Proteins that promote epithelial to mesenchymal transition (EMT) are associated with cancer metastasis. Inhibition of EMT regulators may be a promising approach in cancer therapy. In this study, Thymoquinone (TQ) was used to treat cancer cell lines to investigate its effects on EMT-regulatory proteins and cancer metastasis. We show that TQ inhibited cancer cell growth, migration and invasion in a dose-dependent manner. At the molecular level, TQ treatment decreased the transcriptional activity of the TWIST1 promoter and the mRNA expression of TWIST1, an EMT-promoting transcription factor. Accordingly, TQ treatment also decreased the expression of TWIST1-upregulated genes such as N-Cadherin and increased the expression of TWIST1-repressed genes such as E-Cadherin, resulting in a reduction of cell migration and invasion. TQ treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mice but partially attenuated the migration and invasion in TWIST1-overexpressed cell lines. Furthermore, we found that TQ treatment enhanced the promoter DNA methylation of the TWIST1 gene in BT 549 cells. Together, these results demonstrate that TQ treatment inhibits TWIST1 promoter activity and decreases its expression, leading to the inhibition of cancer cell migration, invasion and metastasis. These findings suggest TQ as a potential small molecular inhibitor of cancer growth and metastasis.

Nuclear receptor coactivator 1 (NCOA1) is overexpressed in a subset of breast cancer and its increased expression positively correlates with disease recurrence and metastasis. Although NCOA1 is known to promote breast cancer metastasis through working with multiple transcription factors to upregulate the expression of Twist1, ITGA5, CSF-1, SDF1 and CXCR4, the role of NCOA1 in breast tumor angiogenesis has not been investigated. In this study, we found that the microvascular density (MVD) was significantly decreased and increased in Ncoa1-knockout and NCOA1-overexpressing mammary tumors, respectively, in several breast cancer mouse models. Knockout or knockdown of NCOA1 in breast cancer cell lines also markedly compromised their capability to induce angiogenesis in Matrigel plugs embedded subcutaneously in mice, while this compromised capability could be rescued by VEGFa treatment. At the molecular level, NCOA1 upregulates VEGFa expression in both mouse mammary tumors and cultured breast cancer cells, and it does so by associating with both c-Fos, which is recruited to the AP-1 site at bp -938 of the VEGFa promoter, and HIF1α, which is recruited to the HIF1α-binding element at bp -979 of the VEGFa promoter, to enhance VEGFa transcription. In 140 human breast tumors, high NCOA1 protein correlates with high MVD and patients with both high NCOA1 and high MVD showed significantly shorter survival time. In summary, this study revealed a novel mechanism that NCOA1 potentiates breast cancer angiogenesis through upregulating HIF1α and AP-1-mediated VEGFa expression, which reinforces the rational of targeting NCOA1 in controlling breast cancer progression and metastasis.

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.

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.

Cisplatin-based concurrent chemoradiotherapy for patients with unresectable, locally advanced esophageal squamous cell carcinoma (ESCC) is associated with significant toxicities that are often intolerable. Prognosis for this subgroup of patients remains poor, and new therapeutic approaches are urgently needed. We investigated the efficacy and safety of paclitaxel combined with erlotinib and concurrent radiotherapy in patients with inoperable ESCC. Erlotinib (150 mg) was administered daily for 60 days beginning at the start of radiotherapy, and paclitaxel (45 mg/m²) was administered weekly along with intensity modulated conformal radiotherapy (60 Gy in 30 fractions). The median follow-up time was 21 months. The associations between EGFR and VEGF expression and treatment outcome were evaluated. Among the 21 patients treated, the overall response rate (CR + PR) was 85.6%. The median LPFS, PFS and OS were: 17.5, 14.3, and 22.9 months, respectively. Treatment-related grade 3 toxicities included esophagitis (two patients) and hypoleukemia (one patient). Grade 4 pulmonary toxicity was observed in one patient. Patients expressing EGFR had longer PFS, while those expressing VEGF or with a history of smoking had worse outcomes. Weekly paclitaxel combined with erlotinib and concurrent radiotherapy shows promise as an effective, tolerated regimen for patients with inoperable ESCC.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that commonly arises through neuroendocrine differentiation (NED) of prostate adenocarcinoma (PAC) after therapy, such as radiation therapy and androgen deprivation treatment (ADT). No effective therapeutic is available for NEPC and its molecular mechanisms remain poorly understood. We have reported that G protein-coupled receptor kinase 3 (GRK3, also called ADRBK2) promotes prostate cancer progression. In this study, we demonstrate that the ADT-activated cAMP response element binding protein (CREB) directly targets and induces GRK3. We show GRK3 expression is higher in NEPC than in PAC cells and mouse models, and it positively correlates with the expression and activity of CREB in human prostate cancers. Notably, overexpression of GRK3 in PAC cells increased the expression of NE markers in a kinase activity dependent manner. Conversely, silencing GRK3 blocked CREB-induced NED in PAC cells, reversed NE phenotypes and inhibited proliferation of NEPC cells. Taken together, these results indicate that GRK3 is a new critical activator of NE phenotypes and mediator of CREB activation in promoting NED of prostate cancer cells.