HIF-inducible miR-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment.

Scientific Reports, Apr 2015

The molecular mechanisms of hypoxia induced breast cell migration remain incompletely understood. Our results show that hypoxia through hypoxia-inducible factor (HIF) brings about a time-dependent increase in the level of an oncogenic microRNA, miR-191 in various breast cancer cell lines. miR-191 enhances breast cancer aggressiveness by promoting cell proliferation, migration and survival under hypoxia. We further established that miR-191 is a critical regulator of transforming growth factor beta (TGFβ)-signaling and promotes cell migration by inducing TGFβ2 expression under hypoxia through direct binding and indirectly by regulating levels of a RNA binding protein, human antigen R (HuR). The levels of several TGFβ pathway genes (like VEGFA, SMAD3, CTGF and BMP4) were found to be higher in miR-191 overexpressing cells. Lastly, anti-miR-191 treatment given to breast tumor spheroids led to drastic reduction in spheroid tumor volume. This stands as a first report of identification of a microRNA mediator that links hypoxia and the TGFβ signaling pathways, both of which are involved in regulation of breast cancer metastasis. Together, our results show a critical role of miR-191 in hypoxia-induced cancer progression and suggest that miR-191 inhibition may offer a novel therapy for hypoxic breast tumors.

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HIF-inducible miR-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment.

Abstract The molecular mechanisms of hypoxia induced breast cell migration remain incompletely understood. Our results show that hypoxia through hypoxia-inducible factor (HIF) brings about a time-dependent increase in the level of an oncogenic microRNA, miR-191 in various breast cancer cell lines. miR-191 enhances breast cancer aggressiveness by promoting cell proliferation, migration and survival under hypoxia. We further established that miR-191 is a critical regulator of transforming growth factor beta (TGFβ)-signaling and promotes cell migration by inducing TGFβ2 expression under hypoxia through direct binding and indirectly by regulating levels of a RNA binding protein, human antigen R (HuR). The levels of several TGFβ pathway genes (like VEGFA, SMAD3, CTGF and BMP4) were found to be higher in miR-191 overexpressing cells. Lastly, anti-miR-191 treatment given to breast tumor spheroids led to drastic reduction in spheroid tumor volume. This stands as a first report of identification of a microRNA mediator that links hypoxia and the TGFβ signaling pathways, both of which are involved in regulation of breast cancer metastasis. Together, our results show a critical role of miR-191 in hypoxia-induced cancer progression and suggest that miR-191 inhibition may offer a novel therapy for hypoxic breast tumors. Introduction Hypoxia (pO2, <5–10 mm Hg) is a regular feature of breast tumor microenvironment and has often been associated with poor prognosis1. It affects a variety of tumor properties such as proliferation, migration, invasion, epithelial to mesenchymal transition, angiogenesis, vascularization and apoptosis2. Besides, it also leads to therapeutic failure by promoting resistance to ionizing radiation and various chemotherapeutic drugs3,4. Thus, understanding of hypoxia signaling has been an active area of research. Specific hypoxia regulated genes such as HIF (hypoxia inducible factor), VEGFA (vascular endothelial growth factor A) and CA9 (carbonic anhydrase 9) have been identified as promising targets for cancer therapy or as diagnostic/prognostic markers in clinical research5,6,7. A major advance in the understanding of hypoxia signaling was made with our discovery that hypoxia, apart from regulating protein coding genes, also regulates a class of small, endogenous, non-coding RNAs called microRNAs (miRNAs)8. Until now, more than 2500 of miRNAs have been discovered in humans, however, functions are known of very few of them. miRNAs play an important role in disease physiology and pathogenesis through fine tuning of gene expression9,10. They are conventionally known to bind to the 3′UTR of target genes and bring about their degradation or translational repression depending on the degree of complementarity with the target site11. However, recent studies suggest that miRNA binding sites are not limited to the 3′UTR but may involve the coding, 5′UTR or promoter regions of the target genes12,13. Additional major revelation has been that miRNAs may induce the target gene expression as well, that may include mechanisms such as their interplay with RNA binding proteins, interaction with promoter elements (TATA box motifs) and 5′UTR of the target genes14,15,16,17,18. These findings suggest the versatility of miRNA mode of action and cellular functions. The repertoire of hypoxia regulated miRNAs (HRMs) may vary depending upon the cellular or physiological context, however, specific HRMs consistently show hypoxia inducibility in diverse contexts8,19. The most prominent and well studied among these is miR-210 that was found to be robustly induced by hypoxia across various cell types20. The hypoxic regulation of HRMs has been found to be HIF dependent, though it is speculated that other hypoxia relevant transcription factors such as p53 and nuclear factor-Kappa B may be involved21. Specific HRMs such as miR-210, miR-373, miR-155 and miR-10b have been shown to regulate diverse functions such as angiogenesis, apoptosis, DNA repair, migration and cell proliferation to fine tune the cellular response to hypoxia22,23,24,25. This corroborates the crucial role of HRMs in mediating cellular adaptation to hypoxia in tumor cells. However, the functions of most of the HRMs remain unknown. Here, we have focused our work on miRNA, miR-191, that was found to be hypoxia inducible in breast cancer26. Higher levels of miR-191 have been reported before in several cancers including breast cancer and it has been shown to function as an oncogenic miRNA in some of these (breast, hepatic, colon and gastric cancers)27. miR-191 was identified as an important player in estrogen signaling that controls various oncogenic properties in breast cancer, though in a context dependent manner26,28. However, its association or functions under hypoxia have not been investigated yet. Our results here identify miR-191 as a HIF regulated miRNA that promotes proliferation, migration and therapeutic resistance under hypoxia. miR-191 overexpres (...truncated)


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Neha Nagpal, Hafiz M. Ahmad, Shibu Chameettachal, Durai Sundar, Sourabh Ghosh, Ritu Kulshreshtha. HIF-inducible miR-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment., Scientific Reports, 2015, Issue: 5, DOI: 10.1038/srep09650