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 (, ), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotype compared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes ( and 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 and the tumourigenicity 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.

Genomic analysis of Pancreatic Neuroendocrine Tumors (PanNETs) has revealed that these tumors often lack mutations in typical cancer-related genes such as the tumor suppressor gene p53 . Instead, PanNET tumorigenesis usually involves mutations in specific PanNET-related genes, such as tumor suppressor gene MEN1 . Using a PanNET mouse model, human tissues and human cell lines, we studied the cross-talk among MEN1, p53 and Notch signaling pathways and their role in PanNETs. Here, we show that reactivation of the early developmental program of islet cells underlies PanNET tumorigenesis by restoring the proliferative capacity of PanNET cells. We investigated the role of INSM1, a transcriptional regulator of islet cells’ development, and revealed that its expression and subcellular localization is regulated by MEN1 and p53 . Both human and mouse data show that loss of MEN1 in a p53 wild-type genetic background results in increased nuclear INSM1 expression and cell proliferation. Additionally, inhibition of Notch signaling in a p53 wild-type background reduces the proliferation of PanNET cells, due to repression of INSM1 transcription and nuclear localization. Our study elucidates the molecular mechanisms governing the interactions of INSM1 with MEN1, p53 and Notch and their roles in PanNET tumorigenesis, suggesting INSM1 as a key transcriptional regulator of PanNET cell proliferation.

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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, betacell 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. Insulinomas (INS) are the most commonly diagnosed functioning pancreatic neuroendocrine tumours in humans and dogs 1,2. They are insulin-producing tumours that can arise from pancreatic beta-cells 1,2. Diagnosis and treatment of human and canine INS can be challenging and prognosis highly variable depending on therapeutic decisions 3-5. Currently, surgical resection of primary localised benign INS is considered the treatment of choice. However, the prognosis after surgery for advanced-stage disease is still poor and adjuvant therapy options are limited 5,6. Thus, better therapeutic tools are urgently required. Traditional benign versus malignant classification of these tumours is often difficult at initial diagnosis. Grade and stage are the major determinants of prognosis in humans 4,7. Similarly, in dogs, TNM stage, increased nuclear size, pleomorphism, Ki67 and DNA content are considered valuable histological features to predict the biological behaviour of INS 6, 8. Still, grading and staging have been so far insufficient to fully assess the degree of malignancy of human and canine INS. In both humans and dogs, these tumours can have heterogeneous microscopic findings and the presence of metastases, mainly located in the liver, represents so far the only definitive feature that characterises individual tumours as malignant 3,8-11. Canine INS are considered typically malignant as they