Pro-Apoptotic and Anti-Invasive Properties Underscore the Tumor-Suppressing Impact of Myoglobin on a Subset of Human Breast Cancer Cells

Abstract

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice

“…The number of Ki67 positive cells in MB+/+ WapCre;Trp53 flox /MB tumors was decreased when compared with MB−/− tumors. Similar model-specific differences in breast cancer cell proliferation between MB+/+ and MB−/− genotypes were observed with SKBR3 and MCF7 cells 16 . While MB in SKBR3 cells was shown to increase proliferation, the opposite influence was observed for MCF7 cells.…”

“…In addition, MB is preferentially expressed in well-differentiated and hormone-receptor-positive tumors, which correlates with a less aggressive tumor phenotype 3 . Parallel to our PyMT/MB mouse model, MB−/− MCF7 cells reveal elevated in vitro invasion and motility rates compared with MB+/+ cells 16 . However, SKBR3 16 and MDA-MB468 14 breast cancer cells demonstrated a faster in vitro migration when cells contained MB, implying that the anti-invasive property of MB must be strictly evaluated in a cell- or tumor-type-specific context.…”

“…However, the MB genotype did not significantly affect this outcome. Yet, in the parallel in vitro study, we were able to show that MB sensitizes breast cancer cells to Doxorubicin 16 . It is, furthermore, of great interest and clinical relevance that Doxorubicin treatment nullified the stronger metastatic propensity of MB-deficient tumors of PyMT/MB mice.…”

“…After washing in TBST buffer, the horseradish peroxidase (HRP) conjugated secondary antibodies were diluted 1:5000 in blocking buffer (donkey-anti-rabbit, NA934V, Amersham/ goat-anti-mouse, sc-2031, Santa Cruz Biotechnology) and added to the membranes, which were placed on a shaker for 1 h. Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher, 34095) was used to detect of the bands in a luminescent image analyzer LAS-3000 (Fujifilm). Antibodies have been previously validated by us 12 , 13 , 16 . We could not provide the full-length membranes for some blots because the membranes were cut before hybridization with the primary antibody.…”

“…In this context, MB may act as a tumor suppressor probably by exerting pro-apoptotic and anti-mitochondrial effects in hypoxic human breast cancer cells 14 or via molecular interaction with the known tumor suppressor p53 15 . In human MCF7 breast cancer cells, MB has been shown to restrict migration and promote apoptotic cell death 16 . However, whether MB in mammary tumors influences the response to chemotherapy is not known.…”