c‐Myc promotes lymphatic metastasis of pancreatic neuroendocrine tumor through VEGFC upregulation

Abstract Pancreatic neuroendocrine tumor (pNET) is a pancreatic neoplasm with neuroendocrine differentiation. pNET in early stage can be treated with surgical resection with long‐term survival, whereas the prognosis of pNET with locoregional or distant metastasis is relatively poor. Lymphangiogenesis is essential for tumor metastasis via the lymphatic system and may overhead distant metastasis. c‐Myc overexpression is involved in tumorigenesis. The role of c‐Myc in lymphangiogenesis is unclear. In this study, we evaluated the mechanism and effect of c‐Myc on lymphangiogenesis of pNET via interaction of lymphatic endothelial cells (LECs) and pNET cells. Lymph node metastasis was evaluated in pNET xenograft mice. Potential target agents to inhibit lymph node metastasis were evaluated in an animal model. We found that vascular endothelial growth factor C (VEGFC) expression and secretion was increased in pNET cell lines with c‐Myc overexpression. c‐Myc transcriptionally upregulates VEGFC expression and the secretion of pNET cells by directly binding to the E‐box of the VEGFC promoter and enhances VEGF receptor 3 phosphorylation and the tube formation of LECs. c‐Myc overexpression is associated with lymph node metastasis in pNET xenograft mice. Combinational treatment with an mTOR inhibitor and c‐Myc inhibitor or VEGFC‐neutralizing chimera protein reduced lymph node metastasis in the mice with c‐Myc overexpression. The mTOR inhibitor acts on lymphangiogenesis by reducing VEGFC expression in pNET cells and inhibiting the tube formation of LECs. In conclusion, mTOR and c‐Myc are important for lymphangiogenesis of pNET and are potential therapeutic targets for prevention and treatment of lymph node metastasis in pNET.


| INTRODUC TI ON
Pancreatic neuroendocrine tumor (pNET) is a rare histologic subtype of pancreatic cancer with the expression of neuroendocrine markers. It accounts for approximately 3% of all pancreatic cancers in Taiwan. 1 The incidence of pNET has significantly increased in the USA and Taiwan in the recent decade. 1,2 Although the localized pNET has a long-term survival (10-year survival rate of 62%-79%), pNET with distant metastasis has a 10-year survival rate approximating 20% according to the Surveillance, Epidemiology, and End Results (SEER) in the USA. 3 There are many factors reported to be associated with the overall survival of pNET, such as tumor grade, Ki-67 index, mitotic rate, age, sex, lymph node status, liver metastasis, and bone metastasis. [4][5][6][7] Lymph node metastasis is an important prognostic factor for tumor recurrence and outcome of cancers. 8 The association of lymph node status with postoperative recurrence or survival in pNET is controversial. [9][10][11][12][13] However, Jiang et al 14

have
shown that the presence of lymph node metastases was significantly associated with decreased disease-free survival (hazard ratio The metastasis of tumor cells occurs through lymphatic and blood vessels. Lymphatic vessels undergoing dynamic changes, such as lymphangiogenesis and lymphatic vessel remodeling, may facilitate metastasis. 8 There are many molecules associated with lymphangiogenesis, including vascular endothelial growth factor C (VEGFC)/D, VEGFA, angiopoietins, growth factors (hepatocyte growth factor (HGF), fibroblast growth factor, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin-like growth factor), inflammatory cytokines, etc. 16 Among them, the VEGFC/VEGF receptor 3 (VEGFR3) and VEGFD/ VEGFR3 axis is known as a major driver of lymphangiogenesis. 8,16 Lymph node metastasis was shown to be associated with the recurrence and disease-related survival of pNET. 14,15 However, the mechanism of lymph node metastasis in pNET was not well understood. We have identified a high percentage of c-Myc overexpression in pNET via Phosphatase and tensin homolog (PTEN)/ liver kinase B1 (LKB1)-dependent and PTEN/LKB1-independent mechanisms previously. 17 In this study, we investigated the association of c-Myc and lymph node metastasis in pNET, delineated the regulatory mechanism, and identified the potential targeted agents against lymph node metastasis in pNET.

| MATERIAL S AND ME THODS
This study was approved by the Institutional Review Board of the National Health Research Institutes.

| Plasmid transfection and lentiviral infection
Human c-Myc overexpression and c-Myc knockdown by shRNA were conducted by a lentiviral infection system according to Addgene's instruction. Briefly, lentiviral suspension was produced from 293T cells by transfecting expressing vector, packaging plasmid, and enveloping plasmid. We added lentiviral suspension to infect pNET cells in growth medium containing polybrene for 24 hours. Then the growth medium of the pNET cells was removed, and fresh growth medium was added to the lentiviral vector-infected pNET cells for another 24 hours. Then, puromycin was added to the growth medium of pNET cells for selecting lentiviral vector-infected cells to obtain stable expression clones.
Mouse c-Myc overexpression and VEGFC promoter-luciferase plasmids transfection were performed by using Turbofect transfection reagent (Thermo) according to the manufacturer's instruction. Briefly, 1 µg of DNA and 2 µL of transfection reagent were mixed in serum-free growth medium and added into a 70%-90% confluent cell layer. Transgene expression of the cells was measured after incubation for 24-48 hours.

| RNA extraction and RT-PCR
Total RNA was isolated from control or c-Myc-overexpressed QGP-1 and NIT-1 cells by using a total RNA mini kit (Geneaid), and a high-capacity cDNA reverse transcription kit (Applied Biosystems Inc) was used to perform reverse transcription according to the manufacturer's protocol. VEGFC expression was examined by using SYBR green PCR master mix, and β-actin

| Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was performed as described previously. 18 Briefly, control, c-Myc-overexpressed, and c-Myc-knocked-down QGP-1 cells were fixed with 1% formaldehyde at 37°C for 10 minutes and washed by cold PBS. The cells were harvested and lysed with RIPA buffer, and the collected cell lysate was sonicated to shear DNA to an average fragment size of 500-1000 bp.
After sonication, the cell lysate was treated with RNase A to remove RNA and with protease K to cleave peptide bonds. Anti-c-Myc (2 μg/ mL) and anti-Rabbit IgG (negative control) (2 μg/mL) antibodies were used for precipitating the protein/DNA complex. DNA fragments were collected and subjected to PCR amplification by using the primers specific for the detection of the −987 to −865 VEGFC promoter region which contained E-box sequence. The sequences for the primers are forward: 5′-GGGAGGGAGGACAAGAACTC-3′ and reverse: 5′-GACCGGCTTTAGAGGTGATG-3′.

| Immunoprecipitation assay
Control, c-Myc-overexpressed, and c-Myc-knocked-down QGP-1 cells were harvested and lysed by RIPA buffer. The cell lysate was incubated with anti-c-Myc (1 μg/mL) and anti-rabbit IgG (negative control; 1 μg/mL) antibodies at 4°C overnight. c-Myc-associated protein complex was pulled down by protein A/G and subjected to Western blot.

| Measurement of VEGFC secretion
QGP-1 and NIT-1 cells transfected with vector control or c-Myc overexpression plasmids were cultured in serum-free growth medium for 24 hours. Then, the growth medium was collected and concentrated 30-fold by an amicon ultra centrifugal filter device (Merck). The concentrated secretion from the cells was used to evaluate the amount of VEGFC secretion from the cells by Western blot.

| Tube formation assay of lymphoepithelial cells (LECs)
Growth factor-reduced Matrigel (BD Biosciences) was coated to each well of prechilled channel slide and incubated at 37°C for

| Pathological examination
Tumor and proximal (inguinal) lymph node from each mouse were har-

| Statistical analysis
The difference of relative luciferase activity and relative tube formation was analyzed by t-test using EXCEL (Microsoft

| c-Myc positively correlates with VEGFC expression in pNET cells
We have found that enlarged lymph node was noted in the xenograft mouse model of pNET cells (QGP-1) with PTEN and/or LKB1 loss (data not shown), which activate c-Myc. 17 In order to know whether c-Myc activation is associated with lymph node metastasis in pNET, we evaluated the effect of c-Myc on VEGFC expression in pNET cells (QGP-1 and NIT-1). Figure 1A shows that the expression of VEGFC was decreased in both cell lines with c-Myc knockdown.
By contrast, Figure 1B shows that VEGFC expression was increased in both cell lines with c-Myc overexpression compared with the cells infected with vector control. The downstream targets of c-Myc, such as E2F1 and TERT, 20,21 were also shown to be positively correlated with c-Myc levels, as presented in Figure S2. The results suggest that VEGFC expression is positively correlated with c-Myc activation. In addition, the VEGFC secretion was increased in both cell lines with c-Myc overexpression, as shown in Figure 1C. The results demonstrate that c-Myc upregulates VEGFC expression in pNET cells.

| c-Myc induces VEGFC expression via transcriptional upregulation
We further evaluated whether c-Myc regulates VEGFC expression at transcriptional level. Figure 2A shows in Figure 2C. The VEGFC promoter activity was abolished when the E-Box sequence was mutated, as shown in Figure 2D.

| c-Myc enhances tube formation of LECs via induction of VEGFC/VEGFR3 interaction
The main function of VEGFC is to trigger lymphangiogenesis via its receptor VEGFR3. We added condition medium derived from QGP-1 cells with or without c-Myc overexpression into LECs. Figure 3A shows that increased VEGFR3 phosphorylation and Prox1, the downstream target of VEGFR3, 26

| c-Myc promotes lymph node metastasis in a xenograft mouse model
We  Figure S4 and Table S1. Figure 4A shows that  Table S2.
There were three mice with high expression of VEGFC in the control group, whereas nine mice had high expression of VEGFC in the group with c-Myc overexpression (P = .02, Fisher's exact test).

| mTOR inhibitor inhibits lymphangiogenic properties of lymphatic endothelial cells
In QGP-1 xenograft mice with c-Myc overexpression, lymph node metastasis was reduced in the group treated with a combination of RAD001 and 10058-F4 or VEGFR3/Fc but not in the group treated with RAD001, 10058-F4, or VEGFR3/Fc alone. The result suggests that mTOR plays an important role for lymph node metastasis in pNET. We then evaluated the effect of the mTOR inhibitor on lymphangiogenesis. Figure 5A shows that RAD001 reduced the phosphorylation of VEGFR3 and the tube formation of LECs. Similar result was also presented in murine LECs (SVEC4-10), as shown in Figure 5B, which was consistent with the result in an animal model.

| D ISCUSS I ON
In this study, we demonstrate that c-Myc overexpression transcriptionally upregulates VEGFC expression in pNET cells, which promotes lymphangiogenesis of pNET cells in vitro and in vivo.
VEGFC is a member of the PDGF family. 27 It is indispensable for embryonic and adult lymphangiogenesis, 28 and it correlates significantly with lymphangiogenesis and the proliferation and migration of vascular endothelial cells. 27 VEGFC is produced by macrophage and granulocytes and promotes lymphangiogenesis at sites of tissue inflammation. 29,30 However, VEGFC can also be secreted by cancer cells themselves and directly promote cancer cell migration and invasion, tumor-associated lymphangiogenesis, and lymphatic metastasis. 8  were involved in tumor growth. c-Myc promotes lymph node metastasis, and the size of lymph node was reduced in the mice treated with the c-Myc inhibitor in our animal study ( Figure 4B).
However, the number of mice with lymph node metastasis was not significantly reduced by treatment with the c-Myc inhibitor or VEGFC-neutralizing recombinant protein alone, but it was reduced by treatment with a combination of the mTOR inhibitor and one of them. The result suggests that mTOR in addition to c-Myc is also an important signal for lymphangiogenesis in pNET. Activation of mTOR pathway was shown to be associated with increased LVD and lymph node metastasis via upregulation of VEGFC in various cancers in in vitro and in vivo models and in human samples. [45][46][47] mTOR inhibitors decreased VEGFC/D expression and diminished lymphangiogenesis and lymph node metastasis in various cancer models. [45][46][47][48] The results demonstrate the role of mTOR activation in lymphangiogenesis by VEGFC in LECs and primary tumors. In our study, RAD001 was shown to decrease the VEGFC expression of QGP-1 cells ( Figure S6). RAD001 suppressed VEGFR3 phosphorylation and inhibited the tube formation of human and murine LECs, as shown in Figure 5. The results suggest that the mTOR inhibitor targets both tumor cells and LECs for inhibition of lymphangiogenesis in pNET.
Activation of the mTOR pathway has been shown in pNET according to gene expression array and immunohistochemistry. 49,50 The association between mTOR and c-Myc in pNET has been shown in our previous study. 17 In the current study, we have  Figure 6.
In conclusion, c-Myc promotes lymphatic metastasis via transcriptional upregulation of VEGFC in pNET, whereas mTOR activation is also important for the lymphangiogenesis of pNET. Combined targeting of mTOR and c-Myc/VEGFC is a potential therapy for prevention and treatment of lymphatic metastasis in pNET.

F I G U R E 6
The putative model of lymphatic metastasis in pancreatic neuroendocrine tumor (pNET). c-Myc can be regulated by PTEN and LKB1 via the AKT/mTOR axis, and it can backregulate PTEN to activate the mTOR pathway. c-Myc can upregulate VEGFC expression and the secretion of QGP-1 cells (pNET) and promotes lymphangiogenesis and lymph node metastasis. mTOR is constitutively activated in pNET and can be negatively regulated by PTEN and/or LKB1. mTOR promotes lymphangiogenesis in pNET via increasing vascular endothelial growth factor C (VEGFC) expression of pNET cells and inhibiting tube formation of lymphoepithelial cells (LECs) in pNET. Combined treatment with an mTOR inhibitor, RAD001, and a c-Myc inhibitor (10058-F4) or a VEGFC-neutralizing chimeric protein (VEGFR/Fc) reduces the lymphangiogenesis and lymph node metastasis of pNET in a mouse model