David Ting, MD
Mass General Cancer Center
Assistant Professor in Medicine
Harvard Medical School
Pancreatic cancer remains one of the most deadly cancers where the vast majority of patients are diagnosed too late and conventional therapies have largely been ineffective, making early detection and novel drug targets greatly needed. RNA sequencing technologies have recently provided unprecedented resolution of how cancer cells behave. Recent analysis of pancreatic tumors has found a significant amount of “non-coding” RNAs being produced in cancer cells, but not in normal tissues that have provided new insight into this disease and has implications as novel early detection biomarkers. In addition, the Ting Laboratory has been utilizing innovative microfluidic chip technologies to capture circulating tumor cells (CTCs) in the blood of pancreatic cancer patients as a means to understand why pancreatic cancers spread so quickly and as a potential non-invasive tool to diagnose our patients earlier.
David Ting, MD
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5% overall survival at 5 years, and therefore, new strategies for early detection and therapeutics are greatly needed. The Ting Laboratory has utilized RNA-sequencing technology to understand the complex transcriptional landscape of PDAC. We have used this technology to identify non-coding sequences (ncRNA) that are differentially expressed in cancer versus normal tissues. This has provided novel insight into the pathogenesis of PDAC and offers a method to identify novel biomarkers and therapeutic targets. In addition, we have been able to capture pancreatic circulating tumor cells (CTCs) with an innovative microfluidic chip technology and successfully applied RNA-sequencing to these cells to understand their role in the metastatic cascade.
Satellite Non-coding RNAs
RNA sequencing of primary PDAC tumors and a variety of normal tissues demonstrated that approximately half of all PDAC transcripts sequenced were unannotated, while nearly all reads in normal pancreas could be aligned, offering a unique opportunity for novel biomarker discovery in PDAC. Initial analysis of this data identified significant transcription emanating from pericentromeric heterochromatic regions of the genome previously thought to be inactive due to heavy epigenetic silencing. Pericentromeric heterochromatin is comprised of large tandem arrays of repetitive elements called satellites and these regions are known to be differentially methylated in a variety of malignancies. Cell line models have demonstrated that the accumulation of satellite transcripts can be induced by DNA demethylation, heat shock, or the induction of apoptosis, and their overexpression disrupts kinetichore formation causing genomic instability. Analysis of all human satellites identified the HSATII satellite as being exquisitely specific for pancreatic cancer compared to normal pancreatic tissue. HSATII expression was confirmed by RNA in situ hybridization (RNA-ISH) and was present in preneoplastic pancreatic intraepithelial neoplasia (PanIN) suggesting satellite expression occurs early in tumorigenesis, which provides for a potential biomarker for early detection. Furthermore, satellite expression correlated with the expression of a set of genes enriched in stem cells suggesting a link between satellites and altered cancer cell fate. We are now trying to understand the biological role of satellites in pancreatic cancer as well as develop RNA-sequencing pipelines to discover other novel ncRNAs.
Pancreatic Circulating Tumor Cells
CTCs are cells that have entered the vasculature and are thought to harbor the precursors of metastasis. Using a novel microfluidic device developed by Daniel Haber’s and Mehmet Toner’s group at MGH, we have been able to isolate pancreatic CTCs and perform RNA sequencing on these rare cells. RNA-sequencing of these pancreatic CTCs has identified aberrant WNT signaling as an important pathway in the metastatic process. In particular, the TAK1 kinase was found to be a key part of WNT signaling in CTCs and confers the ability to resist apoptosis in the setting of non-adherent conditions (i.e. anoikis resistance).
The temporal development of CTCs in tumorigenesis is not well understood, but evidence for CTC shedding in early localized cancers suggests that these cells are heterogeneous and that only a small subset of CTCs have the biological potential to metastasize. To better understand CTC heterogeneity we have now developed methods for RNA-sequencing at single cell resolution. This has provided a complete transcriptome evaluation of these rare cells and we are developing tools to better characterize single cell RNA-sequencing data. The early emergence of CTCs and the opportunity to understand the biology of metastasis in transit offers the potential for developing non-invasive, early detection tools and new strategies to target metastasis.
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