Abstract
Aberrant activation of Ras/Raf/mitogen-activated protein kinase (MAPK) signaling is frequently linked to metastatic prostate cancer (PCa); therefore, the characterization of modulators of this pathway is critical for defining therapeutic vulnerabilities for metastatic PCa. The lysine methyltransferase SET and MYND domain 3 (SMYD3) methylates MAPK kinase kinase 2 (MAP3K2) in some cancers, causing enhanced activation of MAPK signaling. In PCa, SMYD3 is frequently overexpressed and associated with disease severity; however, its molecular function in promoting tumorigenesis has not been defined. We demonstrate that SMYD3 critically regulates tumor-associated phenotypes via its methyltransferase activity in PCa cells and mouse xenograft models. SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition associated behaviors by altering the abundance of the intermediate filament vimentin. Furthermore, activation of the SMYD3-MAP3K2 signaling axis supports a positive feedback loop continually promoting high levels of SMYD3. Our data provide insight into signaling pathways involved in metastatic PCa and enhance understanding of mechanistic functions for SMYD3 to reveal potential therapeutic opportunities for PCa.
INTRODUCTION
Metastatic prostate cancer (PCa) has a 5-year survival rate of less than 30% and is often resistant to treatment (1), indicating an urgent need to improve understanding of mechanisms that drive PCa progression and identify clinically actionable vulnerabilities. Aberrant signaling in critical pathways, such as the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (2), p53 (3), MYC (4), and RAS/RAF/mitogen-activated protein kinase (MAPK) (5, 6) signaling pathways, promotes PCa aggressiveness in vitro and metastatic spread in vivo. Protein lysine methylation signaling is also linked to PCa progression. Numerous lysine methyltransferases (KMTs) are overexpressed in primary tumors and metastatic lesions, including NSD2 (7), G9a (8), and EZH2 (9), a known driver of lethal castration-resistant prostate cancer (CRPC) and a key therapeutic target. Misregulation of these enzymes disrupts the chromatin landscape through altered histone methylation patterns and underlies pathological gene expression programs (10, 11). The protein KMT SET and MYND domain 3 (SMYD3) is also highly expressed in PCa (12–14); however, whether it has a direct molecular role in promoting aggressive phenotypes associated with advanced prostate adenocarcinoma has not been clearly defined.
SMYD3 is predominantly expressed during early embryonic development in mammals, where it regulates myogenesis and differentiation of skeletal and cardiac muscle (15–17). Its abundance is low in most adult tissues; however, it is frequently overexpressed in solid tumors, including prostate, breast, lung, colorectal, and liver cancers, among others (13, 18–20). In these tumor types, SMYD3 overexpression has been implicated in promoting survival, migration, and invasion (12, 18–20); therefore, it is critical to clearly identify the molecular oncogenic mechanisms reliant on SMYD3. In lung and pancreatic ductal adenocarcinomas, the major cellular target for SMYD3’s methyltransferase activity is the MAPK, MAPK kinase kinase 2 (MAP3K2) (18). Methylation at lysine (K) 260 of MAP3K2 by SMYD3 inhibits binding of the deactivating phosphatase protein phosphatase 2A (PP2A), thereby sustaining aberrant activation of the Ras/Raf/MAPK kinase (MEK)/extracellular-regulated kinase (ERK) signaling axis and promoting tumorigenesis (18). In small cell lung cancer, a Ras-independent cancer, SMYD3 methylates another nonhistone substrate, the ubiquitin ligase ring finger protein 113A (RNF113A), promoting resistance to alkylating chemotherapeutics (21). Despite the high expression levels of SMYD3 in other tumor types, including PCa, the prosurvival and prometastatic pathways dependent on SMYD3 signaling have not been clearly defined in these cells.
While activating mutations in the Ras/Raf signaling pathways are not common in PCa, approximately 40% of primary tumors and more than 90% of metastatic tumors show expression changes that lead to pathway activation (2). In addition, a substantial increase in MAPK signaling is found in tissue samples of advanced-staged tumors with high Gleason scores and in distant metastases (5, 6). Notably, the SMYD3 target MAP3K2 shows a fourfold increase in expression in cancerous prostate compared to benign tissue (22). Mouse models combining Ras activation with loss of phosphatase and tensin homolog (Pten), a common genetic lesion in primary prostate tumors, show rapid progression of primary tumors, features of epithelial-mesenchymal transition (EMT), and increased metastatic burden (6, 23). These data suggest that the aberrant activation of the Ras/MAPK pathway may be a critical step in promoting EMT and the development of advanced PCa. These observations have led to efforts to target MAPK signaling to treat metastatic CRPC (mCRPC), particularly using the MEK1/2 inhibitor trametinib (24).
In this study, we demonstrate that SMYD3 overexpression promotes PCa cell aggressiveness by enhancing migration, invasion, and anchorage-independent growth and altering adhesive properties of the cells. This role of SMYD3 is dependent on its catalytic activity and, specifically, its methylation of MAP3K2, which maintains constitutive activation of MEK/ERK signaling and alters the abundance of the EMT protein vimentin, contributing to PCa progression. We identify a positive feedback loop in which MEK/ERK activation positively stabilizes protein levels of SMYD3, continually augmenting its high abundance and oncogenic properties. Last, depletion of SMYD3 or inhibition of its catalytic activity substantially improves survival and decreases metastases in mouse orthotopic xenograft models. Together, these data uncover the SMYD3-MAP3K2 signaling axis as a key regulator of PCa aggression and highlight its potential as a therapeutic target for advanced disease.
RESULTS
SMYD3 expression increases in high-grade PCa
The SMYD3 mRNA is highly expressed in multiple tumor types relative to normal tissue and frequently increases further in advanced disease stages (12–14, 20, 25–28). Evaluation of SMYD3 expression in primary prostate tumors from The Cancer Genome Atlas (TCGA) database (29) compared to tumor-adjacent (TCGA) or normal prostate tissue [Genotype-Tissue Expression (GTEx)] showed a significant increase in SMYD3 mRNA levels in tumor samples (Fig. 1A), similar to additional independent datasets of primary prostate tumors [Fig. 1A (30, 31)]. Tumor samples stratified by Gleason score also showed increased SMYD3 mRNA expression relative to normal tissue, with more SMYD3 mRNA present in higher-grade tumors (Fig. 1B). Analysis of patient cohorts with mCRPC also showed increased relative expression of SMYD3 mRNA in metastatic samples compared to primary tumors (Fig. 1, C to E). We also tested SMYD3 abundance in a genetically engineered mouse model that recapitulates the development of human lethal prostate adenocarcinoma and metastasis through MYC overexpression and loss of Pten in prostatic luminal epithelial cells (Hoxb13-MYC+/− Hoxb13-Cre+/− PtenFl/Fl, or BMPC mice) (32). Primary tumors isolated from these mice showed much higher SMYD3 protein abundance compared to tissue from a normal anterior prostate lobe using anti-SMYD3 immunoblotting (Fig. 1F). In total, these data indicate that SMYD3 expression is up-regulated in prostate tumors, particularly at advanced stages, implicating its misregulation in the progression of PCa.
Loss of SMYD3 abrogates tumor-associated phenotypes of PCa cells in vitro
To determine the role of SMYD3 in oncogenic properties of PCa cells, we generated PC-3 and LNCaP stable cell lines expressing short hairpin SMYD3 (shSMYD3) that targets the 3′ untranslated region (3′UTR) of SMYD3 under control of a doxycycline-inducible promoter (Tet-on). PC-3 cells, derived from bone metastases, are androgen independent and have more aggressive properties than androgen-dependent, metastatic lymph node–derived LNCaP cells; therefore, these two cell lines represent different aspects of disease progression (33–35). We verified knockdown of SMYD3 in these cells using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunofluorescence, which showed no detectable SMYD3 in the presence of doxycycline (Fig. 1, G and H, and fig. S1A). We also generated additional SMYD3 knock-down PC-3 cells using a short hairpin RNA (shRNA) targeting a different sequence of the 3′UTR of SMYD3 [fig. S1B; (18)]. Upon SMYD3 knockdown, cell viability of both PC-3 and LNCaP cells was moderately reduced, although the overall rate of proliferation remained similar either with or without SMYD3 (Fig. 1I). However, SMYD3 depletion did substantially impair the rate of collective cell migration in both PC-3 and LNCaP cells (Fig. 1J and fig. S1, C and 1D), and this reduced rate is independent of doxycycline treatment alone (fig. S1E). Given the moderate reduction in proliferation of SMYD3-depleted cells, we also assayed migration upon cell cycle arrest with aphidicolin treatment and observed that SMYD3 knockdown still resulted in decreased migration rate, indicating that SMYD3 driven changes in migration rate were independent of differences in proliferation (fig. S1F).
We also tested the role for SMYD3 in anchorage-independent growth by measuring colony formation in soft agar. We observed no or very little colony formation of either PC-3 or LNCaP cells lacking SMYD3, while control cells grew efficiently in soft agar (Fig. 1K). This disruption to anchorage-independent growth was independent of doxycycline treatment alone (fig. S1G). Spheroids of PC-3 and LNCaP cells with repressed SMYD3 formed loose aggregates of cells, rather than the compact, three-dimensional structures formed with SMYD3-expressing cells (fig. S1H). To investigate the involvement of SMYD3 in adhesion, we tested PCa cell adhesion to the extracellular matrix component fibronectin, self-adhesion, and adhesion to fibroblasts, which constitute a major component of the prostate tumor microenvironment. In all cases, we observed that loss of SMYD3 substantially increased adhesion to all substrates tested (Fig. 1L and fig. S1, I to K). Last, SMYD3 repression also led to decreased cell invasion capabilities of both PC-3 and LNCaP cells (Fig. 1M and fig. S1, K and L). Together, our findings show that SMYD3 plays a critical role in tumor cell migration, invasion, anchorage-independent growth, as well as cell–extracellular matrix, cell-cell, and cell-fibroblast adhesion in PCa cells in vitro.
The catalytic activity of SMYD3 is required for its tumorigenic properties in PCa cells
Our data show a clear role for SMYD3 in cell migration, adhesion, and invasion of PCa cells; however, it is not known whether these functions rely on the methyltransferase activity of SMYD3 or alternatively, noncatalytic functions of the enzyme. To test this, we generated stable PC-3 and LNCaP cells that ectopically express either wild-type SMYD3 or the catalytically inactive F183A mutant (18) at similar levels upon SMYD3 knockdown in the presence of doxycycline (Fig. 2A and fig. S2, A and B). Compared to wild-type SMYD3, expression of SMYD3F183A mutant slowed collective cell migration of PC-3 and LNCaP cells (Fig. 2B and fig. S2C), similar to the phenotype observed with SMYD3 depletion. Cells expressing SMYD3F183A also showed increased adhesion to fibronectin, other PCa cells, and fibroblasts compared to SMYD3WT cells (Fig. 2C and fig. S2D). Last, similar to loss of endogenous SMYD3, SMYD3F183A decreased invasiveness of PCa cells (Fig. 2D).
