PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors

Abstract

Small intestinal neuroendocrine tumors (SI-NETs) are small, slow growing neoplasms with loss of one copy of chromosome 18 as a common event. Frequently mutated genes on chromosome 18 or elsewhere have not been found so far. The aim of this study was to investigate a possible tumor suppressor role of the transmembrane receptor type tyrosine phosphatase PTPµ (PTPRM at 18p11) in SI-NETs. Immunohistochemistry, quantitative RT-PCR, colony formation assay and quantitative CpG methylation analysis by pyrosequencing were performed. Undetectable/very low levels of PTPRM or aberrant pattern of immunostaining, with both negative and positive areas, were detected in the majority of tumors (33/40), and a significantly reduced mRNA expression in metastases compared to primary tumors was observed. Both the DNA methylation inhibitor 5-aza-2'-deoxycytidine and the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) induced PTPRM expression in CNDT2.5 and KRJ-I SI-NET cells. CpG methylation of upstream regulatory regions, the promoter region and the exon 1/intron 1 boundary was detected by pyrosequencing analysis of the two cell lines and not in the analyzed SI-NETs. Overexpression of PTPRM in the SI-NET cell lines reduced cell growth and cell proliferation and induced apoptosis. The tyrosine phosphatase activity of PTPRM was not involved in cell growth inhibition. The results support a role for PTPRM as a dysregulated candidate tumor suppressor gene in SI-NETs and further analyses of the involved mechanisms are warranted.

Keywords: DNA methylation; PTPRM; SI-NETs; epigenetic; neuroendocrine tumors.

Figure 1

Representative results from immunohistochemical analysis of PTPRM (anti-PTPRM antibody ab111207) in 19 primary tumors and 21 paired metastases. Scale bar, 50 µm. (A) Negative staining of a metastatic tumor, (B) variable heterogeneous pattern of staining, with both negative and positive areas, in a primary tumor and (C) positive staining of a metastatic tumor are shown.

Figure 2

Chromogranin A-positive cells are positively stained for PTPRM in normal small intestinal tissue. (A) Two consecutive tissue sections of the normal small intestine were used for immunohistochemical analysis of Chromogranin A and PTPRM. PTPRM staining was observed only in the presence of the primary antibody (No primary antibody). Scale bar, 50 µm. (B) Immunofluorescent double staining for chromogranin A and PTPRM. Scale bar, 50 µm.

Figure 3

Expression level of PTPRM in primary tumors and metastases (logarithmic scale). Real-time RT-PCR analysis of PTPRM shows significantly reduced expression in metastases (n = 27) compared to paired primary tumors (n = 23). Wilcoxon–Mann–Whitney U test result displayed in box plot (P = 0.001). Whiskers show minimum and maximum values, boxes represent 25–75% data ranges and horizontal lines within boxes are medians. (Primary tumors: lower quartile = −1.1, median = 0, upper quartile = 0.69), (Metastases: lower quartile = −2, median = −1.2, upper quartile = −0.35). (Wilcoxon–Mann–Whitney U test without outliers; Primary tumors (n = 20) and metastases (n = 26), P = 0.002).

Figure 4

Expression of PTPRM, as determined by quantitative real-time RT-PCR, in CNDT2.5 and KRJ-I cells. (A) After inhibition of DNA methylation by 5-aza-2′-deoxycytidine (Aza) for 72 h. (B) After treatment with 3-deazaneplanocin (DZNep) for 72 h. Data shown are means ± s.d. of triplicates.

Figure 5

Quantitative bisulfite pyrosequencing analysis of the PTPRM gene. (A) Schematic structure of the PTPRM gene upstream regulatory region and exon 1. Location of CGI, CGI shores and DNA methylation assays are shown. (B) Methylation analysis of 13 CpG residues in the promoter region. (C) Methylation analysis of 25 CpG residues at the exon1/intron 1 region.

Figure 6

Growth regulatory role of PTPRM in SI-NET cells. (A) Colony formation assay in the adhesive CNDT2.5 cell line. Cells transfected with PTPRM expression vector or an empty vector were selected for puromycin resistance, and colonies were counted after 10 days. Increased expression of PTPRM was measured at mRNA level after transfection. (B) Determination of cell proliferation and apoptosis in CNDT2.5 (adhesive cells) and KRJ-I (suspension cells) transfected with PTPRM expression vector or empty vector for 72 h. Data shown are means ± s.d. of triplicates.

Figure 7

Expression of both PTPRM-wt and PTPRM-mut suppresses colony formation in SI-NET cells. (A) CNDT2.5 cells transfected with empty vector, PTPRM-wt or PTPRM-mut were selected for G418 resistance and colonies were counted after 10 days. PTPRM-mut consisted of a double mutant of PTPRM in which the Cys¹⁰⁹⁵ and Cys¹³⁸⁹ residues were changed to serine in order to disrupt tyrosine phosphatase activity (11). (B) Expression of PTPRM after transfection as determined by quantitative real-time RT-PCR (ANOVA and Bonferroni test were applied). Data shown are means ± s.d. of triplicates. Note that the expression plasmid PTPRM-wt was different from the one used in the experiment of Fig. 6.