Multi-omic investigation of the mechanisms underlying the pathobiology of head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy associated with molecular heterogeneity, locoregional spread, resistance to therapy and relapse after initial treatment. Increasing evidence suggests that master developmental pathways with key roles in adult tissue homeostasis, including Hippo and Wnt/β-catenin signaling, are dysregulated in the initiation and progression of HNSCC. However, a comprehensive investigation into the crosstalk between these pathways is currently lacking, and may prove crucial to the discovery of novel targets for HNSCC therapy. More recent evidence points to the tumor microenvironment, mainly comprising cancer-associated fibroblasts (CAFs), as capable of influencing tumor cell behavior and promoting invasive HNSCC phenotypes. Nonetheless, current methods to screen for CAF markers in tumors are restricted to targeted immunostaining experiments with limited success and robustness across tissue types. The Cancer Genome Atlas network has generated multi-tiered molecular profiles for over 10,000 tumors spanning more than two dozen different cancer types, providing an unprecedented opportunity for the application and development of integrative methods aimed at the in silico interrogation of experimentally-derived signatures. These multi-omic profiles further enable one to link genomic anomalies, including somatic mutations and DNA copy number alterations, with phenotypic effects driven by pathogenic pathway activity. Effectively querying this vast amount of information to help elucidate subsets of functionally and clinically-relevant oncogenic drivers, however, remains an ongoing challenge. To address these issues, I first investigate the effects of oncogenic pathway perturbation in HNSCC using experimental models coupled with in vitro genome-wide transcriptional profiling. Next, I describe a new computational approach for the unbiased identification of CAF markers in HNSCC solely using bulk tumor RNA-sequencing information. Lastly, I have developed Candidate Driver Analysis or CaDrA - a statistical framework that allows one to query genetic and epigenetic alterations for candidate drivers of signature activity within a given disease context. Collectively, this work offers new perspectives on the molecular cues underlying HNSCC development, while simultaneously highlighting the power of integrative genomics methods capable of accelerating the discovery of novel targets for cancer diagnosis and therapy

CaDrA: A Computational Framework for Performing Candidate Driver Analyses Using Genomic Features

The identification of genetic alteration combinations as drivers of a given phenotypic outcome, such as drug sensitivity, gene or protein expression, and pathway activity, is a challenging task that is essential to gaining new biological insights and to discovering therapeutic targets. Existing methods designed to predict complementary drivers of such outcomes lack analytical flexibility, including the support for joint analyses of multiple genomic alteration types, such as somatic mutations and copy number alterations, multiple scoring functions, and rigorous significance and reproducibility testing procedures. To address these limitations, we developed Candidate Driver Analysis or CaDrA, an integrative framework that implements a step-wise heuristic search approach to identify functionally relevant subsets of genomic features that, together, are maximally associated with a specific outcome of interest. We show CaDrA’s overall high sensitivity and specificity for typically sized multi-omic datasets using simulated data, and demonstrate CaDrA’s ability to identify known mutations linked with sensitivity of cancer cells to drug treatment using data from the Cancer Cell Line Encyclopedia (CCLE). We further apply CaDrA to identify novel regulators of oncogenic activity mediated by Hippo signaling pathway effectors YAP and TAZ in primary breast cancer tumors using data from The Cancer Genome Atlas (TCGA), which we functionally validate in vitro. Finally, we use pan-cancer TCGA protein expression data to show the high reproducibility of CaDrA’s search procedure. Collectively, this work demonstrates the utility of our framework for supporting the fast querying of large, publicly available multi-omics datasets, including but not limited to TCGA and CCLE, for potential drivers of a given target profile of interest

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 Huntington's 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

MicroRNAs located in the Hox gene clusters are implicated in huntington\u27s disease pathogenesis

Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington\u27s 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 \u3c 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

Background: MicroRNAs (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. Methods: We 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. Results: We 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. Conclusions: These 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. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0083-3) contains supplementary material, which is available to authorized users

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

<div><p>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.</p></div

PDGFRβ localizes primarily to the surrounding stroma in OSCC.

<p><b>A</b>. PDGFRβ staining is absent in adjacent epithelium and hyperplasia samples, but is present in ulcer and OSCC specimens. Red arrows indicate blood vessels and green arrows indicate fibroblasts. <b>B</b>. Picrosirius red staining of collagen 1 and 3 fibers (left) and immunostaining of PDGFRβ (middle) and additional marker periostin (right) that were identified from the NN analysis.</p

Gene expression profile of the top 50 hits (53 including <i>COL1A1</i>, <i>COL1A2</i> and <i>COL3A1</i>) obtained from the nearest neighbor (NN) gene expression analysis using mean collagen expression as a reference for TCGA OSCC data.

<p>Only samples with known tumor grade are highlighted. Expression values were log₂-transformed after adding a pseudo-count of 1 and standardized per gene prior to their visualization. AE: Adjacent normal epithelium.</p

PDGFRβ Is a Novel Marker of Stromal Activation in Oral Squamous Cell Carcinomas - Fig 5

<p>Pan-cancer NN analysis highlights PDGFRβ as a potential CAF marker in multiple cancer types A. Heatmap of average pairwise Kolmogorov-Smirnoff (KS) D statistics using the top 50 genes in the collagen NN cancer analysis of each of 11 cancer types. <b>B</b>. Venn diagram of top 50 genes in collagen NN ranked list within the five most closely related cancers (based on the average KS D statistic; Fig 5A). Core overlap has 11 genes, which includes PDGFRβ, highlighted in red. ACC: Adrenocortical carcinoma; BLCA: Bladder urothelial carcinoma; COAD: Colon adenocarcinoma; KIRC: Kidney renal clear cell carcinoma; LAML: Acute myeloid leukemia; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; PAAD: Pancreatic adenocarcinoma; PRAD: Prostate adenocarcinoma.</p