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Human neurodegenerative diseases, such as Alzheimer’s disease (AD), are not easily modeled in vitro due to the inaccessibility of brain tissue and the level of complexity required by existing cell culture systems. Three-dimensional (3D) brain organoid systems generated from human pluripotent stem cells (hPSCs) have demonstrated considerable potential in recapitulating key features of AD pathophysiology, such as amyloid plaque- and neurofibrillary tangle-like structures. A number of AD brain organoid models have also been used as platforms to assess the efficacy of pharmacological agents in disease progression. However, despite the fact that stem cell-derived brain organoids mimic early aspects of brain development, they fail to model complex cell-cell interactions pertaining to different regions of the human brain and aspects of natural processes such as cell differentiation and aging. Here, we review current advances and limitations accompanying several hPSC-derived organoid methodologies, as well as recent attempts to utilize them as therapeutic platforms. We additionally discuss comparative benefits and disadvantages of the various hPSC-derived organoid generation protocols and differentiation strategies. Lastly, we provide a comparison of hPSC-derived organoids to primary tissue-derived organoids, examining the future potential and advantages of both systems in modeling neurodegenerative disorders, especially AD.

Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP–TEAD and β-catenin–TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin–TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP–p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional “switch” between pluripotency and initiation of differentiation.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).

Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.

The tumor suppressor gene RASSF1A is inactivated through point mutation or promoter hypermethylation in many human cancers. In this study, we conducted a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Rassf1a-null mice to identify candidate genes that collaborate with loss of Rassf1a in tumorigenesis. We identified 10 genes, including the transcription factor Runx2, a transcriptional partner of Yes-associated protein (YAP1) that displays tumor suppressive activity through competing with the oncogenic TEA domain family of transcription factors (TEAD) for YAP1 association. While loss of RASSF1A promoted the formation of oncogenic YAP1-TEAD complexes, the combined loss of both RASSF1A and RUNX2 further increased YAP1-TEAD levels, showing that loss of RASSF1A, together with RUNX2, is consistent with the multistep model of tumorigenesis. Clinically, RUNX2 expression was frequently downregulated in various cancers, and reduced RUNX2 expression was associated with poor survival in patients with diffuse large B-cell or atypical Burkitt/Burkitt-like lymphomas. Interestingly, decreased expression levels of RASSF1 and RUNX2 were observed in both precursor T-cell acute lymphoblastic leukemia and colorectal cancer, further supporting the hypothesis that dual regulation of YAP1-TEAD promotes oncogenic activity. Together, our findings provide evidence that loss of RASSF1A expression switches YAP1 from a tumor suppressor to an oncogene through regulating its association with transcription factors, thereby suggesting a novel mechanism for RASSF1A-mediated tumor suppression. Cancer Res; 72(15); 3817–27. ©2012 AACR.