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Hypoxia induces thousands of mRNAs and miRNAs to mediate tumor malignancy. However, hypoxia-induced long noncoding RNA (lncRNA) transcriptome and their role in triple-negative breast cancer (TNBC) have not been defined. Here we identified hypoxia-induced lncRNA transcriptome in two human TNBC cell lines by whole transcriptome sequencing. AC093818.1 was one of 26 validated lncRNAs and abundantly expressed in TNBC in vitro and in vivo . 5'- and 3'-rapid amplification of cDNA ends assays revealed that the isoform 2 was a dominant AC093818.1 transcript in TNBC cells and thus referred to as lncIHAT ( lnc RNA i nduced by h ypoxia and a bundant in T NBC). Hypoxia-inducible factor 1 (HIF1) but not HIF2 bound to the hypoxia response element at the promoter of lncIHAT to activate its transcription in hypoxic TNBC cells. LncIHAT promoted TNBC cell survival in vitro and tumor growth and lung metastasis in mice. Mechanistically, lncIHAT was required for the expression of its proximal neighboring oncogenic genes PDK1 and ITGA6 in TNBC cells and tumors. Reexpression of PDK1 and ITGA6 rescued survival and growth of lncIHAT knockdown TNBC cells in vitro . Collectively, these findings uncovered lncIHAT as a new hypoxia-induced oncogenic cis -acting lncRNA in TNBC. IMPLICATIONS: This study systematically identified hypoxia-induced lncRNA transcriptome in TNBC and sheds light on multiple layers of regulatory mechanisms of gene expression under hypoxia., (©2020 American Association for Cancer Research.)

Intratumoral hypoxia, which is associated with breast cancer metastasis and patient mortality, increases the percentage of breast cancer stem cells (BCSCs) but the underlying molecular mechanisms have not been delineated. Here we report that hypoxia-inducible factor 1 (HIF-1) triggers the expression and activity of TAZ, a transcriptional co-activator that is required for BCSC maintenance, through two discrete mechanisms. First, HIF-1 binds directly to the WWTR1 gene and activates transcription of TAZ mRNA. Second, HIF-1 activates transcription of the SIAH1 gene, which encodes a ubiquitin protein ligase that is required for the hypoxia-induced ubiquitination and proteasome-dependent degradation of LATS2, a kinase that inhibits the nuclear localization of TAZ. Inhibition of HIF-1α, TAZ, or SIAH1 expression by short hairpin RNA blocked the enrichment of BCSCs in response to hypoxia. Human breast cancer database analysis revealed that increased expression (greater than the median) of both TAZ and HIF-1 target genes, but neither one alone, is associated with significantly increased patient mortality. Taken together, these results establish a molecular mechanism for induction of the BCSC phenotype in response to hypoxia.

Unlabelled: Targeted therapy against triple-negative breast cancers, which lack expression of the estrogen, progesterone, and HER2 receptors, is not available and the overall response to cytotoxic chemotherapy is poor. One of the molecular hallmarks of triple-negative breast cancers is increased expression of genes that are transcriptionally activated by hypoxia-inducible factors (HIFs), which are implicated in many critical aspects of cancer progression including metabolism, angiogenesis, invasion, metastasis, and stem cell maintenance. Ganetespib is a second-generation inhibitor of heat shock protein 90 (HSP90), a molecular chaperone that is essential for the stability and function of multiple client proteins in cancer cells including HIF-1α. In this study, human MDA-MB-231 and MDA-MB-435 triple-negative breast cancer cells were injected into the mammary fat pad of immunodeficient mice that received weekly intravenous injections of ganetespib or vehicle following the development of palpable tumors. Ganetespib treatment markedly impaired primary tumor growth and vascularization, and eliminated local tissue invasion and distant metastasis to regional lymph nodes and lungs. Ganetespib treatment also significantly reduced the number of Aldefluor-positive cancer stem cells in the primary tumor. Primary tumors of ganetespib-treated mice had significantly reduced levels of HIF-1α (but not HIF-2α) protein and of HIF-1 target gene mRNAs encoding proteins that play key roles in angiogenesis, metabolism, invasion, and metastasis, thereby providing a molecular basis for observed effects of the drug on the growth and metastasis of triple-negative breast cancer., Key Messages: Triple-negative breast cancers (TNBCs) respond poorly to available chemotherapy. TNBCs overexpress genes regulated by hypoxia-inducible factors (HIFs). Ganetespib induces degradation of HSP90 client proteins, including HIF-1α. Ganetespib inhibited TNBC orthotopic tumor growth, invasion, and metastasis. Ganetespib inhibited expression of HIF-1 target genes involved in TNBC progression.

Both preclinical and clinical studies suggest that brief cycles of ischemia and reperfusion in the arm or leg may protect the heart against injury following prolonged coronary artery occlusion and reperfusion, a phenomenon known as remote ischemic preconditioning. Recent studies in mice indicate that increased plasma interleukin-10 (IL-10) levels play an important role in remote ischemic preconditioning induced by clamping the femoral artery for 5 min followed by 5 min of reperfusion for a total of three cycles. In this study, we demonstrate that remote ischemic preconditioning increases plasma IL-10 levels and decreases myocardial infarct size in wild-type mice but not in littermates that are heterozygous for a knockout allele at the locus encoding hypoxia-inducible factor (HIF) 1α. Injection of a recombinant adenovirus encoding a constitutively active form of HIF-1α into mouse hind limb muscle was sufficient to increase plasma IL-10 levels and decrease myocardial infarct size. Exposure of C2C12 mouse myocytes to cyclic hypoxia and reoxygenation rapidly increased levels of IL-10 mRNA, which was blocked by administration of the HIF-1 inhibitor acriflavine or by expression of short hairpin RNA targeting HIF-1α or HIF-1β. Chromatin immunoprecipitation assays demonstrated that binding of HIF-1 to the Il10 gene was induced when myocytes were subjected to cyclic hypoxia and reoxygenation. Taken together, these data indicate that HIF-1 activates Il10 gene transcription and is required for remote ischemic preconditioning.

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that play key roles in breast cancer biology. We hypothesized that interaction of HIF-1 with epigenetic regulators may increase HIF-1 transcriptional activity, and thereby promote breast cancer progression. We report that the histone demethylase jumonji domain containing protein 2C (JMJD2C) selectively interacts with HIF-1α, but not HIF-2α, and that HIF-1α mediates recruitment of JMJD2C to the hypoxia response elements of HIF-1 target genes. JMJD2C decreases trimethylation of histone H3 at lysine 9, and enhances HIF-1 binding to hypoxia response elements, thereby activating transcription of BNIP3, LDHA, PDK1, and SLC2A1, which encode proteins that are required for metabolic reprogramming, as well as LOXL2 and L1CAM, which encode proteins that are required for lung metastasis. JMJD2C expression is significantly associated with expression of GLUT1, LDHA, PDK1, LOX, LOXL2, and L1CAM mRNA in human breast cancer biopsies. JMJD2C knockdown inhibits breast tumor growth and spontaneous metastasis to the lungs of mice following mammary fat pad injection. Taken together, these findings establish an important epigenetic mechanism that stimulates HIF-1-mediated transactivation of genes encoding proteins involved in metabolic reprogramming and lung metastasis in breast cancer.

Cancer cells feature altered glucose metabolism that allows their rapid growth. They consume large amounts of glucose to produce lactate, even in the presence of ample oxygen, which is known as the Warburg effect. Pyruvate kinase M2 (PKM2) contributes to the Warburg effect by previously unknown mechanisms. Hypoxia-inducible factor 1 (HIF-1) mediates PKM2 gene transcription and glucose reprogramming in cancer cells. The recent discovery of novel physical and functional interactions between PKM2 and HIF-1 in cancer cells has provided insight into molecular mechanisms underlying the Warburg effect.