Shuyun Rao

Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor-β Pathway

BACKGROUND & AIMSnDevelopment of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor-beta (TGF-β) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets.nnnMETHODSnWe performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3).nnnRESULTSnWe found somatic mutations in at least 1 gene whose product is a member of TGF-β signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12 of 202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGF-β signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGF-β signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGF-β signaling had shorter survival times than patients with tumors with activation of TGF-β signaling (Pxa0= .0129). Patterns of TGF-β signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, and SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared with cells that expressed normal SPTBN1 (controls).nnnCONCLUSIONSnIn genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGF-β signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; up-regulation of genes in this pathway would contribute to inflammation and fibrosis, whereas down-regulation would indicate loss of TGF-β tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGF-β pathway; agents that block TGF-β should be used only in patients with specific types of HCCs.

Transforming growth factor-β in liver cancer stem cells and regeneration

Cancer stem cells have established mechanisms that contribute to tumor heterogeneity as well as resistance to therapy. Over 40% of hepatocellular carcinomas (HCCs) are considered to be clonal and arise from a stem‐like/cancer stem cell. Moreover, HCC is the second leading cause of cancer death worldwide, and an improved understanding of cancer stem cells and targeting these in this cancer are urgently needed. Multiple studies have revealed etiological patterns and multiple genes/pathways signifying initiation and progression of HCC; however, unlike the transforming growth factor β (TGF‐β) pathway, loss of p53 and/or activation of β‐catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF‐β signaling in gastrointestinal cancers, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support effective targeted therapies for these common and lethal cancers. Moreover, pathways involved in stem cell transformation into gastrointestinal cancers remain largely undefined. Identifying the key mechanisms and developing models that reflect the human disease can lead to effective new treatment strategies. In this review, we dissect the evidence obtained from mouse and human liver regeneration, and mouse genetics, to provide insight into the role of TGF‐β in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477–493)

Alcohol, stem cells and cancer

Dosage, gender, and genetic susceptibility to the effects of alcohol remained only partially elucidated. In this review, we summarize the current knowledge of the mechanisms underlying the role of alcohol in liver and gastrointestinal cancers. In addition, two recent pathways- DNA repair and TGF-β signaling which provide new insights into alcohol in the regulation of cancers and stem cells are also discussed here.

PRAJA is overexpressed in glioblastoma and contributes to neural precursor development

PRAJA, a RING-H2 E3 ligase, is abundantly expressed in brain tissues such as the cerebellum and frontal cortex, amongst others, and more specifically in neural progenitor cells as well as in multiple cancers that include glioblastomas. However, the specific role that Praja plays in neural development and gliomas remains unclear. In this investigation, we performed bioinformatic analyses to examine Praja1 and Praja2 expression across 29 cancer types, and observed raised levels of Praja1 and Praja2 in gliomas with an inverse relationship between Praja1 and apoptotic genes and Praja substrates such as Smad3. We analyzed the role of Praja in the developing brain through loss of function studies, using morpholinos targeting Praja1 in embryonic zebrafish, and observed that Praja1 is expressed prominently in regions enriched with neural precursor cell subtypes. Antisense Praja morpholinos resulted in multiple embryonic defects including delayed neural development likely through increased apoptosis. Further studies revealed high levels of Cdk1 with loss of Praja1 in TGF-β or insulin treated cells, supporting the link between Praja1 and cell cycle regulation. In summary, these studies underscore Prajas role in mammalian brain development and Praja1 deregulation may lead to gliomas possibly through the regulation of cell cycle and/or apoptosis.

Baseline human gut microbiota profile in healthy people and standard reporting template

A comprehensive knowledge of the types and ratios of microbes that inhabit the healthy human gut is necessary before any kind of pre-clinical or clinical study can be performed that attempts to alter the microbiome to treat a condition or improve therapy outcome. To address this need we present an innovative scalable comprehensive analysis workflow, a healthy human reference microbiome list and abundance profile (GutFeelingKB), and a novel Fecal Biome Population Report (FecalBiome) with clinical applicability. GutFeelingKB provides a list of 157 organisms (8 phyla, 18 classes, 23 orders, 38 families, 59 genera and 109 species) that forms the baseline biome and therefore can be used as healthy controls for studies related to dysbiosis. The incorporation of microbiome science into routine clinical practice necessitates a standard report for comparison of an individual’s microbiome to the growing knowledgebase of “normal” microbiome data. The FecalBiome and the underlying technology of GutFeelingKB address this need. The knowledgebase can be useful to regulatory agencies for the assessment of fecal transplant and other microbiome products, as it contains a list of organisms from healthy individuals. In addition to the list of organisms and abundances the study also generated a list of contigs of metagenomics dark matter. In this study, metagenomic dark matter represents sequences that cannot be mapped to any known sequence but can be assembled into contigs of 10,000 nucleotides or higher. These sequences can be used to create primers to study potential novel organisms. All data is freely available from https://hive.biochemistry.gwu.edu/gfkb and NCBI’s Short Read Archive.

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA’s research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.

Abstract 5330: Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases

Hepatocellular carcinoma (HCC) is the 3 rd leading cause of cancer deaths worldwide, and rising in the United States at an alarming rate. Multiple E3 ubiquitin ligases such as the SMURFs and RINGH2 proteins have been identified as negative regulators of the TGF-β pathway. However, to our knowledge, there remains a gap in the integration between genomics, underlying mechanisms and the development of targeted therapeutics harnessing these TGF-β-associated E3 ligases for HCC. The aim of this study is to elucidate the role of E3 ligases in HCC, through TCGA analyses and provide mechanistic insight into these as therapeutic targets for HCC. We first analyzed the 488 hepatocellular cancers and screened for alterations in The Cancer Genome Atlas (TCGA). Immunohistochemistry (IHC), Q-PCR, Western blot analysis were used to validate the expression levels of two of the most highly altered E3 ligases, PRAJA and Keap1 in hepatocellular cancer tissues and cell lines in human and in TGF-β-deficient β2SP +/- mouse models. Inhibition studies of PRAJA and Keap1 were performed by lentivirus shRNA in HCC cell lines, and xenograft studies. From the TCGA data, we observe two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the “activated” TGF-β signature (p=0.0027). We next analyzed 29 TGF-β-related E3 ligases, and observed raised expression of the following: PRAJA1 (12.7% of HCCs), KEAP1 (6.4%), UCHL5 (16.4%), WWP2 (11.8%), WWP1 (10%), Smurf2 (9.1%), Skp2 (9.1%), and Smurf1 (8.2%). Interestingly, expression patterns corresponded with a few TGF-β signaling members regulated by some of these E3 ligases, namely Smad3 (altered in 54%) and β2SP (27%). We identified that PRAJA1 targets Smad3 and β2SP for ubiquitination and degradation. We further observe raised levels of PRAJA (25%) and KEAP1 (70%) in 176 human liver cancers, by IHC, compared to normal controls. Depletion of PRAJA and KEAP1 with either shRNAs or E3 ligase inhibitors, substantially inhibited growth and induced apoptosis through PRAJA/Smad3/β2SP and KEAP1/Nrf signaling in HCC cell lines and xenografts. These results suggest that E3 ligases such as PRAJA1 and KEAP1 may be valuable therapeutic targets for liver cancer in the context of TGF-β signaling, an important approach given that few effective targeted therapeutics are available for this cancer with poor prognosis. Citation Format: Shuyun Rao, Heather Levin, Jian Chen, Rehan Akbani, Jon White, Wilma Jogunoori, Shoujun Gu, Kazufumi Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra. Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases [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 5330. doi:10.1158/1538-7445.AM2017-5330