AGTR1 Is Overexpressed in Neuroendocrine Neoplasms, Regulates Secretion and May Potentially Serve as a Target for Molecular Imaging and Therapy

This study identified and confirmed angiotensin II (ATII) as a strong activator of signaling in neuroendocrine neoplasm (NEN) cells. Expression analyses of the ATII receptor type 1 (AGTR1) revealed an upregulation of mRNA levels (RT-qPCR) and radioligand binding (autoradiography) in small-intestinal (n = 71) NEN tissues compared to controls (n = 25). NEN cells with high AGTR1 expression exhibited concentration-dependent calcium mobilization and chromogranin A secretion upon stimulation with ATII, blocked by AGTR1 antagonism and Gαq inhibition. ATII also stimulated serotonin secretion from BON cells. AGTR1 ligand saralasin was coupled to a near-infrared fluorescent (NIRF) dye and tested for its biodistribution in a nude mouse model bearing AGTR1-positive BON and negative QGP-1 xenograft tumors. NIRF imaging showed significantly higher uptake in BON tumors. This proof of concept establishes AGTR1 as a novel target in NEN, paving the way for translational chelator-based probes for diagnostic PET imaging and radioligand therapy.

UCHL1 loss alters the cell cycle in metastatic pancreatic neuroendocrine tumors

Loss of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) expression by CpG promoter hypermethylation is associated with metastasis in gastroenteropancreatic neuroendocrine tumors; however, the mechanism of how UCHL1 loss contributes to metastatic potential remains unclear. In this study, we first confirmed that the loss of UCHL1 expression on immunohistochemistry was significantly associated with metastatic tumors in a translational pancreatic neuroendocrine tumor (PNET) cohort, with a sensitivity and specificity of 78% and 89%, respectively. To study the mechanism driving this aggressive phenotype, BON and QGP-1 metastatic PNET cell lines, which do not produce UCHL1, were stably transfected to re-express UCHL1. In vitro assays, RNA sequencing and reverse phase protein array (RPPA) analyses were performed comparing empty-vector negative controls and UCHL1-expressing cell lines. UCHL1 re-expression is associated with lower anchorage-independent colony growth in BON cells, lower colony formation in QGP cells and a higher percentage of cells in the G0/G1 cell-cycle phase in BON and QGP cells. On RPPA proteomic analysis, there was an upregulation of cell-cycle regulatory proteins CHK2 (1.2-fold change, P = 0.004) and P21 (1.2-fold change, P = 0.023) in BON cells expressing UCHL1; western blot confirmed upregulation of phosphorylated CHK2 and P21. There were no transcriptomic differences detected on RNA sequencing between empty-vector negative controls and UCHL1-expressing cell lines. In conclusion, UCHL1 loss correlates with metastatic potential in PNETs and its re-expression induces a less aggressive phenotype in vitro, in part by inducing cell-cycle arrest through posttranslational regulation of phosphorylated CHK2. UCHL1 expression should be considered as a functional biomarker in detecting PNETs capable of metastasis.

Temporal response properties of koniocellular (blue-on and blue-off) cells in marmoset lateral geniculate nucleus

Visual perception requires integrating signals arriving at different times from parallel visual streams. For example, signals carried on the phasic-magnocellular (MC) pathway reach the cerebral cortex pathways some tens of milliseconds before signals traveling on the tonic-parvocellular (PC) pathway. Visual latencies of cells in the koniocellular (KC) pathway have not been specifically studied in simian primates. Here we compared MC and PC cells to “blue-on” (BON) and “blue-off” (BOF) KC cells; these cells carry visual signals originating in short-wavelength-sensitive (S) cones. We made extracellular recordings in the lateral geniculate nucleus (LGN) of anesthetized marmosets. We found that BON visual latencies are 10–20 ms longer than those of PC or MC cells. A small number of recorded BOF cells ( n = 7) had latencies 10–20 ms longer than those of BON cells. Within all cell groups, latencies of foveal receptive fields (<10° eccentricity) were longer (by 3–8 ms) than latencies of peripheral receptive fields (>10°). Latencies of yellow-off inputs to BON cells lagged the blue-on inputs by up to 30 ms, but no differences in visual latency were seen on comparing marmosets expressing dichromatic (“red-green color-blind”) or trichromatic color vision phenotype. We conclude that S-cone signals leaving the LGN on KC pathways are delayed with respect to signals traveling on PC and MC pathways. Cortical circuits serving color vision must therefore integrate across delays in (red-green) chromatic signals carried by PC cells and (blue-yellow) signals carried by KC cells.

Antidromic latency of magnocellular, parvocellular, and koniocellular (Blue-ON) geniculocortical relay cells in marmosets

We studied the functional connectivity of cells in the lateral geniculate nucleus (LGN) with the primary visual cortex (V1) in anesthetized marmosets (Callithrix jacchus). The LGN sends signals to V1 along parallel visual pathways called parvocellular (P), magnocellular (M), and koniocellular (K). To better understand how these pathways provide inputs to V1, we antidromically activated relay cells in the LGN by electrically stimulating V1 and measuring the conduction latencies of P (n = 7), M (n = 14), and the “Blue-ON” (n = 5) subgroup of K cells (K-BON cells). We found that the antidromic latencies of K-BON cells were similar to those of P cells. We also measured the response latencies to high contrast visual stimuli for a subset of cells. We found the LGN cells that have the shortest latency of response to visual stimulation also have the shortest antidromic latencies. We conclude that Blue color signals are transmitted directly to V1 from the LGN by K-BON cells.

In vitro activity of ABT-888 (A), 5-FU (F), and dacarbazine (D) in neuroendocrine tumors (NET).

236 Background: Low-grade neuroendocrine tumors (NET) have few cytotoxic chemotherapy options. Data suggests that a combination of temozolomide and a fluoropyrimidine has clinical efficacy. ABT-888 is a novel poly-ADP ribose polymerase (PARP) inhibitor that has been tested in a phase 1 setting with temozolomide, with synergy demonstrated in preclinical models of other tumors. We proposed an in-vitro study of ABT-888 with varying concentrations of dacarbazine (D), and 5-FU (F) on human neuroendocrine cells (BON). Methods: BON cells were incubated with varying concentrations and combinations of ABT-888 (2.5, 5 and 10uM), F (25-100uM), and D (25-100uM) for 96 hours. After incubation, cell growth was measured by MTT rapid colorimetric assay with absorbance reported as mean % control. Western blot analysis was performed for chromogranin A, PARP, γH2AXand XIAP to assess for cell death and on-target effects for the population treated with 5-FU. Combination indices (CI) were calculated using the Chou-Talalay method using CompuSyn. CI’s below 1 signified synergy. Results: ABT-888 alone did not demonstrate any antitumor effect (103%). D alone had antitumor effect (74% at 50uM, 67% at 100uM) which was improved by adding ABT-888 (70% at 50uM D; CI 0.73; p=0.06, 60% at 100 uM D; CI 0.88; p=0.0003). F alone had antitumor effect (82% at 50uM and 71% at 100uM) which was improved by adding 2.5uM ABT-888 (71% at 50uM of F; CI=0.59, and 58% at 100uM of F; CI=0.74), and further enhanced with 5uM of concomitant ABT-888 (56% at 50uM of F; CI=0.68, and 49% at 100uM of F; CI=0.76). Western analysis of lysates showed markers of increased apoptosis, decreased PARP, and decreased expression of CgA. The combination of F+D did not demonstrate increased cytotoxicity with the addition of ABT-888 (58% with/without ABT-888 p=0.61). Conclusions: ABT-888 demonstrated in vitro synergy against BON cells with F or D. The combination of all three compounds (A+F+D) did not demonstrate synergy above F+D. Synergy was statistically significant with increasing doses of cytotoxic compounds which are achievable in vivo with current doses of A, F, and D. The combination of ABT-888 with temozolomide or a fluoropyrimidine merits further study in human clinical trials.

mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.