The link between menin and pleiotrophin in the tumor biology of pancreatic neuroendocrine neoplasms

Abstract MEN1, which encodes menin protein, is the most frequently mutated gene in pancreatic neuroendocrine neoplasms (pNEN). Pleiotrophin (PTN) has been reported as a downstream factor of menin that promotes metastasis in different tumor entities. In this study, the effect of menin and its link to PTN were assessed using features of pNEN cells and the outcome of patients with pNEN. The expression levels of menin and PTN in tissues from patients with pNEN were examined using qRT‐PCR and western blot and compared with their metastasis status. Functional assays, including transwell migration/invasion and scratch wound‐healing assays, were performed on specifically designed CRISPR/Cas9‐mediated MEN1‐knockout (MEN1‐KO) pNEN cell lines (BON1MEN1‐KO and QGP1MEN1‐KO) to study the metastasis of pNEN. Among 30 patients with menin‐negative pNEN, 21 revealed a strong protein expression of PTN. This combination was associated with metastasis and shorter disease‐free survival. Accordingly, in BON1MEN1‐KO and QGP1MEN1‐KO cells, PTN protein expression was positively associated with enhanced cell migration and invasion, which could be reversed using PTN silencing. PTN is a predicting factor of metastatic behavior of menin‐deficient‐pNEN. In vitro, menin is able to both promote and suppress the metastasis of pNEN by regulating PTN expression depending on the tumoral origin of pNEN cells.


| INTRODUC TI ON
Pancreatic neuroendocrine neoplasms (pNEN) are a heterogeneous group of malignancies arising from different cell types within the pancreas. 1 According to the most recent WHO classification, 2 pNEN are categorized into well differentiated pancreatic neuroendocrine tumors (pNET Grade G1, G2 and G3) as well as poorly differentiated pancreatic neuroendocrine carcinoma (pNEC G3). The grading depends on the proliferation rate, which is measured in Ki67-positive cells within 10 high-powered fields (G1: <3%, G2: 3%-20%, G3: >20%) and is the strongest prognostic factor. 3 Further effective prognostic tools are the TNM classification, 4 presenting a strong correlation of size and metastasis with unfavorable prognosis, metastasis in lymph nodes 5 /distant metastasis, 6 as well as markers such as preoperative dysglycemia (blood glucose ≥140 mg% and/or HbA 1c ≥6.5%) 7 or peroperative CRP (>5 mg/L). 8 Furthermore, pNENs in combination with lymph node 9 or distant metastasis 6,10 are associated with worse prognosis.
Pancreatic neuroendocrine neoplasms can be inherited (as in MEN1 syndrome) or developed due to germline mutations within the MEN1 gene that is located on 11q13. 11 MEN1 translates into the protein menin, which is known to be a tumor suppressor in the development of different endocrine tumors. 12,13 However, the vast majority (60%-90%) of pNEN arise sporadically. [14][15][16][17] Frequently occurring genetic alterations in pNEN are LOH of MEN1(50%) 18 and/or MEN1 mutations (40%). 19 MEN1 mutations often include nonsense and missense mutations as well as in-frame deletions distributed across the gene locus. 20,21 Those alterations often result in menin protein degradation and inactivation revealing that menin may play an important role in tumorigenesis and tumor progression of all pNEN. However, the mechanism of how menin inactivation initiates tumorigenesis of pNEN is not well understood.
The heparin-binding growth factor pleiotrophin (PTN) is a secreted 19-kDa regulatory peptide with angiogenic properties that is considered as a proto-oncogene and is overexpressed in various malignancies such as breast, prostate, colon, and skin cancer. [22][23][24][25] Menin has been shown to repress PTN expression in non-small-cell lung cancer 26 and malignant melanoma. 26 As there is currently no literature on the role of PTN in pNEN, this study aimed to evaluate the link between menin and PTN in pNEN. For this purpose, CRISPR/ Cas9 MEN1-knockout BON1 and QGP1 cells were developed and subsequent functional alternations were analyzed. The association between menin and PTN was further examined in pNEN tissue.

| Origin and culture of BON1 and QGP1 cells
The human pNEN cell line BON1, which was derived from a lymph node metastasized site of pNEN, 27 was given as a gift from Dr. M.
Kidd, Yale University School of Medicine and had already been authenticated using short tandem repeat (STR) analysis. The human pNEN cell line QGP1 (pNEN primary) was purchased from the JCRB (Japan). The adherent monolayer BON1 and QGP1 cells were cultured as described previously. 28 Whole exome sequencing of both cell lines has shown significant genetic differences. 29

| Sanger sequencing
Sanger sequencing was used to confirm the synthesized sgRNA-lentiCRISPRv2 vector with the U6 primer before infection of pNEN cells and the MEN1-knockout in CRISPR/Cas9 cell lines using the primer (AAATTGGACAGCTCCGGTGT) (Invitrogen) as previously described. 33

| Quantitative real-time PCR (qRT-PCR)
The mRNA of frozen tissues was extracted and reverse tran- qRT-PCR analysis was performed as previously described. 28 GAPDH was used as an internal reference for normalization and the relative mRNA expression of each gene was analyzed using the ΔΔCT method as recommended. 34

| Protein extraction and western blot analysis
Protein was extracted by RIPA buffer supplemented with complete protease inhibitors (Roche) from frozen pNEN tissue specimen (60-80 mg) or from pNEN cell pellets. Protein expression was assessed as previously described 35

| Scratch wound-healing and migration/ invasion assay
Scratch wound-healing assay was used to assess the cell migration ability. A 10μl pipette tip (Eppendorf) was used to perform a vertical scratch in each well of 6-well plates (Falcon, Colorado, USA) which was filled with more than 90% confluency monolayer cells.
After removing non-adherent cells, the cell-free area was measured.
At ×100 magnification, photographic documentation of cell-free areas was taken every 24 h. The wound-healing rate was calculated using the formula: wound-healing rate (%) = [(width at 0 h − width at 120 h)/(width at 0 h)] × 100.
The migration and invasion potential were assessed by transwell migration/invasion assay as described previously. 28 All experiments were repeated three times.

| Statistical analysis
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software). Relative expression and number of cells were recorded as mean ±SD. n describes the number of replicates. The OS time of patients with pNEN was defined as the time from resection to either death or last follow-up. Survival curves were plotted using the Kaplan-Meier method. The difference between the Kaplan-Meier curves was tested for significance applying the logrank test. For experiments involving MTT assay, transwell migration assay, transwell invasion assay, and scratch wound-healing assay, data from a minimum of three independent experiments were used for statistical analysis. Results from three independent experiments were averaged prior to statistical analysis. A two-tailed, unpaired Student t-test or two-way ANOVA were used to analyze data and determine significant differences. All the analyses were considered statistically significant at a p < 0.05 level and p-values are indicated with asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

| Downregulation of relative MEN1 mRNA expression in metastasized patients with pNEN
Relative mRNA expression levels of MEN1 and PTN were assessed using qRT-PCR in 40 random G1 and G2 pNEN tissues. All samples were divided into two groups regarding their N (lymph node metasta-

| PTN protein expression in meninnegative pNEN
As MEN1 mRNA was downregulated in metastasized pNEN, tumors with negative protein expression of menin (pNEN m− , no detectable protein expression on grayscale analysis using ImageJ software <5%) were analyzed further (n = 30). In all 30 pNEN m− , PTN expression was examined. As shown in Table 1 and Figure 1B

| Impact of menin absence in metastasis of BON1 and QGP1 cells
To determine whether the lack of menin was associated with migration and invasion of pNEN cells, transwell migration and scratch wound-healing assays were performed in both BON1 MEN1-KO and QGP1 MEN1-KO . In transwell migration and invasion assays, cell migration and invasion were markedly accelerated in BON1 MEN1-KO (p < 0.01; Figure 3A,B), but significantly weakened in QGP1 MEN1-KO (p < 0.01; Figure 3C,D). The scratch wound-healing assay further confirmed that the absence of menin significantly promoted the migration abilities of BON1 (p < 0.001; Figure 3E,F), but statistically suppressed cell migration of QGP1 (p < 0.01; Figure 3G,H). Taken together, these data suggested that knockout of MEN1, accelerated cell migration and invasion in the more aggressive BON1 cells, but suppressed the abilities of migration and invasion of QGP1 cells, reflecting the different regulation of menin on metastasis of different pNEN entities.

| Downregulation of PTN on invasion and migration of pNEN cell lines
Having

| DISCUSS ION
As MEN1 is the most frequently mutated gene in sporadic pNEN, 19 this study examined the loss of MEN1 coding protein menin and its

ACK N OWLED G EM ENT
None.

D I SCLOS U R E
No potential conflict of interest relevant to this article was reported.