Abstract 5522: Novel myogenic differentiation transcription factor CASZ1 suppresses rhabdomyosarcoma tumor growth
Authors: Zhihui Liu, Norris Lam, Arnulfo Mendoza, Jun S. Wei, John F. Shern, Marielle Yohe, Javed Khan, Carol J. Thiele
Journal: Cancer Research (2017)

Abstract Disruption of muscle regulatory factors (MRFs) in muscle progenitor cells results in a failure to withdraw from cell cycle and terminal differentiation and is proposed to contribute to rhabdomyosarcoma (RMS) tumorigenesis. Zinc finger transcription factor CASZ1 is a tumor suppressor gene and regulates normal nervous system and heart development. CASZ1 is known to regulate a subset of genes that are involved in skeletal muscle development although the function of CASZ1 in normal myogenesis and RMS tumorigenesis is unknown. CASZ1 levels increase over 20-fold when C2C12 myoblasts differentiate into myotubes. To probe its role in normal myogenesis we performed genetic knockdown or overexpression experiments in C2C12 myoblasts. When CASZ1 expression is silenced using RNAi, there is a significant reduction in myotubes upon induction of differentiation. Realtime PCR shows that knockdown of CASZ1 increases Myf5, a MRF that determines early commitment of muscle precursor cells, but decreases Myogenin (2-fold, p&amp;lt;0.01), a MRF required for terminal differentiation and myotube formation. Knockdown of CASZ1 also decreases late skeletal differentiation genes Acta1, Ckm, Tnnt1 and Tnni2 (&amp;gt; 2-fold, p&amp;lt;0.01). Overexpression of CASZ1 induces expression of Myogenin, Acta1, Ckm and Tnnt1, represses Myf5 and accelerates myotube formation. This indicates that CASZ1 regulates genes important in the transition from early to late myogenic differentiation. To investigate the role of CASZ1 in RMS, we first evaluated the expression of CASZ1 in primary RMS tumors using public available microarray database. CASZ1 mRNA levels are ~1.5-fold lower in embryonal RMS (ERMS) samples compared to normal muscle (p&amp;lt;0.001), while levels of CASZ1 in alveolar (ARMS) are similar to levels in normal muscle. CASZ1 levels increase over 3-fold when RD cells (ERMS) were cultured in differentiation medium (100 nM 12-O-tetradecanoylphorbol-13-acetate, TPA). Knockdown of CASZ1 in the RD cells suppressed TPA induced expression of myogenic markers TNNT1 and TNNI2 while CASZ1 overexpression stimulated expression of TNNT1 and TNNI2 (all p&amp;lt;0.05). An in vivo spontaneous xenograft model showed that overexpression of CASZ1 significantly suppressed RD tumor growth (p&amp;lt;0.005). In RMS patients, NexGen sequencing of 85 tumors identified 4 samples with nonsynonymous single nucleotide variants (SNVs) in CASZ1 that were absent in the 1000 Genomes databases. We engineered these SNVs into CASZ1 construct and transfected them into C2C12 cells. We found that the R25C SNV had impaired nuclear localization and failed to activate skeletal muscle gene transcription (p&amp;lt;0.03). Taken together, our results suggest that the regulation of muscle differentiation program by CASZ1 in C2C12 is integral to proper myogenic differentiation, and genetic variants of CASZ1 disrupt early myogenesis and may contribute to ERMS tumorigenesis and progression. Citation Format: Zhihui Liu, Norris Lam, Arnulfo Mendoza, Jun S. Wei, John F. Shern, Marielle Yohe, Javed Khan, Carol J. Thiele. Novel myogenic differentiation transcription factor CASZ1 suppresses rhabdomyosarcoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2017-5522