Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Abstract

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC–mediated glucose metabolism. Implications: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

GD2 and its biosynthetic enzyme GD3 synthase promote tumorigenesis in prostate cancer by regulating cancer stem cell behavior

“…TMAs were stained for GD2 by immunohistochemistry (IHC) as described previously 27 . Briefly, TMAs were deparaffinized in xylene, rehydrated in descending alcohols and heated in the antigen retrieval solution (vector, #H-3300) in a microwave to unmask the antigens.…”

“…Western blotting of whole cell extracts prepared in Triton-X-100 lysis buffer and quantified using the BCA assay kit (#23225; Thermo Fisher Scientific) was performed as described previously 27 . 50 μg protein aliquots were run on SDS-7.5% polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membrane, and immunoblotted with the indicated antibodies.…”

GD2 and its biosynthetic enzyme GD3 synthase promote tumorigenesis in prostate cancer by regulating cancer stem cell behavior

“…Prostate cancer (PC) patient tissue microarrays and immunohistochemical analysis: Tissue microarrays (TMAs) composed of paired normal prostate and prostate cancer Gleason grades (3, 4 and 5; n = 320) and or of 45 cases of bone and visceral metastatic samples from Rapid Autopsy TMAs were obtained without any identifying patient information from the Prostate Cancer Biorepository Network (PCBN) repository under a materials transfer agreement. TMAs were stained for GD2 by immunohistochemistry (IHC) as described previously (27). Briefly, TMAs were deparaffinized in xylene, rehydrated in descending alcohols and heated in the antigen retrieval solution (vector, cat#H-3300) in a microwave to unmask the antigens.…”

“…Western Blotting: Western blotting of whole cell extracts prepared in Triton-X-100 lysis buffer and quantified using the BCA assay kit (#23225; Thermo Fisher Scientific) was performed as described previously [27]. 50 μg protein aliquots were run on SDS-7.5% polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membrane, and immunoblotted with the indicated antibodies.…”

“…Tumorsphere assay: Tumorsphere cultures were done as described [27] with minor modifications. the tumorsphere medium contained DMEM/F12,Thermo Fisher; #1133032) supplemented with 1% penicillin/streptomycin, 4 μg/ml heparin (Stem cell technologies; #07980), 20 ng/ml Animal-Free Recombinant Human EGF (Peprotech; #AF-100-15), 10 ng/ml Recombinant Human FGF-basic (Peprotech; #100-18B), 1X N-2 supplement (Gibco; #17502-048), 1X B27 supplement (Gibco; #17504-044) and 4% Matrigel (BD Biosciences; #356234].…”