Yixiang Xu

SRC-1 and Twist1 Expression Positively Correlates with a Poor Prognosis in Human Breast Cancer

To evaluate the possible prognostic value of Steroid Receptor Coactivator-1 (SRC-1) and Twist1 expression in human breast cancer, we examined SRC-1 and Twist1 expression using immunohistochemistry on tissue microarray sections containing 137 breast cancer specimens. All patients were followed up for a median of 5 years following surgery. Survival curves were generated using the Kaplan-Meier method. Multivariate analysis was performed using the Cox proportional hazard regression model to assess the prognostic values. The results showed a positive correlation between SRC-1 and Twist1 expression at protein levels (P < 0.001). Also, SRC-1 expression positively correlated with HER2 expression (P = 0.024). The protein expression of Twist1 positively associated with lymph node metastasis (P < 0.001), but inversely correlated with PR status (P = 0.041). Patients with SRC-1 or Twist1-positive expression exhibited poorer overall survival (OS) and disease-free survival (DFS) than did those with SRC-1 or Twist1-negative expression (P < 0.05 for all). In addition, SRC-1-negativeive/Twist1-negative patients had the best OS and DFS (P < 0.01 for both). In multivariate survival analysis, SRC-1 expression, tumor stage, and PR were found to be independent prognostic factors related to OS (P = 0.019, < 0.001 and 0.02, respectively) and Twist1 expression, lymph node status and PR were independent predictors of DFS (P = 0.006, 0.001 and 0.029, respectively). These results suggest that a combined SRC-1/Twist1 expression status could improve the prognostic judgment for breast cancer patients.

The Steroid Receptor Coactivator-3 Is Required for the Development of Castration-Resistant Prostate Cancer

The transcriptional coactivator SRC-3 plays a key role in enhancing prostate cancer cell proliferation. Although SRC-3 is highly expressed in advanced prostate cancer, its role in castration-resistant prostate cancer (CRPC) driven by PTEN mutation is unknown. We documented elevated SRC-3 in human CRPC and in PTEN-negative human prostate cancer. Patients with high SRC-3 and undetectable PTEN exhibited decreased recurrence-free survival. To explore the causal relationship in these observations, we generated mice in which both Pten and SRC-3 were inactivated in prostate epithelial cells (Pten3CKO mice), comparing them with mice in which only Pten was inactivated in these cells (PtenCKO mice). SRC-3 deletion impaired cellular proliferation and reduced tumor size. Notably, while castration of PtenCKO control mice increased the aggressiveness of prostate tumors relative to noncastrated counterparts, deletion of SRC-3 in Pten3CKO mice reversed all these changes. In support of this finding, castrated Pten3CKO mice also exhibited decreased levels of phospho-Akt, S6 kinase (RPS6KB1), and phosphorylated S6 protein (RPS6), all of which mediate cell growth and proliferation. Moreover, these tumors appeared to be more differentiated as evidenced by higher levels of Fkbp5, an AR-responsive gene that inhibits Akt signaling. Lastly, these tumors also displayed lower levels of certain androgen-repressed genes such as cyclin E2 and MMP10. Together, our results show that SRC-3 drives CRPC formation and offer preclinical proof of concept for a transcriptional coactivator as a therapeutic target to abrogate CRPC progression.

NCOA1 Directly Targets M-CSF1 Expression to Promote Breast Cancer Metastasis

In breast cancer, overexpression of the nuclear coactivator NCOA1 (SRC-1) is associated with disease recurrence and resistance to endocrine therapy. To examine the impact of NCOA1 overexpression on morphogenesis and carcinogenesis in the mammary gland (MG), we generated MMTV-hNCOA1 transgenic [Tg(NCOA1)] mice. In the context of two distinct transgenic models of breast cancer, NCOA1 overexpression did not affect the morphology or tumor-forming capability of MG epithelial cells. However, NCOA1 overexpression increased the number of circulating breast cancer cells and the efficiency of lung metastasis. Mechanistic investigations showed that NCOA1 and c-Fos were recruited to a functional AP-1 site in the macrophage attractant CSF1 promoter, directly upregulating colony-simulating factor 1 (CSF1) expression to enhance macrophage recruitment and metastasis. Conversely, silencing NCOA1 reduced CSF1 expression and decreased macrophage recruitment and breast cancer cell metastasis. In a cohort of 453 human breast tumors, NCOA1 and CSF1 levels correlated positively with disease recurrence, higher tumor grade, and poor prognosis. Together, our results define an NCOA1/AP-1/CSF1 regulatory axis that promotes breast cancer metastasis, offering a novel therapeutic target for impeding this process.

Inducible knockout of Twist1 in young and adult mice prolongs hair growth cycle and has mild effects on general health, supporting Twist1 as a preferential cancer target.

Twist1 promotes epithelial-mesenchymal transition, invasion, metastasis, stemness, and chemotherapy resistance in cancer cells and thus is a potential target for cancer therapy. However, Twist1-null mice are embryonic lethal, and people with one Twist1 germline mutant allele develop Saethre-Chotzen syndrome; it is questionable whether Twist1 can be targeted in patients without severe adverse effects. We found that Twist1 is expressed in several tissues, including fibroblasts of the mammary glands and dermal papilla cells of the hair follicles. We developed a tamoxifen-inducible Twist1 knockout mouse model; Twist1 knockout in 6-week-old female mice did not affect mammary gland morphogenesis and function during pregnancy and lactation. In both males and females, the knockout did not influence body weight gain, heart rate, or total lean and fat components. The knockout also did not alter blood pressure in males, although it slightly reduced blood pressure in females. Although Twist1 is not cyclically expressed in dermal papilla cells, knockout of Twist1 at postnatal day 13 (when hair follicles have developed) drastically extended the anagen phase and accelerated hair growth. These results indicate that Twist1 is not essential for maintaining an overall healthy condition in young and adult mice and that loss of function facilitates hair growth in adulthood, supporting Twist1 as a preferential target for cancer therapy.

Tamoxifen inhibits ER-negative breast cancer cell invasion and metastasis by accelerating Twist1 degradation.

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.

Breast tumor cell-specific knockout of Twist1 inhibits cancer cell plasticity, dissemination, and lung metastasis in mice

Significance We found that Twist1 is coexpressed with multiple other epithelial–mesenchymal transition (EMT)-inducing transcription factors (TFs) in small subsets of primary and circulating breast tumor cells. Together, they program a partial EMT and a basal-like phenotype of tumor cells, which most likely are responsible for disseminating into the circulation and metastasizing to the lung in mice. Targeting Twist1 largely diminishes all the cellular features and phenotypes of breast tumor cells, indicating Twist1 may be a target for inhibiting breast cancer metastasis. These findings are in favor of the role of partial EMT in breast cancer progression toward the metastatic state. Twist1 is an epithelial–mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) Twist1 or tumor cell-specific Twist1 knockout (Twist1TKO). Twist1 knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in ∼6% of the advanced WT tumor cells. Most of these Twist1+ cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ERα and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin+/vimentin+). In advanced Twist1TKO tumor cells, Twist1 knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1TKO tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1TKO tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.

Nuclear receptor NR4A1 is a tumor suppressor down-regulated in triple-negative breast cancer

The nuclear receptor (NR) superfamily contains hormone-inducible transcription factors that regulate many physiological and pathological processes through regulating gene expression. NR4A1 is an NR family member that still does not have an identified endogenous ligand, and its role in cancer is also currently unclear and controversial. In this study, we aimed to define the expression profiles and specific role of NR4A1 in the highly malignant triple-negative breast cancer (TNBC), which still lacks available targeted therapies. Bioinformatic analysis revealed a decrease of NR4A1 mRNA expression in human TNBC samples. Semi-quantitative analysis of NR4A1 protein expression by immunohistochemistry also identified a progressive NR4A1 reduction during the development of mouse basal-like mammary tumors and a significant NR4A1 downregulation in human TNBC samples. Furthermore, the expression levels of NR4A1 in human TNBC were negatively associated with tumor stage, lymph node metastasis and disease recurrence. Moreover, ectopic expression of NR4A1 in MDA-MB-231, a TNBC cell line with little endogenous NR4A1, inhibited the proliferation, viability, migration and invasion of these cells, and these inhibitions were associated with an attenuated JNK1-AP-1-cyclin D1 pathway. NR4A1 expression also largely suppressed the growth and metastasis of these cell-derived tumors in mice. These results demonstrate that NR4A1 is downregulated in TNBC and restoration of NR4A1 expression inhibits TNBC growth and metastasis, suggesting that NR4A1 is a tumor suppressor in TNBC.The nuclear receptor (NR) superfamily contains hormone-inducible transcription factors that regulate many physiological and pathological processes through regulating gene expression. NR4A1 is an NR family member that still does not have an identified endogenous ligand, and its role in cancer is also currently unclear and controversial. In this study, we aimed to define the expression profiles and specific role of NR4A1 in the highly malignant triple-negative breast cancer (TNBC), which still lacks available targeted therapies. Bioinformatic analysis revealed a decrease of NR4A1 mRNA expression in human TNBC samples. Semi-quantitative analysis of NR4A1 protein expression by immunohistochemistry also identified a progressive NR4A1 reduction during the development of mouse basal-like mammary tumors and a significant NR4A1 downregulation in human TNBC samples. Furthermore, the expression levels of NR4A1 in human TNBC were negatively associated with tumor stage, lymph node metastasis and disease recurrence. Moreover, ectopic expression of NR4A1 in MDA-MB-231, a TNBC cell line with little endogenous NR4A1, inhibited the proliferation, viability, migration and invasion of these cells, and these inhibitions were associated with an attenuated JNK1–AP-1–cyclin D1 pathway. NR4A1 expression also largely suppressed the growth and metastasis of these cell-derived tumors in mice. These results demonstrate that NR4A1 is downregulated in TNBC and restoration of NR4A1 expression inhibits TNBC growth and metastasis, suggesting that NR4A1 is a tumor suppressor in TNBC.

The histone demethylase Kdm3a is required for normal epithelial proliferation, ductal elongation and tumor growth in the mouse mammary gland

Histone modification alters chromatin architecture to regulate gene transcription. KDM3A is a histone demethylase in the JmjC domain-containing protein family. It removes di- and mono- methyl residues from di- or mono-methylated lysine 9 of histone H3 (H3K9me2/me1). Recent studies have shown that Kdm3a plays an important role in self-renewal of embryonic stem cells, spermatogenesis, metabolism, sex determination and tumor angiogenesis. However, its role in mammary gland development and breast carcinogenesis remains unclear. In this study, we found that Kdm3a is expressed in the mouse mammary gland epithelial cells. Knockout of Kdm3a significantly increased H3K9me2/me1 levels in these epithelial cells, which correlated with markedly decreased mammary gland ductal elongation and branching in the intact knockout virgin mice. Furthermore, estrogen replacement in the ovariectomized Kdm3a knockout mice couldn’t rescue the retarded ductal growth. Moreover, transplantation of KO mammary gland pieces to wild type recipient mice showed slower ductal growth compared with that of WT gland pieces. Consistently, knockout of Kdm3a also reduced the proliferation rates and cyclin D1 expression in the mammary gland epithelial cells. In addition, Kdm3a knockout did not significantly change the latency of the polyoma middle T oncogene-induced mammary gland tumorigenesis. Tumor growth, however, was slowed which might be due to the decrease in cyclin D1 expression and tumor cell proliferation. We also found that Kdm3a binds and activates the cyclin D1 promoter. These results demonstrate that Kdm3a plays an important intrinsic role in promoting mammary gland ductal growth and tumor growth probably through enhancing cyclin D1 expression and cell proliferation.

Abstract 2694: SRC-1 promotes tumor angiogenesis through up-regulation of HIF1α-mediated VEGFa expression.

Steroid receptor coactivator 1 (SRC-1) belongs to the p160 coactivator family which also contains SRC-2 (TIF2) and SRC-3 (AIB1/pCIP/ACTR/RAC3). These coactivators interact with nuclear receptors and other transcription factors to up-regulate gene expression. Accumulated evidence shows that SRC-1 exerts its promotive role in breast cancer progression and metastasis by up-regulating transcription of multiple genes important for tumor cell epithelial-mesenchymal transition (EMT), migration, invasion and endocrine therapy resistance. However, its role in tumor angiogenesis has not been examined. In our recent studies using SRC-1 KO/PyMT and WT/PyMT mammary tumor cell lines established in our lab, we found that injection of SRC-1 KO/PyMT cells to matrigel plug formed under the back skin in SCID mice induced less blood vessel formation compared with that of WT/PyMT tumor cells. Accordingly, injection of human breast cancer MDA-MB-231 cells with stable knockdown of SRC-1 by shRNA to the matrigel plug also induced less neovascularization. Meanwhile, VEGFa mRNA level is significantly reduced in mammary tumors from SRC-1 KO/PyMT transgenic mice. On the contrary, in SRC-1-overexpressed mammary tumors from MMTV/TVA/PyMT mice, the expression of VEGFa was dramatically increased. In agreement with these findings, in vitro studies revealed that VEGFa mRNA levels are much lower in two of the SRC-1 KO/PyMT tumor cell lines compared with WT/PyMT cell lines. Knockdown of SRC-1 in WT/PyMT cells and human MDA-MB-231 cells reduced VEGFa levels, while adenovirus-mediated re-expression of SRC-1 in SRC-1 KO/PyMT cells significantly potentiated VEGFa expression. Furthermore, promoter analyze revealed that SRC-1 could interact with HIF-1α and AP-1 to potentiate the VEGFa promoter activity. We also found that both SRC-1 and HIF-1α are associated with a VEGFa promoter region by ChIP assays. These results indicate that SRC-1 can promote breast cancer progression and metastasis through enhancing tumor angiogenesis. Targeting SRC-1 may be a feasible strategy for inhibiting breast tumor angiogenesis and metastasis. Citation Format: Li Qin, Yixiang Xu, Zhen Feng, Yelin Wu, Yan Xu, Lan Liao, Jianming Xu. SRC-1 promotes tumor angiogenesis through up-regulation of HIF1α-mediated VEGFa expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2013-2694 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1399: SRC-1 up-regulates CSF-1 expression to potentiate breast cancer metastasis

Steroid receptor coactivator-1 (SRC-1/NCOA1) has been reported to be overexpressed in a subset of human breast cancer (BC) and its overexpression correlates with Her2 expression, disease recurrence and endocrine therapy resistance. To determine the role and underlying mechanisms of SRC-1 overexpression in BC progression and metastasis, a transgenic mouse model with overexpression of human SRC-1 in mouse mammary epithelial cells was created. In vivo data revealed that overxpression of SRC-1 potentiated BC metastasis to lung in both MMTV/Neu and MMTV/TVA/PyMT tumor models. While SRC-1 overexpression did not obviously accelerate tumor growth in these BC mouse models, the number of tumor cells invaded into blood vessels and metastasis index in lung tissue significantly increased in these mice. In addition to the elevated Twist expression and activation of Her2 and AKT driven by SRC-1 overexpression in the tumors, CSF-1 level was significantly increased in SRC-1 overexpressed tumors. We also found that CSF-1 expression was decreased in two PyMT/SRC-1 KO tumor cell lines compared with the two PyMT/WT control cell lines. Knockdown of SRC-1 in PyMT/WT cells or human BC cells reduced CSF-1 levels, while adenoviral-mediated expression of SRC-1 in PyMT/SRC-1 KO cells or human BC cells significantly promoted CSF-1 expression. Furthermore SRC-1 was found to interact with transcription factors, c-jun and c-fos, to activate the csf-1 promoter. Both SRC-1 and c-fos were found to associate with the csf-1 promoter at AP-1 binding sites in human BC cells. This study provided compelling evidence that overexpression of SRC-1 promotes BC metastasis. Targeting SRC-1 might be a potential therapeutic strategy for controlling BC metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1399. doi:1538-7445.AM2012-1399