Metastatic pancreatic neuroendocrine tumors have decreased somatostatin expression and increased Akt signaling

Abstract

Background: Patients with pancreatic neuroendocrine tumors often present with metastases, which reduce survival. Molecular features associated with pancreatic neuroendocrine tumor tumorigenesis have been reported, but mechanisms of metastasis remain incompletely understood.

Methods: RNA sequencing was performed on primary and metastatic pancreatic neuroendocrine tumors from 43 patients. Differentially expressed genes were identified, and quantitative polymerase chain reaction used to confirm expression differences. BON cells were transfected with short interfering RNAs and short hairpin RNAs to create knockdowns. Expression changes were confirmed by quantitative polymerase chain reaction, cell viability assessed, and protein levels evaluated by Western blot and immunofluorescence.

Results: Nodal and hepatic metastases had decreased expression of somatostatin compared with primary tumors (P = .003). Quantitative polymerase chain reaction in a validation cohort confirmed 5.3-fold lower somatostatin expression in hepatic metastases (P = .043) with no difference in somatostatin receptor, synaptophysin, or chromogranin A expression. Somatostatin knockdown in BON cells increased cell metabolic activity, viability, and growth. Somatostatin-knockdown cells had significantly higher levels of phosphorylated Akt protein and higher mTOR compared with controls.

Conclusion: Pancreatic neuroendocrine tumor metastases have lower expression of somatostatin than primary tumors, and somatostatin knockdown increased growth in pancreatic neuroendocrine tumor cell lines. This was associated with increased activation of Akt, identifying this pathway as a potential mechanism by which loss of somatostatin expression promotes the metastatic phenotype.

Figure 1. Somatostatin expression is lower in PNET metastases compared to primary tumors

(a) Comparison of somatostatin expression in primary PNETs, nodal metastases, and hepatic metastases from 43 patients by RNA sequencing. (b) Comparison of somatostatin expression between matched primary and hepatic metastases (n = 16) by RNA sequencing from the same cohort of patients as in (a). (c-f) Comparison of gene expression in primary PNETs and hepatic metastases by qPCR in a different validation cohort of 12 patients for somatostatin (c), somatostatin receptor 2 (d), synaptophysin (e), and chromogranin A (f). * p < .05 Abbreviations: lymph node (LN), metastasis (met), pancreatic neuroendocrine tumor (PNET)

Figure 2. Comparison of gene expression in BON SST-knockdown cells

(a-c) Comparison between SST-knockdown and control BON cells of expression of somatostatin (a), synaptophysin (b), and somatostatin receptor 2 (c) by qPCR. BON cells were reverse-transfected with two siRNA against SST to create KD 1 and KD 2 with a scrambled siRNA as a control. Data are from three independent experiments. * p < .05, ** p < .01. Abbreviations: knockdown (KD), short interfering RNA (siRNA), somatostatin (SST)

Figure 3. SST knockdown increases cell viability and growth in BON cells

(a) Immunofluorescence image of SST-knockdown and control BON cells. Knockdown was accomplished by reverse-transfection with two siRNA against SST and a scrambled siRNA as a control. (b) Cell viability of SST-knockdown and control cells at day 3 and 6 post-transfection. Cell viability was determined using alamarBlue assay. (c) Immunofluorescence image of stable SST-knockdown and control cells. Knockdown was accomplished by infection with lentivirus encoding two shRNAs against SST and a scrambled shRNA as a control. (d) Cell count of stable SST-knockdown and control cells, determined by manual counting using a hemocytometer every two days. Data represent mean ± SEM from triplicates. Abbreviations: short interfering RNA (siRNA), short hairpin RNA (shRNA), somatostatin (SST)

Figure 4. SST knockdown increases phosphorylation of Akt

Representative Western blots (repeated 3 or more times) of SST-knockdown and control BON cells. Protein was collected at day 5 after siRNA transfection for Western blot analysis. (a) Blots were incubated with antibodies against phosphor-Akt (Ser473), total Akt, and GAPDH. (b) Relative levels of phosphor-Akt, normalized to GAPDH, between SST-knockdown and control cells. (c) Blots were incubated with antibodies against phosphor-mTOR (Ser2448), total mTOR, and GAPDH. (c) Relative levels of phosphor-mTOR, normalized to GAPDH, between SST-knockdown and control cells. Quantification performed by ImageJ analysis. Data represent mean ± SD and are representative of 3 or more experiments. * p < .05. Abbreviations: short interfering RNA (siRNA), knockdown (KD), somatostatin (SST)