Vinay K. Kartha

MicroRNAs Located in the Hox Gene Clusters Are Implicated in Huntington's Disease Pathogenesis

Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntingtons disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value<0.05). Three of these, miR-196a-5p, miR-196b-5p and miR-615-3p, were expressed at near zero levels in control brains. Expression was verified for all five miRNAs using reverse transcription quantitative PCR and all but miR-1247-5p were replicated in an independent sample (8HD/8C). Ectopic miR-10b-5p expression in PC12 HTT-Q73 cells increased survival by MTT assay and cell viability staining suggesting increased expression may be a protective response. All of the miRNAs but miR-1247-5p are located in intergenic regions of Hox clusters. Total mRNA sequencing in the same samples identified fifteen of 55 genes within the Hox cluster gene regions as differentially expressed in HD, and the Hox genes immediately adjacent to the four Hox cluster miRNAs as up-regulated. Pathway analysis of mRNA targets of these miRNAs implicated functions for neuronal differentiation, neurite outgrowth, cell death and survival. In regression models among the HD brains, huntingtin CAG repeat size, onset age and age at death were independently found to be inversely related to miR-10b-5p levels. CAG repeat size and onset age were independently inversely related to miR-196a-5p, onset age was inversely related to miR-196b-5p and age at death was inversely related to miR-615-3p expression. These results suggest these Hox-related miRNAs may be involved in neuroprotective response in HD. Recently, miRNAs have shown promise as biomarkers for human diseases and given their relationship to disease expression, these miRNAs are biomarker candidates in HD.

miR-10b-5p expression in Huntington’s disease brain relates to age of onset and the extent of striatal involvement

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that recognize sites of complementarity of target messenger RNAs, resulting in transcriptional regulation and translational repression of target genes. In Huntington’s disease (HD), a neurodegenerative disease caused by a trinucleotide repeat expansion, miRNA dyregulation has been reported, which may impact gene expression and modify the progression and severity of HD.MethodsWe performed next-generation miRNA sequence analysis in prefrontal cortex (Brodmann Area 9) from 26 HD, 2 HD gene positive, and 36 control brains. Neuropathological information was available for all HD brains, including age at disease onset, CAG-repeat size, Vonsattel grade, and Hadzi-Vonsattel striatal and cortical scores, a continuous measure of the extent of neurodegeneration. Linear models were performed to examine the relationship of miRNA expression to these clinical features, and messenger RNA targets of associated miRNAs were tested for gene ontology term enrichment.ResultsWe identified 75 miRNAs differentially expressed in HD brain (FDR q-value <0.05). Among the HD brains, nine miRNAs were significantly associated with Vonsattel grade of neuropathological involvement and three of these, miR-10b-5p, miR-10b-3p, and miR-302a-3p, significantly related to the Hadzi-Vonsattel striatal score (a continuous measure of striatal involvement) after adjustment for CAG length. Five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-10b-3p, and miR-106a-5p) were identified as having a significant relationship to CAG length-adjusted age of onset including miR-10b-5p, the mostly strongly over-expressed miRNA in HD cases. Although prefrontal cortex was the source of tissue profiled in these studies, the relationship of miR-10b-5p expression to striatal involvement in the disease was independent of cortical involvement. Correlation of miRNAs to the clinical features clustered by direction of effect and the gene targets of the observed miRNAs showed association to processes relating to nervous system development and transcriptional regulation.ConclusionsThese results demonstrate that miRNA expression in cortical BA9 provides insight into striatal involvement and support a role for these miRNAs, particularly miR-10b-5p, in HD pathogenicity. The miRNAs identified in our studies of postmortem brain tissue may be detectable in peripheral fluids and thus warrant consideration as accessible biomarkers for disease stage, rate of progression, and other important clinical characteristics of HD.

A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. Implications: This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer. Mol Cancer Res; 13(6); 957–68. ©2015 AACR.

PDGFRβ Is a Novel Marker of Stromal Activation in Oral Squamous Cell Carcinomas.

Carcinoma associated fibroblasts (CAFs) form the main constituents of tumor stroma and play an important role in tumor growth and invasion. The presence of CAFs is a strong predictor of poor prognosis of head and neck squamous cell carcinoma. Despite significant progress in determining the role of CAFs in tumor progression, the mechanisms contributing to their activation remain poorly characterized, in part due to fibroblast heterogeneity and the scarcity of reliable fibroblast surface markers. To search for such markers in oral squamous cell carcinoma (OSCC), we applied a novel approach that uses RNA-sequencing data derived from the cancer genome atlas (TCGA). Specifically, our strategy allowed for an unbiased identification of genes whose expression was closely associated with a set of bona fide stroma-specific transcripts, namely the interstitial collagens COL1A1, COL1A2, and COL3A1. Among the top hits were genes involved in cellular matrix remodeling and tumor invasion and migration, including platelet-derived growth factor receptor beta (PDGFRβ), which was found to be the highest-ranking receptor protein genome-wide. Similar analyses performed on ten additional TCGA cancer datasets revealed that other tumor types shared CAF markers with OSCC, including PDGFRβ, which was found to significantly correlate with the reference collagen expression in ten of the 11 cancer types tested. Subsequent immunostaining of OSCC specimens demonstrated that PDGFRβ was abundantly expressed in stromal fibroblasts of all tested cases (12/12), while it was absent in tumor cells, with greater specificity than other known markers such as alpha smooth muscle actin or podoplanin (3/11). Overall, this study identified PDGFRβ as a novel marker of stromal activation in OSCC, and further characterized a list of promising candidate CAF markers that may be relevant to other carcinomas. Our novel approach provides for a fast and accurate method to identify CAF markers without the need for large-scale immunostaining experiments.

Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis

Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

Functional and genomic analyses reveal therapeutic potential of targeting β-catenin/CBP activity in head and neck cancer

BackgroundHead and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by tumor heterogeneity, locoregional metastases, and resistance to existing treatments. Although a number of genomic and molecular alterations associated with HNSCC have been identified, they have had limited impact on the clinical management of this disease. To date, few targeted therapies are available for HNSCC, and only a small fraction of patients have benefited from these treatments. A frequent feature of HNSCC is the inappropriate activation of β-catenin that has been implicated in cell survival and in the maintenance and expansion of stem cell-like populations, thought to be the underlying cause of tumor recurrence and resistance to treatment. However, the therapeutic value of targeting β-catenin activity in HNSCC has not been explored.MethodsWe utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between β-catenin and cAMP-responsive element binding (CREB)-binding protein (CBP) using the small molecule inhibitor ICG-001. We generated and annotated in vitro treatment gene expression signatures of HNSCC cells, derived from human oral squamous cell carcinomas (OSCCs), using microarrays. We validated the anti-tumorigenic activity of ICG-001 in vivo using SCC-derived tumor xenografts in murine models, as well as embryonic zebrafish-based screens of sorted stem cell-like subpopulations. Additionally, ICG-001-inhibition signatures were overlaid with RNA-sequencing data from The Cancer Genome Atlas (TCGA) for human OSCCs to evaluate its association with tumor progression and prognosis.ResultsICG-001 inhibited HNSCC cell proliferation and tumor growth in cellular and murine models, respectively, while promoting intercellular adhesion and loss of invasive phenotypes. Furthermore, ICG-001 preferentially targeted the ability of subpopulations of stem-like cells to establish metastatic tumors in zebrafish. Significantly, interrogation of the ICG-001 inhibition-associated gene expression signature in the TCGA OSCC human cohort indicated that the targeted β-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival.ConclusionsCollectively, our results identify β-catenin/CBP interaction as a novel target for anti-HNSCC therapy and provide evidence that derivatives of ICG-001 with enhanced inhibitory activity may serve as an effective strategy to interfere with aggressive features of HNSCC.

CaDrA: A computational framework for performing candidate driver analyses using binary genomic features

Identifying complementary genetic drivers of a given phenotypic outcome is a challenging task that is important to gaining new biological insight and discovering targets for disease therapy. Existing methods aimed at achieving this task lack analytical flexibility. We developed Candidate Driver Analysis or CaDrA, a framework to identify functionally-relevant subsets of binary genomic features that, together, are associated with a specific outcome of interest. We evaluate CaDrA’s sensitivity and specificity for typically-sized multi-omic datasets, and demonstrate CaDrA’s ability to identify both known and novel drivers of oncogenic activity in cancer cell lines and primary tumors.

Abstract 803: Targeting β-catenin/CBP signaling in OSCC

Objectives: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype. Methods: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24 hi CD29 hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24 low CD29 low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice. Results: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24 hi CD2 hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24 low CD29 low . Treatment of CD44+CD24 hi CD29 hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy. Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000. Citation Format: Khalid Alamoud, Khikmet Sadykov, Vinay Kartha, Stefano Monti, Anna Belkina, Jennifer Snyder-Cappione, Sara Pai, Maria Kukuruzinska. Targeting β-catenin/CBP signaling in OSCC [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 803. doi:10.1158/1538-7445.AM2017-803