A Twist1-MLL-WDR5-HOTTIP Complex Regulates HOXA9 Chromatin to Facilitate Metastasis of Prostate Cancer

Abstract

The Twist1 transcription factor induces a transcriptional program resulting in cellular phenotypic changes, the epithelial-mesenchymal transition (EMT), during cancer progression. Twist1 is overexpressed in prostate cancer (PCa) specimens correlating with metastatic disease. We have demonstrated previously that Twist1 activates transcription of HOXA9 which contributes to the induction of a Twist1-dependent metastatic phenotype in PCa. Twist1 is canonically known as a transcription factor, but recent data suggest an epigenetic role for Twist1 in gene regulation. During embryogenesis, the histone methyltransferase complex, MLL-WDR5 and the lncRNA HOTTIP, regulate the expression of the HOXA cluster of genes by H3K4 chromatin methylation. Interestingly, PCa whole exome sequencing has shown frequent mutations in MLL2. Taken together, we hypothesized that Twist1 cooperates with the MLL-WDR5-HOTTIP (MWH) complex to drive expression of HOXA9 mediating PCa metastasis. Isogenic PCa cell lines using parental Myc-CaP and PC3 cells were created. Stable overexpression and knockdown (KD) experiments were performed with retroviral vectors. Transient KD with siRNA used commercial liposomal transfection reagents. Molecular biology procedures such as co-immunoprecipitation (IP), RNA IP (RIP), qPCR, Western and chromatin-IP (ChIP) were performed as stated below. We also used in vitro assays that mimic the various stages of cancer progression to metastasis as below. The MSKCC cBioPortal was used to perform PCa expression analysis. Twist1 could interact with components of the MWH complex as shown by Twist1 co-IP of WDR5 and RIP of HOTTIP. Twist1 overexpression also upregulated expression of the lncRNA HOTTIP in PCa cells. Functionally, WDR5 KD reduced cellular migration and invasion in Twist1 overexpressing cells. Similarly, KD against the lncRNA component HOTTIP also reduced Twist1-induced migration. Importantly, WDR5 or HOTTIP knockdown could abrogate Twist1-induced HOXA9 expression as shown by reduced HOXA9 message and protein levels. ChIP analysis of the HOXA9 promoter revealed that Twist1-overexpressing PCa cells had increased H3K4me3 levels specific for MWH activity. Last, TWIST1and genes encoding MWH complex components were co-overexpressed in PCa samples. Twist1 plays key roles during development and is a master transcriptional regulator of the EMT that promotes cancer metastasis. We demonstrated three important findings in Twist1-overexpressing PCa: (1) Twist1 forms a complex with the MWH methyltransferase complex; (2) the MWH complex activates HOXA9 expression by H3K4me3 chromatin modification of HOXA9 promoter region; and (3) the MWH complex is required for full Twist1-induced pro-metastatic behaviors.