CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides, and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE prostate cancer (NEPC) tumors and cell lines compared to normal prostate epithelia, and that its expression is independent of androgen regulation. Knockdown of CYB561 in androgen-deprived LNCaP cells dampened NE differentiation potential and transdifferentiation-induced increase in iron levels. In NEPC PC-3 cells, depletion of CYB561 reduced the secretion of growth-promoting factors, lowered intracellular ferrous iron concentration, and mitigated the highly aggressive nature of these cells in complementary assays for cancer hallmarks. These findings demonstrate the role of CYB561 in facilitating transdifferentiation and maintenance of NE phenotype in CRPC through its involvement in neuropeptide biosynthesis and iron metabolism pathways.

Krüppel-like factor 9 (KLF9) links hormone dysregulation and circadian disruption to breast cancer pathogenesis.

BACKGROUND: Circadian disruption is an emerging driver of breast cancer (BCa), with epidemiological studies linking shift work and chronic jet lag to increased BCa risk. Indeed, several clock genes participate in the gating of mitotic entry, regulation of DNA damage response, and epithelial-to-mesenchymal transition, thus impacting BCa etiology. Dysregulated estrogen (17ß-estradiol, E2) and glucocorticoid (GC) signaling prevalent in BCa may further contribute to clock desynchrony by directly regulating the expression and cycling dynamics of genes comprising the local breast oscillator. In this study, we investigated the tumor suppressor gene, Krüppel-like factor 9 (KLF9), as an important point of crosstalk between hormone signaling and the circadian molecular network, and further examine its functional role in BCa. METHODS: Through meta-analysis of publicly available RNA- and ChIP-sequencing datasets from BCa tumor samples and cell lines, and gene expression analysis by RT-qPCR and enhancer- reporter assays, we elucidated the molecular mechanism behind the clock and hormone regulation of KLF9. Lentiviral knockdown and overexpression of KLF9 in three distinct breast epithelial cell lines (MCF10A, MCF7 and MDA-MB-231) was generated to demonstrate the role of KLF9 in orthogonal assays on breast epithelial survival, proliferation, apoptosis, and migration. RESULTS: We determined that KLF9 is a direct GC receptor target in mammary epithelial cells, and that induction is likely mediated through coordinate transcriptional activation from multiple GC-responsive enhancers in the KLF9 locus. More interestingly, rhythmic expression of KLF9 in MCF10A cells was abolished in the highly aggressive MDA-MB-231 line. In turn, forced expression of KLF9 altered the baseline and GC/E2-responsive expression of several clock genes, indicating that KLF9 may function as a regulator of the core clock machinery. Characterization of the role of KLF9 using complementary cancer hallmark assays in the context of the hormone-circadian axis revealed that KLF9 plays a tumor-suppressive role in BCa regardless of molecular subtype. KLF9 potentiated the anti-tumorigenic effects of GC in E2 receptor + luminal MCF7 cells, while it restrained GC-enhanced oncogenicity in triple-negative MCF10A and MDA-MB-231 cells. CONCLUSIONS: Taken together, our findings support that dysregulation of KLF9 expression and oscillation in BCa impinges on circadian network dynamics, thus ultimately affecting the BCa oncogenic landscape.

Synergistic gene regulation by thyroid hormone and glucocorticoid in the hippocampus.

The hippocampus is considered the center for learning and memory in the brain, and its development and function is greatly affected by the thyroid and stress axes. Thyroid hormone (TH) and glucocorticoids (GC) are known to have a synergistic effect on developmental programs across several vertebrate species, and their effects on hippocampal structure and function are well-documented. However, there are few studies that focus on the processes and genes that are cooperatively regulated by the two hormone axes. Cross-regulation of the thyroid and stress axes in the hippocampus occurs on multiple levels such that TH can regulate the expression of the GC receptor (GR) while GC can modulate tissue sensitivity to TH by controlling the expression of TH receptor (TR) and enzymes involved in TH biosynthesis. Thyroid hormone and GC are also known to synergistically regulate the transcription of genes associated with neuronal function and development. Synergistic gene regulation by TH and GC may occur through the direct, cooperative action of TR and GR on common target genes, or by indirect mechanisms involving gene regulatory cascades activated by TR and GR. In this chapter, we describe the known physiological effects and underlying molecular mechanisms of TH and GC synergistic gene regulation in the hippocampus.

Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer.

Glucocorticoids (GCs; eg, hydrocortisone [CORT]) are routinely used as chemotherapeutic, anti-emetic, and palliative agents in breast cancer (BCa) therapy. The effects of GC signaling on BCa progression, however, remain a contentious topic as GC treatment seems to be beneficial for receptor-positive subtypes but elicits unfavorable responses in triple-negative BCa (TNBC). The mechanistic basis for these conflicting effects of GC in BCa is poorly understood. In this study, we sought to decipher the molecular mechanisms that govern the GC-dependent induction of the tumor suppressor ERRFI1 gene, an inhibitor of epidermal growth factor receptor (EGFR) signaling, and characterize the role of the GC-ERRFI1 regulatory axis in TNBC. Treatment of TNBC cell lines with a protein synthesis inhibitor or GC receptor (GR) antagonist followed by gene expression analysis suggests that ERRFI1 is a direct GR target. Using in silico analysis coupled with enhancer-reporter assays, we identified a putative ERRFI1 enhancer that supports CORT-dependent transactivation. In orthogonal assays for cell proliferation, survival, migration, and apoptosis, CORT mostly facilitated an oncogenic phenotype regardless of malignancy status. Lentiviral knockdown and overexpression of ERRFI1 showed that the CORT-enhanced oncogenic phenotype is restricted by ERRFI1 in the normal breast epithelial model MCF10A and to a lesser degree in the metastatic TNBC line MDA-MB-468. Conversely, ERRFI1 conferred pro-tumorigenic effects in the highly metastatic TNBC model MDA-MB-231. Taken together, our findings suggest that the progressive loss of the GC-dependent regulation and anti-tumorigenic function of ERRFI1 influences BCa progression and may contribute to the unfavorable effects of GC therapy in TNBC.

Coordinated transcriptional regulation by thyroid hormone and glucocorticoid interaction in adult mouse hippocampus-derived neuronal cells.

The hippocampus is a well-known target of thyroid hormone (TH; e.g., 3,5,3'-triiodothyronine-T3) and glucocorticoid (GC; e.g., corticosterone-CORT) action. Despite evidence that TH and GC play critical roles in neural development and function, few studies have identified genes and patterns of gene regulation influenced by the interaction of these hormones at a genome-wide scale. In this study we investigated gene regulation by T3, CORT, and T3 + CORT in the mouse hippocampus-derived cell line HT-22. We treated cells with T3, CORT, or T3 + CORT for 4 hr before cell harvest and RNA isolation for microarray analysis. We identified 9 genes regulated by T3, 432 genes by CORT, and 412 genes by T3 + CORT. Among the 432 CORT-regulated genes, there were 203 genes that exhibited an altered CORT response in the presence of T3, suggesting that T3 plays a significant role in modulating CORT-regulated genes. We also found 80 genes synergistically induced, and 73 genes synergistically repressed by T3 + CORT treatment. We performed in silico analysis using publicly available mouse neuronal chromatin immunoprecipitation-sequencing datasets and identified a considerable number of synergistically regulated genes with TH receptor and GC receptor peaks mapping within 1 kb of chromatin marks indicative of hormone-responsive enhancer regions. Functional annotation clustering of synergistically regulated genes reveal the relevance of proteasomal-dependent degradation, neuroprotective effect of growth hormones, and neuroinflammatory responses as key pathways to how TH and GC may coordinately influence learning and memory. Taken together, our transcriptome data represents a promising exploratory dataset for further study of common molecular mechanisms behind synergistic TH and GC gene regulation, and identify specific genes and their role in processes mediated by cross-talk between the thyroid and stress axes in a mammalian hippocampal model system.

Deciphering the regulatory logic of an ancient, ultraconserved nuclear receptor enhancer module.

Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5-6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection.

Embryonic lethality in Dear gene-deficient mice: new player in angiogenesis.

The dual endothelin-1/angiotensin II receptor (Dear) binds endothelin-1 (ET-1) and angiotensin II (ANG II) with equal affinities in the Dahl S/JRHS rat strain. To elucidate its physiological significance within the context of multiple receptor isoforms and diverse ET-1 and ANG II functions spanning blood pressure regulation, tumor proliferation, and angiogenesis, we characterized mouse Dear and Dear-deficient mice. Unlike null mutant models of ET-1, ANG II, and all other ET-1 and ANG II receptors, Dear(-/-) deficiency results in impaired angiogenesis, dysregulated neuroepithelial development, and embryonic lethality by embryonic day 12.5. Interestingly, mouse Dear does not bind ANG II, similar to Dahl R/JRHS rat Dear, but binds ET-1 and vascular endothelial growth factor (VEGF) signal peptide (VEGFsp) with equal affinities, suggesting a putative novel multifunction for VEGFsp and a parsimonious mechanism for coordination of VEGF-induced and Dear-mediated pathways. Consistent with its developmental angiogenic role, Dear inhibition results in decreased tumor growth in B16-F10 melanoma cell-induced subcutaneous tumor in female Dear(+/-)/C57BL6BC10 mice, but not in males (age 3.5 mo), and in 127Cs radiation-induced orthotopic mammary tumors in Sprague-Dawley female rats (age range 3-6.5 mo). Altogether, the data identify Dear as a new player in angiogenesis during development downstream to, and nonredundant with, VEGF-mediated pathways, as well as a putative modulator of tumor angiogenesis acting within a gender-specific paradigm.