The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer

Abstract

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Figure 1

MMSET is overexpressed in PCa cell lines and influences global histone methylation. (A) Nuclear extracts from prostate cell lines were immunoblotted with the indicated antibodies, and quantified by densitometry. MMSET levels were normalized to histone H4 and represented as relative to levels in BPH1 cells. (B) RWPE-1 and BPH1 cells were infected with a control retrovirus, or viruses harboring MMSET I, MMSET II, or SET domain mutant MMSET II (Y1118A). Nuclear proteins were immunoblotted as indicated. (C–D) PCa DU145, PC-3 and 22Rv1 cell lines (C), and non-transformed RWPE-1 and BPH1 cells (D) were transfected with the indicated siRNAs. Nuclear extracts were prepared 6 days after transfection and immunoblotted with the indicated antibodies.

Figure 2

MMSET knockdown decreases anchorage-dependent and independent proliferation, migration, and invasion of metastatic DU145 PCa cells. (A) Proliferation curves of DU145 cells transfected with the indicated siRNAs. Proliferation was measured by MTT conversion. Absorbance values are represented relative to time 0 (mean +/− SD of 3 independent biological replicates; *p<0.05). (B) Soft-agar colony formation. Cells were transfected in biological triplicates with the indicated siRNAs. The mean colony number per well +/− SD is presented (**p<0.01). (C–D) Quantification of one of three representative, independent migration (C) and invasion (D) experiments. The mean number of migrating or invading cells per field +/− SD of is presented (**p<0.01). (E) Representative fields of invasion assays (200x magnification).

Figure 3

Overexpression of MMSET promotes invasion and migration in non-transformed RWPE-1 cells. (A) Proliferation curves of RWPE-1 cells infected with a control retrovirus or a virus harboring wild-type or SET domain mutant MMSET. Proliferation was measured by MTT conversion. Absorbance values (mean +/− SD of three independent experiments) are represented as relative to time 0. (B) Soft-agar colony formation. Cells were infected in biological triplicates with retroviruses described in (A), and seeded in soft agar. The mean colony number per well +/− SD is presented. (C–D) Quantification of one of three representative, independent migration (C) and invasion (D) experiments. The mean number of cells per field +/− SD is presented (**p<0.01; ***p<0.001). (E) Representative fields of invasion assay (200x magnification).

Figure 4

MMSET regulates EMT. (A) RWPE-1 cells were infected with control retrovirus or a virus harboring wild-type or SET domain mutant MMSET. Cytoplasmic fibroblast-like elongations are indicated with arrowheads (400x magnification). (B) Total protein extracts from RWPE-1 cells described in (A) were immunoblotted as indicated. (C) The expression of mesenchymal (vimentin, fibronectin 1, N-cadherin) and epithelial (desmoplakin, DSC3, occludin) markers was analyzed by real time PCR in cells described in (A). Gene expression normalized to GAPDH, from three independent experiments (+/−SD), is presented relative to that observed in cells transduced with control retrovirus (*p<0.05; **p<0.01).

Figure 5

MMSET regulates TWIST1 expression in PCa. (A) RWPE-1 cells were infected with control retrovirus or virus containing MMSET. TWIST1 expression was analyzed by real time PCR, normalized to GAPDH and presented as relative to that obtained in control cells (mean +/− SD from three independent experiments; ***p<0.001). Total protein extracts from these cells were immunoblotted as indicated. (B) DU145 cells were transfected with the indicated siRNAs, and analyzed as in (A); (**p<0.01). (C) MMSET and TWIST1 mRNA expression in normal (N) and malignant (T) prostate specimens was extracted from deposited gene expression datasets (–39), analyzed in Oncomine and presented as box and whisker plots. Statistically significant differences are indicated with the p-value.

Figure 6

TWIST1 expression mediates MMSET-induced EMT, migration and invasion. (A) RWPE-1 cells infected with control retrovirus (MMSET-) or MMSET-harboring retrovirus (MMSET+), were transfected with siRNAs targeting TWIST1, or a control siRNA (Scr). TWIST1 expression was analyzed by real time PCR, normalized to GAPDH, and presented relative to that observed in cells transduced with control retrovirus (mean +/− SD from three independent experiments; **p<0.01). (B) Photomicrographs of cells described in (A) were taken 4 days after transfection (400x magnification). (C) Total protein extracts from cells described in (A) were immunoblotted with the indicated antibodies. (D–E) Quantification of one of three representative, independent migration (D) and invasion (E) experiments of cells described in (A). The mean number of migrating or invading cells per field +/−SD is presented (**p<0.01).

Figure 7

MMSET binding across the TWIST1 locus correlates with high levels of H3K36me2. Chromatin from RWPE-1 cells infected with control retrovirus or a virus expressing MMSET (A), or from DU145 cells transfected with the indicated siRNAs (B), was immunoprecipitated with anti-MMSET or anti-H3K36me2 antibodies. The purified DNA was analyzed by real time PCR using primers amplifying regions across the TWIST1 locus. Results are presented as percentage of total input DNA precipitated. The average +/− SD of three independent experiments is presented (*p<0.05; **p<0.01).