HOTAIR is a long noncoding RNA (lncRNA) that is transcribed from the antisense strand of HOXC gene locus in chromosome 12. HOTAIR coordinates with chromatin modifying enzymes and regulates gene silencing. It is overexpressed in various carcinomas including breast cancer. Herein, we demonstrated that HOTAIR is crucial for cell growth and viability and its knockdown induced apoptosis in breast cancer cells. We also demonstrated that HOTAIR is transcriptionally induced by estradiol (E2). Its promoter contains multiple functional estrogen-response-elements (EREs). Estrogen receptors (ERs) along with various ER-coregulators such as histone methylases MLL1 and MLL3 and CBP/p300 bind to the promoter of HOTAIR in an E2-dependent manner. Level of histone H3K4-trimethylation, histone acetylation and RNA polymerase II recruitment is enriched at the HOTAIR promoter in presence of E2. Knockdown of ERs and MLLs down regulated the E2-induced HOTAIR expression. Thus, similar to protein coding gene transcription, E2-induced transcription of antisense transcript HOTAIR is coordinated via ERs and ER-coregulators and this mechanism of HOTAIR over expression potentially contributes towards breast cancer progression.

In non–small cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumor suppressor STK11 encoding the kinase LKB1 result in aggressive tumors prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant (KL) cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analyzed tumor metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards an activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KL mutant tumors. KL cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). GFPT2 inhibition selectively reduced KL tumor cell growth in culture, xenografts and genetically-modified mice. Our results define a new metabolic vulnerability in KL tumors and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

HOXC10 is a critical player in the development of spinal cord, formation of neurons, and associated with human leukemia. We found that HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen (17b-estradiol, E 2 ). The HOXC10 promoter contains several estrogen response elements (ERE1-7, half-sites). A luciferase-based reporter assay showed that ERE1 and ERE6 of HOXC10 promoter are E 2 responsive. ERa and ERb play critical roles in E 2 -mediated activation of HOXC10. Knockdown of ERa and ERb downregulated E 2 -induced HOXC10 expression. ERa and ERb bind to ERE1 and ERE6 regions in an E 2 -dependent manner. Additionally, knockdown of histone methylases MLL3 and MLL4 (but not MLL1 and MLL2) diminished E 2 -induced expression of HOXC10. MLL3 and MLL4 were bound to the ERE1 and ERE6 regions of HOXC10 promoter in an E 2 -dependent manner. Overall, we demonstrated that HOXC10 is overexpressed in breast cancer, and it is an E 2 -responsive gene. Histone methylases MLL3 and MLL4, along with ERs, regulate HOXC10 gene expression in the presence of E 2 .

Homeobox containing gene HOXC6 is a critical player in mammary gland development, milk production and is overexpressed in breast and prostate cancer. We demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). HOXC6 promoter contains two putative estrogen-response elements (EREs), termed as ERE11/2 and ERE21/2. Promoter analysis using luciferase based reporter assay demonstrated that both EREs are responsive to E2, ERE11/2 being more responsive than ERE21/2. Estrogen receptors, ERα and ERβ, bind to these EREs in an E2-dependent manner and antisense-mediated knockdown of ERs suppressed the E2-dependent activation of HOXC6 expression. Similarly, knockdown of histone methylases, MLL2 and MLL3, decreased E2-mediated activation of HOXC6. However, depletion of MLL1 or MLL4 showed no significant effect. MLL2 and MLL3 were bound to the HOXC6 EREs in an E2-dependent manner. In contrast, MLL1 and MLL4 that were bound to the HOXC6 promoter in the absence of E2, decreased upon exposure to E2. MLL2 and MLL3 play key roles in histone H3K4-trimethylation and recruitment of general transcription factors and RNAP II in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors N-CoR and SAFB1 were bound in the HOXC6 promoter in absence of E2 and that binding were decreased upon E2-treatment indicating their critical roles in suppressing HOXC6 gene expression under non-activated condition. Knockdown of either ERα or ERβ abolished E2-dependent recruitment of MLL2 and MLL3 into the HOXC6 promoter demonstrating key roles of ERs in recruitment of these MLLs into HOXC6 promoter. Overall, our studies demonstrated that HOXC6 is an estrogen-responsive gene and histone methylases MLL2 and MLL3, in coordination with ERα and ERβ, transcriptionally regulate HOXC6 in an E2-dependent manner.

HOXB9 is a homeobox-containing gene and is critical for the development of mammary gland and sternum. HOXB9 is also regulated by estrogen and is critical for angiogenesis. We investigated the biochemical roles of HOXB9 and its homeodomain in cell-cycle progression and tumorigenesis. Our studies demonstrated that HOXB9 is overexpressed in breast cancer tissue. HOXB9 overexpression stimulated 3D formation in soft agar assay. HOXB9 binds to the promoters of various tumor growth and angiogenic factors and regulates their expression. The homeodomain of HOXB9 plays crucial roles in transcriptional regulation of tumor growth factors and also in 3D colony formation, indicating crucial roles of the HOXB9 homeodomain in tumorigenesis. Overall, we demonstrated that HOXB9 is a critical regulator of tumor growth factors and is associated with tumorigenesis.