Abdulraheem Alshareef

The PI3K/AKT/c‐MYC Axis Promotes the Acquisition of Cancer Stem‐Like Features in Esophageal Squamous Cell Carcinoma

The importance of intratumoral heterogeneity has been highlighted by the identification and characterization of cancer stem cells (CSCs). Based on the differential responsiveness to a Sox2 reporter, SRR2, we had found a novel dichotomy in esophageal squamous cell carcinoma (ESCC) cells, with reporter‐responsive (RR) cells showing more CSC‐like features than reporter‐unresponsive (RU) cells. Specifically, RR cells exhibited significantly higher tumorsphere formation capacity, proportions of CD44High cells, chemoresistance to cisplatin, and tumorigenic potential in vivo. H2O2, a potent inducer of oxidative stress and reactive oxygen species, was found to induce a conversion from RU to RR cells; importantly, converted RR cells acquired CSC‐like features. The PI3K/AKT/c‐MYC signalling axis is important in this context, since pharmacologic blockade of PI3K‐AKT or siRNA knockdown of c‐MYC effectively inhibited the RR phenotype and its associated CSC‐like features, as well as the H2O2‐induced RU/RR conversion. In a cohort of 188 ESCC patient samples, we found a significant correlation between strong c‐MYC expression and a short overall survival (p = .009). In conclusion, we have described a novel intratumoral heterogeneity in ESCC. The identification of the PI3K/AKT/c‐MYC axis as a driver of CSC‐like features carries therapeutic implications. Stem Cells 2016;34:2040–2051

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

Loss of miR-200b promotes invasion via activating the Kindlin-2/integrin β1/AKT pathway in esophageal squamous cell carcinoma: An E-cadherin-independent mechanism

Our previous studies have shown that loss of miR-200b enhances the invasiveness of esophageal squamous cell carcinoma (ESCC) cells. However, whether the miR-200-ZEB1/2-E-cadherin regulatory cascade, a master regulator of epithelial-to-mesenchymal transition (EMT), is involved in the regulation of ESCC invasion remains elusive. Here, we show that miR-200b represses ESCC cell invasion in vivo without altering the expression of E-cadherin and vimentin, two surrogate markers of EMT. However, an inverse correlation was observed between the expression levels of miR-200b and ZEB1/2 in both ESCC cell lines (n = 7, P < 0.05) and ESCC tumor samples (n = 88, P < 0.05). Methylation of E-cadherin gene was found to block the regulation of E-cadherin by the miR-200b-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b in ESCC. We revealed that miR-200b suppresses the integrin β1-AKT pathway via targeting Kindlin-2 to mitigate ESCC cell invasiveness. In two independent cohorts of ESCC samples (n = 20 and n = 53, respectively), Kindlin-2 expression positively correlated with the activation status of both the integrin signaling pathway and the PI3K-AKT signaling pathway (both P < 0.01). These data highlight that suppression of the Kindlin-2-integrin β1-AKT regulatory axis is an alternative mechanism underlying the tumor suppressor function of miR-200b in ESCC.

The opposing function of STAT3 as an oncoprotein and tumor suppressor is dictated by the expression status of STAT3β in esophageal squamous cell carcinoma

Purpose: STAT3 is known to have both oncogenic and tumor suppressive effects, but the regulation of these opposing effects is elusive. We hypothesized that STAT3β, one of the two STAT3 isoforms, is the key determinant in this context. Experimental Design: The prognostic significance of STAT3β and phospho-STAT3αY705 (pSTAT3αY705) was evaluated in 286 cases of patients with esophageal squamous cell carcinoma (ESCC). STAT3β-induced changes in the chemosensitivity to cisplatin and 5-fluorouracil were assessed both in vitro and in vivo. STAT3β-induced changes in the frequency of cancer stem cells were evaluated using Hoechst and CD44 staining. How STAT3β regulates STAT3α was determined using immunoprecipitation, confocal microscopy, DNA-binding, and chromatin immunoprecipitation-PCR. Results: STAT3β expression is an independent protective prognostic marker in patients with ESCC, which strongly correlated with longer overall survival (P = 0.0009) and recurrence-free survival (P = 0.0001). STAT3β significantly decreased the cancer stem cell population, and sensitized ESCC cells to cisplatin and 5-fluorouracil in tumor xenografts. Mechanistically, STAT3β markedly attenuated the transcription activity of STAT3α via inducing STAT3α:STAT3β heterodimers. However, the heterodimer formation decreased the binding between STAT3α and PTPN9 (better known as PTP-MEG2), a protein tyrosine phosphatase, thereby promoting the phosphorylation of STAT3αY705 and enhancing its nuclear translocation and DNA binding. Correlating with this, high STAT3β expression converts the prognostic value of pSTAT3αY705 from unfavorable to favorable in patients with ESCC. Conclusions: STAT3β suppresses chemoresistance and cancer stemness by blocking the transcriptional activity of STAT3α. The paradoxical increase in pSTAT3αY705 induced by STAT3β carries important implications as to how the biologic and prognostic significance of STAT3 in cancers should be interpreted. Clin Cancer Res; 22(3); 691–703. ©2015 AACR.

The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers

Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners.

Oxidative stress induces the acquisition of cancer stem-like phenotype in breast cancer detectable by using a Sox2 regulatory region-2 (SRR2) reporter

We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expression of 3 signature genes of RR cells (CD133, GPR49 and MUC15). Lastly, the H2O2-induced RU/RR conversion was detectable in a SCID mouse xenograft model and primary tumor cells. To conclude, the H2O2-induced RU/RR conversion has provided a novel model to study the acquisition of cancer stemness and plasticity.

Disheveled proteins promote cell growth and tumorigenicity in ALK-positive anaplastic large cell lymphoma.

Our previous oligonucleotide array studies revealed that ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL) express high levels of the disheveled proteins (Dvls), a family of proteins that is integral to the Wnt signaling pathways. In this study, we assessed whether the Dvls are important in the pathogenesis of ALK(+)ALCL. By Western blotting, Dvl-2 and Dvl-3 were found to be highly expressed in ALK(+)ALCL cell lines and patient samples. The higher molecular weight forms, consistent with phosphorylated/active Dvl proteins, were observed in these lysates. siRNA knock-down of Dvls did not affect the Wnt canonical pathway, as assessed by the β-catenin protein levels and nuclear localization. In contrast, the same treatment led to changes in the transcriptional activity of NFAT and the phosphorylation status of Src, both of which are known to be regulated by the Wnt non-canonical signaling pathways in other cell types. Coupled with these biochemical changes, there was a significant decrease in cell growth and soft agar colony formation. NPM-ALK, the oncogenic tyrosine kinase characteristic of ALK(+)ALCL, was found to bind to the Dvls and enhance their tyrosine phosphorylation. In conclusion, our data suggest that the Dvls contribute to the pathogenesis of ALK(+)ALCL via signaling in the Wnt non-canonical pathways. To our knowledge, this is the first report demonstrating a physical and functional interaction between the Dvls and an oncogenic tyrosine kinase.

miR-200b induces cell cycle arrest and represses cell growth in esophageal squamous cell carcinoma

miR-200b is a pleiotropically acting microRNA in cancer progression, representing an attractive therapeutic target. We previously identified miR-200b as an invasiveness repressor in esophageal squamous cell carcinoma (ESCC), whereas further understanding is warranted to establish it as a therapeutic target. Here, we show that miR-200b mitigates ESCC cell growth by inducing G2-phase cell cycle arrest and apoptosis. The expression/activation of multiple key cell cycle regulators such as CDK1, CDK2, CDK4 and Cyclin B, and the Wnt/β-Catenin signaling are modulated by miR-200b. We identified CDK2 and PAF (PCNA-associated factor), two important tumor-promoting factors, as direct miR-200b targets in ESCC. Correlating with the frequent loss of miR-200b in ESCC, both CDK2 and PAF levels are significantly increased in ESCC tumors compared to case-matched normal tissues (n = 119, both P < 0.0001), and correlate with markedly reduced survival (P = 0.007 and P = 0.041, respectively). Furthermore, CDK2 and PAF are also associated with poor prognosis in certain subtypes of breast cancer (n = 1802) and gastric cancer (n = 233). Although CDK2 could not significantly mediate the biological function of miR-200b, PAF siRNA knockdown phenocopied while restored expression of PAF abrogated the biological effects of miR-200b on ESCC cells. Moreover, PAF was revealed to mediate the inhibitory effects of miR-200b on Wnt/β-Catenin signaling. Collectively, the pleiotropic effects of miR-200b in ESCC highlight its potential for therapeutic intervention in this aggressive disease.

Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays

IntroductionAberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets.MethodsWe used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples.ResultsWe found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples.ConclusionsIn conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.

DDX17 (P72), a Sox2 binding partner, promotes stem-like features conferred by Sox2 in a small cell population in estrogen receptor-positive breast cancer.

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy, we tested our hypothesis that Sox2, which is a key regulator of the RR phenotype, is under the control of its binding partners. In this study, we focused on DDX17, known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation, we confirmed the binding between DDX17 and Sox2, although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells, it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR, DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2, as well as 3 of its specific gene targets including MUC15, CCND1 and CD133. Correlating with these findings, siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude, DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells, DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes.