“Pulsed Hypoxia” Gradually Reprograms Breast Cancer Fibroblasts into Pro-Tumorigenic Cells via Mesenchymal–Epithelial Transition

Abstract

Hypoxia arises in most growing solid tumors and can lead to pleotropic effects that potentially increase tumor aggressiveness and resistance to therapy through regulation of the expression of genes associated with the epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET). The main goal of the current work was to obtain and investigate the intermediate phenotype of tumor cells undergoing the hypoxia-dependent transition from fibroblast to epithelial morphology. Primary breast cancer fibroblasts BrC4f, being cancer-associated fibroblasts, were subjected to one or two rounds of “pulsed hypoxia” (PH). PH induced transformation of fibroblast-shaped cells to semi-epithelial cells. Western blot analysis, fluorescent microscopy and flow cytometry of transformed cells demonstrated the decrease in the mesenchymal markers vimentin and N-cad and an increase in the epithelial marker E-cad. These cells kept mesenchymal markers αSMA and S100A4 and high ALDH activity. Real-time PCR data of the cells after one (BrC4f_Hyp1) and two (BrC4f_Hyp2) rounds of PH showed consistent up-regulation of TWIST1 gene as an early response and ZEB1/2 and SLUG transcriptional activity as a subsequent response. Reversion of BrC4f_Hyp2 cells to normoxia conditions converted them to epithelial-like cells (BrC4e) with decreased expression of EMT genes and up-regulation of MET-related OVOL2 and c-MYC genes. Transplantation of BrC4f and BrC4f_Hyp2 cells into SCID mice showed the acceleration of tumor growth up to 61.6% for BrC4f_Hyp2 cells. To summarize, rounds of PH imitate the MET process of tumorigenesis in which cancer-associated fibroblasts pass through intermediate stages and become more aggressive epithelial-like tumor cells.

KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer

“…Notably, MET in TME fibroblasts is a fundamental process in ontogenesis and oncogenesis. It has been demonstrated that hypoxia can reprogram the fibroblast genome towards epithelial-like cells through the EMT pathway, leading cancer-associated fibroblasts to undergo intermediate states and become more aggressive epithelial-like tumor cells ( 88 ). The role of the KDM7A-DT regulation in hypoxia-associated cancer progression mechanisms is perspective in the contexts of basic tumor biology and clinical needs.…”

Identification of a novel cancer-associated fibroblasts gene signature based on bioinformatics analysis to predict prognosis and therapeutic responses in breast cancer

“…Furthermore, hypoxia inhibited the tumor-suppressive function of various immune cells, thereby contributing to immunotherapy resistance [ 39 , 40 ]. It is worth noting that hypoxia can trigger angiogenesis and EMT, further exacerbating immunosuppression [ [41] , [42] , [43] , [44] ]. The CIBERSORT analysis showed that high CAFs risk scores were positively correlated to Tregs and M2 macrophage polarization, and the ssGSEA analysis showed that high CAFs risk scores were negatively correlated to CD8 + T cells and TILs, which might be the mechanism leading to immunotherapy resistance in patients with high-risk scores.…”

Understanding human aging and the fundamental cell signaling link in age-related diseases: the middle-aging hypovascularity hypoxia hypothesis

“…These changes occur during embryogenesis (type 1 EMT), wound healing, tissue regeneration, fibrosis (type 2 EMT) and in cancer, where they contribute to cell stemness (plasticity), drug resistance, immune escape, and metastasis (type 3 EMT) ( Manfioletti and Fedele, 2022 ). EMT can be seen as a period of a bet-hedging—evolutionary cellular adaptation ( Jolly et al, 2018 ; Capp and Thomas, 2022 ; Jain et al, 2022 ), which diversifies mesenchymal phenotypes (cancer stemness) and increases their survival ( Mortezaee and Majidpoor, 2022 ) in changing hypoxia-induced pathophysiology ( Lee et al, 2020 ; Papale et al, 2020 ; Nushtaeva et al, 2023 ; Zhang et al, 2023 ).…”

“…EMT is reversal by the mesenchymal-to-epithelial transition (MET) ( Yang J. et al, 2020 ; Manfioletti and Fedele, 2022 ), which can be synonymous to type 2 EMT, a reparative-associated fibrotic process in response to chronic inflammation ( Marconi et al, 2021 ). The chief candidate for EMT reversal (regeneration/rejuvenation) would be the timely amelioration of hypoxia ( López-Novoa and Nieto, 2009 ; Lee et al, 2020 ; Papale et al, 2020 ; Nushtaeva et al, 2023 ; Zhang et al, 2023 ) before the establishment of dysregulation–degeneration hypoxic cell biology ( Phua, 2021 ; Rando and Jones, 2021 ) and cellular senescence ( Welford and Giaccia, 2011 ; Barabutis et al, 2018 ; Lacroix et al, 2020 ; Otero-Albiol and Carnero, 2021 ). In a review article, new approaches to alleviating hypoxia through the modulation of the vascular state in the tumor microenvironment offer promise for ovarian cancer immunotherapeutic strategies ( Klemba et al, 2020 ).…”