Comparative analysis of the occupancy of Histone H3 Lysine 4 methylation in the cells treated with TGFß and Interferonγ.

In this current study, we have compared our H3K4me3 Chip-Sequencing data in PC3 cells in response to 6 h and 24 h TGFß stimulation with the IFNγ stimulated/unstimulated HeLa S3 cells Since both TGFß and IFNγ play an essential role in tumorigenesis both as a tumor promoter and tumor suppressor and known to antagonize each other's signalling, it would be of utmost importance to find out the regions undergoing histone modification changes in response to TGFß and IFNγ and compare them to explore the genes common to both as well as the specific for each ligand. Our study has compared the genes showing H3K4me3 occupancy in response to both TGFß and IFNγ. Several genes were found to be shared between the TGFß and IFNγ. DAVID Functional enrichment analysis in the TGFß and IFNγ dataset revealed association of genes with different biological processes such as miRNA-mediated gene silencing, positive regulation of ERK cascade, hypoxia-induced apoptosis repression, translational regulation and molecular functions such as TGFßR activity, GPCR activity, TGFß binding activity. Further analysis of these genes can reveal fascinating insights into epigenetic regulation by growth factor stimulation.

GSK-J4: An H3K27 histone demethylase inhibitor, as a potential anti-cancer agent.

Aberrant epigenetic modifications are emerging as potent drivers of tumor initiation and progression. The deregulation of H3K27me3 marks has shown to play an important role in cancer progression in several cancers. The H3K27me3 mark is associated with gene silencing. The reversible nature of these epigenetic aberrations makes them an important target for treating cancer. GSK-J4 is a histone demethylase inhibitor that inhibits the JMJD3/UTX enzyme, which results in the upregulation of H3K27me3 levels. In this review, the anti-cancer properties of GSK-J4 have been summarized, the various molecular pathways targeted, in-vivo studies, and drug combination studies in different cancer models. GSK-J4 targeted pathways like apoptosis, cell cycle, invasion, migration, DNA damage repair, metabolism, oxidative stress, stemness, etc. GSK-J4 is a promising candidate alone and in combination with other conventional anti-cancer drugs against different cancer types.

Combination Treatment of a Phytochemical and a Histone Demethylase Inhibitor-A Novel Approach towards Targeting TGFß-Induced EMT, Invasion, and Migration in Prostate Cancer.

Minimizing side effects, overcoming cancer drug resistance, and preventing metastasis of cancer cells are of growing interest in current cancer therapeutics. Phytochemicals are being researched in depth as they are protective to normal cells and have fewer side effects. Hesperetin is a citrus bioflavonoid known to inhibit TGFß-induced epithelial-to-mesenchymal transition (EMT), migration, and invasion of prostate cancer cells. Targeting epigenetic modifications that cause cancer is another class of upcoming therapeutics, as these changes are reversible. Global H3K27me3 levels have been found to be reduced in invasive prostate adenocarcinomas. Combining a demethylase inhibitor and a known anti-cancer phytochemical is a unique approach to targeting cancer to attain the aforementioned objectives. In the current study, we used an H3K27 demethylase (JMJD3/KDM6B) inhibitor to study its effects on TGFß-induced EMT in prostate cancer cells. We then gave a combined hesperetin and GSK-J4 treatment to the PC-3 and LNCaP cells. There was a dose-dependent increase in cytotoxicity and inhibition of TGFß-induced migration and invasion of prostate cancer cells after GSK-J4 treatment. GSK-J4 not only induced trimethylation of H3K27 but also induced the trimethylation of H3K4. Surprisingly, there was a reduction in the H3K9me3 levels. GSK-J4 alone and a combination of hesperetin and GSK-J4 treatment effectively inhibit the important hallmarks of cancer, such as cell proliferation, migration, and invasion, by altering the epigenetic landscape of cancer cells.

Global Gene Expression Regulation Mediated by TGFß Through H3K9me3 Mark.

Background: Epigenetic alterations play an important part in carcinogenesis. Different biological responses, including cell proliferation, migration, apoptosis, invasion, and senescence, are affected by epigenetic alterations in cancer. In addition, growth factors, such as transforming growth factor beta (TGFß) are important regulators of tumorigenesis. Our understanding of the interplay between the epigenetic bases of tumorigenesis and growth factor signaling in tumorigenesis is rudimentary. Some studies suggest a link between TGFß signaling and the heterochromatinizing histone mark H3K9me3. There is evidence for signal-dependent interactions between R-Smads and histone methyltransferases. However, the effects of TGFß signaling on genome wide H3K9me3 landscape remains unknown. Our research examines TGFß -induced genome-wide H3K9me3 in prostate cancer. Method: Chromatin-Immunoprecipitation followed by sequencing was performed to analyze genome-wide association of H3K9me3 epigenetic mark. DAVID Functional annotation tool was utilized to understand the involvement of different Biological Processes and Molecular Function. MEME-ChIP tool was also used to analyze known and novel DNA-binding motifs. Results: H3K9me3 occupancy appears to increase at intronic regions after short-term (6 hours) TGFß stimulation and at distal intergenic regions during long-term stimulation (24 hours). We also found evidence for a possible association of SLC transporters with H3K9me3 mark in presence of TGFß during tumorigenesis. No direct correlation was found between the occupancy of H3K9me3 mark and the expression of various genes. The epigenetic mechanisms-mediated regulation of gene expression by TGFß was concentrated at promoters rich in SRY and FOXJ3 binding sites. Conclusion: Our results point toward a positive association of oncogenic function of TGFß and the H3K9me3 mark and provide a context to the role of H3K9me3 in TGFß-induced cell migration and cell adhesion. Interestingly, these functions of TGFß through H3K9me3 mark regulation seem to depend on transcriptional activation in contrast to the conventionally known repressive nature of H3K9me3.

Global Histone H3 Lysine 4 Trimethylation (H3K4me3) Landscape Changes in Response to TGFß.

TGFß expression acts as a biomarker of poor prognosis in prostate cancer. It plays a dual functional role in prostate cancer. In the early stages of the tumor, it acts as a tumor suppressor while at the later stages of tumor development, it promotes metastasis. The molecular mechanisms of action of TGFß are largely understood through the canonical and non-canonical signal transduction pathways. Our understanding of the mechanisms that establish transient TGFß stimulation into stable gene expression patterns remains incomplete. Epigenetic marks like histone H3 modifications are directly linked with gene expression and they play an important role in tumorigenesis. In this report, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) to identify the genome-wide regions that undergo changes in histone H3 Lysine 4 trimethylation (H3K4me3) occupancy in response to TGFß stimulation. We also show that TGFß stimulation can induce acute epigenetic changes through the modulation of H3K4me3 signals at genes belonging to special functional categories in prostate cancer. TGFß induces the H3K4me3 on its own ligands like TGFß, GDF1, INHBB, GDF3, GDF6, BMP5 suggesting a positive feedback loop. The majority of genes were found to be involved in the positive regulation of transcription from the RNA polymerase II promoter in response to TGFß. Other functional categories were intracellular protein transport, brain development, EMT, angiogenesis, antigen processing, antigen presentation via MHC class II, lipid transport, embryo development, histone H4 acetylation, positive regulation of cell cycle arrest, and genes involved in mitotic G2 DNA damage checkpoints. Our results link TGFß stimulation to acute changes in gene expression through an epigenetic mechanism. These findings have broader implications on epigenetic bases of acute gene expression changes caused by growth factor stimulation.

Smad7 Enhances TGF-ß-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells.

Transforming growth factor ß (TGF-ß) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-ß to control TGF-ß signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-ß induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.

TGF-ß promotes PI3K-AKT signaling and prostate cancer cell migration through the TRAF6-mediated ubiquitylation of p85α.

Transforming growth factor-ß (TGF-ß) is a pluripotent cytokine that regulates cell fate and plasticity in normal tissues and tumors. The multifunctional cellular responses evoked by TGF-ß are mediated by the canonical SMAD pathway and by noncanonical pathways, including mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3'-kinase (PI3K)-protein kinase B (AKT) pathway. We found that TGF-ß activated PI3K in a manner dependent on the activity of the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). TRAF6 polyubiquitylated the PI3K regulatory subunit p85α and promoted the formation of a complex between the TGF-ß type I receptor (TßRI) and p85α, which led to the activation of PI3K and AKT. Lys63-linked polyubiquitylation of p85α on Lys513 and Lys519 in the iSH2 (inter-Src homology 2) domain was required for TGF-ß-induced activation of PI3K-AKT signaling and cell motility in prostate cancer cells and activated macrophages. Unlike the activation of SMAD pathways, the TRAF6-mediated activation of PI3K and AKT was not dependent on the kinase activity of TßRI. In situ proximity ligation assays revealed that polyubiquitylation of p85α was evident in aggressive prostate cancer tissues. Thus, our data reveal a molecular mechanism by which TGF-ß activates the PI3K-AKT pathway to drive cell migration.

TGFß-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1.

High levels of transforming growth factor-ß (TGFß) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFß is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFß conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TßRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFß also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TßRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFß that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFß-TRAF6-p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3ß by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells.

APC and Smad7 link TGFß type I receptors to the microtubule system to promote cell migration.

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3ß (GSK-3ß). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-ß (TGFß) and is known to inhibit various TGFß-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFß stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3ß kinases to facilitate local TGFß/p38-dependent inactivation of GSK-3ß, accumulation of ß-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFß type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFß.

TRAF6 ubiquitinates TGFß type I receptor to promote its cleavage and nuclear translocation in cancer.

Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.

The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates embryonic development and tissue homeostasis; however, aberrations of its activity occur in cancer. TGF-beta signals through its Type II and Type I receptors (TbetaRII and TbetaRI) causing phosphorylation of Smad proteins. TGF-beta-associated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, was originally identified as an effector of TGF-beta-induced p38 activation. However, the molecular mechanisms for its activation are unknown. Here we report that the ubiquitin ligase (E3) TRAF6 interacts with a consensus motif present in TbetaRI. The TbetaRI-TRAF6 interaction is required for TGF-beta-induced autoubiquitylation of TRAF6 and subsequent activation of the TAK1-p38/JNK pathway, which leads to apoptosis. TbetaRI kinase activity is required for activation of the canonical Smad pathway, whereas E3 activity of TRAF6 regulates the activation of TAK1 in a receptor kinase-independent manner. Intriguingly, TGF-beta-induced TRAF6-mediated Lys 63-linked polyubiquitylation of TAK1 Lys 34 correlates with TAK1 activation. Our data show that TGF-beta specifically activates TAK1 through interaction of TbetaRI with TRAF6, whereas activation of Smad2 is not dependent on TRAF6.

TGFbeta1-induced activation of ATM and p53 mediates apoptosis in a Smad7-dependent manner.

ATM, a DNA-damage sensitive kinase and p53, are frequently inactivated in a variety of cancers as they together with gammaH2AX are critical guardians against DNA damage. Here, we report of a functional cross-talk between the cytokine TGFbeta and p53, leading to apoptosis of epithelial cells, involving Smad7, a TGFbeta target gene p38 MAP kinase, and ATM. Using ectopic expression of p53, siRNA for Smad7, p38alpha-/- deficient cells and specific inhibitors, we show that TGF-beta induces apoptosis via ATM and p53 in epithelial cells. Intriguingly, Smad7 act as a scaffold protein to promote functional interactions between p38, ATM and p53 upon TGFbeta treatment, facilitating their activation. Smad7 colocalizes with gammaH2AX in DNA damage foci and was required for proper cell cycle checkpoints to prevent genetic instability. Our data imply that Smad7 plays a crucial role upstream of ATM and p53 to protect the genome from insults evoked by extracellular stress.

The length of the transcript encoded from the Kcnq1ot1 antisense promoter determines the degree of silencing.

The underlying mechanisms linking antisense RNA, chromatin architecture and gene expression have not been fully elucidated. Here we show that long transcripts encoded from the Kcnq1ot1 antisense promoter silence the flanking genes more efficiently than short antisense transcripts. Interestingly, the antisense RNA-mediated deposition of inactive chromatin-specific histone modifications was higher with the longer antisense transcripts than with the shorter antisense transcripts. The kinetic studies of expression and chromatin remodeling of overlapping and nonoverlapping genes in response to antisense transcription revealed that the overlapping gene was rapidly silenced due to decrease in the occupancy of basal transcription machinery and simultaneous enrichment of its promoter with inactive chromatin modifications. The nonoverlapping gene, initially enriched with histone modifications specific to active chromatin, was subsequently silenced. Surprisingly, the flanking sequences were initially enriched with H3K9 monomethylation, as compared to di- and trimethylation, with a subsequent shift to trimethylated H3K9 enrichment. Our data provide a new perspective into antisense RNA-mediated gene silencing, and, more importantly, provide an explanation for why the antisense transcripts encoded from imprinting control regions are of significant length.

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region.

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

Bidirectional silencing and DNA methylation-sensitive methylation-spreading properties of the Kcnq1 imprinting control region map to the same regions.

The mechanisms underlying the phenomenon of genomic imprinting are poorly understood. Accumulating evidence suggests that imprinting control regions (ICR) associated with the imprinted genes play an important role in creation of imprinted expression domains by propagating parent-of-origin-specific epigenetic modifications. We have recently documented that the Kcnq1 ICR unidirectionally blocks enhancer-promoter communications in a methylation-dependent manner in Hep-3B and Jurkat cell lines. In this report we show that the Kcnq1 ICR harbors bidirectional silencing and methylation-sensitive methylation-spreading properties in a lineage-specific manner. We fine map both of these functions to two critical regions, and loss of one these regions results in loss of silencing as well as methylation spreading. The cell type-specific functions of the Kcnq1 ICR suggest binding of cell type-specific factors to various cis elements within the ICR. Fine mapping of the silencing and methylation-spreading functions to the same regions explains the fact that the silencing factors associated with this region primarily repress the neighboring genes and that methylation occurs as a consequence of silencing.

The kinetics of deregulation of expression by de novo methylation of the h19 imprinting control region in cancer cells.

Epigenetic lesions are common in neoplasia and range from hypermethylation of subsets of CpG islands to loss of imprinting. By exploiting an episomal model system and the strong de novo methylation capacity of a human cancer cell line, we show that an H19 minigene rapidly becomes methylated and silenced, mimicking the inactivation of the maternal H19 allele in a range of cancers. Although the H19 imprinting control region (ICR) initially displayed methylation protection, it eventually succumbed to the pressure mounted by the de novo methylation machinery of the JEG-3 cells. Importantly, we were able to visualize the kinetics of the loss of the H19 ICR chromatin insulator function in association with chromatin compaction. Our results document that a strong de novo methylation machinery leads to loss of methylation privilege states of H19 ICR to functionally manifest loss of insulator function in a matter of only a few days in human cancer cells.