Tumor suppressor CASZ1(1p36.22) is often lost in poor prognosis neuroblastomas (NB) with an undifferentiated histopathology. Differentiation of NB cells upon CASZ1 restoration indicates CASZ1 loss may contribute to the undifferentiated phenotype during NB tumorigenesis. How CASZ1 functions during normal mammalian development is unknown. Using a murine embryonic stem cell (mESC) embryogenesis model, we investigated the effects of CASZ1 haploinsufficiency and CASZ1 reconstitution on differentiation programs.
Differentiation in mESCs, wild-type (WT) and Casz1 haploinsufficient (CAS+/- mESCs), was evaluated using mESC embryoid body (EB) neurogenesis and adipogenesis assays. For rescue experiments, CAS+/- mESCs were transfected with a bacterial artificial chromosome containing the full human CASZ1 genomic locus (CASBAC). Gene expression was evaluated by qPCR and western blot.
Analysis of CAS+/- mESCs revealed pluripotency gene expression was relatively unaffected by CASZ1 loss, but there is higher Myc mRNA (∼5.5 fold) compared to WT mESCs. During neural differentiation Myc is aberrantly expressed as evidenced by increased protein levels compared to the mESC state. Four days after LIF withdrawal, CAS+/- EBs have ∼2.5 fold higher MYC levels as compared to WT EBs that have half the MYC levels as compared to the mESC state. Coincident with aberrant MYC expression CAS+/- mESCs exhibit a ∼2.8 fold increase in Gata4 mRNA (endoderm lineage) and a ∼3.2 fold increase in Gata2 mRNA (mesoderm lineage) compared to WT mESCs. Functionally, CAS+/- mESCs retain adipogenesis potential. However after neural induction, CAS+/- EBs exhibit decreased neuroectoderm lineage commitment with neural lineage marker Pax6 mRNA levels some ∼8.9 fold less than WT EBs. Neurogenesis potential is lost as evidenced by a failure to extend ßIII tubulin positive neuritic processes. However, CASBAC mESCs restore Myc regulation, lineage commitment, and neurogenesis potential. Four days after LIF withdrawal, CASBAC EBs have ∼0.6 fold MYC levels as compared to CASBAC mESCs, much like WT EBs. Lineage markers Gata4 and Gata2 also normalize to ∼0.7 and ∼0.5 times WT EB levels, respectively. Pax6 levels in CASBAC EBs are also restored to ∼1.8 fold WT EB levels. Most noticeable is the restoration of full neurogenesis potential as evidenced by ßIII tubulin positive neuritic processes.
The data shows CAS+/- mESCs are unable to implement a neural differentiation program. We propose that CASZ1 haploinsufficiency causes misalignment of spatial and temporal transcription factor networks necessary for proper neurogenesis. Perturbation of neurogenesis results in latent proliferative states in neural precursors, which may contribute to tumorigenesis and the undifferentiated histopathology of poor prognosis NBs.
Citation Format: Stanley He, Ryan Virden, Zhihui LIu, Carol J. Thiele. CASZ1 haploinsufficiency in murine embryonic stem cells causes increased MYC expression, aberrant lineage commitment, and prevents neurogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5040. doi:10.1158/1538-7445.AM2013-5040