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Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

ABSTRACT: Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1? and estrogen receptor alpha (ER?) and a negative correlation between HIF-1? and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ER? under hypoxic conditions (1% O2). Here, we compared the impact of hypoxia on the ER? signaling pathway for T47D cells in a 2D and 3D culture format. In the 2D format, the cells were cultured as monolayers. In the 3D format, paper-based scaffolds supported cells suspended in a collagen matrix. Using ELISA, Western blot, and immunofluorescence measurements, we show that hypoxia differentially regulates ER? protein levels in a culture environment-dependent manner. In the 2D format, the protein levels are significantly decreased in hypoxia. In the 3D format, the protein levels are maintained in hypoxia. Hypoxia reduced ER? transcriptional activation in both culture formats. These results highlight the importance of considering tissue dimensionality for in vitro studies. They also show that ER? protein levels in hypoxia are not an accurate indicator of ER? transcriptional activity, and confirm that a positive stain for ER? in a clinical sample may not necessarily indicate hormone sensitivity.

SUBMITTER: Whitman NA

PROVIDER: S-EPMC6688900 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

Whitman Nathan A NA Lin Zhi-Wei ZW Kenney Rachael M RM Albertini Leonardo L Lockett Matthew R MR

Archives of biochemistry and biophysics 20190601

Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1α and estrogen receptor alpha (ERα) and a negative correlation between HIF-1α and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ERα under hypoxic conditions (1% O<sub>2</sub>). Here, we compared the impact of hypoxia on the ERα signaling pathway fo ...[more]

PMID: 31163125

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Project description:BACKGROUND: The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). METHODS: The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. RESULTS: We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. CONCLUSION: This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

Dataset Information

Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

Publications

Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

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