Baiqu Huang

SOX4 induces epithelial-mesenchymal transition and contributes to breast cancer progression

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with breast cancer progression and metastasis. Here, we report that ectopic overexpression of SOX4 in immortalized human mammary epithelial cells is sufficient for acquisition of mesenchymal traits, enhanced cell migration, and invasion, along with epithelial stem cell properties defined by the presence of a CD44(high)/CD24(low) cell subpopulation. SOX4 positively regulated expression of known EMT inducers, also activating the TGF-β pathway to contribute to EMT. SOX4 itself was induced by TGF-β in mammary epithelial cells and was required for TGF-β-induced EMT. Murine xenograft experiments showed that SOX4 cooperated with oncogenic Ras to promote tumorigenesis in vivo. Finally, in clinical specimens of human breast cancer, we found that SOX4 was abnormally overexpressed and correlated with the triple-negative breast cancer subtype (ER(-)/PR(-)/HER2(-)). Our findings define an important function for SOX4 in the progression of breast cancer by orchestrating EMT, and they implicate this gene product as a marker of poor prognosis in this disease.

Lifespan extension and elevated hsp gene expression in Drosophila caused by histone deacetylase inhibitors

SUMMARY The heat shock proteins (Hsps) play a positive role in lifespan determination, and histone acetylation has been shown to be involved in transcription of hsp genes in Drosophila. To further determine if hsp22 and hsp70 expression is correlated with lifespan, and if histone acetylation participates in this process, RNA levels for hsp22 and hsp70 were analyzed throughout the lifespan in the long-lived and short-lived iso-female lines. The results showed that hsp22 and hsp70 RNA levels were higher in long-lived line than in short-lived line and that the long-lived flies responded more rapidly to heat but were more tolerant to high temperature. Moreover, we investigated the influences of histone acetylation modification on longevity and on hsp gene expression by using histone deacetylase (HDAC) inhibitors TSA and BuA. The results demonstrated that both inhibitors were able to extend the lifespan and promote hsp22 and hsp70 expression. However, the optimal concentrations of these inhibitors, and probably the mechanisms of their actions, vary with the genetic background. In addition, we showed that HDAC inhibitors caused the hyperacetylation of core histone H3, implicating the involvement of chromatin modulation in hsp gene transcription. These data suggested a close correlation among histone acetylation, hsp gene expression and longevity in D. melanogaster.

Histone Deacetylase Inhibitor Trichostatin A Potentiates Doxorubicin-Induced Apoptosis by Up-regulating PTEN Expression

The tumor suppressor gene PTEN is a major negative regulator of the PI3K/Akt cellular survival pathway. Overexpression of PTEN by adenoviral transfection increases doxorubicin‐induced apoptosis. Whereas doxorubicin‐induced apoptosis can be potentiated by the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), the mechanisms underlying this process remain unclear. The aim of this work was to investigate whether changes in PTEN expression are involved in TSA/doxorubicin‐induced apoptosis.

p300 plays a role in p16 INK4a expression and cell cycle arrest

As a cyclin-dependent kinase inhibitor, p16INK4a plays a key role in cell cycle progression and cellular differentiation, and its expression is frequently altered in human cancers through epigenetically mediated transcriptional silencing. In this report, we demonstrate that p300 was able to induce cell cycle arrest, and this process was reversed by p16INK4a silencing by RNA interference in HeLa cells. We also show that p300 was involved in activation of p16INK4a expression in 293T cells. Specifically, p300 cooperated with Sp1 to stimulate both p16INK4a promoter activity and mRNA expression. Co-immunoprecipitation and mammalian two-hybrid assays revealed that p300 and Sp1 formed a complex through interaction between the Q domain of p300 and the N-terminal domain of Sp1. The chromatin immunoprecipitation assays verified that p300 was recruited to p16INK4a promoter, and the histone acetyltransferase domain of p300 participated in p16INK4a activation through inducing hyperacetylation of histone H4 at p16INK4a gene. These data suggest that p300 plays a critical role in transcriptional regulation of p16INK4a and in cell cycle arrest.

FOXC1, a target of polycomb, inhibits metastasis of breast cancer cells

Polycomb group (PcG) proteins have recently been shown related to cancer development. The PcG protein EZH2 is involved in progression of prostate and breast cancers, and has been identified as a molecular marker in breast cancer. Nevertheless, the molecular mechanism by which PcG proteins regulate cancer progression and malignant metastasis is still unclear. PcG proteins methylate H3K27 in undifferentiated epithelial cells, resulting in the repression of differentiation genes such as HOX. FOXC1 is a member of the Forkhead box transcription factor family, which plays an important role in differentiation, and is involved in eye development. We discovered in this study that the expression of FOXC1 gene was negatively correlated to that of PcG genes, i.e., Bmi1, EZH2, and SUZ12, in MCF-7 and MDA-MB-231 cells. To investigate the regulatory effects of PcG proteins on FOXC1 gene, the two cell lines were transfected with either expression plasmids or siRNA plasmids of Bmi1, EZH2, and SUZ12, and we found that PcGs, especially EZH2, could repress the transcription of FOXC1 gene. Chromatin immunoprecipitation (ChIP) assay showed that histone methylation and acetylation modifications played critical roles in this regulatory process. When FOXC1 was stably transfected into MDA-MB-231 cells, the migration and invasion of the cells were repressed. Moreover, the tumorigenicity and the spontaneous metastatic capability regulated by FOXC1 were determined by using an orthotropic xenograft tumor model of athymic mice with the FOXC1-MDA-MB-231HM and the GFP-MDA-MB-231HM cells, and the results showed that FOXC1 in MDA-MB-231HM cells inhibited migration and invasion in vitro and reduced the pulmonary metastasis in vivo. Data presented in this report contribute to the understanding of the mechanisms by which EZH2 participates in tumor development.

PRMT7 Induces Epithelial-to-Mesenchymal Transition and Promotes Metastasis in Breast Cancer

Epithelial-to-mesenchymal transition (EMT) enables metastasis. E-cadherin loss is a hallmark of EMT, but there remains an incomplete understanding of the epigenetics of this process. The protein arginine methyltransferase PRMT7 functions in various physiologic processes, including mRNA splicing, DNA repair, and neural differentiation, but its possible roles in cancer and metastasis have not been explored. In this report, we show that PRMT7 is expressed at higher levels in breast carcinoma cells and that elevated PRMT7 mediates EMT and metastasis. PRMT7 could inhibit the expression of E-cadherin by binding to its proximal promoter in a manner associated with altered histone methylation, specifically with elevated H4R3me2s and reduced H3K4me3, H3Ac, and H4Ac, which occurred at the E-cadherin promoter upon EMT induction. Moreover, PRMT7 interacted with YY1 and HDAC3 and was essential to link these proteins to the E-cadherin promoter. Silencing PRMT7 restored E-cadherin expression by repressing H4R3me2s and by increasing H3K4me3 and H4Ac, attenuating cell migration and invasion in MDA-MB-231 breast cancer cells. Overall, our results define PRMT7 as an inducer of breast cancer metastasis and present the opportunity for applying PRMT7-targeted therapeutics to treat highly invasive breast cancers.

SOX7, down-regulated in colorectal cancer, induces apoptosis and inhibits proliferation of colorectal cancer cells

The sex-determining region Y-box 7 (Sox7) is a member of high mobility group (HMG) transcription factor family, essential for embryonic development and endoderm differentiation. Deregulation of Wnt signaling pathway is a hallmark of colorectal cancer. Our results showed that the expression level of SOX7 was frequently down-regulated in human colorectal cancer cell lines and in primary colorectal tumor tissues, and the SOX7 silencing was partially due to the aberrant DNA methylation of the gene. Restoration of SOX7 induced colorectal cancer cell apoptosis, inhibited cell proliferation and colony formation. In addition, SOX7 efficiently suppressed beta-catenin-mediated transcriptional activity.

The transcription factor ZBP‐89 suppresses p16 expression through a histone modification mechanism to affect cell senescence

The transcription factor ZBP‐89 has been implicated in the induction of growth arrest and apoptosis. In this article, we demonstrate that ZBP‐89 was able to restrain senescence in NCI‐H460 human lung cancer cells, through epigenetically regulating p16INK4a expression. Specifically, our results indicate that knockdown of ZBP‐89 by RNA interference stimulated cellular senescence in NCI‐H460 cells, as judged by the senescence‐associated β‐galactosidase activity assay and senescence‐associated heterochromatin foci assay, and this process could be reversed by RNA interference‐mediated p16INK4a silencing. We also show that histone deacetylase (HDAC) 3 and HDAC4 inhibited p16INK4a promoter activity in a dose‐dependent manner. Furthermore, chromatin immunoprecipitation assays verified that HDAC3 was recruited to the p16INK4a promoter by ZBP‐89 through an epigenetic mechanism involving histone acetylation modification. Moreover, immunofluorescence and coimmunoprecipitation assays revealed that ZBP‐89 and HDAC3 formed a complex. These data suggest that ZBP‐89 and HDAC3, but not HDAC4, can work coordinately to restrain cell senescence by downregulating p16INK4a expression through an epigenetic modification of histones.

CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression.

Epithelial-mesenchymal transition is a change of cellular plasticity critical for embryonic development and tumor metastasis. CDK5 is a proline-directed serine/threonine kinase playing important roles in cancer progression. Here we show that CDK5 is commonly overexpressed and significantly correlated with several poor prognostic parameters of breast cancer. We found that CDK5 participated in TGF-β1-induced EMT. In MCF10A, TGF-β1 upregulated the CDK5 and p35 expression, and CDK5 knockdown inhibited TGF-β1-induced EMT. CDK5 overexpression also exhibited a potential synergy in promoting TGF-β1-induced EMT. In mesenchymal breast cancer cells MDA-MB-231 and BT549, CDK5 knockdown suppressed cell motility and tumorigenesis. We further demonstrated that CDK5 modulated cancer cell migration and tumor formation by regulating the phosphorylation of FAK at Ser-732. Therefore, CDK5-FAK pathway, as a downstream step of TGF-β1 signaling, is essential for EMT and motility in breast cancer cells. This study implicates the potential value of CDK5 as a molecular marker for breast cancer.

Vitamin D stimulates apoptosis in gastric cancer cells in synergy with trichostatin A/sodium butyrate-induced and 5-aza-2′-deoxycytidine-induced PTEN upregulation.

Previous studies have shown an anticancer effect of vitamin D, but the mechanisms underlying this action have not been fully explored. Here we show that 1,25‐dihydroxyvitamin D3 (VD3, the active form of vitamin D) significantly promoted apoptosis in the undifferentiated gastric cancer cell line HGC‐27, and this was accompanied by a concurrent increase in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on VD3 treatment. In contrast, knockdown of PTEN expression by stable transfection of PTEN small interfering RNA greatly decreased the apoptosis rate. We further demonstrated that VD3 induced PTEN expression through vitamin D receptor. In addition, our evidence showed that vitamin D receptor, Egr‐1 and p300 induced PTEN expression in a synergistic fashion. Furthermore, we found that the histone deacetylase inhibitors trichostatin A and sodium butyrate and the methylation inhibitor 5‐aza‐2′‐deoxycytidine played important roles in vitamin D‐induced apoptosis through PTEN upregulation. The data presented in this article suggest potential benefits of vitamin D in gastric cancer therapies in association with the use of trichostatin A/sodium butyrate and 5‐aza‐2′‐deoxycytidine.