The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms

Department of Gastroenterological Surgery, Yale University School of Medicine, New Haven, CT, USA.
Cancer (Impact Factor: 4.89). 05/2012; 118(10):2763-75. DOI: 10.1002/cncr.26592
Source: PubMed


Although gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki-67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP-NEN biomarker.
Functional assays were undertaken to investigate the mechanistic role of INA in the pancreatic BON cell line. Expression levels of INA were investigated in 50 pancreatic NENs (43 primaries, 7 metastases), 43 small intestinal NENs (25 primaries, 18 metastases), normal pancreas (n = 10), small intestinal mucosa (n = 16), normal enterochromaffin (EC) cells (n = 9), mouse xenografts (n = 4) and NEN cell lines (n = 6) using quantitative polymerase chain reaction, Western blot, and immunostaining analyses.
In BON cells, decreased levels of INA messenger RNA and protein were associated with the inhibition of both proliferation and mitogen-activated protein kinase (MAPK) signaling. INA was not expressed in normal neuroendocrine cells but was overexpressed (from 2-fold to 42-fold) in NEN cell lines and murine xenografts. In pancreatic NENs, INA was overexpressed compared with pancreatic adenocarcinomas and normal pancreas (27-fold [P = .0001], and 9-fold [P = .02], respectively). INA transcripts were correlated positively with Ki-67 (correlation coefficient [r] = 0.5; P < .0001) and chromogranin A (r = 0.59; P < .0001). INA distinguished between primary tumors and metastases (P = .02), and its expression was correlated with tumor size, infiltration, and grade (P < .05).
INA is a novel NEN biomarker, and its expression was associated with MAPK signaling and proliferation. In clinical samples, elevated INA was correlated with Ki-67 and identified malignancy. INA may provide additional biologic information relevant to delineation of both pancreatic NEN tumor phenotypes and clinical behavior.

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    • "To investigate mechanistic roles and the importance of NAP1L1 in proliferation, we used the adherent human metastasized pancreatic neuroendocrine neoplasm (NEN) cell line, BON [33], authenticated by STR (Genetica DNA Laboratories, Cincinnati, OH, USA). The cells were cultivated in medium containing RPMI 1640, Hams’ F12 (Gibco, Grand Island, NY, USA; 1:1 ratio), 10% FBS and penicillin/streptomycin (100 IU/ml) in 75 cm2 flasks (Sarstedt, Newton, NC, USA) as described [32]. "
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    ABSTRACT: Background: The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57(Kip2) (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57(Kip2). Results: NAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic (mammalian) target of rapamycin pathway proteins and their phosphorylation (p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57 (Kip2) promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57(Kip2) (transcript and protein) was upregulated. For methylation of the p57 (Kip2) promoter, NAP1L1 bound directly to the promoter (-164 to +21, chromatin immunoprecipitation). In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57(Kip2) (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed. Conclusion: NAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of p57 (Kip2) promoter methylation.
    Full-text · Article · Jul 2014 · Epigenetics & Chromatin
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    • "Messenger RNA was extracted and converted to cDNA from small pieces (~20mg) of tissue or cell line lysates (1x106 cells) as described [27] using TRIZOL® (Invitrogen, Carlsbad, CA) and the High Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA). Transcript levels of CgA (exons 1-6; corresponding amino acids 1-251 and >85% of the coding region; see Figure 1) and prohormone convertase (PCSK1) expression were quantified using Assays-on-DemandTM products and the ABI 7900 Sequence Detection System (both Applied Biosystems) according to the manufacturers’ instructions. "
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    No preview · Article · Apr 2012 · Neuroendocrinology
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