Autocrine GMCSF Signaling Contributes to Growth of HER2+ Breast Leptomeningeal Carcinomatosis.

Leptomeningeal carcinomatosis (LC) occurs when tumor cells spread to the cerebrospinal fluid-containing leptomeninges surrounding the brain and spinal cord. LC is an ominous complication of cancer with a dire prognosis. Although any malignancy can spread to the leptomeninges, breast cancer, particularly the HER2+ subtype, is its most common origin. HER2+ breast LC (HER2+ LC) remains incurable, with few treatment options, and the molecular mechanisms underlying proliferation of HER2+ breast cancer cells in the acellular, protein, and cytokine-poor leptomeningeal environment remain elusive. Therefore, we sought to characterize signaling pathways that drive HER2+ LC development as well as those that restrict its growth to leptomeninges. Primary HER2+ LC patient-derived ("Lepto") cell lines in coculture with various central nervous system (CNS) cell types revealed that oligodendrocyte progenitor cells (OPC), the largest population of dividing cells in the CNS, inhibited HER2+ LC growth in vitro and in vivo, thereby limiting the spread of HER2+ LC beyond the leptomeninges. Cytokine array-based analyses identified Lepto cell-secreted GMCSF as an oncogenic autocrine driver of HER2+ LC growth. LC/MS-MS-based analyses revealed that the OPC-derived protein TPP1 proteolytically degrades GMCSF, decreasing GMCSF signaling and leading to suppression of HER2+ LC growth and limiting its spread. Finally, intrathecal delivery of neutralizing anti-GMCSF antibodies and a pan-Aurora kinase inhibitor (CCT137690) synergistically inhibited GMCSF and suppressed activity of GMCSF effectors, reducing HER2+ LC growth in vivo. Thus, OPC suppress GMCSF-driven growth of HER2+ LC in the leptomeningeal environment, providing a potential targetable axis. SIGNIFICANCE: This study characterizes molecular mechanisms that drive HER2+ leptomeningeal carcinomatosis and demonstrates the efficacy of anti-GMCSF antibodies and pan-Aurora kinase inhibitors against this disease.

Inhibition of Jumonji Histone Demethylases Selectively Suppresses HER2+ Breast Leptomeningeal Carcinomatosis Growth via Inhibition of GMCSF Expression.

HER2+ breast leptomeningeal carcinomatosis (HER2+ LC) occurs when tumor cells spread to cerebrospinal fluid-containing leptomeninges surrounding the brain and spinal cord, a complication with a dire prognosis. HER2+ LC remains incurable, with few treatment options. Currently, much effort is devoted toward development of therapies that target mutations. However, targeting epigenetic or transcriptional states of HER2+ LC tumors might efficiently target HER2+ LC growth via inhibition of oncogenic signaling; this approach remains promising but is less explored. To test this possibility, we established primary HER2+ LC (Lepto) cell lines from nodular HER2+ LC tissues. These lines are phenotypically CD326+CD49f-, confirming that they are derived from HER2+ LC tumors, and express surface CD44+CD24-, a cancer stem cell (CSC) phenotype. Like CSCs, Lepto lines showed greater drug resistance and more aggressive behavior compared with other HER2+ breast cancer lines in vitro and in vivo. Interestingly, the three Lepto lines overexpressed Jumonji domain-containing histone lysine demethylases KDM4A/4C. Treatment with JIB04, a selective inhibitor of Jumonji demethylases, or genetic loss of function of KDM4A/4C induced apoptosis and cell-cycle arrest and reduced Lepto cell viability, tumorsphere formation, regrowth, and invasion in vitro. JIB04 treatment of patient-derived xenograft mouse models in vivo reduced HER2+ LC tumor growth and prolonged animal survival. Mechanistically, KDM4A/4C inhibition downregulated GMCSF expression and prevented GMCSF-dependent Lepto cell proliferation. Collectively, these results establish KDM4A/4C as a viable therapeutic target in HER2+ LC and spotlight the benefits of targeting the tumorigenic transcriptional network. SIGNIFICANCE: HER2+ LC tumors overexpress KDM4A/4C and are sensitive to the Jumonji demethylase inhibitor JIB04, which reduces the viability of primary HER2+ LC cells and increases survival in mouse models.

Astrocytic IGFBP2 and CHI3L1 in cerebrospinal fluid drive cortical metastasis of HER2+breast cancer.

The brain is often reported as the first site of recurrence among breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Although most HER2+tumors metastasize to the subcortical region of the brain, a subset develops in the cortical region. We hypothesize that factors in cerebrospinal fluid (CSF) play a critical role in the adaptation, proliferation, and establishment of cortical metastases. We established novel cell lines using patient biopsies to model breast cancer cortical and subcortical metastases. We assessed the localization and growth of these cells in vivo and proliferation and apoptosis in vitro under various conditions. Proteomic analysis of human CSF identified astrocyte-derived factors that support the proliferation of cortical metastases, and we used neutralizing antibodies to test the effects of inhibiting these factors both in vivo and in vitro. The cortical breast cancer brain metastatic cells exhibited greater proliferation than subcortical breast cancer brain metastatic cells in CSF containing several growth factors that nourish both the CNS and tumor cells. Specifically, the astrocytic paracrine factors IGFBP2 and CHI3LI promoted the proliferation of cortical metastatic cells and the formation of metastatic lesions. Disruption of these factors suppressed astrocyte-tumor cell interactions in vitro and the growth of cortical tumors in vivo. Our findings suggest that inhibition of IGFBP2 and CHI3LI signaling, in addition to existing treatment modalities, may be an effective therapeutic strategy targeting breast cancer cortical metastasis.

HOXA5 Expression Is Elevated in Breast Cancer and Is Transcriptionally Regulated by Estradiol.

HOXA5 is a homeobox-containing gene associated with the development of the lung, gastrointestinal tract, and vertebrae. Here, we investigate potential roles and the gene regulatory mechanism in HOXA5 in breast cancer cells. Our studies demonstrate that HOXA5 expression is elevated in breast cancer tissues and in estrogen receptor (ER)-positive breast cancer cells. HOXA5 expression is critical for breast cancer cell viability. Biochemical studies show that estradiol (E2) regulates HOXA5 gene expression in cultured breast cancer cells in vitro. HOXA5 expression is also upregulated in vivo in the mammary tissues of ovariectomized female rats. E2-induced HOXA5 expression is coordinated by ERs. Knockdown of either ERα or ERß downregulated E2-induced HOXA5 expression. Additionally, ER co-regulators, including CBP/p300 (histone acetylases) and MLL-histone methylases (MLL2, MLL3), histone acetylation-, and H3K4 trimethylation levels are enriched at the HOXA5 promoter in present E2. In summary, our studies demonstrate that HOXA5 is overexpressed in breast cancer and is transcriptionally regulated via estradiol in breast cancer cells.

Histone methylase MLL1 coordinates with HIF and regulate lncRNA HOTAIR expression under hypoxia.

Hypoxia signaling plays a critical role in tumor growth, angiogenesis, metastasis cancer, and aging. Under hypoxia, hypoxia-inducible factors (HIFs) are stabilized and they coordinate the process of hypoxia-induced gene expression and cell signaling leading to increased tumor growth. Recent studies indicate that non-coding RNAs which are closely associated with cancer are abnormally expressed under hypoxia. Here, we have investigated the transcriptional regulation of a cancer associated long non-coding RNA (lncRNA), HOTAIR, under hypoxic conditions. Our studies demonstrate that HOTAIR expression is upregulated under hypoxia in colon cancer and several other types of cancer cells. HOTAIR transcription is regulated by HIF1α which binds to the hypoxia response elements (HRE) present in the HOTAIR promoter under hypoxia. HIF1α knockdown results in decreased HOTAIR expression under hypoxia. Along with HIF1α, histone methylases MLL1 and histone acetylase p300 are enriched at the HOTAIR promoter under hypoxia. The levels of H3K4-trimethylation and histone acetylation are also enriched at the HOTAIR promoter. Furthermore, knockdown of MLL1 downregulated the hypoxia-induced HOTAIR expression, indicating key roles of MLL1 in hypoxia-induced HOTAIR expression. Overall, our studies demonstrate that histone methyl-transferase MLL1 coordinates with HIF1α and histone acetyltransferase p300 and regulate hypoxia-induced HOTAIR expression. The hypoxia-induced upregulation of HOTAIR expression may contribute to its roles in tumorigenesis.

Cooperation of neurotrophin receptor TrkB and Her2 in breast cancer cells facilitates brain metastases.

BACKGROUND: Patients with primary breast cancer that is positive for human epidermal growth factor receptor 2 (Her2+) have a high risk of developing metastases in the brain. Despite gains with systemic control of Her2+ disease using molecular therapies, brain metastases remain recalcitrant to therapeutic discovery. The clinical predilection of Her2+ breast cancer cells to colonize the brain likely relies on paracrine mechanisms. The neural niche poses unique selection pressures, and neoplastic cells that utilize the brain microenvironment may have a survival advantage. METHODS: Tropomyosin-related kinase B (TrkB), Her2, and downstream targets were analyzed in primary breast cancer, breast-to-brain metastasis (BBM) tissues, and tumor-derived cell lines using quantitative real-time PCR, western blot, and immunohistochemical assessment. TrkB function on BBM was confirmed with intracranial, intracardiac, or mammary fat pad xenografts in non-obese diabetic/severe combined immunodeficiency mice. The function of brain-derived neurotrophic factor (BDNF) on cell proliferation and TrkB/Her2 signaling and interactions were confirmed using selective shRNA knockdown and selective inhibitors. The physical interaction of Her2-TrkB was analyzed using electron microscopy, co-immunoprecipitation, and in silico analysis. Dual targeting of Her2 and TrkB was analyzed using clinically utilized treatments. RESULTS: We observed that patient tissues and cell lines derived from Her2+ human BBM displayed increased activation of TrkB, a neurotrophin receptor. BDNF, an extracellular neurotrophin, with roles in neuronal maturation and homeostasis, specifically binds to TrkB. TrkB knockdown in breast cancer cells led to decreased frequency and growth of brain metastasis in animal models, suggesting that circulating breast cancer cells entering the brain may take advantage of paracrine BDNF-TrkB signaling for colonization. In addition, we investigated a possible interaction between TrkB and Her2 receptors on brain metastatic breast cancer cells, and found that BDNF phosphorylated both its cognate TrkB receptor and the Her2 receptor in brain metastatic breast cancer cells. CONCLUSION: Collectively, our findings suggest that heterodimerization of Her2 and TrkB receptors gives breast cancer cells a survival advantage in the brain and that dual inhibition of these receptors may hold therapeutic potential.

Astrocyte-induced Reelin expression drives proliferation of Her2+ breast cancer metastases.

Breast cancer metastasis to the brain develops after a clinical latency of years to even decades, suggesting that colonization of the brain is the most challenging step of the metastatic cascade. However, the underlying mechanisms used by breast cancer cells to successfully colonize the brain's microenvironment remain elusive. Reelin is an archetypal extracellular glycoprotein that regulates migration, proliferation, and lamination of neurons. It is epigenetically silenced in various cancers, and its expression in multiple myelomas is linked to poor patient survival. We found that Reelin expression was low in primary breast cancer tissue. However, its expression was significantly higher in Her2+ breast cancers metastasizing to the brain. In particular, Reelin was highly expressed in the tumor periphery adjacent to surrounding astrocytes. This augmented Reelin expression was seen in Her2+ metastases, but not in triple negative (TN) primary tumors or in TN breast to brain metastasis cells co-cultured with astrocytes. Furthermore, the elevated expression was sustained in Her2+ cells grown in the presence of the DNA methyltransferase inhibitor 5-azacytidine, indicating epigenetic regulation of Reelin expression. The relative growth and rate of spheroids formation derived from Her2+ primary and BBM cells co-cultured with astrocytes were higher than those of TN primary and BBM cells, and knockdown of both Reelin and Her2 suppressed the astrocyte-induced growth and spheroid forming ability of Her2+ cells. Collectively, our results indicate that within the neural niche, astrocytes epigenetically regulate Reelin expression and its interaction with Her2 leading to increased proliferation and survival fitness.

The Long Non-coding RNA HIF1A-AS2 Facilitates the Maintenance of Mesenchymal Glioblastoma Stem-like Cells in Hypoxic Niches.

Long non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia-inducible lncRNA, upregulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming. Among the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Downregulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs' speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context.

Endocrine disrupting chemical, bisphenol-A, induces breast cancer associated gene HOXB9 expression in vitro and in vivo.

HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development and is associated with breast and other types of cancer. Here, we demonstrate that HOXB9 expression is transcriptionally regulated by estradiol (E2), in vitro and in vivo. We also demonstrate that the endocrine disrupting chemical bisphenol-A (BPA) induces HOXB9 expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of ovariectomized (OVX) rats. Luciferase assay showed that estrogen-response-elements (EREs) in the HOXB9 promoter are required for BPA-induced expression. Estrogen-receptors (ERs) and ER-co-regulators such as MLL-histone methylase (MLL3), histone acetylases, CBP/P300, bind to the HOXB9 promoter EREs in the presence of BPA, modify chromatin (histone methylation and acetylation) and lead to gene activation. In summary, our results demonstrate that BPA exposure, like estradiol, increases HOXB9 expression in breast cells both in vitro and in vivo through a mechanism that involves increased recruitment of transcription and chromatin modification factors.

Glucose-based regulation of miR-451/AMPK signaling depends on the OCT1 transcription factor.

In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5' AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment.

Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer.

HOXC6 is a homeobox-containing gene associated with mammary gland development and is overexpressed in variety of cancers including breast and prostate cancers. Here, we have examined the expression of HOXC6 in breast cancer tissue, investigated its transcriptional regulation via estradiol (E2) and bisphenol-A (BPA, an estrogenic endocrine disruptor) in vitro and in vivo. We observed that HOXC6 is differentially over-expressed in breast cancer tissue. E2 induces HOXC6 expression in cultured breast cancer cells and in mammary glands of Sprague Dawley rats. HOXC6 expression is also induced upon exposure to BPA both in vitro and in vivo. Estrogen-receptor-alpha (ERα) and ER-coregulators such as MLL-histone methylases are bound to the HOXC6 promoter upon exposure to E2 or BPA and that resulted in increased histone H3K4-trimethylation, histone acetylation, and recruitment of RNA polymerase II at the HOXC6 promoter. HOXC6 overexpression induces expression of tumor growth factors and facilitates growth 3D-colony formation, indicating its potential roles in tumor growth. Our studies demonstrate that HOXC6, which is a critical player in mammary gland development, is upregulated in multiple cases of breast cancer, and is transcriptionally regulated by E2 and BPA, in vitro and in vivo.

Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol.

Enhancer of Zeste homolog 2 (EZH2), a methyltransferase specific to histone 3 lysine 27, is a critical player in gene silencing and is overexpressed in breast cancer. Our studies demonstrate that EZH2 is transcriptionally induced by estradiol in cultured breast cancer cells and in the mammary glands of ovariectomized rats. EZH2 promoter contains multiple functional estrogen-response elements. Estrogen receptors (ERs) and ER coregulators such as mixed lineage leukemia (MLL) histone methylases (MLL2 and MLL3) and histone acetyltransferase CBP/P300 bind to the EZH2 promoter in the presence of estradiol and regulate estradiol-induced EZH2 expression. EZH2 expression is also increased upon exposure to estrogenic endocrine disrupting chemicals (EDCs) such as bisphenol-A (BPA) and diethylstilbestrol (DES). Similar to estradiol, BPA and DES-induced EZH2 expression is coordinated by ERs, MLLs and CBP/P300. In summary, we demonstrate that EZH2 is transcriptionally regulated by estradiol in vitro and in vivo, and its expression is potentially dysregulated upon exposure to estrogenic EDCs.

Light influences how the fungal toxin deoxynivalenol affects plant cell death and defense responses.

The Fusarium mycotoxin deoxynivalenol (DON) can cause cell death in wheat (Triticum aestivum), but can also reduce the level of cell death caused by heat shock in Arabidopsis (Arabidopsis thaliana) cell cultures. We show that 10 µg mL(-1) DON does not cause cell death in Arabidopsis cell cultures, and its ability to retard heat-induced cell death is light dependent. Under dark conditions, it actually promoted heat-induced cell death. Wheat cultivars differ in their ability to resist this toxin, and we investigated if the ability of wheat to mount defense responses was light dependent. We found no evidence that light affected the transcription of defense genes in DON-treated roots of seedlings of two wheat cultivars, namely cultivar CM82036 that is resistant to DON-induced bleaching of spikelet tissue and cultivar Remus that is not. However, DON treatment of roots led to genotype-dependent and light-enhanced defense transcript accumulation in coleoptiles. Wheat transcripts encoding a phenylalanine ammonia lyase (PAL) gene (previously associated with Fusarium resistance), non-expressor of pathogenesis-related genes-1 (NPR1) and a class III plant peroxidase (POX) were DON-upregulated in coleoptiles of wheat cultivar CM82036 but not of cultivar Remus, and DON-upregulation of these transcripts in cultivar CM82036 was light enhanced. Light and genotype-dependent differences in the DON/DON derivative content of coleoptiles were also observed. These results, coupled with previous findings regarding the effect of DON on plants, show that light either directly or indirectly influences the plant defense responses to DON.

Bisphenol-A and diethylstilbestrol exposure induces the expression of breast cancer associated long noncoding RNA HOTAIR in vitro and in vivo.

Antisense transcript, long non-coding RNA HOTAIR is a key player in gene silencing and breast cancer and is transcriptionally regulated by estradiol. Here, we have investigated if HOTAIR expression is misregulated by bisphenol-A (BPA) and diethylstilbestrol (DES). Our findings demonstrate BPA and DES induce HOTAIR expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of rat. Luciferase assay showed that HOTAIR promoter estrogen-response-elements (EREs) are induced by BPA and DES. Estrogen-receptors (ERs) and ER-coregulators such as MLL-histone methylases (MLL1 and MLL3) bind to the HOTAIR promoter EREs in the presence of BPA and DES, modify chromatin (histone methylation and acetylation) and lead to gene activation. Knockdown of ERs down-regulated the BPA and DES-induced expression of HOTAIR. In summary, our results demonstrate that BPA and DES exposure alters the epigenetic programming of the HOTAIR promoters leading to its endocrine disruption in vitro and in vivo.

Antisense oligonucleotide mediated knockdown of HOXC13 affects cell growth and induces apoptosis in tumor cells and over expression of HOXC13 induces 3D-colony formation.

HOXC13 is a homeobox containing gene that plays crucial roles in hair development and origin of replication. Herein, we investigated the biochemical functions of HOXC13 and explored its potential roles in tumor cell viability. We have designed a phosphorothioate based antisense-oligonucleotide that specifically knockdown HOXC13 in cultured cells. Cell viability and cytotoxicity assays demonstrated that HOXC13 is essential for cell growth and viability. Antisense-mediated knockdown of HOXC13 affected the cell viability and induced apoptosis in cultured tumor cells. HOXC13 regulates the expression of cyclins and antisense-mediated knockdown of HOXC13 resulted in cell cycle arrest and apoptosis in colon cancer cells. Finally over expression of HOXC13 resulted in 3D-colony formation in soft-agar assay indicating its potential roles in cell proliferation and tumorigenesis.

Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol.

HOTAIR (HOX antisense intergenic RNA) is a long noncoding RNA (lncRNA) that is transcribed from the antisense strand of homeobox C 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 (mixed lineage leukemia 1) and MLL3 and CREB-binding protein/p300 bind to the promoter of HOTAIR in an E2-dependent manner. Level of histone H3 lysine-4 trimethylation, histone acetylation, and RNA polymerase II recruitment is enriched at the HOTAIR promoter in the presence of E2. Knockdown of ERs and MLLs downregulated 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 overexpression potentially contributes towards breast cancer progression.

MLL histone methylases regulate expression of HDLR-SR-B1 in presence of estrogen and control plasma cholesterol in vivo.

High-density lipoprotein receptors scavenger receptor class B type I [HDLR-SR-B1 (SR-B1)] is a key player in reverse cholesterol transport and maintaining blood cholesterol. We demonstrated that human SR-B1 is transcriptionally activated by 17ß-estradiol (E2) in HEPG2 and JAR cells. SR-B1 promoter contains multiple estrogen response elements (ERE half-sites) along with some Sp1 binding sites. Knockdown of estrogen receptor (ER)α and ERß down-regulated E2-induced SR-B1 expression. ERs were bound to SR-B1 promoter EREs in an E2-dependent manner. Along with ERs, mixed-lineage leukemia (MLL) histone methylases, especially MLL1 and MLL2, play key roles in E2-mediated SR-B1 activation. MLL1 and MLL2 bind to SR-B1 promoter in an E2-dependent manner and control the assembly of transcription pre-initiation complex and RNA polymerase II (RNAPII) recruitment. ERs and MLLs play critical roles in determining the cholesterol uptake by steroidogenic tissues/cells, and their knockdown suppressed the E2-induced cholesterol uptake efficiencies of the cells. Intriguingly, MLL2 knockdown in mice resulted in a 33% increase in plasma cholesterol level and also reduced SR-B1 expression in mice liver, demonstrating its crucial functions in controlling plasma cholesterol in vivo.

Homeodomain-containing protein HOXB9 regulates expression of growth and angiogenic factors, facilitates tumor growth in vitro and is overexpressed in breast cancer tissue.

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.

HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen via involvement of histone methylases MLL3 and MLL4.

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 (17ß-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. ERα and ERß play critical roles in E(2)-mediated activation of HOXC10. Knockdown of ERα and ERß downregulated E(2)-induced HOXC10 expression. ERα and ERß 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).

HOXC6 Is transcriptionally regulated via coordination of MLL histone methylase and estrogen receptor in an estrogen environment.

Homeobox (HOX)-containing gene HOXC6 is a critical player in mammary gland development and milk production, and is overexpressed in breast and prostate cancers. We demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). HOXC6 promoter contains two putative estrogen response elements (EREs), termed as ERE1(1/2) and ERE2(1/2). Promoter analysis using luciferase-based reporter assay demonstrated that both EREs are responsive to E2, with ERE1(1/2) being more responsive than ERE2(1/2). Estrogen receptors (ERs) ERα and ERß 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 the 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 H3 lysine-4 trimethylation and in the recruitment of general transcription factors and RNA polymerase II in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors N-CoR and SAFB1 were bound in the HOXC6 promoter in the absence of E2, and that binding was decreased upon E2 treatment, indicating their critical roles in suppressing HOXC6 gene expression under nonactivated conditions. Knockdown of either ERα or ERß abolished E2-dependent recruitment of MLL2 and MLL3 into the HOXC6 promoter, demonstrating key roles of ERs in the recruitment of these mixed lineage leukemias into the HOXC6 promoter. Overall, our studies demonstrated that HOXC6 is an E2-responsive gene, and that histone methylases MLL2 and MLL3, in coordination with ERα and ERß, transcriptionally regulate HOXC6 in an E2-dependent manner.