Elham Barazeghi

Background: Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing but highly metastatic, with most patients presenting metastases at diagnosis. Radical surgery remains the only potential curative option when feasible. Consequently, there is a critical need for novel therapeutic strategies that can limit tumor progression and enable more personalized treatment approaches in combination with current clinical practices. In this study, we evaluated the impact of metformin on SI-NET cell growth in vivo, characterized the associated microRNA expression profile, and identified potential driver genes modulated by metformin treatment. Methods: A total of 22 SI-NET xenograft mouse models were established using CNDT2.5 and GOT1 cells. Mice were treated with metformin (2.56 mg/mL in drinking water) or water as control for 4 weeks. To explore the molecular impact of metformin, both small-RNA and total-RNA sequencing were performed on the dissected xenograft tumors. Proliferation and apoptosis were further evaluated by immunohistochemistry. Results: In vivo treatment of SI-NET cells with metformin led to a reduction in tumor size in both CNDT2.5 and GOT1 xenograft models. Our sequencing analyses identified seven altered microRNAs and 1,776 differentially expressed genes in metformin-treated tumors compared to controls. To uncover potential driver genes in SI-NETs affected by metformin, we compared the differentially expressed genes from GOT1 xenograft model with those identified by comparing single-cell RNA profile of enterochromaffin cells to SI-NETs. This novel approach revealed a set of significantly regulated genes, including those involved in tumor proliferation, apoptosis, and metastasis, as well as genes related to voltage-gated calcium channels and signal transduction. Conclusions: Our novel findings support further investigation of metformin as a potential therapeutic agent in clinical trials for SI-NET patients, and suggest that identified miRNAs should be assessed as potential predictive biomarkers for metformin treatment. This study highlights novel candidate driver genes affected by metformin, which are associated with key cellular processes and enterochromaffin cell’s function, offering insights into the underlying mechanisms in SI-NETs.

Background: Several studies have attempted to identify the initiating drivers of small intestinal neuroendocrine tumor (SI-NET) development and the molecular mechanisms underlying their progression and metastatic spread. Previous gene expression studies have used bulk microarrays or RNA sequencing to compare tumor tissue with normal intestinal mucosa. However, the intestine comprises multiple distinct cell types, and bulk analyses are limited by this cellular heterogeneity, which can confound tumor-specific signals. Methods: We performed single-cell RNA sequencing on primary SI-NETs and paired normal mucosa from two patients to directly compare tumor cells with their cells of origin, the enterochromaffin (EC) cells. To minimize type I errors, we applied a two-step validation strategy by overlapping differentially expressed genes with an external single-cell dataset and cross-referencing candidate genes for enteroendocrine expression in the Human Protein Atlas. Results: For further distinction and characterization, ECs were subdivided into serotonergic and non-serotonergic clusters. This analysis revealed that the SI-NET cells are transcriptionally more similar to serotonergic ECs, consistent with serum metabolite profiles derived from clinical parameters. Our analyses uncovered a loss-of-expression program characterized by regulators of epithelial differentiation and in parallel, a gain-of-expression program displayed neuronal signaling gene induction, implicating functional reprogramming toward neuronal-like properties. Together, these specific losses and gains suggest that our patient-derived SI-NETs undergo adaptation through both loss of enteroendocrine functions and acquisition of neurobiological-promoting signaling pathways. Conclusions: These findings nominate candidate drivers for further functional validation and highlight potential therapeutic strategies in our patient cohort, including restoring suppressed Notch signaling and targeting aberrant neuronal signaling networks. However, even with a two-step validation procedure, the modest cohort size limits statistical power and generalizability, particularly for the proposed association to a serotonergic phenotype. Larger, multi-patient single-cell studies are required to confirm these mechanisms and establish their clinical relevance.

Small intestinal neuroendocrine tumors (SI-NETs) represent the most frequent neoplasms of the small intestine, with loss of chromosome 18 as the most common genetic aberration. To date, no highly frequently mutated genes have been identified on chromosome 18 or elsewhere. This study aimed to explore the potential role of methyl-CpG binding domain protein 2 (MBD2), located at 18q21.2, as a tumor suppressor gene in SI-NETs. By immunohistochemistry, we found undetectable or very low levels of MBD2 expression in 64% of the analyzed SI-NETs, and overall, very low levels of mRNA and protein expression were observed. Overexpression of MBD2 in the SI-NET cell line, GOT1, reduced cell growth and induced apoptosis, though cell migration remained unaffected. In addition, MBD2 expression decreased cell proliferation and migration capacity of the neuroendocrine cells NCI-H727 but showed no effect on apoptosis. Furthermore, MBD2 expression in both cell lines was found to upregulate E-cadherin but downregulate the levels of N-cadherin and vimentin in GOT1 and NCI-H72, respectively, as determined by western blot analysis. These results suggest that MBD2 plays a role in inhibiting the epithelial-mesenchymal transition process and may function as a potential tumor suppressor gene in SI-NETs. Future studies to elucidate the underlying mechanisms are warranted.

Several studies have attempted to find the initiating drivers of small intestinal neuroendocrine tumors (SI-NET) development and the molecular mechanisms driving progression and metastatic spread. For gene expression studies using bulk microarrays and RNA sequencing, researchers commonly use normal intestinal mucosa as a control. The intestine is made up of several different cell types, and using bulk RNA-seq may generate findings that reflect factors other than the tumor transformation. This could potentially contribute to the lack of discoverable treatments and prevention strategies for SI-NETs. We performed scRNA-seq on tissue from two patients that had tumor resection surgery and separated the EC cells from the normal intestinal mucosa and used specific markers to compare them against SI-NET tumors cells. We pinpointed new genes associated with chr18 haploinsufficiency but also others with loss or gain of expression that have not previously been associated with SI-NETs, and which could be potential targets for further functional genomic studies.