in p53, KRAS and PIK3CA genes. SV40 expression showed a significant association with presence of p53 mutation (p<0.0001.) as well as p53 overexpression (p<0.0001). In addition, SV40 expression was associated with overexpression of c-MET (p=0.0111) and IGF-1(p=0.0334). EOC patients with SV40 expression had a poor progression free survival (PFS) of 13.2 months as compared to 20 months with absent SV40 expression (p=0.0980). In the multivariate analysis using Cox Proportional Hazard model for multiple factors like age, FIGO stage, grade, the relative risk was 1.70 for high SV40 expression (95% CI 1.10 – 2.69; p=0.0227) and 2.32 for high stage group III-IV (95% CI 1.33 – 3.90; p=0.0038). Thus, SV40 over expression was an independent prognostic marker in EOC. Altogether, our data highlights the prognostic utility of SV40 as a prognostic biomarker and confirms the well-known effect of SV40 on down-regulation or inactivation of p53, and activation of c-MET/IGF-1 in the epithelial ovarian carcinogenesis. 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 2711. doi:10.1158/1538-7445.AM2011-2711

Abstract 2104: Frequent inactivation of A20 and its prognostic significance in Middle Eastern colorectal carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway in hematolymphoid neoplasms. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. However, role of A20 gene in CRC is unknown. Therefore, we sought to elucidate the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. We investigated the genetic and epigenetic abnormalities of A20 gene in CRC. In a tissue microarray cohort of 434 CRC, we studied loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH. We also screened 116 random CRC and 14 CRC cell lines for mutations in A20 gene and A20 promoter methylation. Of the 116 CRC samples, A20 deletions were seen in 15.3% and incidence of A20 mutation was 2.5%; most of the mutations (4/5) were missense mutations. A20 promoter hyper-methylation was seen in 50.8% and was correlated with loss of protein expression (p=0.0079). A20 inactivation, defined as loss or reduced expression of protein was observed in 63% of CRC and A20 inactivation was an independent prognostic marker for poor survival in all CRC. In addition A20 expression retained its prognostic value in the following subgroups: Stage III; Stage II and III; and Stage III and IV. A20 was inactivated in majority of the CRC due to promoter methylation and can be targeted to modulate NF-κb expression. 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 2104. doi:10.1158/1538-7445.AM2011-2104