SMYD3: An Oncogenic Driver Targeting Epigenetic Regulation and Signaling Pathways.
ABSTRACT: SMYD3 is a member of the SMYD lysine methylase family and plays an important role in the methylation of various histone and non-histone targets. Aberrant SMYD3 expression contributes to carcinogenesis and SMYD3 upregulation was proposed as a prognostic marker in various solid cancers. Here we summarize SMYD3-mediated regulatory mechanisms, which are implicated in the pathophysiology of cancer, as drivers of distinct oncogenic pathways. We describe SMYD3-dependent mechanisms affecting cancer progression, highlighting SMYD3 interplay with proteins and RNAs involved in the regulation of cancer cell proliferation, migration and invasion. We also address the effectiveness and mechanisms of action for the currently available SMYD3 inhibitors. The findings analyzed herein demonstrate that a complex network of SMYD3-mediated cytoplasmic and nuclear interactions promote oncogenesis across different cancer types. These evidences depict SMYD3 as a modulator of the transcriptional response and of key signaling pathways, orchestrating multiple oncogenic inputs and ultimately, promoting transcriptional reprogramming and tumor transformation. Further insights into the oncogenic role of SMYD3 and its targeting of different synergistic oncogenic signals may be beneficial for effective cancer treatment.
Project description:Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.
Project description:SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription.
Project description:SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.
Project description:The coordinated expression of myogenic regulatory factors, including MyoD and myogenin, orchestrates the steps of skeletal muscle development, from myoblast proliferation and cell-cycle exit, to myoblast fusion and myotubes maturation. Yet, it remains unclear how key transcription factors and epigenetic enzymes cooperate to guide myogenic differentiation. Proteins of the SMYD (SET and MYND domain-containing) methyltransferase family participate in cardiac and skeletal myogenesis during development in zebrafish, Drosophila and mice. Here, we show that the mammalian SMYD3 methyltransferase coordinates skeletal muscle differentiation in vitro. Overexpression of SMYD3 in myoblasts promoted muscle differentiation and myoblasts fusion. Conversely, silencing of endogenous SMYD3 or its pharmacological inhibition impaired muscle differentiation. Genome-wide transcriptomic analysis of murine myoblasts, with silenced or overexpressed SMYD3, revealed that SMYD3 impacts skeletal muscle differentiation by targeting the key muscle regulatory factor myogenin. The role of SMYD3 in the regulation of skeletal muscle differentiation and myotube formation, partially via the myogenin transcriptional network, highlights the importance of methyltransferases in mammalian myogenesis.
Project description:Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.
Project description:SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFβ-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGFβ-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGFβ-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.
Project description:The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 ?-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.
Project description:SET and MYND domain containing 3 (SMYD3) is a histone methyltransferase (HMT) and transcription factor, which serves important roles in carcinogenesis. Numerous downstream target genes of SMYD3 have been identified in previous studies. However, the downstream microRNA (miRNA) s regulated by SMYD3 are yet to be elucidated. In the present study, the results of miRNA microarray demonstrated that 30 miRNA expression profiles were upregulated, whilst 24 miRNAs were downregulated by >2.0-fold in the SMYD3-overexpressed MCF-7 breast cancer cells. The HMT activity was demonstrated to be essential for SMYD3-mediated transactivation of miR-200c-3p and the overexpression of miR-200c-3p inhibited the transactivation effects of SMYD3 on myocardin-related transcription factor-A-dependent migration-associated genes. To our best knowledge, the current study is the first to report on the transcriptional regulation of SMYD3 on miRNAs, and miR-200c may be a downstream negative regulator of the SMYD3-mediated pathway in the migration of breast cancer cells. These results may provide a novel theoretical basis to understand the mechanisms underlying the initiation, progression, diagnosis, prevention and therapy of breast cancer.
Project description:The AKT/mTOR pathway is critical for bladder cancer (BC) pathogenesis and is hyper-activated during BC progression. In the present study, we identified a novel positive feedback loop involving oncogenic factors histone methyltransferase SMYD3, insulin-like growth factor-1 receptor (IGF-1R), AKT, and E2F-1. SMYD3 expression was significantly up-regulated in BC tumors and positively associated with histological grade, lymph node metastasis, and shorter patient survival. Depletion of SMYD3 inhibited BC cell proliferation, colony formation, migration, invasion, and xenograft tumor growth. Mechanistically, SMYD3 inhibition led to the diminished AKT/mTOR signaling activity, thereby triggering deleterious effects on BC cells. Furthermore, SMYD3 directly activates the expression of IGF-1R, a critical activator of AKT in BC, by inducing hyper-methylation of histone H3-K4 and subsequent chromatin remodeling in the IGF-1R promoter region. On the other hand, E2F-1, a downstream factor of the AKT pathway, binds to the E2F-1 binding motifs at the SMYD3 promoter and consequently induces SMYD3 transcription and expression. Thus, SMYD3/IGF-1R/AKT/E2F-1 forms a positive feedback loop leading to the hyper-activated AKT signaling. Our findings provide not only profound insights into SMYD3-mediated oncogenic activity but also present a unique avenue for treating BC by directly disrupting this signaling circuit.
Project description:The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-L-homocysteine at 2.8?Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.