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.
The HMGA2 protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unraveled. In this study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved Caspase-3 was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the upregulation of p53, Bax and cleaved Caspase-9, downregulation of Bcl-2; as well as release of Cytochrome C from the mitochondria. We further demonstrate that the shRNA-mediated Apaf1 silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved Caspase-3 level and a decline of cell death ratio.Our results also reveal that ?H2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the upregulation of cleaved Caspase-2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Furthermore, we found that the Wnt pathway was inhibited after HMGA2 overexpression, with a concurrent downregulation of the IAP factor survivin. Overall, this study unraveled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.
The epithelial-mesenchymal transition (EMT) is one of the main mechanisms contributing to the onset of cancer metastasis, and has proven to be associated with breast cancer progression. SHON is a novel secreted hominoid-specific protein we have previously identified; it is specifically expressed in all human cancer cell lines tested and is oncogenic for human mammary carcinoma cells. Here, we show that ectopic overexpression of SHON in immortalized human mammary epithelial cells is sufficient for cells to acquire the mesenchymal traits, as well as the enhanced cell migration and invasion, along with the epithelial stem cell properties characterized by increased CD44(high) /CD24(low) subpopulation and mammosphere-forming ability. Moreover, we demonstrate that SHON positively activates the autocrine transforming growth factor-? (TGF-?) pathway to contribute to EMT, while SHON itself is induced by TGF-? in mammary epithelial cells. These data are in favor of a SHON-TGF?-SHON-positive feedback loop that regulates EMT program in breast cancer progression. Finally, examination of the human clinic breast cancer specimens reveals that tumor cells may extracellularly release SHON protein to promote the cancerization of surrounding cells. Together, our findings define an important function of SHON in regulation of EMT via TGF-? signaling, which is closely associated with the invasive subtypes of human breast cancer.
The cellulase and xylanase genes of filamentous Trichoderma fungi exist under carbon catabolite repression mediated by the regulator carbon catabolite repressor (CREI). Our objective was to find the role of CREI in a cellulase-hyperproducing mutant of Trichoderma koningii, and address whether enzyme production can be further improved by silencing the cre1 gene. cre1 partially silenced strains were constructed to improve enzyme production in T. koningii YC01, a cellulase-hyperproducing mutant. Silencing of cre1 resulted in derepression of cellulase gene expression in glucose-based cultivation. The cre1 interference strain C313 produced 2.1-, 1.4-, 0.8-, and 0.8-fold higher amounts of filter paper activity, ?-1,4-exoglucanase activity (?-nitrophenyl-?-D-cellobioside as substrate), ?-1,4-endoglucanase activity (sodium carboxymethyl cellulose as substrate), and xylanase activity, respectively, than the control strain, suggesting that silencing of cre1 resulted in enhanced enzyme production capability. In addition, downregulation of cre1 resulted in elevated expression of another regulator of xylanase and cellulase expression, xyr1, indicating that CREI also acted as a repressor of xyr1 transcription in T. koningii under inducing conditions. These results show that RNAi is a feasible method for analyzing the regulatory mechanisms of gene expression and improving xylanase and cellulase productivity in T. koningii.
DNA-damaging agents are able to induce irreversible cell growth arrest and senescence in some types of tumour cells, thus contributing to the static feature of cancer. However, senescent tumour cells may re-enter the cell cycle, leading to tumour relapse. Understanding the mechanisms that control the viability of senescent cells may be critical for tumour suppression. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which contribute to the stability of the senescent state. However, it is unclear whether SAHF formation is universal in tumour cells. We report that the DNA-damaging agents doxorubicin and 7-ethyl-10-hydroxycamptothecin were able to induce the formation of SAHF in some tumour cell types, and this induction was accompanied by activation of the retinoblastoma protein pathway. By contrast, tumour cells in which the retinoblastoma protein pathway could not be activated by doxorubicin or 7-ethyl-10-hydroxycamptothecin failed to form SAHF. In parallel, tumour cells with deficient retinoblastoma protein were also unable to form SAHF. In addition, we show that the mitogen-activated protein kinase p38 pathway was involved in tumour cell SAHF formation in response to doxorubicin and 7-ethyl-10-hydroxycamptothecin. Furthermore, HMG box transcription factor 1 (HBP1), a downstream target of the mitogen-activated protein kinase p38-mediated senescence pathway, was required for SAHF formation. Taken together, the results of the present study highlight the roles of the mitogen-activated protein kinase p38/retinoblastoma protein pathway in tumour cell SAHF formation in response to DNA-damaging agents, and provide new insights into the mechanisms of DNA damage-mediated tumour suppression.
Inactivation of the tumor suppressor p53 and activation of the oncogene Ras are the two most pivotal events in tumor development. However, potential intersection between p53 and Ras activity during an EMT process, which plays a crucial role during malignant tumor progression, remains elusive. Here, we report that increased expression of wild type p53 suppressed H-Ras(V12)-induced EMT phenotypes and restrained stem cell properties, through downregulation of MEK-ERK signaling pathways. In vivo experiments showed that p53 was able to inhibit H-Ras(V12)-induced tumor growth of human mammary epithelial cells. This study elucidates a novel correlation between the tumor suppressor gene p53 and the oncogene Ras in regulating EMT program, and expands the knowledge about the function of p53 in EMT process.
We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.
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-?1-induced EMT. In MCF10A, TGF-?1 upregulated the CDK5 and p35 expression, and CDK5 knockdown inhibited TGF-?1-induced EMT. CDK5 overexpression also exhibited a potential synergy in promoting TGF-?1-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-?1 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.
The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3-end and 5-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%-86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (?nha1 and ?nhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.
Cellular senescence is an irreversible form of cell cycle arrest that provides a barrier to neoplastic transformation. The integrity of the Rb (Retinoblastoma) pathway is necessary for the formation of the senescence-associated heterochromatin foci (SAHF) that offers a molecular basis for the stability of the senescent state. Surprisingly, although high mobility group A2 protein (HMGA2) can promote tumorigenesis and inhibit Rb function in tumor cells, high-level expression of HMGA2 is sufficient to induce SAHF formation in primary cells. It therefore becomes significant to determine whether Rb protein is necessary in HMGA2-induced SAHF formation. In this study, we established the cellular senescence and SAHF assembly WI38 cell model by ectopic expression of HMGA2, in which typical senescent markers were seen, including notable upregulation of p53, p21 and p16, and elevated SA-?-galactosidase staining together with downregulation of E2F target genes. We then showed that the Rb pathway inhibitor E7 protein was able to partly abolish the ability of SAHF formation after HMGA2 expression in WI38 cells, indicating that Rb is a crucial factor for HMGA2-induced SAHF formation. However, Rb depletion did not completely rescue the cell growth arrest induced by HMGA2, suggesting that Rb is not an exclusive pathway for HMGA2-induced senescence in WI38 cells.
To investigate whether enzyme production can be enhanced in the Trichoderma reesei industrial hyperproducer strain RUT C30 by manipulation of cellulase regulation, the positive regulator Xyr1 was constitutively expressed under the control of the strong T. reesei pdc promoter, resulting in significantly enhanced cellulase activity in the transformant during growth on cellulose. In addition, constitutive expression of xyr1 combined with downregulation of the negative regulator encoding gene ace1 further increased cellulase and xylanase activities. Compared with RUT C30, the resulting transformant exhibited 103, 114, and 134 % greater total secreted protein levels, filter paper activity, and CMCase activity, respectively. Surprisingly, strong increases in xyr1 basal expression levels resulted in very high levels of CMCase activity during growth on glucose. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression, and suggest an attractive new single-step approach for increasing total cellulase productivity in T. reesei.
Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. The latest studies revealed that aggressive breast cancer, especially the triple-negative breast cancer (TNBC) subtype was frequently associated with apparent EMT, but the mechanisms are still unclear. NEDD9/HEF1/Cas-L is a member of the Cas protein family and was identified as a metastasis marker in multiple cancer types. In this study, we wished to discern the role of NEDD9 in breast cancer progression and to investigate the molecular mechanism by which NEDD9 regulates EMT and promotes invasion in triple-negative breast cancer. We showed that expression of NEDD9 was frequently upregulated in TNBC cell lines, and in aggressive breast tumors, especially in TNBC subtype. Knockdown of endogenous NEDD9 reduced the migration, invasion and proliferation of TNBC cells. Moreover, ectopic overexpression of NEDD9 in mammary epithelial cells led to a string of events including the trigger of EMT, activation of ERK signaling, increase of several EMT-inducing transcription factors and promotion of their interactions with the E-cadherin promoter. Data presented in this report contribute to the understanding of the mechanisms by which NEDD9 promotes EMT, and provide useful clues to the evaluation of the potential of NEDD9 as a responsive molecular target for TNBC chemotherapy.
We obtained 5 positive novel histone deacetylase inhibitors (HDACIs) from a polyoxometalate (POM) library by using a cell-based screening system targeting the p21 gene promoter. Among them, PAC-320, a new tri-organic-tin-substitute germanotungstate, displayed remarkable extracellular inhibitory activity. Meanwhile, the crystal structure of PAC-320 was characterized by X-ray crystallography. PAC-320 could stably exist under physiological conditions as revealed by UV spectrum, CV and TG. PAC-320 possessed a strong inhibitory effect to intracellular HDAC activity. More significantly, PAC-320 inhibited the growth of a variety of cancer cells, and exhibited remarkable anticancer effect in a hepatocarcinoma H22 cell mice model. This study revealed, for the first time, that the HDAC inhibitory activity is a mechanism by which POMs exert their anticancer effect.
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.
BMP4 (bone morphogenetic protein 4) is a multifunctional cytokine known to exert its biological effects through a variety of signalling pathways. The diverse function of BMP4 appears to be due to multiple pathways activated by BMP4 itself. Our previous studies have demonstrated that BMP4 is able to drive lung cancer cells into a process of premature senescence; however, the signalling pathways, as well their interplays and roles associated with this process, are not well understood. To address these questions, in the present study we investigated the signalling and molecular mechanisms underlying the BMP4-induced senescence, and our data demonstrated that p38 MAPK (mitogen-activated protein kinase) and Smad pathways were necessary for this process. Meanwhile, the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which is required for senescence, was not activated by BMP4 in the lung cancer cell line NCI-H460. We also showed that the BMP4-responsive R-Smads (receptor-regulated Smads), i.e. Smad1 and Smad5, were necessary for the up-regulation of p16(INK)?(a) and p21(WAF)¹(/cip)¹ and for the induction of premature senescence. Furthermore, we found that activation of the p38 MAPK pathway by BMP4 was essential for the full activation of transcription potential of Smad1/5. Overall, the results of the present study implicate a complex co-operation between p38 MAPK and Smad pathways in BMP4-mediated premature senescence.
Both RASSF2A (Ras-associated family 2A) and p300 are implicated in apoptosis. However, little is known about the interrelationship between these two proteins in induction of apoptosis. Here we show that p300 was able to induce late apoptosis through up-regulation of RASSF2A in human gastric cancer cells SGC-7901 (p53-mutant). Our data demonstrated that p300 stimulated RASSF2A expression in 293T cells in cooperation with the transcription factor Sp1. Results of ChIP (chromatin immunoprecipitation) assays revealed that p300 induced histones H3 and H4 hyperacetylation at RASSF2A promoter. Moreover, p300 and Sp1 reciprocally facilitated their binding to RASSF2A promoter. Overall, data arising from this study indicate that Sp1-mediated RASSF2A gene transcription is activated by p300 through histone acetylation, and this activation plays an important role in inducing late apoptosis.
Melanoma differentiation-associated gene/interleukin-24 (mda-7/IL-24) is a cytokine that can activate monocytes and T helper 2 cells. The expression of mda-7/IL-24 gradually fades with the progression of melanoma, and it is undetectable at the metastatic stage. Ectopic expression of mda-7/IL-24 selectively suppresses growth and induces apoptosis in cancer cells with little harm to normal cells. However, the transcriptional regulation of the mda-7/IL-24 gene has not been extensively studied. In this study, we show that the expression of mda-7/IL-24 was upregulated by the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBu), whereas it was downregulated by HDAC4. We also found that the histone acetylation level and the binding of the transcriptional factor Sp1 to the mad-7 promoter were reduced upon HDAC4 treatment. Moreover, the HDAC inhibitor TSA induced histone hyperacetylation and stimulated Sp1 binding to the mda-7/IL-24 promoter, which in turn enhanced the expression of mda-7/IL-24. Therefore, we conclude that histone acetylation modification plays an important role in the regulation of mda-7/IL-24 and that the transcription factor Sp1 participates in this process.
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.
HOXB13 is a homeodomain protein implicated to play a role in growth arrest in AR (androgen receptor)-negative prostate cancer cells. Expression of HOXB13 is restricted to the AR-expressing prostate cells. In this report, we demonstrate that the HDAC inhibitor NaB (sodium butyrate) was able to induce cell growth arrest and to increase HOXB13 expression in AR-negative prostate cancer cells. We also show that both HDAC4 and YY1 participated in the repression of HOXB13 expression through an epigenetic mechanism involving histone acetylation modification. Specifically, co-immunoprecipitation assays revealed that HDAC4 and YY1 formed a complex. The chromatin immunoprecipitation (ChIP) assays verified that HDAC4 was recruited to HOXB13 promoter by YY1. Moreover, promoter truncation and point mutation studies determined that the two proximal YY1 binding sites on the HOXB13 promoter were essential for the recruitments of YY1 and HDAC4. Data presented in this report suggest that YY1 and HDAC4 affected cell growth by repressing transcriptional regulation of HOXB13 through an epigenetic modification of histones.
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 p(16INK4a) 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 beta-galactosidase activity assay and senescence-associated heterochromatin foci assay, and this process could be reversed by RNA interference-mediated p16(INK4a) silencing. We also show that histone deacetylase (HDAC) 3 and HDAC4 inhibited p16(INK4a) promoter activity in a dose-dependent manner. Furthermore, chromatin immunoprecipitation assays verified that HDAC3 was recruited to the p16(INK4a) 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 p16(INK4a) expression through an epigenetic modification of histones.
Mesenchymal stem cells (MSCs) possess self-renewal and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms such as histone modifications could be critical for determining the fate of stem cells. In this study, full human genome promoter microarrays and expression microarrays were used to explore the roles of histone modifications (H3K9Ac and H3K9Me2) upon the induction of MSC osteogenic differentiation. Our results revealed that the enrichment of H3K9Ac was decreased globally at the gene promoters, whereas the number of promoters enriched with H3K9Me2 was increased evidently upon osteogenic induction. By a combined analysis of data from both ChIP-on-chip and expression microarrays, a number of differentially expressed genes regulated by H3K9Ac and/or H3K9Me2 were identified, implicating their roles in several biological events, such as cell cycle withdraw and cytoskeleton reconstruction that were essential to differentiation process. In addition, our results showed that the vitamin D receptor played a trans-repression role via alternations of H3K9Ac and H3K9Me2 upon MSC osteogenic differentiation. Data from this study suggested that gene activation and silencing controlled by changes of H3K9Ac and H3K9Me2, respectively, were crucial to MSC osteogenic differentiation.
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.
Cell senescence, an irreversible cell cycle arrest, reflects a safeguard program that limits the capacity of uncontrolled cell proliferation. Treatment of tumor cells with certain chemotherapeutic agents activates premature senescence to decrease the tumorigenecity. Here we show that sublethal concentrations of adriamycin could induce premature senescence in lung cancer cells. Adriamycin treatment resulted in the up-regulation of BMP4, which is underexpressed in NSCLC (non-small cell lung cancers). Moreover, the BMP4-Smad pathway played a key role in mediating adriamycin-induced senescence. Overexpression of BMP4 was able to induce premature senescence in lung cancer cells and this process required the participation of cyclin/cyclin-dependent kinase (cdk) inhibitors p16(INK4a) and p21(WAF1/cip1). We also show that increases of p16(INK4a) and p21(WAF1/cip1) expression in response to BMP4 were mediated by the Smad signaling pathway. Furthermore, our data revealed that p300 was recruited to P16(INK4a) and P21(WAF1/cip1) promoters by Smad1/5/8 to induce the hyperacetylation of histones H3 and H4 at the promoters. The present study provides useful clues to the evaluation of the potentiality of BMP4 as a responsive molecular target for cancer chemotherapy.
The human pituitary tumor transforming gene (hPTTG) serves as a marker for malignancy grading in several cancers. hPTTG is involved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase (HAT) p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation (ChIP) analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found that treatment of 293T cells with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylation modification in the regulation of hPTTG.
The tumor suppressor p16(INK4A) (p16) blocks the cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein. We describe here a novel aspect of the posttranslational control that has an important functional consequence on p16 protein. We first discovered that the p16 protein was methylated in various cell lineages. We then determined that the arginine 22, 131 and 138 of p16 were the main methylation sites. Western blotting and TUNEL analyses revealed that the p16 protein bearing these point mutations induced a higher apoptosis ratio than wild-type p16 in A549 cells. Furthermore, co-immunoprecipitation assays suggested that decrease of p16 arginine methylation level promoted the association of p16 with CDK4. Additionally, we determined that the protein arginine methyltransferase 6 (PRMT6) was responsible for the p16 arginine methylation. Results from flow cytometric analysis demonstrated that PRMT6 overexpression counteracted the cell cycle arrest at G1 phase induced by wild-type p16 in A549 cells. We also provided evidence that PRMT6 was able to interact with p16, and that the intensity of p16-CDK4 association was reduced upon PRMT6 overexpression. Together, data presented in this report establish that methylation at specific arginine residues of p16 protein by PRMT6 may be critical for the activity of p16.
All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.
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-? pathway to contribute to EMT. SOX4 itself was induced by TGF-? in mammary epithelial cells and was required for TGF-?-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.
Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to induce apoptosis of cancer cells, and a significant number of genes have been identified as potential effectors responsible for HDAC inhibitor-induced apoptosis. However, the mechanistic actions of these HDAC inhibitors in this process remain largely undefined. We here report that the treatment of LNCap prostate cancer cells with HDAC inhibitor trichostatin A (TSA) resulted in downregulation of the Jumonji domain-containing protein 2B (JMJD2B). We also found that the TSA-mediated decrease in survivin expression in LNCap cells was partly attributable to downregulation of JMJD2B expression. This effect was attributable to the promoted degradation of survivin protein through inhibition of Cyclin B1/Cdc2 complex-mediated survivin Thr34 phosphorylation. Consequently, knockdown of JMJD2B enhanced TSA-induced apoptosis by regulating the Cyclin B1-dependent survivin degradation to potentiate the apoptosis pathways.
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