Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells

Abstract

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.

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

“…Unsupervised clustering of these DEGs showed that normal pancreatic tissues clustered with primary INS lesions from patients bearing early stage INS (TNM stage I), and clustered separately from primary INS lesions from patients bearing advanced stage disease (TNM stage II–IV). When comparing primary INS with INS-metastatic lymph nodes, only 164 DEG were identified, suggesting less transcriptomic variances between these tissues 21 . Unsupervised clustering showed that primary INS from patients with high-grade INS (Grade 2) clustered together with the INS-metastatic lymph nodes.…”

“…Differences in gene expression of pairwise comparisons were organised as follows: (i) normal pancreas against primary INS; (ii) primary INS against INS-metastatic lymph nodes; (iii) normal lymph nodes against INS-metastatic lymph nodes. The normalised gene counts for differentially expressed genes were analysed for hierarchical clustering analysis with the R package “heatmap2” 21 , 23 .…”

“…Since the completion of the canine genome sequencing 19 , various RNA-sequencing (RNA-seq) studies have identified gene signature characteristics of malignancy in a variety of cancers occurring in dogs such as meningioma 13 , mammary carcinoma 16 , melanoma 20 and bladder carcinoma 18 , providing additional information about the oncogenesis of human tumours. The close resemblance of canine INS to human malignant INS with regard to clinical signs, histopathology and disease progression, makes canine INS an interesting study model for human malignant INS 21 . For instance, our recent study comparing human and canine INS cell lines identified the Notch pathway as a key regulator of stemness and chemoresistance both in vitro and in vivo, providing a rationale for focused further research on this druggable target for INS in both species 2 .…”

Emerging agents that target signaling pathways in cancer stem cells

“…Additionally, the γ-secretase inhibitor RO4929097 can significantly inhibit Notch target genes Hes1 and Hey1, and it is in a phase II clinical setting to treat breast cancer, ovarian cancer, and renal cell carcinoma [67]. The combination of RO4929097 and 5-FU can decrease the proportion of the CSC subgroup with insulinomas (INS) [68]. DAPT (GSI-IX) is a new type of Notch1 inhibitor that was initially used to treat Alzheimer's disease, and increasing number of studies have shown that it can inhibit CSCs [69].…”

Novel Targets in Pancreatic Cancer Therapy - Current Status and Ongoing Translational Efforts

“…Likewise, Notch signaling has now long been found to be altered and pathophysiologically involved in pancreatic carcinogenesis. Various approaches to pharmacologically target oncogenic Notch signaling in different cancers including pancreatic cancer have been developed, including the application of γ-secretase inhibitors and antagonists against Notch ligands [73,74,75], and some of these endeavors are still underway at various stages of preclinical as well as early clinical evaluation [76,77,78,79,80,81,82]. …”