Research ArticleExperimental Studies

PTPN3 Could Βe a Therapeutic Target of Pancreatic Cancer

HIDEYA ONISHI, NAOYA IWAMOTO, KEITA SAKANASHI, SATOKO KOGA, YASUHIRO OYAMA, KOSUKE YANAI, KATSUYA NAKAMURA, SHUNTARO NAGAI, AKIKO FUJIMURA, KAZUNORI NAKAYAMA, KEIGO OZONO and AKIO YAMASAKI
Anticancer Research June 2022, 42 (6) 2869-2874; DOI: https://doi.org/10.21873/anticanres.15768

Abstract

Background/Aim: Recently, protein tyrosine phosphatase non-receptor type 3 (PTPN3) has gained attention. However, the role of PTPN3 in cancer has not been fully elucidated. In the present study, we analyzed the role of PTPN3 in pancreatic cancer and investigated whether PTPN3 could be a new therapeutic target for pancreatic cancer. Materials and Methods: Two pancreatic ductal adenocarcinoma (PDAC) cell lines were used as target cells. Cell proliferation was investigated using cell counting and a xenograft mouse model. Migration and invasion were analyzed using Transwell inserts. Activation-related signaling molecules were examined by western blotting. Results: PTPN3 contributes to the proliferation, migration, and invasion of PDAC cells in vitro. PTPN3 promotes tumor growth in a mouse xenograft model, an action mediated partially through the MAPK pathway. Conclusion: PTPN3 could be a new therapeutic target for pancreatic cancer.

Key Words:

Pancreatic cancer is one of the deadliest cancers worldwide. Many therapeutic approaches have been tested, but their effects are limited. Therefore, the development of therapies targeting new molecules is required.

Protein tyrosine phosphatase (PTP) non-receptor type 3 (PTPN3) is a tyrosine phosphatase that negatively regulates certain tyrosine kinase signaling pathways (1). Recently, we showed that PTPN3 is highly expressed in activated lymphocytes and that PTPN3 acts as an immune checkpoint to regulate proliferation, migration, and cytotoxicity in lymphocytes (2). Therefore, the development of cancer therapies targeting PTPN3 in activated lymphocytes is promising.

However, PTPN3 is also expressed in cancers and its biological significance remains controversial. For example, PTPN3 inhibits the proliferation and migration of non-small cell lung cancer cells (3). However, in small cell lung cancer, we have shown that PTPN3 induces proliferation and invasiveness (4). Other researchers have also shown that PTPN3 increases the malignant traits of glioma and ovarian cancer, and angiogenesis in gastric cancer (5-8). However, the biological significance of PTPN3 in pancreatic cancer has not yet been examined.

In the present study, we examined the biological role of PTPN3 in pancreatic cancer and evaluated whether PTPN3 could function as a novel therapeutic target for pancreatic cancer.

Materials and Methods

Cell culture and reagents. Two human pancreatic ductal adenocarcinoma (PDAC) cell lines (SUIT-2 and PANC-1) were maintained in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY, USA) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

Western blot analysis. Western blotting was performed as previously described (9). Protein samples (50 μg) were separated by electrophoresis on a sodium dodecyl sulfate (SDS)-sulfate-polyacrylamide gel and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). The protein-transferred membranes were incubated overnight at 4°C with primary antibodies against PTPN3 (1:200, sc-9789, Santa Cruz Biotechnology, Dallas, TX, USA), AKT (1:1,000, sc-8312, Santa Cruz Biotechnology), pAKT (1:1,000, sc-101629, Santa Cruz Biotechnology), ERK (1:1,000, No. 9102, Cell Signaling Technology, Danvers, MA, USA), and pERK (1:1,000, No. 9101, Cell Signaling Technology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added, and the membranes were incubated for 1 h at room temperature. The antibodies against α-tubulin (1:1,000, Sigma-Aldrich, St. Louis, MO, USA) were used as protein loading controls.

RNA interference. ON-TARGETplus™ SMARTpool siRNA targeting PTPN3 (L-009372) and a negative control siRNA (ON-TARGETplus™ Control non-targeting siRNA, D-001810) were purchased from Dharmacon (Lafayette, CO, USA). Cells (0.2×106 cells/well) seeded in six-well plates were transfected with 100 nM siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

The nucleotide sequence of the short hairpin RNA (shRNA) targeting PTPN3 was as follows: shPTPN3, CAATCAGAAGCA GAATCCTGCTATA. The oligonucleotides were ligated into a plasmid vector (pcDNA™6.2-GW/Em-miR, #K4934-00; Thermo Fisher Scientific) and used as PTPN3 shRNA. Cells were used for experiments 2 days after transfection.

Migration and invasion assay. Invasion assays were performed using a Matrigel-coated Transwell insert, as previously described (9). Briefly, cells (2×105) were placed in the upper chamber and incubated for 18 h. The cells that migrated to the lower side of the filter were fixed and stained with Diff-Quik reagent (Sysmex, Kobe, Japan) and counted under a light microscope (Nikon Eclipse TE 300, Nikon, Tokyo, Japan). The migration assay was performed using a non-Matrigel-coated Transwell insert.

Cell proliferation assay. All PDAC cell lines were seeded in 96-well plates at 5,000 cells/well and cultured for 16 h. The cell numbers were counted using a light microscope on days 2 and 3.

In vivo xenograft tumor model. Five-week-old female athymic nude mice (BALB/c nu/nu) were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and acclimated for one week. All experimental procedures were approved by the Animal Care and Use Committee of Kyushu University (permit number A29-067-0). All mice were housed and maintained in a specific pathogen-free animal facility at Kyushu University, and efforts were made to minimize the number of animals used and their suffering. All experiments were performed in strict accordance with the Guidelines for the Proper Conduct of Animal Experiments (the Science Council of Japan). SUIT-2 cells transfected with PTPN3 siRNA or control siRNA were subcutaneously implanted into the flanks of nude mice (1×106 cells in Matrigel per mouse; n=3 in each treatment group). Tumor size was measured twice each week, and volume was calculated as follows: A × B2 × 0.5, where A is the longest diameter and B is the smallest of the two perpendicular diameters of the tumor.

Immunohistochemistry. Tumors recovered from the mice were fixed in formalin and embedded in paraffin. Immunohistochemistry was performed as previously described (10). The following primary antibodies were used: PTPN3 (1:50, sc-515181; Santa Cruz Biotechnology) and Ki67 (1:50, sc-15402; Santa Cruz Biotechnology). Histofine Simple Stain MAX-PO (M or R; Nichirei Bioscience, Tokyo, Japan) was used as the secondary antibody.

Statistical analyses. Data are presented as the mean±standard deviation (SD). Calculations were performed using JMP software (version 12.0; SAS Institute, Cary, NC, USA) or Microsoft Excel software (Microsoft, Redmond, WA, USA). The Student’s t-test was used to compare continuous variables between pairs of groups. Statistical significance was set at p<0.05.

Results

PTPN3 induces proliferation of PDAC cells. First, we investigated whether PTPN3 is involved in the proliferation of PDAC cells. Two days after transfection with siRNA or shRNA, cells were re-plated and proliferation was estimated by cell counting using a light microscope. PTPN3 siRNA-transfected PDAC cells and PTPN3 shRNA-transfected PDAC cells showed a significantly reduced proliferation compared to control cells in both cell lines (Figure 1). These results suggest that PTPN3 induces PDAC cell proliferation.

Figure 1.
Figure 1.

PTPN3 inhibition decreases proliferation in pancreatic ductal adenocarcinoma (PDAC) cells. Cell numbers at the indicated days in two PDAC cell lines transfected with control siRNA, PTPN3 siRNA, control shRNA, and PTPN3 shRNA were counted using light microscopy. Data are presented as means±standard deviations. *Significantly different at p<0.05.

PTPN3 induces the migration and invasion of PDAC cells. Next, we analyzed whether PTPN3 was involved in the migration and invasion of PDAC cells. In these experiments, the number of cells that migrated from the upper to lower side was counted. Analysis of migration indicated that the number of PTPN3 siRNA-transfected PDAC cells was approximately 20% lower than that of control cells in both cell lines (Figure 2A). Analysis of invasion indicated that the number of PTPN3 siRNA-transfected PDAC cells decreased by approximately 40% compared to that of control cells in both cell lines (Figure 2B). These results suggest that PTPN3 induces the migration and invasion of PDAC cells.

Figure 2.
Figure 2.

PTPN3 inhibition decreases migration and invasion in pancreatic ductal adenocarcinoma (PDAC) cells. A) The migration abilities of PDAC cells transfected with control siRNA or PTPN3 siRNA were estimated using a Transwell insert. B) The invasive abilities of PDAC cells transfected with control siRNA or PTPN3 siRNA were estimated using a Matrigel invasion assay. Data are presented as means±standard deviations. *Significantly different at p<0.05.

PTPN3 induces tumor growth in vivo. Next, we confirmed the in vivo results of PTPN3 promoting activity in PDAC cells using a xenograft mouse model. In this experiment, the mice were divided into two groups: a control siRNA-transfected SUIT2 cell-injected group (control group) and a PTPN3 siRNA-transfected SUIT2 cell-injected group (PTPN3 siRNA group). Each siRNA-transfected SUIT2 cell line was subcutaneously administered in both flank areas, and tumor formation and volume were checked for two weeks. Five of the six areas showed tumor formation in both groups. A representative image is shown in Figure 3A. The tumor volume in the PTPN3 siRNA group was significantly lower than that in the control group (Figure 3B). Immunohistochemical staining of the tumor showed that PTPN3 expression was decreased in the PTPN3 siRNA group, and that Ki67 expression, which reflects cell proliferation, was decreased in the PTPN3 siRNA group (Figure 3C).

Figure 3.
Figure 3.

PTPN3 induces tumor proliferation in vivo. SUIT-2 cells transfected with PTPN3-targeting siRNA or non-targeting control siRNA were subcutaneously implanted into bilateral flank regions (1.0×106 cells in 50 μl of RPMI) of BALB/c nude mice (n=6). A) Representative photos of mice (2.0 weeks after tumor injection) are shown. Arrow shows tumor. B) The tumor size was determined on the indicated days. Data are presented as means±standard deviations. *Significantly different at p<0.05. C) PTPN3 and Ki67 immunostainings were performed using tumors from mice. Original magnification is 400×.

MAPK pathway is involved in PTPN3-induced cell proliferation and tumor growth of PDAC. Next, we investigated the pathway that contributes to PTPN3-induced proliferation. First, we confirmed that PTPN3 expression in PTPN3 siRNA-transfected cells decreased by >80% in both cell lines (Figure 4). Phosphorylated ERK in PTPN3 siRNA-transfected PDAC cells was significantly decreased compared to that in the control, whereas phosphorylated AKT was not affected by PTPN3 inhibition in both cell lines (Figure 4). These results suggest that the MAPK pathway is involved in the effect of PTPN3 in PDAC cells.

Figure 4.
Figure 4.

MAPK pathway is involved with the cell activation of pancreatic ductal adenocarcinoma (PDAC) by PTPN3. The protein expression of PTPN3, AKT, ERK and the phosphorylation of AKT and ERK were examined by western blot. α-tubulin was used as a loading control.

Discussion

The results showing that PTPN3 inhibition decreases proliferation, migration, and invasion in pancreatic cancer cells suggest that PTPN3 inhibition could be a therapeutic strategy for pancreatic cancer. Previously, we showed that PTPN3 inhibition leads to the upregulation of the cytotoxicity of activated lymphocytes (2). When we consider systemic therapy for pancreatic cancer, PTPN3 inhibition could have dual effects on lymphocyte activation and direct tumor suppression. However, there are no specific PTPN3 inhibitors. PTP has 23 isoforms, and pan-PTP inhibitors exist; however, pan-PTP inhibitors do not affect the activation of lymphocytes (2). Therefore, the development of a PTPN3-specific inhibitor is urgently required.

In this study, we showed that NF-κB induces the expression of PTPN3, whereas TGF-β decreases its expression (11). NF-κB is activated during lymphocyte activation. Since NF-κB induces PTPN3 expression and PTPN3 inhibits the over-activation of lymphocytes, the homeostasis of lymphocyte activation may be regulated in this way. In addition, the cancer microenvironment (CME) is where activated lymphocytes fight cancer. In CME, TGF-β is secreted by cancer cells and migration of lymphocytes is affected. TGF-β may be involved in the down-regulation of NF-κB and PTPN3 expression.

PTPN11 is a downstream regulator of PD-1 signaling (12). This suggests that PTPN3 inhibitors could be used as new cancer therapeutic agents. Consistent with the relation between PTPN11/PD-1, PTPN3 may also be regulated by receptor-mediated signaling. Research in this direction may hold promise.

PTPN3 leads to dephosphorylation of tyrosine kinases because it is a tyrosine phosphatase. Interestingly, in pancreatic cancer, PTPN3 leads to the induction of malignant phenotypes such as proliferation and invasiveness of cancer cells. We have reported that PTPN3 augments the proliferation, migration, and invasion of lung neuroendocrine tumors and that PTPN3 promotes the phosphorylation of tyrosine kinase through the expression of calcium voltage-gated channel subunit alpha 1G (4). PTPN3 may also accelerate the phosphorylation of tyrosine kinases through a certain molecule in pancreatic cancer. Further research in this direction will be essential to examine whether PTPN3 can be a therapeutic target.

In the present study, we examined the biological role of PTPN3 in pancreatic cancer and elucidated that PTPN3 could be a new therapeutic target for pancreatic cancer.

Acknowledgements

The Authors thank Ms. Emi Onishi for her technical assistance. This study was supported by JSPS KAKENHI Grant Numbers JP 20K09180, JP 21K08672, and JP 21K09583. The Authors would like to thank Editage (www.editage.com) for English language editing.

Footnotes

  • Authors’ Contributions

    Conception and design: Onishi H, Yanai K, Nagai S; data acquisition and analysis: Koga S, Oyama Y, Nakayama K, Yamasaki A; writing and original draft preparation: Onishi H; critical review and editing: Iwamoto N, Sakanashi K, Ozono K; funding acquisition: Nakamura K, Fujimura A, Onishi H.

  • Conflicts of Interest

    The Authors declare no financial or commercial conflicts of interest regarding this study.

  • Received April 20, 2022.
  • Revision received May 12, 2022.
  • Accepted May 13, 2022.

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PTPN3 Could Βe a Therapeutic Target of Pancreatic Cancer
HIDEYA ONISHI, NAOYA IWAMOTO, KEITA SAKANASHI, SATOKO KOGA, YASUHIRO OYAMA, KOSUKE YANAI, KATSUYA NAKAMURA, SHUNTARO NAGAI, AKIKO FUJIMURA, KAZUNORI NAKAYAMA, KEIGO OZONO, AKIO YAMASAKI
Anticancer Research Jun 2022, 42 (6) 2869-2874; DOI: 10.21873/anticanres.15768
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