Neuropilin-2 contributes to tumor progression in preclinical models of small intestinal neuroendocrine tumors.

The identification of novel regulators of tumor progression is a key challenge to gain knowledge on the biology of small intestinal neuroendocrine tumors (SI-NETs). We recently identified the loss of the axon guidance protein semaphorin 3F as a protumoral event in SI-NETs. Interestingly the expression of its receptor neuropilin-2 (NRP-2) was still maintained. This study aimed at deciphering the potential role of NRP-2 as a contributor to SI-NET progression. The role of NRP-2 in SI-NET progression was addressed using an approach integrating human tissue and serum samples, cell lines and in vivo models. Data obtained from human SI-NET tissues showed that membranous NRP-2 expression is present in a majority of tumors, and is correlated with invasion, metastatic abilities, and neovascularization. In addition, NRP-2 soluble isoform was found elevated in serum samples from metastatic patients. In preclinical mouse models of NET progression, NRP-2 silencing led to a sustained antitumor effect, partly driven by the downregulation of VEGFR2. In contrast, its ectopic expression conferred a gain of aggressiveness, driven by the activation of various oncogenic signaling pathways. Lastly, NRP-2 inhibition led to a decrease of tumor cell viability, and sensitized to therapeutic agents. Overall, our results point out NRP-2 as a potential therapeutic target for SI-NETs, and will foster the development of innovative strategies targeting this receptor. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dependence receptor TrkC is a putative colon cancer tumor suppressor.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.

Antitumor effect of everolimus in preclinical models of high-grade gastroenteropancreatic neuroendocrine carcinomas.

BACKGROUND/AIMS: While the range of therapeutic options for well-differentiated gastroenteropancreatic neuroendocrine tumors has recently increased with the emergence of targeted therapies, such as mTOR inhibitors, there is no recent progress in the treatment of poorly differentiated neuroendocrine carcinomas (PDNECs). Since PDNECs have been shown to strongly express mTOR pathway components, the aim of the present study was to assess the antitumor effect of the mTOR inhibitor everolimus in preclinical models of PDNECs. METHODS: The expression of mTOR pathway components and their response to everolimus were assessed in two neuroendocrine cell lines: STC-1 and GluTag. A xenograft model of intrahepatic dissemination in the nude mouse, based on the intrasplenic injection of either STC-1 and GluTag tumor cells, was used. Animals were started on everolimus treatment 3 days after injection. The effects of treatment on tumor growth, proliferative capacities, apoptosis and in situ expression of mTOR pathway components were assessed. RESULTS: The expression of mTOR pathway components was comparable in STC-1 and GluTag cells and in human PDNECs and could be inhibited in vitro by everolimus. In vivo, the tumor volume of STC-1 and GluTag xenografts was significantly reduced in treated animals (6.05 ± 1.84% as compared to 21.76 ± 3.88% in controls). Everolimus treatment also induced a significant decrease in Ki67 index and in the phosphorylation levels of the two major effectors of mTOR, p70S6K and 4E-BP1. CONCLUSION: Our experimental data suggest that mTOR inhibition could be considered a therapeutic option for high-grade gastroenteropancreatic neuroendocrine tumors.

Effects of somatostatin and octreotide on the interactions between neoplastic gastroenteropancreatic endocrine cells and endothelial cells: a comparison between in vitro and in vivo properties.

BACKGROUND/AIMS: Experimental studies in vitro suggest that somatostatin and some of its analogues used in clinical practice, such as octreotide, may have potent antiangiogenic properties. However, the clinical transposition of these data is difficult. METHODS: To address this issue, we designed a comparative study of the effects of somatostatin and octreotide on the interactions between neoplastic endocrine cells and endothelial cells in several in vitro and in vivo experimental models, including primary cultures of human umbilical vein endothelial cells (HUVEC), indirect cocultures between HUVEC and the somatostatin-producing endocrine cell line STC-1, and an animal model of intrahepatic dissemination of STC-1 cells. RESULTS: 10(-8)M octreotide markedly inhibited both basal and VEGF-stimulated HUVEC proliferation, had no effect on endothelial cell migration, but inhibited endothelial tubule formation. HUVEC cocultured with the somatostatin- and VEGF-producing STC-1 cells presented a markedly decreased proliferation, a slightly increased motility and an increased capacity of tubule formation; in this system, the inhibition of endothelial cell proliferation was abolished by neutralizing anti-somatostatin but was restored in the presence of anti-VEGF antibodies. This suggests that somatostatin is able to antagonize the effects of VEGF on endothelial cell proliferation but not on endothelial cell sprouting. Finally, no significant effect of octreotide on tumor growth and intratumoral microvascular density was detected in an experimental model of intrahepatic dissemination of STC-1 cells. CONCLUSION: The in vitro antiangiogenic effects of somatostatin and its analogues are likely to be efficiently counterbalanced in the tumor microenvironment by the concomitant release of proangiogenic factors like VEGF.

VEGF secretion by neuroendocrine tumor cells is inhibited by octreotide and by inhibitors of the PI3K/AKT/mTOR pathway.

Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as somatostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1alpha under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

HIP/PAP, a member of the reg family, is expressed in glucagon-producing enteropancreatic endocrine cells and tumors.

Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) protein, a member of the reg family, is constitutively expressed by some specialized epithelial cell subsets in the digestive tract and the pancreas. We performed a detailed analysis of the expression of HIP/PAP protein in normal digestive endocrine cells according to their localization, lineage, and differentiation stage, and in digestive endocrine tumors according to their site of origin and hormonal profile. In both adult and fetal normal tissues, HIP/PAP expression was detected only in endocrine cells of the small intestine, ascending colon, and pancreas. Two different expression patterns were identified: (a) a strong cytoplasmic labeling observed in the endocrine cells of the digestive mucosa and the outer rim of Langerhans islets specialized in the synthesis of glucagon and glucagon-like peptides; (b) a weak cytoplasmic immunoreactivity observed in the other pancreatic endocrine cell populations. HIP/PAP expression was detected in 36 of the 184 cases of digestive endocrine tumors examined; 32 of these cases (89%) were pancreatic. The 2 patterns observed in the normal state were retained: (a) a strong labeling was observed in 5% to 100% of tumor cells in 26 tumors, all expressing glucagon or glucagon-like peptides; (b) a weak labeling was present in 10 tumors, presenting various hormonal profiles. In conclusion, a strong expression of HIP/PAP is characteristic of glucagon-producing normal and neoplastic enteropancreatic endocrine cells. Our results lend further support to the concept that members of the reg family play regulatory roles in various endocrine cell populations and that their expression in endocrine cells is lineage-specific.

Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

The role of angiogenesis in endocrine liver metastases: an experimental study.

Liver metastases are a major adverse event during the evolution of digestive endocrine tumors. However, little is known about their natural history and the determinants of their growth. In particular, whereas liver endocrine metastases, like their primary counterparts, are hypervascular, the role of tumor-associated angiogenesis has been little explored. We therefore designed an experimental model to study the intrahepatic growth of tumor endocrine cells; murine enteroendocrine STC-1 cells were injected into the spleen of nude mice to obtain their hepatic dissemination through the portal vein. Three stages of intrahepatic tumor growth were identified. Engraftment stage, until day 4 after intrasplenic injection of STC-1 cells, was avascular. Early growth, until day 17, resulted in small, infralobular nodules. Late growth, after day 17, was characterized by the development of large nodules associated with peritumoral vessels and containing abnormal intratumoral vessels. To test the effects of potentially anti-angiogenic agents on tumor growth, we then used STC-1 cells stably transfected with the endostatin-coding sequence. Intrahepatic tumor volume showed no significant change at days 4 and 8, but a dramatic decrease at day 28 (9.7 +/- 1.7% of liver tissue versus 25.2 +/- 2.4% in controls), because of a markedly lower number of large nodules (11 +/- 1.8% versus 42 +/- 5.8%) likely to result from an increased apoptotic index (39.4 +/- 5.6% versus 18.3 +/- 3.4). Our results suggest that active angiogenesis is not necessary for the engraftment and early growth of endocrine cells metastatic to the liver but is required at a later stage of progression.

Endothelial cell differentiation in hepatocellular adenomas: implications for histopathological diagnosis.

BACKGROUND/AIMS: Little information is available about the patterns of endothelial cell differentiation observed in hepatocellular adenomas. We therefore aimed to analyze the endothelial cell immunophenotype in a large series of these tumors and evaluate its possible diagnostic relevance. METHODS: The expression of continuous and sinusoidal endothelial cell markers and of extracellular matrix proteins was analyzed by immunoperoxidase in 56 adenomas, as compared with 30 cases of focal nodular hyperplasia (FNH), 2 cases of telangiectatic FNH and 40 cases of hepatocellular carcinoma (HCC). RESULTS: Twenty-eight adenomas (50%) presented a sinusoidal pattern of endothelial cell differentiation, characterized by the expression of specific sinusoidal endothelial cell markers and the presence of a subendothelial matrix resembling the normal perisinusoidal matrix. Eleven tumors (19.5%) presented a continuous pattern of endothelial cell differentiation. Seventeen tumors (30.5%) showed a mixed pattern. Twenty-eight FNH presented a sinusoidal pattern of endothelial cell differentiation; all HCC presented a continuous pattern of endothelial cell differentiation. CONCLUSIONS: In hepatocellular adenomas, intra-tumoral vessels usually present a sinusoidal pattern of endothelial cell differentiation. However, the vascular phenotypic heterogeneity observed in our study questions the potential relevance of endothelial cell immunophenotyping for the differential diagnosis between hepatocellular adenomas and well differentiated HCC.

Expression, regulation, and function of alpha V integrins in hepatocellular carcinoma: an in vivo and in vitro study.

The expression of alpha V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of alpha V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of alpha V integrins in hepatocellular carcinoma. We first analyzed the expression of alpha V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. alpha V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 alpha V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study alpha V integrin regulation and function. HepG2 and Hep3B cells expressed alpha V integrin chain and used alpha V beta 1 and alpha V beta 5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta significantly increased the expression levels of alpha V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an alpha V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor.

Relationship between vascular development and vascular differentiation during liver organogenesis in humans.

BACKGROUNDS/AIMS: The complex vascular architecture characteristic of the normal adult liver is progressively acquired during the fetal life. In this study, we aimed to evaluate the relationship between angiogenesis and vascular differentiation during liver organogenesis. METHODS: We studied, in 51 fetuses of different gestational ages, the expression of markers of endothelial cell differentiation, integrins, pro- and anti-angiogenic extracellular matrix components, vascular endothelial growth factor (VEGF) and its receptors. RESULTS: Three main stages in the development of the vascular architecture of the liver were identified: (a) from 5 to 10 gestation weeks (GW), no evidence of de novo angiogenesis was detected; the vessels present in the liver primordium were the precursors of portal veins and sinusoids, deriving from preexisting vessels; (b) from 10 to 25 GW, angiogenesis and vasculogenesis resulted in the development of, respectively, arteries and intra-portal capillaries, while portal veins and hepatic sinusoids followed a differentiation process; (c) after 25 GW, little changes were detected in the various vascular compartments. The maximal expression of VEGF and its receptors was from 5 to 25 GW. CONCLUSIONS: The development of the hepatic vascular architecture is a multistep process combining angiogenesis, vasculogenesis and vascular differentiation, regulated by specific growth and differentiation factors including VEGF.

Low microvessel density is an unfavorable histoprognostic factor in pancreatic endocrine tumors.

BACKGROUND AND AIMS: In many malignant tumors, intratumoral microvascular density (MVD) has been suggested to be a prognostic parameter. We aimed to provide a quantitative evaluation of intratumoral microvascular density in a large series of resected endocrine tumors of the pancreas and to evaluate the potential prognostic significance of this parameter. METHODS: Eighty-two tumors from 77 patients have been studied. MVD was evaluated by 2 observers after CD34 immunostaining and correlated with the following parameters: WHO classification, hormonal profile, tumor size, vascular endothelial growth factor expression, occurrence of metastasis, duration of survival. RESULTS: MVD ranged from 5 to 92 vessels/field. MVD was significantly higher in well-differentiated benign endocrine tumors than in tumors of uncertain behavior and in carcinomas. No close correlation was found between MVD and the hormonal profile. MVD was significantly higher in tumors characterized by the following histoprognostic parameters: size <2 cm, proliferation index <2%, no evidence of metastasis. No close correlation was observed between MVD and VEGF expression. Finally, a MVD <30 vessels/field was associated with the occurrence of metastasis in tumors <2 cm and/or with a proliferation index <2% and with a significantly shorter survival after surgery. CONCLUSIONS: The quantitative analysis of microvessel density in pancreatic endocrine tumors may identify patients who, despite favorable conventional histoprognostic factors, are at risk of unfavorable evolution.