Influence of hypoxia on differentiation and proliferation of non-malignant and tumor cells

University dissertation from Molecular Medicine

Abstract: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state. Despite expression of EPOR we show here that rhEPO stimulation of breast cancer cells has no apparent effect on cell growth in vitro. When knocking down EPOR we found a decreased proliferation in ERα+ breast cancer cells only. This inhibitory effect on proliferation appeared to be mediated via a decrease in ERα-activity. EPOR knockdown further improved Tamoxifen effects on ERα-activity in one of the cell lines tested. These results are in accordance with our previous observation that high EPOR expression in tumors correlates to impaired Tamoxifen response in breast cancer patients. We previously demonstrated that small cell lung carcinoma (SCLC) cells lack expression of HIF-2α protein, whereas HIF-1α is expressed at both acute and prolonged hypoxia. Searching for a role of HIF-1 in hypoxic SCLC cells, we investigated metabolic pathways and HIF-1 dependence. When repressing HIF1A expression, the ATP levels were not affected and SCLC cell viability did not decrease by glucose deprivation. In contrast, glutamine withdrawal drastically decreased the number of cells and increased cell death, still in a HIF-independent fashion. The tested SCLC cells are MYC or MYCL amplified and MYC overexpression is known to stimulate glutamine metabolism. Our results might further suggest that Myc overexpression overrides the need of HIF-1α expression and activity in the response of SCLC cells to hypoxia by inducing glutaminolysis and lipogenesis.

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