The nuclear receptor coactivator-6 (NCOA6, AIB3, PRIP, ASC-2, TRBP, RAP250 or NRC) is a co-activator for nuclear hormone receptors and certain other transcription factors. NCOA6 plays an important role in embryonic development, adipocyte differentiation, metabolism and breast carcinogenesis. The human and mouse NCOA6 genes had 15 and 14 previously identified exons, respectively. This study further identified an alternatively spliced exon 11b (E11b) in human or E10b in mouse, which codes a short polypeptide and a Stop codon, resulting in splicing variants lacking the last four exon-coded polypeptide. Analyses of mouse testis NCOA6 mRNAs identified four alternatively spliced variants, NCOA6-α (without E10b), -β (without E10a and E10b), -γ (with E10a and E10b) and -δ (without E10a but with E10b). These isoforms were detected in multiple mouse tissues and in MDA-MB-435 human cells. NCOA6-α and -β are mainly located in the nucleus; NCOA6-γ is located in both cytoplasm and nucleus; and NCOA6-δ is mainly located in mitochondria. The C-terminus coded by the last four exons was responsible for locating NCOA6-α and -β into the nucleus. The human E11a or mouse E10a-coded region is responsible for distributing NCOA6-γ in both cytoplasm and nucleus, while the region coded by E8-E9 in human or E7-E8 in mouse is responsible for directing NCOA6-δ to mitochondria. Our assays also demonstrated that NCOA6-α and -β could significantly enhance estrogen receptor α-mediated transcription, but NCOA6-γ and -δ were unable to do so. These results suggest that the diverse physiological function of NCOA6 may be mediated by multiple isoforms expressed in different tissues and localized in different subcellular compartments.

Kdm3b is a JmjC domain-containing histone H3 (H3) demethylase and its physiological functions are largely unknown. In this study, we found that Kdm3b protein is highly expressed in multiple cell types in the mouse testes, including Leydig cells, Sertoli cells, spermatogonia and spermatocytes at different differentiation stages. We also observed Kdm3b protein in the epithelial cells of the caput epididymis, prostate and seminal vesicle. Breeding tests revealed that the number of pups produced by the breeding pairs with Kdm3b knockout (Kdm3bKO) males and wild type (WT) females was reduced 68% because of the decreased number of litters when compared with the breeding pairs with WT males and females. Further analysis demonstrated that Kdm3bKO male mice produced 44% fewer number of mature sperm in their cauda epididymides, displaying significantly reduced sperm motility. No significant differences in the circulating concentration of testosterone and the expression levels of androgen receptor and its representative target genes in the testis were observed. However, the circulating levels of 17β-estradiol, a modulator of sperm maturation and male sexual behaviors, was markedly reduced in Kdm3bKO male mice. Strikingly, abrogation of Kdm3b in male mice significantly increased the latencies to mount, intromit and ejaculate and decreased the number of mounts and intromissions, largely due to their loss of interest in female odors. These findings indicate that Kdm3b is required for normal spermatogenesis and sexual behaviors in male mice.

FGFR1 is a receptor tyrosine kinase deregulated in certain breast cancers (BCs) with a poor prognosis. Although FGFR1-activated phosphorylation cascades have been mapped, the key genes regulated by FGFR1 in BC are largely unclear. FOXQ1 is an oncogenic transcription factor. Although we found that activation of FGFR1 robustly upregulated FOXQ1 mRNA, how FGFR1 regulates FOXQ1 gene expression and whether FOXQ1 is essential for FGFR1-stimulated cell proliferation are unknown. Herein, we confirmed that activation of FGFR1 robustly upregulated FOXQ1 mRNA and protein in BC cells. Knockdown of FOXQ1 blocked the FGFR1 signaling-stimulated BC cell proliferation, colony formation, and xenograft tumor growth. Inhibition of MEK or ERK1/2 activities, or knockout of ERK2 but not ERK1 suppressed the FGFR1 signaling-promoted FOXQ1 gene expression. Inhibition of ERK2 in ERK1 knockout cells blocked, while ectopic expression of FOXQ1 in ERK2 knockout cells rescued the FGFR1-signaling-promoted cell growth. Mechanistically, c-FOS, an early response transcription factor upregulated by the FGFR1-MEK-ERK2 pathway, bound to the FOXQ1 promoter to mediate the FGFR1 signaling-promoted FOXQ1 expression. These results indicate that the FGFR1-ERK2-c-FOS-FOXQ1 regulatory axis plays an essential role in the FGFR1 signaling-promoted BC growth. Targeting ERK2 and FOXQ1 should block BC growth caused by a deregulated FGFR1 signaling.

Kdm3b is a Jumonji C domain-containing protein that demethylates mono- and di-methylated lysine 9 of histone H3 (H3K9me1 and H3K9me2). Although the enzyme activity of Kdm3b is well characterized in vitro, its genetic and physiological function remains unknown. Herein, we generated Kdm3b knockout (Kdm3bKO) mice and observed restricted postnatal growth and female infertility in these mice. We found that Kdm3b ablation decreased IGFBP-3 expressed in the kidney by 53% and significantly reduced IGFBP-3 in the blood, which caused an accelerated degradation of IGF-1 and a 36% decrease in circulating IGF-1 concentration. We also found Kdm3b was highly expressed in the female reproductive organs including ovary, oviduct and uterus. Knockout of Kdm3b in female mice caused irregular estrous cycles, decreased 45% of the ovulation capability and 47% of the fertilization rate, and reduced 44% of the uterine decidual response, which were accompanied with a more than 50% decrease in the circulating levels of the 17beta-estradiol. Importantly, these female reproductive phenotypes were associated with significantly increased levels of H3K9me1/2/3 in the ovary and uterus. These results demonstrate that Kdm3b-mediated H3K9 demethylation plays essential roles in maintenance of the circulating IGF-1, postnatal somatic growth, circulating 17beta-estradiol, and female reproductive function.

Neuroendocrine tumor cells (NETCs) are commonly observed in prostate cancer. Their presence is associated with castration resistance, metastasis and poor prognosis. Cellular and molecular mechanisms for NETC initiation and growth are unknown. TRAMP mice develop heterogeneous adenocarcinomas induced by expression of the SV40-T/t oncogene in prostate epithelial cells. Here, we demonstrate prostate tumors in TRAMP mice with a mixed genetic background are characterized mostly by atypical hyperplasia (AH) containing steroid receptor coactiator-3-positive, androgen receptor-positive and synaptophysin-negative (SRC-3+/AR+/Syp-) cells. Few SRC-3+/AR-/Syp+ NETCs are present in their prostates. We generated TRAMP mice in which SRC-3 was specifically ablated in AR+/Syp- prostatic epithelial cells (termed PE3KOT mice). In these animals, we observed a substantial reduction in SRC-3-/AR+/Syp- AH tumor growth. There was a corresponding increase in SRC-3-/AR+/Syp- phyllodes lesions, suggesting SRC-3 knockout can convert aggressive AH tumors with mostly epithelial tumor cells into less aggressive phyllodes lesions with mostly stromal tissue. Surprisingly, PE3KOT mice developed many more SRC-3+/AR-/Syp+ NETCs versus control TRAMP mice, indicating SRC-3 expression was retained in NETCs. In contrast, TRAMP mice with global SRC-3 knockout did not develop any NETC, indicating SRC-3 is required for developing NETC. Analysis of cell-differentiating markers revealed that these NETCs might not be derived from the mature AR-/Syp+ neuroendocrine cells or the AR+/Syp- luminal epithelial tumor cells. Instead, these NETCs might originate from the SV40-T/t-transformed intermediate/progenitor epithelial cells. In summary, SRC-3 is required for both AR+/Syp- AH tumor growth and AR-/Syp+ NETC development, suggesting SRC-3 is a target for inhibiting aggressive prostate cancer containing NETCs.

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.

The androgen and androgen receptor (AR)-regulated gene expression plays important roles in normal prostate and prostate cancer development, and AR transcriptional control of genes is mediated by transcriptional coactivators, including the three members of the steroid receptor coactivator (SRC) family, SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2) and SRC-3 (AIB1, ACTR/RAC3/NCOA3). SRC-1 and SRC-3 are overexpressed in multiple human endocrine cancers and knockdown of either one of them in prostate cancer cell lines impedes cellular proliferation. Knockout of SRC-3 in mice suppresses the progression of spontaneous prostate carcinogenesis. In this study, we investigated SRC-1 contribution to prostate cancer in vivo by deleting the SRC-1 gene in TRAMP mice, which contain the probasin promoter-driven SV40 T/t antigen transgene. In assessing tumor mass of mice at various ages, we found that initiation and progression of prostate cancer induced by SV40 T/t antigens were unaltered in SRC-1(-/-) mice versus WT mice. Primary tumor histology and metastasis to distant lymph nodes were also similar in these mice at all time points assessed. These results demonstrate that the role of SRC-1 in mouse prostate carcinogenesis is nonessential and different from the essential contribution of SRC-3 that is required for prostate cancer progression and metastasis in mice. Interestingly, we observed that during prostate tumorigenesis SRC-1 expression was relatively constant, while SRC-3 expression was significantly elevated. Therefore, the loss of SRC-1 function may be compensated by SRC-3 overexpression during prostate tumorigenesis in SRC-1(-/-) mice.

The steroid receptor coactivator-3 (SRC-3), also known as AIB1, ACTR, p/CIP and NCOA3, is a transcriptional coactivator for nuclear receptors and certain other transcription factors. SRC-3 is widely expressed and plays important physiological functions and pathogenic roles in breast and prostate cancers. SRC-3 knockout (SRC-3(-/-)) mice display genetic background-dependent embryonic lethality and multiple local and systemic abnormalities. Since both the partial lethality and the systemic effects caused by global SRC-3 knockout interfere with downstream investigation of tissue-specific function of SRC-3, we have generated floxed SRC-3 (SRC-3(f/f)) mice with conditional alleles carrying loxP sites in introns 10 and 12 by a gene-targeting strategy. The two SRC-3(f/f) mouse lines (A and B) are indistinguishable from wild type mice. To test if deletion of the floxed exons 11 and 12 for SRC-3 nuclear receptor interaction domains and disruption of its downstream sequence for transcriptional activation domains would inactivate SRC-3 function, SRC-3(f/f) mice were crossbred with EIIa-Cre mice to generate SRC-3(d/d) mice with germ line deletion of the floxed SRC-3 gene. Both lines of SRC-3(d/d) mice exhibited growth retardation and low IGF-I levels, which was similar to that observed in SRC-3(-/-) mice. The line A SRC-3(d/d) mice showed normal viability, while line B SRC-3(d/d) mice showed partial lethality similar to SRC-3-/- mice, probably due to variable distributions of genetic background during breeding. These results demonstrate that the floxed SRC-3 mouse lines have been successfully established. These mice will be useful for investigating the cell type- and developmental stage-specific functions of SRC-3.

Goblet cell loss, which leads to the reduction of mucin secretion, is a hallmark of ulcerative colitis (UC). We previously reported that steroid receptor coactivator 3 (SRC-3), a transcriptional coactivator, contributes to host defense against Citrobacter rodentium by recruiting neutrophils, suggesting a role of SRC-3 in intestine homeostasis. However, the biological role of SRC-3 in UC remains unclear. Here, we showed that SRC-3-/- mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis compared with wild-type mice after oral administration of 2% DSS dissolved in drinking water. After oral administration of 2% DSS, SRC-3-/- mice displayed higher mortality rate, significant body weight loss, and higher clinical symptom scores compared to wild-type mice. SRC-3-/- mice suffered a severe loss of mature colonic goblet cells, leading to more severe histopathology and more proinflammatory cytokine production. Mechanistically, SRC-3-/- mice exhibited a decreased expression of transcription factor KLF4 in the colons, which is responsible for colonic goblet cell differentiation and maturation. At the molecular level, SRC-3 cooperated with c-Fos to promote KLF4 expression at the transcriptional level. These results demonstrate that SRC-3 can ameliorate DSS-induced colitis by inhibiting inflammation and promoting colonic goblet cell differentiation and maturation through enhancing the expression of transcriptional factor KLF4, which is responsible for colonic goblet cell differentiation and maturation.

Steroid receptor coactivator 3 (SRC-3) is a member of the p160 SRC family. This factor can interact with multiple nuclear hormone receptors and transcription factors to regulate the expression of their target genes. Although many physiological roles of SRC-3 have been revealed, its role in atherosclerosis is not clear. In this study, we found that SRC-3-/-ApoE-/- mice have reduced atherosclerotic lesions and necrotic areas in their aortas and aortic roots compared with SRC-3+/+ApoE-/- mice after Western diet (WD) feeding for 12 weeks. RNA-Seq and Western blot analyses of the aorta revealed that SRC-3 was required for maintaining the expression of ICAM-1, which was required for macrophage recruitment and atherosclerosis development. siRNA-mediated knockdown of SRC-3 in endothelial cells significantly reduced WD-induced atherosclerotic plaque formation. Additionally, treatment of ApoE-/- mice with SRC-3 inhibitor bufalin prevented atherosclerotic plaque development. SRC-3 deficiency reduced aortic macrophage recruitment. Accordingly, ICAM-1 expression was markedly decreased in the aortas of SRC-3-/-ApoE-/- mice and ApoE-/- mice with endothelial SRC-3 knockdown mediated by AAV9-shSRC-3 virus. Mechanistically, SRC-3 coactivated NF-κB p65 to increase ICAM-1 transcription in endothelial cells. Collectively, these findings demonstrate that inhibiting SRC-3 ameliorates atherosclerosis development, at least in part through suppressing endothelial activation by decreasing endothelial ICAM-1 expression via reducing NF-κB signaling.

Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.

The prostate epithelium is composed of basal (BC), luminal (LEC), and neuroendocrine (NEC) cells. It is unclear how many subtypes of BCs in the prostate and which subtype of BCs contains the main stem cell niche in the adult prostate. Here we report seven BC subpopulations according to their p63, cytokeratin 14 (K14) and K5 expression patterns, including p63-positive/K14-negative/K5-negative (p63+/K14-/K5-), p63-/K14+/K5-, p63-/K14-/K5+, p63+/K14+/K5-, p63+/K14-/K5+, p63-/K14+/K5+, and p63+/K14+/K5+ BCs. We generated a p63-CreERT2 knock-in mouse line that expresses tamoxifen-inducible Cre activity in the p63-expressing cells, including the prostate BCs. We then crossbred this line with ROSA26R mice, and generated p63-CreERT2×ROSA26R bi-genic mice harboring the Cre-activated β-galactosidase reporter gene. We treated these bi-genic mice with tamoxifen to mark the p63+ BCs at different ages or under different hormonal conditions, and then traced the lineage differentiation of these genetically labeled BCs. We discovered that these p63+ BCs contain self-renewable stem cells in culture and efficiently differentiated into LECs, NECs and BCs in the postnatal, adult and re-generating mouse prostates. Therefore, BC population contains heterogeneous BCs that express different combinations of the p63, K14 and K5 differentiation markers. Because K14+ and K5+ BCs were previously shown to be extremely inefficient to produce LECs in adulthood, we propose that the p63+/K5-/K14- subpopulation of BCs contains most stem-like cells, especially in adult animals.

Noncompaction cardiomyopathy (NCC) is a congenital heart disease that causes ventricular dysfunction and high mortality rate in children. The mechanisms responsible for NCC are still unknown. The steroid receptor coactivator-1 (SRC-1) and SRC-3 are transcriptional coactivators for nuclear hormone receptors and certain other transcription factors that regulate many genes in development and organ function. However, the roles of SRC-1/3 in heart morphogenesis, function and NCC occurrence are unknown. This study aims to examine the spatial and temporal expression patterns of SRC-1/3 in the heart and investigate the specific roles of SRC-1/3 in heart development, function and NCC occurrence. Immunochemical analysis detected SRC-1/3 expressions in the proliferating cardiomyocytes of mouse heart at prenatal and neonatal stages, while these expressions disappeared within two weeks after birth. Through generating and characterizing mouse lines with global or cardiomyocyte-specific knockouts of SRC-1/3, we found ablation of SRC-1/3 in the myocardial lineage resulted in prominent trabeculae, deep intertrabecular recesses and thin ventricular wall and septum. These developmental defects caused a failure of trabecular compaction, decreased internal ventricular dimension, reduced cardiac ejection fraction and output and led to a high rate of postnatal mortality. Collectively, these structural and functional abnormalities closely simulate the phenotype of NCC patients. Further molecular analysis of cardiomyocytes in vivo and in vitro revealed that SRC-1/3 directly up-regulate cyclin E2, cyclin B1 and myocardin to promote cardiomyocyte proliferation and differentiation. In conclusion, SRC-1/3 are required for cardiomyocyte proliferation and differentiation at earlier developmental stages, and their dysfunction causes NCC-like abnormalities in the hearts of newborn and adult mice.