PD-L1 Is an Independent Prognostic Marker in Middle Eastern PTC and Its Expression Is Upregulated by BRAFV600E Mutation.

PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating BRAFV600E in PTC cell lines. PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with aggressive clinico-pathological parameters. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with BRAF mutation and patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival compared to patients with BRAF mutation alone. In vitro analysis showed high expression of PD-L1 in BRAF-mutated PTC cell lines compared to a BRAF wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in BRAF-mutated cell lines without affecting cell growth. Knockdown of PD-L1 in BRAF-mutated cell lines significantly decreased the cell growth and induced apoptosis. Our data suggest that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard treatment.

CHD4 Predicts Aggressiveness in PTC Patients and Promotes Cancer Stemness and EMT in PTC Cells.

Chromodomain-helicase-DNA-binding protein 4 (CHD4), a core subunit of the nucleosome remodeling and deacetylation (NuRD) complex is highly expressed in several cancers. However, its role in the pathogenesis and progression of papillary thyroid carcinoma (PTC) has not been investigated. We investigated the prognostic significance of CHD4 in a large cohort of Middle Eastern PTC patients and explored the functional role of CHD4 in regulating cancer stemness and EMT in PTC cells. CHD4 overexpression was observed in 45.3% (650/1436) of PTCs, and was associated with aggressive clinico-pathological parameters and worse outcome. Functional analysis using PTC cell lines showed that forced expression of CHD4 promoted cell proliferation, spheroid growth, migration, invasion and progression of epithelial to mesenchymal transition (EMT) in PTC cells whereas its knockdown reversed the effect. Methylation of E-cadherin was associated with loss of expression in CHD4 expressing cells, while CHD4 depletion reactivated E-cadherin expression. Most importantly, knockdown of mesenchymal transcriptional factors, Snail1 or Zeb1, attenuated the spheroid growth in CHD4 expressing PTC cells, showing a potential link between EMT activation and stemness maintenance in PTC. These findings suggest that CHD4 might be a promising therapeutic target in the treatment of patients with an aggressive subtype of PTC.

Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer.

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. Despite current therapeutic and surgical options, advanced EOC shows poor prognosis. Identifying novel molecular therapeutic targets is highly needed in the management of EOC. Krupple-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in a variety of cancer types. However, its role and expression in EOC is not fully illustrated. Immunohistochemical analysis was performed to assess KLF5 protein expression in 425 primary EOC samples using tissue microarray. We also addressed the function of KLF5 in EOC and its interaction with signal transducer and activator of transcription 3 (STAT3) signaling pathway. We found that KLF5 overexpressed in 53% (229/425) of EOC samples, and is associated with aggressive markers. Forced expression of KLF5 enhanced cell growth in low expressing EOC cell line, MDAH2774. Conversely, knockdown of KLF5 reduced cell growth, migration, invasion and progression of epithelial to mesenchymal transition in KLF5 expressing cell lines, OVISE and OVSAHO. Importantly, silencing of KLF5 decreased the self-renewal ability of spheroids generated from OVISE and OVSAHO cell lines. In addition, downregulation of KLF5 potentiated the effect of cisplatin to induce apoptosis in these cell lines. These data reveals the pro-tumorigenic role of KLF5 in EOC and uncover its role in activation of STAT3 signaling pathway, suggesting the importance of KLF5 as a potential therapeutic target for EOC therapy.

APC truncating mutations in Middle Eastern Population: Tankyrase inhibitor is an effective strategy to sensitize APC mutant CRC To 5-FU chemotherapy.

Colorectal Cancer (CRC) is highly heterogeneous for which prognosis is dependent mainly on clinical staging. There is a need to stratify subpopulations of CRC on molecular basis to better predict outcome and therapy response. Truncating mutations in adenomatous polyposis coli (APC) are well-described events in CRC carcinogenesis. Clinical and genotypic characterization of Middle Eastern CRC based on presence and type of APC was determined in 412 CRC tumors using modern next generation sequencing. APC truncating mutations were identified in 58.2% (240/412) of CRCs. Overall, mutation was significant predictor of superior overall survival. Further, the type of APC mutations (short or long) did not have impact on clinical outcome. However, in vitro analysis showed difference between CRC cell lines carrying short truncating APC vs CRC cells that carry long truncating APC mutation in response to 5-flourouracil (5-FU). Importantly, we were able to overcome the resistance to 5-FU seen in CRC cells carrying short APC by tankyrase inhibitor, XAV939, thereby inhibiting Wnt/ß-catenin signaling cascade. Overall, our results showed that APC mutation status plays an important role in predicting overall survival in Middle Eastern population. Furthermore, in vitro data showed that selective targeting of APC mutated CRC by tankyrase inhibitor can be an effective strategy to overcome 5-FU resistance in CRC cells.

Retraction Note: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism.

This article has been retracted.

Prognostic significance of DNMT3A alterations in Middle Eastern papillary thyroid carcinoma.

BACKGROUND: Thyroid cancer is the second most common cancer affecting Saudi women after breast cancer, with papillary thyroid carcinoma (PTC) accounting for 80-90% of thyroid cancers. DNA methyltransferases affect DNA methylation, and it is thought that they play an important role in the malignant transformation of various cancers. METHODS: We sought to evaluate the frequency of DNA methyltransferase 3A (DNMT3A) alterations in a large cohort of >1000 PTC cases using exome sequencing, capture sequencing, immunohistochemistry and methylation-specific polymerase chain reaction. We also performed in vitro analysis to investigate the role of DNMT3A methylation in PTC cell lines. RESULTS: DNMT3A pathogenic mutations were noted in 1.2% (12/1013) of PTC cases. Reduced/loss of DNMT3A expression was seen in 59.8% (579/968) of PTC cases and was significantly associated with the DNMT3A mutation (p = 0.0120). DNMT3A alterations (mutation and/or loss of expression) were associated with aggressive clinical parameters and a poor outcome. The promoter region of the DNMT3A gene was methylated in 57.1% of PTC cases tested and was significantly associated with reduced DNMT3A protein expression (p = 0.0253). Treatment of the methylated PTC cell line with 5-aza-2'-deoxycytidine resulted in demethylation of the DNMT3A gene, leading to restoration of its expression. Demethylation significantly potentiated the TRAIL-mediated apoptosis in PTC cells. Interestingly, silencing of DNMT3A using siRNA suppressed TRAIL-mediated apoptosis. CONCLUSION: These findings suggest that DNMT3A alterations play an important role in PTC pathogenesis and demethylation agents can be used to restore the function of DNMT3A in a subset of patients with PTC.

TGFß-induced SMAD4-dependent Apoptosis Proceeded by EMT in CRC.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. In Saudi Arabia, colorectal cancer is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFß/Smad4 signaling pathway in initiation and progression of colorectal cancer is well documented. This study examined the role of TGFß/Smad4 signaling pathway in a large cohort of Saudi patients with colorectal cancer, followed by in vitro analysis to dissect the dual role of TGFß on inducing epithelial-to-mesenchymal transition (EMT) and apoptosis. Our study demonstrated high frequency of Smad4 alterations with low expression of Smad4 protein identifying a subgroup of aggressive colorectal cancer to be an independent marker for poor prognosis. Functional studies using colorectal cancer cells show that TGFß induces Smad4-dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1, was essential for TGFß-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in colorectal cancer cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFß-induced apoptosis. Furthermore, TGFß/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4 alterations were found in the Saudi patients with colorectal cancer. Functional study results indicate that TGFß induces Smad4-dependent EMT followed by apoptosis in colorectal cancer cells.

Roles of ROS, Nrf2, and autophagy in cadmium-carcinogenesis and its prevention by sulforaphane.

Environmental and occupational exposures to cadmium increase the risk of various cancers, including lung cancer. The carcinogenic mechanism of cadmium, including its prevention remains to be investigated. Using fluorescence and electron spin resonance spin trapping, the present study shows that in immortalized lung cells (BEAS-2BR cells), exposure cadmium generated reactive oxygen species (ROS). Through ROS generation, cadmium increased the protein level of TNF-α, which activated NF-κB and its target protein COX-2, creating an inflammatory microenvironment. As measured by anchorage-independent colony formation assay, cadmium induced malignant cell transformation. Inhibition of ROS by antioxidants inhibited transformation, showing that ROS were important in the mechanism of this process. The inflammatory microenvironment created by cadmium may also contribute to the mechanism of the transformation. Using tandem fluorescence protein mCherry-GFP-LC3 construct, the present study shows that cadmium-transformed cells had a property of autophagy deficiency, resulting in accumulation of autophagosomes and increased p62. This protein upregulated Nrf2, which also upregulated p62 through positive feed-back mechanism. Constitutive Nrf2 activation increased its downstream anti-apoptotic proteins, Bcl-2 and Bcl-xl, resulting in apoptosis resistance. In untransformed BEAS-2BR cells, sulforaphane, a natural compound, increased autophagy, activated Nrf2, and decreased ROS. In cadmium-transformed BEAS-2BR cells, sulforaphane restored autophagy, decreased Nrf2, and decreased apoptosis resistance. In untransformed cells, this sulforaphane induced inducible Nrf2 to decrease ROS and possibly malignant cell transformation. In cadmium-transformed cells, it decreased constitutive Nrf2 and reduced apoptosis resistance. The dual roles of sulforaphane make this natural compound a valuable agent for prevention against cadmium-induced carcinogenesis.

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.

FoxM1 and ß-catenin predicts aggressiveness in Middle Eastern ovarian cancer and their co-targeting impairs the growth of ovarian cancer cells.

Epithelial ovarian cancer (EOC) is a highly lethal disease with poor prognosis especially in advanced stage tumor. Emerging evidence has reported that aberrant upregulation of FoxM1 and ß-catenin are closely associated with aggressiveness of human cancer. However, interplay between these factors in the aggressiveness of EOC is not fully illustrated. In this study, we show that FoxM1 is frequently increased in Middle Eastern EOC and associated with high proliferative index (p = 0.0007) and high grade tumor (p = 0.0024). Interestingly, FoxM1 is significantly associated with elevated nuclear ß-catenin and the concomitant increase of FoxM1 and ß-catenin is associated with advanced stage of EOC by immunohistochemical analysis of 261 samples of Saudi patients with EOC. Functional analysis showed that ß-catenin is a direct transcriptional target of FoxM1 in EOC cell lines. FoxM1 inhibition either by specific inhibitor, thiostrepton or siRNA suppressed ß-catenin expression, whereas overexpression of FoxM1 increased nuclear ß-catenin expression. We identified two FoxM1 binding sites in the ß-catenin promoter that specifically bound to FoxM1 protein. Down-regulation of FoxM1 using thiostrepton induced apoptosis and inhibited cell migration/invasion in EOC cells. Moreover, co-inhibition of FoxM1 by thiostrepton and ß-catenin by FH535 significantly and synergistically inhibited EOC cell growth in vitro and in vivo. Collectively, our findings confer that co-targeting FoxM1/ß-catenin signaling cascade may be a promising molecular therapeutic choice in advanced EOC.

Downregulation of SKP2 in Papillary Thyroid Cancer Acts Synergistically With TRAIL on Inducing Apoptosis via ROS.

Context and Objective: S-phase kinase protein 2 (SKP2) is an F-box protein with proteasomal properties and has been found to be overexpressed in a variety of cancers. However, its role in papillary thyroid cancer (PTC) has not been fully elucidated. Experimental Design: SKP2 expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of >1000 PTC samples. In vitro and in vivo studies were performed using proteasome inhibitor bortezomib and proapoptopic death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) either alone or in combination on PTC cell lines. Results: SKP2 was overexpressed in 45.5% of PTC cases and was significantly associated with extrathyroidal extension (P = 0.0451), distant metastasis (P = 0.0435), and tall cell variant (P = 0.0271). SKP2 overexpression was also directly associated with X-linked inhibitor of apoptosis protein overexpression (P < 0.0001) and Bcl-xL overexpression (P = 0.0005) and inversely associated with death receptor 5 (P < 0.0001). The cotreatment of bortezomib and TRAIL synergistically induced apoptosis via mitochondrial apoptotic pathway in PTC cell lines. Furthermore, bortezomib and TRAIL synergistically induced reactive oxygen species (ROS) generation and caused death receptor 5 upregulation through activation of the extracellular signal-regulated kinase-C/EBP homologous protein signaling cascade. Finally, bortezomib treatment augmented the TRAIL-mediated anticancer effect on PTC xenograft tumor growth in nude mice. Conclusion: These data suggest that SKP2 is a potential therapeutic target in PTC and that a combination of bortezomib and TRAIL might be a viable therapeutic option for the treatment of patients with aggressive PTC.

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Patients with aggressive breast cancer (BC) subtypes usually don't have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly, nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.

MED12 is recurrently mutated in Middle Eastern colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognising the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients. DESIGN: In the discovery phase of the study, we conducted whole genome sequencing of tumour and corresponding germline DNA in 27 patients with CRC. In addition to known driver mutations, we identified three MED12 somatic mutations. In the replication phase, we employed a next-generation sequencing approach to capture and sequence MED12 and other candidate genes in a larger sample of 400 patients with CRC and confirmed the enrichment for recurrent MED12 mutations. RESULTS: In order to gain insight into a plausible biological mechanism for the potential role of MED12 mutations in CRC, we studied CRC cell lines that differ substantially in the expression level of MED12, and found the latter to be correlated inversely with transforming growth factor (TGF)-ß signalling and directly with apoptosis in response to chemotherapeutic agents. Importantly, these correlations were replicated when MED12 expression was experimentally manipulated. CONCLUSIONS: Our data expand the recently described role of MED12 as a tumour suppressor in other cancers to include CRC, and suggest TGF-ß signalling as a potential mediator of this effect.

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

BACKGROUND: Breast 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. METHODS: We 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. RESULTS: XIAP 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. CONCLUSION: These 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.

Quercetin inhibits Cr(VI)-induced malignant cell transformation by targeting miR-21-PDCD4 signaling pathway.

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Inhibition of Cr(VI)-induced carcinogenesis by a dietary antioxidant is a novel approach. Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables, possesses potent antioxidant and anticancer properties. MicroRNA-21 (miR-21) is a key oncomiR significantly elevated in the majority of human cancers that exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (PDCD4). The present study examined the effect of quercetin on the inhibition of Cr(VI)-induced malignant cell transformation and the role of miR-21-PDCD4 signaling involved. Our results showed that quercetin decreased ROS generation induced by Cr(VI) exposure in BEAS-2B cells. Chronic Cr(VI) exposure induced malignant cell transformation, increased miR-21 expression and caused inhibition of PDCD4, which were significantly inhibited by the treatment of quercetin in a dose dependent manner. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of quercetin showed reduced tumor incidence compared to Cr(VI) alone treated group. Stable knockdown of miR-21 and overexpression of PDCD4 or catalase in BEAS-2B cells suppressed Cr(VI)-induced malignant transformation and tumorigenesis. Taken together, these results demonstrate that quercetin is able to protect BEAS-2B cells from Cr(VI)-induced carcinogenesis by targeting miR-21-PDCD4 signaling.