Biology of Virus-associated Cancers

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

We have completed all analysis and the manuscript for solid organ transplant recipient (SOTR) bladder cancers. This study identified DNA viruses in over half of all tumors with BK polyomavirus (BKPyV) being the most abundant. BKPyV was found clonally integrated through microhomology-mediated end joining in many tumors, similar to HPV and MCPyV-associated cancers. Furthermore, integration associated with focal amplifications of the tumor genome and inactivation of the viral helicase domain. BKPyV+ tumors showed significant transcriptional changes consistent with the activity of the viral Large T antigen. Overall, this study provides highly detailed molecular characterization of SOTR bladder cancers and highlight more viral involvement in these tumors than previously appreciated. The manuscript is being resubmitted to eLife for review. To experimentally study the interplay between BKPyV and bladder cancer we have implemented several cellular models. First, using bladder cancer cell lines, we have identified several lines that are deficient for viral replication, which may provide insights into how tumors may lose viral gene expression in late-stage tumors, and one cell line highly susceptible for infection. Second, we've used a spontaneously immortalized urothelial cell line (HBLAK), which retains a normal morphology and ability to differentiate into stratified urothelium, to study virus infections in three dimensions. From these combined experiments, we've identified transcription factors (FOXA1 and GATA3) associated with cellular differentiation and overexpressed in luminal subtype bladder cancers that seem to associate with BKPyV replication. We have generated an HBLAK cell line overexpressing FOXA1 and preliminary experiments suggest that this can enhance BKPyV replication. Ongoing experiments are validating the mechanism by which this transcription factor is increasing viral replication To study genomic mutagenesis and instability caused by viral infection, We have also engineered bladder cells to remove the primary mutagenic enzymes responsible for the somatic mutations observed in bladder cancer and in BKPyV evolution (APOBEC3A and APOBEC3B). These lines will be used in with native infection and exogenous expression of viral oncogenes in both monolayer cultures and organotypic culture to study both host and viral mutagenesis and carcinogenic potential. In the above-mentioned cellular models, we are also using chromatin conformation sequencing in conjunction with knockdown of factors that control chromatin looping (CTCF, WAPL, RAD21) to study how these affect polyomavirus replication and erroneous integration into the host genome. While these models are used to study events prior and during integration, we are using newly derived virus-positive Merkel cell carcinoma cell lines to study how integrated viral DNAs in established cancers interact with host chromatin and affect cellular gene expression and chromatin conformation. Lastly, we have completed total RNA sequencing and whole genome sequencing of sebaceous neoplasm specimens, another cancer highly enriched in immunosuppressed patients, but without a clear infectious etiology. Preliminary analysis of these data suggest there are distinct viral genera present at different stages of tumorigenesis. Detailed analysis is ongoing.