Yu-Ting Chou

Coexpression of Oct4 and Nanog Enhances Malignancy in Lung Adenocarcinoma by Inducing Cancer Stem Cell–Like Properties and Epithelial–Mesenchymal Transdifferentiation

Epithelial-mesenchymal transition (EMT), a critical process of cancer invasion and metastasis, is associated with stemness property of cancer cells. Though Oct4 and Nanog are homebox transcription factors essential to the self-renewal of stem cells and are expressed in several cancers, the role of Oct4/Nanog signaling in tumorigenesis is still elusive. Here microarray and quantitative real-time PCR analysis showed a parallel, elevated expression of Oct4 and Nanog in lung adenocarcinoma (LAC). Ectopic expressions of Oct4 and Nanog in LACs increased the percentage of CD133-expressing subpopulation and sphere formation, enhanced drug resistance, and promoted EMT. Ectopic expressions of Oct4 and Nanog activated Slug and enhanced the tumor-initiating capability of LAC. Furthermore, double knockdown of Oct4 and Nanog suppressed the expression of Slug, reversed the EMT process, blocked the tumorigenic and metastatic ability, and greatly improved the mean survival time of transplanted immunocompromised mice. The immunohistochemical analysis demonstrated that expressions of Oct4, Nanog, and Slug were present in high-grade LAC, and triple positivity of Oct4/Nanog/Slug indicated a worse prognostic value of LAC patients. Our results support the notion that the Oct4/Nanog signaling controls epithelial-mesenchymal transdifferentiation, regulates tumor-initiating ability, and promotes metastasis of LAC.

The PML isoform IV is a negative regulator of nuclear EGFR’s transcriptional activity in lung cancer

Epidermal growth factor receptor (EGFR) is a membrane-bound receptor tyrosine kinase, which can transduce intracellular signals responsible for cell proliferation. It is frequently overexpressed and/or constitutively activated in non-small cell lung cancer and thus is considered as a major cause of this disease. Recently, EGFR has been found in the nucleus where the nuclear EGFR (nEGFR) can function as a transcription factor activating the transcription of genes such as cyclin D1 gene (CCND1), which is essential for cell proliferation. Nevertheless, how nEGFRs transcriptional activity is regulated remains unclear. Promyelocytic leukemia protein (PML) is a tumor suppressor, which is lost in various cancers including lung cancer. However, the role of PML in the suppression of lung cancer growth is still unclear. When we investigated the role of PML in the regulation of lung cancer cell growth, we found that PML isoform IV (PMLIV) preferentially represses the growth of lung cancer cells bearing constitutively active EGFR. Besides, when growing in the EGFR activating conditions, the growth of EGFR wild-type bearing A549 cells has been repressed by PMLIV overexpression. We also found that PMLIV can interact physically with nEGFR and represses the transcription of nEGFR target genes. We showed that PMLIV is recruited by nEGFR to the target promoters and reduces the promoter histone acetylation level via HDAC1. Together, our results suggest that PMLIV interacts with nEGFR upon EGFR activation and represses the transcription of nEGFR target genes such as CCND1 and thus leading to inhibition of the lung cancer cell growth.

Epigenetic switch between SOX2 and SOX9 regulates cancer cell plasticity

Cell differentiation within stem cell lineages can check proliferative potential, but nodal pathways that can limit tumor growth are obscure. Here, we report that lung cancer cell populations generate phenotypic and oncogenic plasticity via a switch between differentiation programs controlled by SOX2 and SOX9, thus altering proliferative and invasive capabilities. In lung cancer cells, SOX2 bound the EPCAM promoter to induce EpCAM-p21Cip1-cyclin A2 signaling, encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound the SLUG promoter to induce SLUG-mediated cell invasion with a spindle-like phenotype. Pharmacologic inhibition of HDAC elevated a SOX9-positive cell population from SOX2-positive cells, whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on the SOX9 promoter. In clinical specimens, the expression of SOX2 and SOX9 correlated negatively and positively with lung tumor grade, respectively. Our findings identify SOX2 and SOX9 as nodal epigenetic regulators in determining cancer cell plasticity and metastatic progression. Cancer Res; 76(23); 7036-48. ©2016 AACR.

α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic cancers due to advanced stage presentation, peritoneal dissemination, and refractory ascites at diagnosis. We investigated the role of α2,3-sialyltransferase type I (ST3GalI) by analyzing human ovarian cancer datasets and human EOC tissue arrays. We found that high expression of ST3GalI was associated with advanced stage EOC. Transwell migration and cell invasion assays showed that high ST3GalI expression enhanced migration of EOC cells. We also observed that there was a linear relation between ST3GalI expression and epidermal growth factor receptor (EGFR) signaling in EOC patients, and that high ST3GalI expression blocked the effect of EGFR inhibitors. Co-Immunoprecipitation experiments demonstrated that ST3GalI and EGFR were present in the same protein complex. Inhibition of ST3GalI using a competitive inhibitor, Soyasaponin I (SsaI), inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted MOSEC cells. Further, SsaI synergistically enhanced the anti-tumor effects of EGFR inhibitor on EOC cells. Our study demonstrates that ST3GalI regulates ovarian cancer cell migration and peritoneal dissemination via EGFR signaling. This suggests α2,3-linked sialylation inhibitors in combination with EGFR inhibitors could be effective agents for the treatment of EOC.

Brain metastases in patients with non-small cell lung cancer: the role of mutated- EGFRs with an exon 19 deletion or L858R point mutation in cancer cell dissemination

Non-small cell lung cancer (NSCLC) patients tend to develop brain metastases (BM), but the link between BM occurrence and driver mutations in NSCLC is not very clear. We explored whether activating mutations of epidermal growth factor receptors (EGFRs) in exon 19 deletion or L858R predict BM in NSCLC. A retrospective multivariable logistic regression analysis of 384 patients demonstrated that the presence of mutated-EGFRs was associated with overall BM (OR=2.24, P=0.001) compared to that of wild-type EGFR (WT-EGFR). Moreover, the time-to-event analysis model considering death as a competing risk revealed that, irrespective of survival, mutated-EGFRs predicted subsequent BM (SBM) in stage IIIB-IV patients (37.1% vs. 10.6%, HR=2.98, P=0.002) after adjusting for age (HR=2.00, P=0.012), gender, histological subtype, and smoking history. Notably, the younger mutated-EGFR subgroup was at a higher risk for SBM compared to the older WT-EGFR one (58.1% vs.10.9%, HR=6.57, P<0.001). Additionally, EGFR exon 19 deletion, despite having a slightly longer overall survival (20.6 vs. 14.2 months, P=0.368), was comparable to L858R mutation in predicting SBM (39.5% vs. 34.5%, HR=0.91, P=0.770). In vitro, the overexpression of mutated-EGFRs induced morphological changes towards a mesenchymal-like phenotype and promoted mobility in lung cancer cells. Clinically, mutated-EGFR NSCLC displayed a higher proportion of vimentin-positive expression (75.3% vs. 51.2%; P=0.007) and a shorter median time to SBM (23.5 months vs. not reached, P=0.017) than WT-EGFR NSCLC. These results suggest that NSCLC patients carrying mutated-EGFRs may require a higher frequency of brain imaging assessments than those with WT-EGFR to facilitate earlier SBM detection during follow-up.Non-small cell lung cancer (NSCLC) patients tend to develop brain metastases (BM), but the link between BM occurrence and driver mutations in NSCLC is not very clear. We explored whether activating mutations of epidermal growth factor receptors (EGFRs) in exon 19 deletion or L858R predict BM in NSCLC. A retrospective multivariable logistic regression analysis of 384 patients demonstrated that the presence of mutated-EGFRs was associated with overall BM (OR=2.24, P=0.001) compared to that of wild-type EGFR (WT-EGFR). Moreover, the time-to-event analysis model considering death as a competing risk revealed that, irrespective of survival, mutated-EGFRs predicted subsequent BM (SBM) in stage IIIB-IV patients (37.1% vs. 10.6%, HR=2.98, P=0.002) after adjusting for age (HR=2.00, P=0.012), gender, histological subtype, and smoking history. Notably, the younger mutated-EGFR subgroup was at a higher risk for SBM compared to the older WT-EGFR one (58.1% vs.10.9%, HR=6.57, P<0.001). Additionally, EGFR exon 19 deletion, despite having a slightly longer overall survival (20.6 vs. 14.2 months, P=0.368), was comparable to L858R mutation in predicting SBM (39.5% vs. 34.5%, HR=0.91, P=0.770). In vitro, the overexpression of mutated-EGFRs induced morphological changes towards a mesenchymal-like phenotype and promoted mobility in lung cancer cells. Clinically, mutated-EGFR NSCLC displayed a higher proportion of vimentin-positive expression (75.3% vs. 51.2%; P=0.007) and a shorter median time to SBM (23.5 months vs. not reached, P=0.017) than WT-EGFR NSCLC. These results suggest that NSCLC patients carrying mutated-EGFRs may require a higher frequency of brain imaging assessments than those with WT-EGFR to facilitate earlier SBM detection during follow-up.

The role of EpCAM in tumor progression and the clinical prognosis of endometrial carcinoma

OBJECTIVE EpCAM is a transmembrane glycoprotein that functions as an epithelial marker in endometrial tissues. However, the correlation between EpCAM and endometrial carcinoma (EC) is not clear. METHODS This study investigated the association between EpCAM and EC. Immunohistochemistry staining and bioinformatics analysis disclosed the clinical importance of low EpCAM expression. The migratory ability of cells expressing low EpCAM levels was studied in transwell invasion assays in vitro and an orthotopic intra-uterine tumor injection model in vivo. The Connectivity MAP was used to identify drugs that effectively inhibit cells with low EpCAM expression. RESULTS According to immunohistochemistry analysis results, low EpCAM expression was associated with an advanced stage and lymph node metastasis in patients with endometrioid EC, and high EpCAM expression favored survival. EpCAM silencing promoted cell invasion, and EpCAM re-expression in EpCAM-silenced EC cells attenuated their invasiveness. EpCAM suppression in an orthotopic uterine implantation model promoted the lymph node metastasis of EC cells. According to quantitative PCR and promoter reporter analyses, estrogen receptor alpha signaling regulated EpCAM expression by enhancing its promoter activity. As shown in the Connectivity MAP analysis, transamin inhibited the invasiveness of EpCAM-silenced EC cells. CONCLUSIONS The loss of EpCAM may increase the malignancy of EC, and these findings provide new insights into the prognostic role of EpCAM in patients with EC.

OCT4B mediates hypoxia-induced cancer dissemination

Hypoxia, the reduction of oxygen levels in cells or tissues, elicits a set of genes to adjust physiological and pathological demands during normal development and cancer progression. OCT4, a homeobox transcription factor, is essential for self-renewal of embryonic stem cells, but little is known about the role of OCT4 in non-germ-cell tumorigenesis. Here, we report that hypoxia stimulates a short isoform of OCT4, called OCT4B, via a HIF2α-dependent pathway to induce the epithelial–mesenchymal transition (EMT) and facilitate cancer dissemination. OCT4B overexpression decreased epithelial barrier properties, which led to an increase in cell migration and invasion in lung cancer cells. OCT4B knockdown attenuated HIF2α-induced EMT and inhibited cancer dissemination in cell-line and animal models. We observed that OCT4B bound the SLUG promoter and enhanced its expression, and SLUG silencing inhibited OCT4B-mediated EMT, accompanied with decreased cell migration and invasion. Correlation analysis revealed that OCT4B expression was significantly associated with the SLUG level in lung tumors. These results provide novel insights into OCT4B-mediated oncogenesis in cancer dissemination.

Abstract 4155: Crosstalk of SOX2 and epithelial-to-mesenchymal transition on EGFR-TKI resistance

EGFR-tyrosine kinase inhibitors (TKIs) have been shown to produce profound therapeutic responses in lung adenocarcinoma harboring EGFR mutations. Despite this initial response, patients with EGFR-mutated lung adenocarcinoma ultimately developed resistance to EGFR-TKIs. Although both SOX2 and epithelial-to-mesenchymal (EMT) have been linked to the EGFR-TKI resistance in lung adenocarcinoma, the interplay between SOX2 and EMT in EGFR-TKI resistance is not known. Here, we report that cancer plasticity switched by SOX2 and EMT regulates EGFR-TKI resistance. We found that the selection of EGFR-TKI resistant cells induced EMT while attenuating SOX2 expression. We observed that EGFR-TKI treatment induced SOX2 expression in EGFR-mutated lung cancer cells, and SOX2 silencing encouraged EGFR-TKI resistance. Preselection of EGFR-mutated lung cancer cells with the EMT feature endowed cells with EGFR-TKI resistance but suppressed SOX2 expression. We found that TGF-β induced EMT but attenuated SOX2 expression, resulting in increased EGFR-TKI resistance. Pharmacological inhibition of HDACs in EGFR-mutated cells attenuated SOX2 expression but induced EMT, causing increased EGFR-TKI resistance. Enriching SOX2 expression in EGFR-mutated cells attenuated EMT and rendered cells more sensitive to EGFR-TKIs. Correlation analysis exhibited negative correlations between SOX2 and EMT markers in primary non-small cell lung cancer. Kaplan-Meier analysis revealed that a SOX2 low/Vimentin high signature predicted a poor survival in EGFR-mutated lung adenocarcinoma. Our findings support the notion that the cancer plasticity regulated by SOX2 and EMT plays a critical role in EGFR-TKI resistance, and SOX2 and Vimentin can function as prognostic biomarkers in lung cancer progression. Citation Format: An-Chun Lee, Yu-Fan Chiu, Ming-Han Kuo, Yuan-Hung Wang, Yu-Ting Chou. Crosstalk of SOX2 and epithelial-to-mesenchymal transition on EGFR-TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2017-4155

Abstract 3408: Oncogenic SOX2 signaling in bladder cancer

The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway. We found that SOX2, a master transcriptional factor controlling self-renewal of stem cells, was highly expressed in a subgroup of transitional cell carcinoma of bladder cancer, correlating with the advanced pathological grade. Moreover, SOX2 expression was associated with poor overall survival and recurrence-free survival outcomes in patients with bladder cancer. Knockdown of SOX2 in SOX2-high 5637 bladder cancer cells attenuated cell growth. Consistently, ectopic expression of SOX2 in SOX2-low T24 bladder cancer cells endowed cells with increased cell proliferation. Spheroid assay showed that SOX2 expression encouraged spheroid-forming ability of bladder cancer cells in low serum condition, demonstrating the involvement of SOX2 signaling in the maintenance of stemness in bladder cancer cells. Immunoblotting revealed that the expression of SOX2 induced phosphorylation of AKT in the serum-free condition, supporting that SOX2 regulates bladder cancer cell survival. Gene expression microarray analysis showed that SOX2 expression induced IGF2, which was further confirmed by Q-PCR analysis in bladder cancer cells. Chi-square test assay showed that IGF2 expression correlated with the advanced tumor stages in bladder cancer. Pharmacological inhibition of IGF2 signaling attenuated cell growth in SOX2-postive bladder cancer cells. Our findings support the notion that SOX2-IGF2 signaling axis confers aggressiveness in bladder cancer cells with potentials as biomarkers and therapeutic targets for bladder cancer intervention. Citation Format: Chia-Chang Wu, Yu-Fan Chiu, Yu-Ting Chou, Yuan-Hung Wang. Oncogenic SOX2 signaling in bladder cancer. [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 3408.

Abstract 2852: CITED2, an emerging regulator in hypoxia- and anoikis- mediated cancer cell growth

In solid tumors, hypoxia and anoikis are two common phenomena occurred in the central of region, inducing a series of genes to switch metabolic pathways, prevent apoptosis and promote continuous growth of cancer cells. Herein, we reported that CITED2 (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain 2) responded to hypoxia and anoikis induction, preventing lung cancer cells from cell cycle arrest. We found that hypoxia induced CITED2 through a HIF2α dependent pathway; moreover, three-dimensional (3D) growth of lung cancer cells induced the expression of CITED2. 3D growth condition elicited anoikis-mediated cell cycle arrest, which was further potentiated by hypoxia stimulation. Knockdown of CITED2 or HIF2α in lung cancer cells enhanced anoikis, causing G1/S cell cycle arrest. Soft agar analysis displayed that both HIF2α and CITED2 are necessary for anchorage-independent cell growth under the hypoxic condition. Spheroid assay indicated that HIF2α-CITED2 signaling axis plays an important role in spheroid formation. Pearson correlation analysis showed that CITED2 was associated with HIF2α expression in lung adenocarcinoma. Kaplan Meier analysis revealed that CITED2 expression was correlated with poor survival outcomes in patients with lung adenocarcinoma. Thus, our findings support the notion that CITED2 plays a critical role in hypoxia- and anokis- mediated cancer cell growth with the potential as a prognostic biomarker for predicting lung cancer progression. Citation Format: Ming-Han Kuo, Chia Ee Chan, Yu-Ting Chou. CITED2, an emerging regulator in hypoxia- and anoikis- mediated cancer cell growth. [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 2852.