The Role and Molecular Mechanisms of Subtype-specific lncRNAs in Epithelial Mesenchymal Transition and Tumor Progression

Abstract

Breast cancer and ovarian cancer are two major health concerns for females worldwide, responsible for 15.5% and 4.7% cancer-related deaths in women, respectively. Among these, basal-like triple-negative breast cancer (TNBC) and mesenchymal-like ovarian cancer (OV) represent particularly aggressive subtypes characterized by high metastatic potential and poor prognoses, traits closely linked to epithelial-to-mesenchymal transition (EMT) and cancer stemness. Despite the known critical role of EMT in tumor progression, the contribution of subtype-specific long non-coding RNAs (lncRNAs) to EMT regulation remains largely unexplored. Notably, this knowledge gap is especially pronounced in mesenchymal-like OV, where subtype-specific lncRNAs have not been identified. By integrating lncRNA and mRNA expression profiles from TCGA-BRCA and TCGA-OV datasets, we constructed a comprehensive regulatory network and identified master regulatory lncRNAs mediating EMT in TNBC and mesenchymal-like OV. In breast cancer, we identified a TNBC-specific lncRNA, LINC01235, which acts as a master regulator of EMT-related genes. LINC01235 upregulation predicts poor survival in breast cancer patients. Depletion of LINC01235 impaired tumor cell growth, invasive migration, EMT and mammosphere formation. In addition, inducible LINC01235 knockdown inhibited tumor progression in MDA-MB-468-derived xenografts. RNA sequencing identified PEG10 as a major downstream target of LINC01235, and we demonstrated that PEG10 is critical for LINC01235-mediated TNBC stemness properties. Mechanistically, the CRISPR-assisted RNA-protein interaction detection method (CARPID) identified RPL22 as a LINC01235-interacting protein. RNA immunoprecipitation (RIP) assay further demonstrated that RPL22 binds with both PEG10 mRNA and LINC01235. Depletion of LINC01235 or RPL22 decreased the half-life of PEG10 mRNA, suggesting that RPL22 and LINC01235 promote the stability of PEG10 mRNA. These findings demonstrated that LINC01235 promotes EMT via the RPL22-PEG10 axis. In OV, we identified ZFHX4-AS1 as a mesenchymal subtype-specific master regulator driving EMT. The overexpression of ZFHX4-AS1 serves as an indicator of unfavorable patient prognosis. Silencing ZFHX4-AS1 reduced OV cell viability, invasive migration, and stemness properties in vitro, and suppressed tumor peritoneal metastasis in vivo. By performing in silico computational analysis and experimental validations, we uncovered ZFHX4, the antisense protein-coding gene of ZFHX4-AS1, as a downstream effector of ZFHX4-AS1. We showed that ZFHX4-AS1 promotes ZFHX4 mRNA stability. Human antigen R (HuR), an RNA stabilizing protein predicted by starBase to interact with both ZFHX4 mRNA and ZFHX4-AS1, was validated by RIP assays. HuR knockdown reduced mRNA and protein levels of ZFHX4. These data support our model that HuR facilitates the interactions of ZFHX4-AS1 and ZFHX4 mRNA to promote its stability, resulting in upregulation of ZFHX4 and OV progression. To further investigate the clinical relevance of ZFHX4-AS1, we performed pan-cancer analysis in 33 types of cancers using gene expression data and survival data from the TCGA database. We discovered a notable and statistically significant positive correlation between the expression levels of ZFHX4-AS1 and ZFHX4 across all 33 cancer types. Our analysis also revealed that ZFHX4-AS1 serves as a prognostic indicator and risk factor in ten types of cancer. In summary, our study uncovered two novel, subtype-specific lncRNAs that drive cancer stemness and progression, which will contribute to the development of effective targeted therapeutics.

Date of Award	26 Sept 2025
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Rebecca Y M CHIN (Supervisor)