Maggie Cam

Hepatic gene expression during treatment with peginterferon and ribavirin: Identifying molecular pathways for treatment response.

The reasons for hepatitis C treatment failure remain unknown but may be related to different host responses to therapy. In this study, we compared hepatic gene expression in patients prior to and during peginterferon and ribavirin therapy. In the on‐treatment group, patients received either ribavirin for 72 hours prior to peginterferon alpha‐2a injection or peginterferon alpha‐2a for 24 hours, prior to biopsy. The patients were grouped into rapid responders (RRs) with a greater than 2‐log drop and slow responders (SRs) with a less than 2‐log drop in hepatitis C virus RNA by week 4. Pretreatment biopsy specimens were obtained from a matched control group. The pretreatment patients were grouped as RRs or SRs on the basis of the subsequent treatment response. Gene expression profiling was performed with Affymetrix microarray technology. Known interferon‐stimulated genes (ISGs) were induced in treated patients. In the pretreatment group, future SRs had higher pretreatment ISG expression than RRs. On treatment, RRs and SRs had similar absolute ISG expression, but when it was corrected for the baseline expression with the pretreatment group, RRs showed a greater fold change in ISGs, whereas SRs showed a greater change in interferon (IFN)‐inhibitory pathways. The patients pretreated with ribavirin had heightened induction of IFN‐related genes and down‐regulation of genes involved in IFN inhibition and hepatic stellate cell activation. Conclusion: These data suggest that ISG inducibility is important for the treatment response and that ribavirin may improve outcomes by enhancing hepatic gene responses to peginterferon. Collectively, these mechanisms may provide a molecular basis for the improved efficacy of combination therapy. (HEPATOLOGY 2007.)

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR–Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer

Familial nonmedullary thyroid cancer accounts for 3 to 9% of all cases of thyroid cancer, but the susceptibility genes are not known. Here, we report a germline variant of HABP2 in seven affected members of a kindred with familial nonmedullary thyroid cancer and in 4.7% of 423 patients with thyroid cancer. This variant was associated with increased HABP2 protein expression in tumor samples from affected family members, as compared with normal adjacent thyroid tissue and samples from sporadic cancers. Functional studies showed that HABP2 has a tumor-suppressive effect, whereas the G534E variant results in loss of function.

Histone Demethylase Jumonji D3 (JMJD3) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

Identification of novel RNA isoforms of LMNA

ABSTRACT The nuclear lamina is a proteinaceous meshwork situated underneath the inner nuclear membrane and is composed of nuclear lamin proteins, which are type-V intermediate filaments. The LMNA gene gives rise to lamin A and lamin C through alternative splicing. Mutations in LMNA cause multiple diseases known as laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder caused by a point mutation that activates a cryptic 5′ splice site in exon 11, resulting in a 150 bp deletion in the LMNA mRNA and the production of the dominant lamin A isoform progerin. During RNA sequencing analysis of wild type and HGPS patient skin fibroblasts, we discovered two novel LMNA isoforms. LMNAΔ447 and LMNAΔ297 use an alternative 3′ splice acceptor site in the 3′ untranslated region, and either the HGPS cryptic 5′ splice site in exon 11 or the wild type 5′ splice site. Both isoforms are present at low levels in HGPS patient and wild type cells in multiple cell types. We validate and quantify the expression levels of these novel isoforms in HGPS and wild type fibroblasts. Overexpression of either LMNAΔ447 or LMNAΔ297 is not sufficient to induce the typical HGPS cellular disease phenotypes and no significant difference in the two isoforms were found between young and old fibroblasts. These results identify and characterize two novel RNA isoforms of LMNA produced through alternative splicing.

ONC201 kills breast cancer cells in vitro by targeting mitochondria

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Abstract LB-173: The Thailand initiative in genomics and expression research for liver cancer (TIGER-LC): Defining novel subtypes of hepatocellular carcinoma and cholangiocarcinoma

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent two major histological cancer subtypes confined within the liver. They are clinically and biologically heterogeneous, and are highly resistant to treatment, which makes them the second most lethal cancer for men in the world. In Thailand, liver cancer represents the primary cause of cancer-related death and is a major health problem, especially in north-eastern area of Thailand where liver fluke (O. viverrini) is endemic and approximately 70% of liver cancers are CCA. While HBV and HCV are major etiological factors for HCC globally, liver fluke infection is a major etiological factor for CCA in Thailand. These unique risk factor patterns provide an opportunity to study cancer heterogeneity and its unique tumor biology. The Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) consortium was established to identify genomic and expression factors that may modify HCC and CCA susceptibility and progression. In a Phase I study, we determined molecular subtypes of HCC and CCA. We performed genomic profiling of 398 surgical specimens derived from 199 liver cancer patients. We employed the Affymetrix Human Transcriptome Array 2.0 to examine transcriptome profiles. Unsupervised Consensus Clustering (cCluster), Subclass Mapping (SM) and Gene Set Enrichment Analysis (GSEA) algorithms were used to analyze transcriptome data. The results were validated in 247 Asian HCC cases and 104 Caucasian CCA cases. We found that the Thai HCC cases consisted of 3 stable subgroups (C1-C3), while the Thai CCA cases contained 4 stable subgroups (C1-C4) based on gene expression patterns determined by cCluster. SM analysis revealed that CCA-C1 and HCC-C1 subtypes shared a similar gene expression matrix, as did CCA-C2 and HCC-C2 for a separate pattern. Interestingly, patients in both CCA-C1 and HCC-C1 had a poor prognosis, while those in CCA-C2 and HCC-C2 had a good prognosis. These prognostic subtypes were validated in an independent Asian HCC cohort but not in a Caucasian CCA cohort. GSEA revealed that among 17 significantly altered canonical pathways in the C1 subtype, 8 are related to mitotic checkpoint signaling. In contrast, the main signaling pathways associated with the C2 subtype were related to cytokine and chemokine signaling. We found that certain mitotic checkpoint genes are highly activated only in C1, but not in the C2 subtype. These results are consistent with the hypothesis that CCA and HCC from Asian populations consist of molecularly-similar tumor subgroups with similar prognostic impacts and unique tumor biology and that the C1 subtype may be sensitive to mitotic checkpoint blockage. Our ability to rigorously classify and validate both HCC and CCA using these tools may represent a new avenue for the development of targeted therapeutic interventions. Citation Format: The TIGER-LC Consortium, Jittiporn Chaisaingmongkol, Anuradha Budhu, Hien Dang, Siritida Rabibhadana, Benjarath Pupacdi, Marshonna Forgues, Vajarabhongsa Bhudhisawasdi, Nirush Lertprasertsuke, Anon Chotirosniramit, Chawalit Pairojkul, Chirayu U. Auewarakul, Thaniya Sricharunrat, Kannika Phornphutkul, Suleeporn Sangrajrang, Maggie Cam, Ping He, Stephen M. Hewitt, Xiaolin Wu, Snorri S. Thorgeirsson, Paul S. Meltzer, Christopher A. Loffredo, Robert H. Wiltrout, Curtis C. Harris, Chulabhorn Mahidol, Mathuros Ruchirawat, Xin W. Wang. The Thailand initiative in genomics and expression research for liver cancer (TIGER-LC): Defining novel subtypes of hepatocellular carcinoma and cholangiocarcinoma. [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 LB-173. doi:10.1158/1538-7445.AM2015-LB-173

Transcriptional Alterations in Hereditary and Sporadic Nonfunctioning Pancreatic Neuroendocrine Tumors According to Genotype.

Nonfunctioning pancreatic neuroendocrine tumors (NFPanNETs) may be sporadic or inherited because of germline mutations associated with von Hippel‐Lindau disease (VHL) or multiple endocrine neoplasia type 1 (MEN1). The clinical behavior of NFPanNETs is difficult to predict, even in tumors of the same stage and grade. The authors analyzed genotype‐specific patterns of transcriptional messenger RNA (mRNA) levels of NFPanNETs to understand the molecular features that determine PanNET phenotype.

Abstract 4390: The Thailand initiative in genomics and expression research in liver cancer: Race related common molecular subtypes among Asian hepatocellular carcinoma and cholangiocarcinoma identified by integrated genomics

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are two distinct histological liver cancers. They are clinically and biologically heterogeneous and highly resistant to treatment, making liver cancer the second most lethal malignancy in the world. In Thailand, liver cancer represents the primary cause of cancer-related death and is a major health problem. While HBV and HCV are major etiological factors for HCC globally, liver fluke infection (O. viverrini) is a major etiological factor for ICC in Thailand, especially in north-eastern Thailand where O. viverrini is endemic and approximately 70% of liver cancers are ICC. These unique risk factor patterns provide an opportunity to study cancer heterogeneity and unique liver tumor biology. The Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC) consortium was established to identify genomic and expression factors that may modify HCC and ICC susceptibility and progression. Here, we determined molecular subtypes and features of HCC and ICC through systems integration of genomic, transcriptomic and metabolic profiles. We performed genome wide profiling of 398 surgical specimens derived from 199 Thai liver cancer patients. We employed the Affymetrix Human Transcriptome Array 2.0, the Affymetrix Genome-Wide Human SNP Array 6.0, Metabolon9s DiscoveryHD4 platform and Exome Sequencing to examine transcriptome profiles, somatic copy number alterations (SCNA), cancer metabolic profiles and mutation patterns, respectively. The results were validated in 847 independent Asian or Caucasian HCC or ICC cases. Transcriptomic analyses revealed that Thai HCC consisted of 3 stable subgroups (C1-C3), while Thai ICC contained 4 stable subgroups (C1-C4). Interestingly, HCC-C1 and ICC-C1 subtypes shared a similar gene expression matrix, as did HCC-C2 and ICC-C2, which correlated with patient survival. These prognostic subtypes were validated in independent Asian HCC and ICC cohorts, but not in Caucasian patients, and were associated with tumor biology rather than etiology. GSEA revealed that the C1 subtype is enriched for mitotic checkpoint anomalies, while the C2 subtype is related to cytokine and chemokine signaling. We found that the C1 subtype encompassed a higher degree of SCNA when compared to the C2 subtype, suggesting an association with a genomic instability phenotype. Further analysis showed that the C2 subtype is linked to an increased body mass index, inflammatory responses and unique tumor metabolic activities. HCC and ICC from Asian populations, while clinically treated as separate entities, share common subtypes with similar actionable drivers which can be targeted to improve precision therapy. Citation Format: Anuradha Budhu, Jittiporn Chaisaingmongkol, Hien Dang, Siritida Rabibhadana, Benjarath Pupacdi, So Mee Kwon, Marshonna Forgues, Yotsawat Pomyen, Vajarabhongsa Bhudhisawasdi, Nirush Lertprasertsuke, Anon Chotirosniramit, Chawalit Pairojkul, Chirayu U. Auewarakul, Thaniya Sricharunrat, Kannika Phornphutkul, Suleeporn Sangrajrang, Maggie Cam, Ping He, Stephen M. Hewitt, Xiaolin Wu, Snorri S. Thorgeirsson, Joshua J. Waterfall, Yuelin J. Zhu, Jennifer Walling, Holly S. Stevenson, Daniel Edelman, Paul S. Meltzer, Christopher A. Loffredo, Robert H. Wiltrout, Curtis C. Harris, Chulabhorn Mahidol, Mathuros Ruchirawat, Xin W. Wang. The Thailand initiative in genomics and expression research in liver cancer: Race related common molecular subtypes among Asian hepatocellular carcinoma and cholangiocarcinoma identified by integrated genomics [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 4390. doi:10.1158/1538-7445.AM2017-4390