Transcriptomic analysis by RNA sequencing characterises malignant progression of canine insulinoma from normal tissue to metastatic disease.

Insulinomas (INS) are the most common human and canine functioning pancreatic neuroendocrine tumours. The long-term prognosis for malignant INS is poor, because micrometastases are frequently missed during surgery. As human and canine malignant INS share clinical and histopathological features, dogs have been proposed as models for INS research. Using RNA-sequencing, we conducted a pilot study to better understand the underlying molecular mechanisms of canine INS. Normal canine pancreas and lymph node control tissues were compared with primary INS and INS-metastatic lymph nodes, revealing more than 3,000 genes differentially expressed in normal pancreas compared to primary INS. Only 164 genes were differentially expressed between primary INS and INS-metastatic lymph nodes. Hierarchical clustering analysis demonstrated similar genetic profiles in normal pancreas and early clinical stage primary INS, whereas late clinical stage primary INS resembled the genetic profile of INS-metastatic lymph nodes. These findings suggest that markers of malignant behaviour could be identified at the primary site of the disease. Finally, using the REACTOME pathways database, we revealed that an active collagen metabolism, extracellular matrix remodelling, beta-cell differentiation and non-beta-cell trans-differentiation might cause disease progression and hyperinsulinism in INS, identifying major pathways worthy of future research in this currently poorly controlled disease.

Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells.

Insulinomas (INS) are the most common neuroendocrine pancreatic tumours in humans and dogs. The long-term prognosis for malignant INS is still poor due to a low success rate of the current treatment modalities, particularly chemotherapy. A better understanding of the molecular processes underlying the development and progression of INS is required to develop novel targeted therapies. Cancer stem cells (CSCs) are thought to be critical for the engraftment and chemoresistance of many tumours, including INS. This study was aimed to characterise and target INS CSCs in order to develop novel targeted therapies. Highly invasive and tumourigenic human and canine INS CSC-like cells were successfully isolated. These cells expressed stem cell markers (OCT4, SOX9, SOX2, CD133 and CD34), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotype in vivo compared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes (NOTCH2 and HES1) were overexpressed in INS CSC-like cells. Protein analysis showed an active NOTCH2-HES1 signalling in INS cell lines, especially in cells resistant to 5-FU. Inhibition of the Notch pathway, using a gamma secretase inhibitor (GSI), enhanced the sensitivity of INS CSC-like cells to 5-FU. When used in combination GSI and 5-FU, the clonogenicity in vitro and the tumourigenicity in vivo of INS CSC-like cells were significantly reduced. These findings suggested that the combined strategy of Notch signalling inhibition and 5-FU synergistically attenuated enriched INS CSC populations, providing a rationale for future therapeutic exploitation.