Shaying Zhao

Cell-Cycle Control of Developmentally Regulated Transcription Factors Accounts for Heterogeneity in Human Pluripotent Cells

Summary Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.

Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer

Spontaneously occurring canine mammary cancer represents an excellent model of human breast cancer, but is greatly understudied. To better use this valuable resource, we performed whole-genome sequencing, whole-exome sequencing, RNA-seq, and/or high-density arrays on twelve canine mammary cancer cases, including seven simple carcinomas and four complex carcinomas. Canine simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Canine complex carcinomas, which are characterized by proliferation of both luminal and myoepithelial cells and are rare in human breast cancer, seem to lack genomic abnormalities. Instead, these tumors have about 35 chromatin-modification genes downregulated and are abnormally enriched with active histone modification H4-acetylation, whereas aberrantly depleted with repressive histone modification H3K9me3. Our findings indicate the likelihood that canine simple carcinomas arise from genomic aberrations, whereas complex carcinomas originate from epigenomic alterations, reinforcing their unique value. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major cell lineage of the mammary gland. Canine simple carcinomas, which faithfully represent human breast carcinomas at the molecular level, provide indispensable models for basic and translational breast cancer research.

Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency

Summary Here we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a “window of opportunity” for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase.

Frequent alteration of the tumor suppressor gene APC in sporadic canine colorectal tumors.

Sporadic canine colorectal cancers (CRCs) should make excellent models for studying the corresponding human cancers. To molecularly characterize canine CRC, we investigated exonic sequence mutations of adenomatous polyposis coli (APC), the best known tumor suppressor gene of human CRC, in 23 sporadic canine colorectal tumors, including 8 adenomas and 15 adenocarcinomas, via exon-resequencing analysis. As a comparison, we also performed the same sequencing analysis on 10 other genes, either located at human 5q22 (the same locus as APC) or 18q21 (also frequently altered in human CRC), or known to play a role in human carcinogenesis. We noted that APC was the most significantly mutated gene in both canine adenomas and adenocarcinomas among the 11 genes examined. Significantly, we detected large deletions of ≥10 bases, many clustered near the mutation cluster region, as well as single or two base deletions in ∼70% canine tumors of both subtypes. These observations indicate that like in the human, APC is also frequently altered in sporadic colorectal tumors in the dog and its alteration is an early event in canine colorectal tumorigenesis. Our study provides further evidence demonstrating the molecular similarity in pathogenesis between sporadic human and canine CRCs. This work, along with our previous copy number abnormality study, supports that sporadic canine CRCs are valid models of human CRCs at the molecular level.

Canine spontaneous head and neck squamous cell carcinomas represent their human counterparts at the molecular level.

Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better understand and utilize this valuable resource, we performed a pilot study that represents its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers recapitulate many molecular features of human HNSCC. These include analogous genomic copy number abnormality landscapes and sequence mutation patterns, recurrent alteration of known HNSCC genes and pathways (e.g., cell cycle, PI3K/AKT signaling), and comparably extensive heterogeneity. Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial–mesenchymal transition genes TWIST1 and SNAI1 are also prominent in these canine tumors. This pilot study, along with a rapidly growing body of literature on canine cancer, reemphasizes the potential value of spontaneous canine cancers in HNSCC basic and translational research.

Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not APC mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, Bacteroides uniformis, a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.

Nucleosome positioning changes during human embryonic stem cell differentiation

ABSTRACT Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content.

SEG - A Software Program for Finding Somatic Copy Number Alterations in Whole Genome Sequencing Data of Cancer

As next-generation sequencing technology advances and the cost decreases, whole genome sequencing (WGS) has become the preferred platform for the identification of somatic copy number alteration (CNA) events in cancer genomes. To more effectively decipher these massive sequencing data, we developed a software program named SEG, shortened from the word “segment”. SEG utilizes mapped read or fragment density for CNA discovery. To reduce CNA artifacts arisen from sequencing and mapping biases, SEG first normalizes the data by taking the log2-ratio of each tumor density against its matching normal density. SEG then uses dynamic programming to find change-points among a contiguous log2-ratio data series along a chromosome, dividing the chromosome into different segments. SEG finally identifies those segments having CNA. Our analyses with both simulated and real sequencing data indicate that SEG finds more small CNAs than other published software tools.

Abstract A07: Spontaneous canine mammary cancers: A much-needed model for human basal-like breast cancer and an ideal system to understand myoepithelial cells

Spontaneous canine mammary cancer represents an excellent model of human breast cancer but is greatly understudied. To better utilize this valuable resource, we performed the first comprehensive characterization of the genome, transcriptome and epigenome of 12 canine mammary cancer cases, including 7 simple carcinomas and 4 complex carcinomas (1). Simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Notably, these tumors closely cluster with human basal-like breast carcinomas from TCGA (2), but not with other intrinsic subtypes, in PAM50 classification with human breast carcinomas. Furthermore, these tumors match the basal-like cancer profile, including MYC amplification, PTEN deletion, BRCA1 mutation, etc. Our results indicate canine simple carcinomas could serve as a much-needed spontaneous cancer model of basal-like breast cancer by effectively bridging the gap between preclinical research and human trials, speeding up drug discovery for the worst subtype of breast cancer. Canine complex carcinomas, characterized by proliferation of both luminal and myoepithelial cells (rare in human breast cancer), appear to lack genomic abnormalities. Instead, these tumors have ~35 chromatin-modification genes downregulated, and are abnormally enriched with active histone modification H4-acetylation while depleted with repressive histone modification H3K9me3. Our findings indicate that these tumors likely originate from epigenomic alterations instead of genomic lesions. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major yet significantly understudied cell lineage of the mammary gland. References: 1. Liu D, Xiong H, Ellis AE, Northrup NC, Rodriguez CO, Jr., O9Regan RM, et al. Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer. Cancer research. 2014. 2. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61-70. Citation Format: Deli Liu, Huan Xiong, Angela E. Ellis, Nicole C. Northrup, Carlos O. Rodriguez Jr, Ruth M. O9Regan, Stephen Dalton, Shaying Zhao. Spontaneous canine mammary cancers: A much-needed model for human basal-like breast cancer and an ideal system to understand myoepithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A07.

Abstract 5147: Canine spontaneous head and neck squamous cell carcinomas represent their human counterparts at the molecular level

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnSpontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better utilize this valuable resource, we performed its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers faithfully recapitulate many key molecular features of human HNSCC. These include similar genomic copy number abnormality landscapes, analogous sequence mutation patterns, and recurrent alteration of known HNSCC genes (e.g., MYC, CDKN2A) and pathways (e.g., cell cycle, mitogenic signaling, TGFβ signaling). Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial-mesenchymal transition genes TWIST1 and SNAI are also prominent in these canine tumors.nnThe strong dog-human molecular homology fundamentally justifies the use of our novel dog-human comparison strategy for driver-passenger discrimination for HNSCC. This cross-species comparison will significantly advance beyond the current findings with the massive genomic data of the cancer genome atlas (TCGA), as exemplified by our pilot study on human 8q, a recurrently amplified site in human HNSCC.nnHNSCC represents the sixth leading cancer by incidence in humans; thus developing effective therapeutic interventions is important. While our understanding of the biology of HNSCC has been greatly advanced over past decades, translating these findings into clinical success has been frustratingly slow. A significant obstacle is the lack of translational model that can bridge the gap between preclinical research and human clinical trials. Our study indicates that spontaneous canine HSCC can effectively bridge this gap, significantly accelerating anti-HNSCC drug discovery.nnNote: This abstract was not presented at the meeting.nnCitation Format: Deli Liu, Huan Xiong, Angela E. Ellis, Nicole C. Northrup, Dong M. Shin, Shaying Zhao. Canine spontaneous head and neck squamous cell carcinomas represent their human counterparts at the molecular level. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5147. doi:10.1158/1538-7445.AM2015-5147