TXMicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti-/pre-miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR-7, miR-194 and miR-449b, or overexpressing miR-204, repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR-204, and two binding sites in the 3 0 -untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR-204-FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression-and metastasis-related miRNAs offers a novel potential therapeutic strategy for the disease.Endometrial cancer is a common cause of gynecological cancer death. The most dominant subtype, endometrioid endometrial cancer (EEC), accounts for >80% of this cancer. Menopause and unopposed estrogenic stimulation are typical risk factors. Patients are generally treated with surgery, radiation, chemotherapy or hormone therapy. Patients with early stage disease have 5-year survival rates over 80%, however, about 15-20% develop metastasis. 1 These patients and those with advanced stage disease or recurrence have poor prognosis due to limitation of effective treatment. 2 Understanding the pathogenesis of this disease may provide insights for the development of novel therapeutic strategies.MicroRNAs (miRNAs) are small noncoding RNA molecules of 19-24 nucleotides that regulate gene expression posttranscriptionally through imperfect base pairing with the 3 0 -untranslated region (3 0 UTR) of target mRNAs, causing transcript degradation and translational inhibition. 3 Approximately 20-30% of all genes are targeted by miRNAs and a single miRNA may target as many as 200 genes. 4 In human cancers, >50% of the miRNA genes are located in chromosomal fragile sites, minimal regions containing loss of heterozygosity, minimal amplicons or common breakpoint regions. 5 DNA ...

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Hepatocellular carcinoma (HCC) is the third leading cause of all cancer-related deaths. Its rapid proliferative nature and HCC specific therapy such as transarterial chemoembolization (TACE) that block arterial blood supply to the tumor frequently induce hypoxic microenvironments i.e. regions with oxygen deprivation. Hypoxia has emerged as a major driving force to promote aggressive phenotypes and stemness of cancer cells in solid tumors. We aimed to identify the key contributors to HCC progression in hypoxic microenvironment. From the analysis of transcriptomic profiles of human HCCs in both our in-house RNA-sequencing (RNA-seq) data and TCGA data as well as HCC cells treated with hypoxia and normoxia, we identified Ephrin-A3 (EFNA3) as a potential hypoxia-inducible oncogene. EFNA3 is a membrane-bound ligand which can activate multiple receptor tyrosine kinases of the Eph family. This family of ligands and receptors are frequently dysregulated in multiple cancer types and have implications in stem cell plasticity. We further validated that EFNA3 was significantly overexpressed in HCCs as compared with their non-tumorous liver samples using qRT-PCR in a separate cohort of HCCs (p<0.0001, n=97). Upon clinicopathological correlation, overexpression of EFNA3 was significantly correlated with the presence of venous invasion (p=0.004) and more advanced tumor stage (p=0.026). In concordance, TCGA-LIHC dataset revealed significantly higher expression of EFNA3 in HCC samples with vascular invasion (p=0.0094) and advanced tumor stage (p=0.0002). Furthermore, we confirmed the upregulation of EFNA3 upon hypoxia treatment in multiple HCC cell lines. Functionally, stable knockdown of EFNA3 using short hairpin RNA approach significantly reduced proliferation and migratory rates of HCC cells. In addition, orthotopic liver injection model of the EFNA3 knockdown HCC cells demonstrated a significant reduction of the growth of the primary tumors. There was also a reduced incidence of distant metastasis to lungs in vivo. Using sphere formation assay to test self-renewal ability in vitro, EFNA3-knockdown HCC cells showed significantly reduced self-renewal ability. In addition, HCC cells positive/high for stemness markers (EpCAM, CD13, CD24, CD44, CD47, CD133) isolated from our PDTX model showed a consistently higher EFNA3 mRNA expression as compared with the negative/low cells. By flow cytometric analysis, the EFNA3 KD MHCC-97L demonstrated a decreased expression of CD47. Conclusion: We identified EFNA3 as a potential hypoxia-inducible oncogene in HCC and demonstrated its critical role in tumor progression, metastasis and cancer stemness of HCC. Based on our results, EFNA3 may be an attractive therapeutic target to counteract hypoxia-specific disease progression. Citation Format: Abdullah Husain, Yung Tuen Chiu, Daniel Wai Ho, Karen Man Sze, Lo Kong Chan, Yu Man Tsui, Carmen Chak Wong, Irene Oi Ng. EFNA3, a key functional mediator of hypoxic microenvironment in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2431.

We investigated the mutational landscape of mTOR signaling cascade in hepatocellular carcinomas (HCCs) with chronic hepatitis B (HBV) background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyper-activation in hepatocarcinogenesis. We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of TSC1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony formation assay in HCC cell lines and the treatment was further tested using our patient derived tumor xenograft (PDTX) models. Results: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n = 18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leading to more aggressive tumor behavior. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony forming ability upon Rapamycin treatment. With the use of the biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hyper-sensitivity towards Rapamycin treatment. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyper-activation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signaling, with potential significance of effective specific drug therapy. Citation Format: Daniel Wai Ho, Lo Kong Chan, Yung Tuen Chiu, Iris Ming Xu, Ronnie Poon, Tan To Cheung, Chung Ngai Tang, Victor Tang, Irene Lo, Polly Lam, Derek Yau, Miao Xin Li, Chun Ming Wong, Irene O. L. Ng. TSC1/2 mutations define a molecular subset of HCC with aggressive behavior and treatment implication [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 4389. doi:10.1158/1538-7445.AM2017-4389