ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis
The mammalian orthologue of the Drosophila ecdysoneless (ECD) mRNA and protein are overexpressed in breast cancer, and its overexpression correlates with poor prognosis and short patient survival, particularly in ERBB2/HER2-positive breast cancer (BC). This work investigates the co-operative oncogenic mechanism of ECD and ERBB2 by deriving transgenic mice overexpressing ECD and/or ERBB2 (huHER2) in mammary epithelium under MMTV promoter (ECD+HER2Tg), as well as human mammary immortal epithelial cell lines (hMECs) overexpressing ECD and/or ERBB2. While huHER2Tg mice developed more homogenous solid nodular carcinomas, double transgenic mice (ECD; huHER2Tg) developed heterogenous and histologically aggressive mammary tumors with basal-like phenotype and epithelial mesenchymal transition (EMT) features, similar to ECDTg tumors.  Importantly, transcriptomic profile of ECD; huHER2Tg tumors revealed upregulation of two major oncogenic pathways, unfolded protein response (UPR) and glycolysis. Consistently, ECD; huHER2Tg tumors exhibited increased mRNA levels of cancer stem cell markers and EMT related genes.  Further, hMECs expressing both ECD and ERBB2 as compared to ECD or ERBB2 expressing cells showed significant increase in oncogenic traits, such as enhanced migration, anchorage independence, mammosphere, and organoid formation. RNA sequencing analysis of single and double transfectants showed significant upregulation of glycolysis and UPR pathways in ECD+ERBB2 expressing cells as compared to ECD or ERBB2 overexpressing hMECs.  Given our recent finding that ECD directly binds to RNA, we demonstrate mRNAs of three key glycolytic enzymes (LDHA, PKM2 and HK2) and mRNA of a major UPR regulated gene GRP78 directly associated with ECD protein using RNA immunoprecipitation assay. Higher expression of glycolytic mRNAs and GRP78 in ECD+ERBB2-expressing cells was due to increased mRNA stability. Lastly, we show an increase in glucose uptake and enhanced glycolytic rate in ECD+ERBB2-overexpressing cells as compared to ECD- or ERBB2-overexpressing hMECs.  Taken together, our findings support a co-operative role of ECD and ERBB2 in oncogenesis by enhancing two major oncogenic pathways, UPR and glycolysis. These results provide experimental proof and mechanistic insights to our previous findings that ECD is overexpressed in ERBB2-positive BC patients, and its' overexpression correlates with poor prognosis and short survival in patients. Overall design: RNA-seq profiling of Vector expressing, ECD overexpressing, ERBB2 overexpressing and ECD+ERBB2 overexpressing 76NTERT human mammary epithelial cells. In adition, RNAseq profiling of ECDTg mouse mammary tumors, huHER2Tg mouse mammary tumors and ECD; huHER2Tg mouse mammary tumors