Exon Array Analysis of Head and Neck Cancers Identifies a Hypoxia Related Splice Variant of LAMA3 Associated with a Poor Prognosis

The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes. The analysis identified a splice variant of LAMA3 (Laminin α 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative RT-PCR. In a series of 59 prospectively collected head and neck tumours, expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants

Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments

Investigating Molecular Drivers of Human Papillomavirus (HPV)-Related Oropharyngeal Cancer

There has been a growing epidemic of human papillomavirus (HPV)-induced oropharyngeal squamous cell carcinoma (OPSCC) for the last few decades. This is an underappreciated disease that can have a devastating effect on the lives of otherwise healthy young patients. Patients with HPV-positive OPSCC generally have a good prognosis, but still 20-30% fail to respond to therapy or later recur for unknown reasons. Our hypothesis is that the type of interactions between viral DNA and the human host DNA may determine a patient’s disease progression. HPV can remain its circular episomal shape, but often linearizes and integrates into the human genome, either into intergenic loci or into genes. The process of HPV integration is of particular interest as a potential driver of HPV-positive OPSCC because it is thought to be a marker of disease progression in cervical cancer and is reported in a large proportion of head and neck tumors with estimates ranging from 50-70%. Integration can lead to large structural variations, disrupt cellular genes and alter gene expression both locally and genome wide, but the exact effects of HPV integration on OPSCC progression are unclear. Previous studies that assessed survival differences between HPV integration positive versus negative patients demonstrated mixed results, so we aimed to clarify whether this process impacts patient outcomes. Using a polymerase chain reaction (PCR)-based approach, we found that HPV-positive, integration-positive patients had higher levels of the HPV oncogenes E6/E7 and a survival advantage over HPV-positive, integration-negative patients. The underlying mechanism for this improved outcome is unclear, but this work provides evidence that HPV integration could serve as a prognostic biomarker. We also utilized this methodology to investigate the clonal nature of HPV integration events, as it is clear that this process affects cell biology given the survival differences we discovered, but how cells containing these viral-human fusions may be selected for during tumor evolution is unclear. We explored the clonality of integration events in bilateral HPV+ tonsillar tumors and found evidence that these tumors often form as a result of clonal expansion from one tonsil to another given that we found shared integration sites across samples. These results indicate that integration events provide a survival advantage to tumor cells which are then selected for and expanded such that they are able to metastasize elsewhere. Finally, to overcome limitations of previous integration calling methodologies, we optimized a new targeted capture sequencing and analysis pipeline called SearcHPV. Through integrated analysis of HPV+ models by SearcHPV and genome-wide linked read sequencing, we demonstrated that HPV integration sites were found not only adjacent to known cancer-related genes such as TP63 and MYC, but also near regions of large structural variation in the tumor genome. Further, analysis of SearcHPV-assembled junction contigs demonstrated that the tool can be used to accurately identify viral-host junction sequences and showed that viral integration occurs through a variety of DNA repair mechanisms including non-homologous end joining, alternative end joining and microhomology mediated repair. Together, these studies highlight HPV genomic integration as an important contributor to cancer progression, and with new tools available, we believe the field is now primed to make major advances in the understanding of HPV-driven pathogenesis, some of which may lead to the development of novel biomarkers and/or treatment paradigms.PHDCancer BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/168121/1/lpinatti_1.pd

Exon array data analysis using Affymetrix power tools and R statistical software

The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform

Exon Array Analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Treatment of non-small cell lung cancer with novel targeted therapies is a major unmet clinical need. Alternative splicing is a mechanism which generates diverse protein products and is of functional relevance in cancer.</p> <p>Results</p> <p>In this study, a genome-wide analysis of the alteration of splicing patterns between lung cancer and normal lung tissue was performed. We generated an exon array data set derived from matched pairs of lung cancer and normal lung tissue including both the adenocarcinoma and the squamous cell carcinoma subtypes. An enhanced workflow was developed to reliably detect differential splicing in an exon array data set. In total, 330 genes were found to be differentially spliced in non-small cell lung cancer compared to normal lung tissue. Microarray findings were validated with independent laboratory methods for <it>CLSTN1</it>, <it>FN1</it>, <it>KIAA1217</it>, <it>MYO18A</it>, <it>NCOR2</it>, <it>NUMB</it>, <it>SLK</it>, <it>SYNE2</it>, <it>TPM1</it>, (in total, 10 events) and <it>ADD3</it>, which was analysed in depth. We achieved a high validation rate of 69%. Evidence was found that the activity of FOX2, the splicing factor shown to cause cancer-specific splicing patterns in breast and ovarian cancer, is not altered at the transcript level in several cancer types including lung cancer.</p> <p>Conclusions</p> <p>This study demonstrates how alternatively spliced genes can reliably be identified in a cancer data set. Our findings underline that key processes of cancer progression in NSCLC are affected by alternative splicing, which can be exploited in the search for novel targeted therapies.</p

Gene expression analysis of head and neck cancer development

Microarray analysis was performed on 32 head and neck keratinocytes cultures using Affymetrix U133A/B genechips. The panel of cultures included normal cells, mortal and immortal cultures of dysplastic keratinocytes and mortal and immortal cultures from carcinomas, all grown to a standard protocol. The overall GEP revealed that many of the well-established HNSCC molecular markers associated with motility and invasion were up-regulated in the mortal cells, particularly in the mortal carcinomas. Immortal NHSCC cells showed elevated expression of cell-cycle markers and loss of differentiation markers. In addition, a small number of common changes in gene expression in all the carcinomas, regardless of replicative fate, were identified. This included several transcription factors. A series of 49 novel gene expression changes consistently associated with immortality in dysplastic keratinocytes and SCCs were identified. The list included genes involves in cell cycle control, signalling, cellular metabolism and maintenance of cellular structure. Validation of the expression of these genes by western blot demonstrated that, in general, the protein expression of genes agreed with the RNA expression level from the microarray data. However, some heterogeneity was evident. The mortal and immortal gene expression signatures were validated by IHC in the tumours from which the cultures were derived. The tumours that gave rise to immortal cell cultures demonstrated a relatively uniform pattern of staining in relation to the novel markers of immortality. However, those tumours which gave rise to mortal cultures exhibited significant heterogeneity of gene expression pattern, with areas characteristic of both the mortal and immortal phenotype present. These novel markers give us further insight into the mechanisms and importance of keratinocytes immortalization. Surrogate markers of immortality could therefore be valuable for assessment of prognosis and therapy if confirmed in larger in vivo studies

Molecular genetic investigations of renal cell carcinoma predisposition

Renal Cell Carcinomas (RCC) are a diverse group of histologically and genetically distinct renal neoplasms accounting for 2.4% of all cancers worldwide. While a majority of RCC cases are sporadic in nature, a proportion are due to genetic predisposition caused by syndromic and non-syndromic conditions. Inherited renal cell carcinoma is associated with alterations in genes such as VHL, MET, FH, and FLCN and identification of these genes has been critical to understanding the molecular biology of both inherited and sporadic RCC, informing both clinical management and treatment. Despite the large number of known genes which are linked to RCC predisposition, most individuals with features of RCC predisposition do not harbour variants in known inherited RCC genes, suggesting additional unknown causes of heritability have yet to be uncovered. This study has utilised a range of genomic sequencing methodologies, scaling from single gene to whole genome sequencing, on individuals with features of renal cell carcinoma predisposition in order to identify novel causes of heritability associated with RCC. Multiple genomic sequencing approaches in these individuals has uncovered a range of potential genetic features that could be associated with predisposition to RCC, including genes not previously known to be associated with RCC, discovery of new molecular mechanisms of genetic inheritance for known RCC predisposition syndromes, and provided innovative methods for the identification and characterisation of molecular alterations in specific inherited RCC subtypes

Establishing the Role of Vitamin D signalling in Immunity and Melanoma Specific Survival

1α,25-dihydroxyvitamin D3 signals via its canonical nuclear receptor: Vitamin D Receptor (VDR). While higher levels of serum vitamin D have been reported to be associated with thinner primary melanomas and better outcome, increased VDR expression has been associated with decreased tumour progression and improved prognosis in melanoma primaries. However, the genomic basis of this effect remains to be explored and a causal mechanism is yet to be established. To address this question, I have used microarray data from a cohort of 703 treatment-naïve primary melanomas from the Leeds Melanoma Cohort (LMC) and corresponding clinical data. In the LMC primary melanomas, serum vitamin D was not significantly associated with melanoma survival. However, tumour VDR expression was significantly (and independently) protective for melanoma death in both the LMC and the TCGA metastatic melanoma datasets. Tumour VDR expression was found to be significantly positively correlated with genes enriched for ECM organization, TNF signalling, IFNg signalling, IL12-mediated signalling and NFkB signalling, which are predominantly immune-related. Concordantly, VDR expression was lower in tumours graded by the pathologist as having no immune infiltrate, compared to tumours with brisk and nonbrisk immune infiltrate. Additionally, VDR correlated positively with imputed immune cells scores. Conversely, the negatively correlated genes were enriched for Mitotic Prophase, Wnt signalling pathway, Mitochondrial translation, citric acid cycle and oxidative phosphorylation, which are predominantly proliferation-related. Of particular interest among the negatively correlated pathways was the Wnt/b-catenin signalling pathway. Functional validation using an in vivo tail-vein metastasis assay revealed that murine melanoma cells stably expressing VDR produced significantly fewer pulmonary metastases compared to control cells with null VDR expression. VDR-expressing cells also had significantly lower expression of Wnt/b-catenin signalling genes compared to control cells. These findings indicate that vitamin D-VDR signalling contributes to control of pro-proliferative and immunosuppressive Wnt/b-catenin signalling in melanoma and that this is associated with less proliferative, less metastatic disease and stronger host immune responses