We here report a 41-year-old patient with tuberous sclerosis and a known germline mutation in the TSC2 gene. During ultrasound tumour screening done for unexplained varicella reactivation a tumour of the left kidney and a lesion in the pancreatic tail were discovered. Pathologic examination of the surgical removed tumours revealed an angiomyolipma of the left kidney and a well-differentiated neuroendocrine tumour of the pancreas (pT2N0cM0,V0L0) with a proliferative index of 5% (G2). According to guidelines no adjuvant therapy was given and the patient was entered into our 6-monthly follow-up scheme. 12 months after the successful removal of the primary tumour the patient developed four new small lesions in the liver with a MRI-morphology coherent with neuroendocrine tumour metastasis.

GEP-NETs are heterogeneous tumors originating from the pancreas (panNET) or the intestinal tract. Only a few patients with NETs are amenable to curative tumor resection, and for most patients, only palliative treatments to successfully control the disease or manage symptoms remain, such as with synthetic somatostatin (SST) analogs (SSAs), such as octreotide (OCT) or lanreotide (LAN). However, even cells expressing low levels of SST receptors (SSTRs) may exhibit significant responses to OCT, which suggests the possibility that SSAs signal through alternative mechanisms, e.g., transforming growth factor (TGF)-β. This signaling mode has been demonstrated in the established panNET line BON but not yet in other permanent (i.e., QGP) or primary (i.e., NT-3) panNET-derived cells. Here, we performed qPCR, immunoblot analyses, and cell counting assays to assess the effects of SST, OCT, LAN, and TGF-β1 on neuroendocrine marker expression and cell proliferation in NT-3, QGP, and BON cells. SST and SSAs were found to regulate a set of neuroendocrine genes in all three cell lines, with the effects of SST, mainly LAN, often differing from those of OCT. However, unlike NT-3 cells, BON cells failed to respond to OCT with growth arrest but paradoxically exhibited a growth-stimulatory effect after treatment with LAN. As previously shown for BON, NT-3 cells responded to TGF-β1 treatment with induction of expression of SST and SSTR2/5. Of note, the ability of NT-3 cells to respond to TGF-β1 with upregulation of the established TGF-β target gene SERPINE1 depended on cellular adherence to a collagen-coated matrix. Moreover, when applied to NT-3 cells for an extended period, i.e., 14 days, TGF-β1 induced growth suppression as shown earlier for BON cells. Finally, next-generation sequencing-based identification of microRNAs (miRNAs) in BON and NT-3 revealed that SST and OCT impact positively or negatively on the regulation of specific miRNAs. Our results suggest that primary panNET cells, such as NT-3, respond similarly as BON cells to SST, SSA, and TGF-β treatment and thus provide circumstantial evidence that crosstalk of SST and TGF-β signaling is not confined to BON cells but is a general feature of panNETs.

Pancreatic neuroendocrine neoplasms (PanNENs) are the second most common malignancy of the pancreas. Surgery remains the only curative treatment for localized disease. For patients with inoperable advanced or metastatic disease, few targeted therapies are available, but their efficacy is unpredictable and variable. Exploiting prior knowledge on pathogenetic processes involved in PanNEN tumorigenesis, we tested buparlisib (PI3K inhibitor) and ribociclib (CDK4/6 inhibitor), as single agents or in combination, in different preclinical models. First, we used cell lines representative of well-differentiated (INS-1E, NT-3) and poorly differentiated (BON-1) PanNENs. The combination of buparlisib with ribociclib reduced the proliferation of 2D and 3D spheroid cultures more potently than the individual drugs. Buparlisib, but not ribociclib, induced apoptosis. The anti-proliferative activity of the drugs correlated with downstream target inhibition at mRNA and protein levels. We then tested the drugs on primary islet microtissues from a genetic PanNET animal model (Men1-defective mice) and from wild-type mice: the drug combination was effective against the former without altering islet cell physiology. Finally, we treated PanNET patient-derived islet-like 3D tumoroids: the combination of buparlisib with ribociclib was effective in three out of four samples. Combined targeting of PI3K and CDK4/6 is a promising strategy for PanNENs spanning various molecular and histo-pathological features.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, due in part to early invasion and metastasis, which in turn involves epithelial–mesenchymal transition (EMT) of the cancer cells. Prompted by the discovery that two PDAC cell lines of the quasi-mesenchymal subtype (PANC-1, MIA PaCa-2) exhibit neuroendocrine differentiation (NED), we asked whether NED is associated with EMT. Using real-time PCR and immunoblotting, we initially verified endogenous expressions of various NED markers, i.e., chromogranin A (CHGA), synaptophysin (SYP), somatostatin receptor 2 (SSTR2), and SSTR5 in PANC-1 and MIA PaCa-2 cells. By means of immunohistochemistry, the expressions of CHGA, SYP, SSTR2, and the EMT markers cytokeratin 7 (CK7) and vimentin could be allocated to the neoplastic ductal epithelial cells of pancreatic ducts in surgically resected tissues from patients with PDAC. In HPDE6c7 normal pancreatic duct epithelial cells and in epithelial subtype BxPC-3 PDAC cells, the expression of CHGA, SYP, and neuron-specific enolase 2 (NSE) was either undetectable or much lower than in PANC-1 and MIA PaCa-2 cells. Parental cultures of PANC-1 cells exhibit EM plasticity (EMP) and harbor clonal subpopulations with both M- and E-phenotypes. Of note, M-type clones were found to display more pronounced NED than E-type clones. Inducing EMT in parental cultures of PANC-1 cells by treatment with transforming growth factor-β1 (TGF-β1) repressed epithelial genes and co-induced mesenchymal and NED genes, except for SSTR5. Surprisingly, treatment with bone morphogenetic protein (BMP)-7 differentially affected gene expressions in PANC-1, MIA PaCa-2, BxPC-3, and HPDE cells. It synergized with TGF-β1 in the induction of vimentin, SNAIL, SSTR2, and NSE but antagonized it in the regulation of CHGA and SSTR5. Phospho-immunoblotting in M- and E-type PANC-1 clones revealed that both TGF-β1 and, surprisingly, also BMP-7 activated SMAD2 and SMAD3 and that in M- but not E-type clones BMP-7 was able to dramatically enhance the activation of SMAD3. From these data, we conclude that in EMT of PDAC cells mesenchymal and NED markers are co-regulated, and that mesenchymal–epithelial transition (MET) is associated with a loss of both the mesenchymal and NED phenotypes. Analyzing NED in another tumor type, small cell carcinoma of the ovary hypercalcemic type (SCCOHT), revealed that two model cell lines of this disease (SCCOHT-1, BIN-67) do express CDH1, SNAI1, VIM, CHGA, SYP, ENO2, and SSTR2, but that in contrast to BMP-7, none of these genes was transcriptionally regulated by TGF-β1. Likewise, in BIN-67 cells, BMP-7 was able to reduce proliferation, while in SCCOHT-1 cells this occurred only upon combined treatment with TGF-β and BMP-7. We conclude that in PDAC-derived tumor cells, NED is closely linked to EMT and TGF-β signaling, which may have implications for the therapeutic use of TGF-β inhibitors in PDAC management.

Chemotherapy with cisplatin (CDDP) and etoposide (ETO) is the standard treatment for gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Despite high initial response rates, treatment-related toxicity and acquired resistance remain significant clinical challenges. No molecularly targeted therapies are currently established for this aggressive cancer. Here, we evaluated Aurora kinase inhibition with alisertib (ALI) as a synthetic lethality strategy in ARID1A-deficient GEP-NEC. Subcutaneous and orthotopic xenograft models were established in immunodeficient mice using the GEP-NEC-derived NT-38 cell line, and the therapeutic efficacy of ALI was compared with CDDP + ETO. Tumor responses were assessed by immunohistochemistry, Western blotting, and RNA sequencing. To confirm ARID1A dependency, transient knockdown was induced in the pancreatic neuroendocrine NT-18LM cell line, demonstrating that ALI sensitivity is significantly enhanced in ARID1A-deficient cells. ALI achieved antitumor efficacy comparable to chemotherapy and was well tolerated, with minimal weight loss relative to CDDP + ETO. Distant metastases, an early feature of GEP-NEC, developed in five animals over six weeks, two of which were treated with ALI and three controls. Transcriptomic profiling revealed convergence of both treatments on signal transduction, focal adhesion, receptor tyrosine kinase, and VEGFA-VEGFR2 signaling, while ALI uniquely enriched pathways related to pancreatic secretion, lipid metabolism, protein processing in the endoplasmic reticulum, and extracellular matrix organization. These findings establish Aurora kinase inhibition as mechanistically distinct and selectively effective in ARID1A-deficient GEP-NEC. Given its efficacy, favorable tolerability, and ARID1A-dependent specificity, ALI may represent a promising alternative to platinum-based chemotherapy, offering a strong rationale for further development of mechanism-driven combination strategies for GEP-NEC.