Inhibition of PTPN3 Expressed in Activated Lymphocytes Enhances the Antitumor Effects of Anti-PD-1 Therapy in Head and Neck Cancer, Especially in Hypoxic Environments.

In the tumor microenvironment, wherein cytotoxic lymphocytes interact with cancer cells, lymphocyte exhaustion, an immune checkpoint inhibitor target, is promoted. However, the efficacy of these inhibitors is limited, and improving response rates remains challenging. We previously reported that protein tyrosine phosphatase nonreceptor type (PTPN) 3 is a potential immune checkpoint molecule for activated lymphocytes and that PTPN3 inhibition should be a focus area for cancer immunotherapy development. Therefore, in this study, we focused on PTPN3-suppressive therapy in terms of lymphocyte exhaustion under hypoxic conditions, which are a cancer microenvironment, and investigated measures for improving the response to anti-programmed death receptor (PD)-1 antibody drugs. We found that PTPN3 expression was upregulated in activated lymphocytes under hypoxic conditions, similar to the findings for other immune checkpoint molecules, such as PD-1, T cell immunoglobulin mucin-3, and lymphocyte-activation gene-3; furthermore, it functioned as a lymphocyte exhaustion marker. In addition, PTPN3-suppressed activated lymphocytes promoted the mammalian target of rapamycin (mTOR)-Akt signaling pathway activation and enhanced proliferation, migration, and cytotoxic activities under hypoxic conditions. Furthermore, PTPN3 suppression in activated lymphocytes increased PD-1 expression and enhanced the antitumor effects of anti-PD-1 antibody drugs against head and neck cancer in vitro and in vivo. These results suggest that the suppression of PTPN3 expression in activated lymphocytes enhances the therapeutic effect of anti-PD-1 antibody drugs in head and neck cancer, especially under hypoxic conditions that cause lymphocyte exhaustion.

PTPN3 inhibition contributes to the activation of the dendritic cell function to be a promising new immunotherapy target.

PURPOSE: In a previous study, protein tyrosine phosphatase non-receptor type (PTPN) 3 was identified as an immune checkpoint molecule in lymphocytes, and its potential as a novel target for cancer immunotherapy was anticipated. However, evaluation of dendritic cell (DC) function as antigen-presenting cells is critical for the development of immunotherapy. In this study, we aimed to analyze the biological effect of PTPN3 on DCs induced from human peripheral blood monocytes obtained from healthy individuals. METHODS: We used short-interfering RNA to knock down PTP3 in DCs. For DC maturation, we added cancer cell lysate and tumor necrosis factor-α/interferon-α to immature DCs. In the cytotoxic assay, the target cancer cells were SBC5, unmatched with DCs from healthy human leukocyte antigen (HLA)-A24, or Sq-1, matched with DCs. Enzyme-linked immunosorbent assay was used to determine the amount of cytokines. To examine the intracellular signaling system, intracellular staining was used. RESULTS: PTPN3 knockdown significantly increased the number of DCs, expression of CD80 and chemokine receptor (CCR)7, and production of interleukin-12p40/p70 in mature DCs. In the HLA-A24-restricted DC and human lung squamous cell carcinoma cell cytotoxic assay, inhibition of PTPN3 expression in mature DCs induced cytotoxic T lymphocytes with increased production of INF-γ and granzyme B, and enhanced toxicity against cancer cells and migration to cancer. Furthermore, inhibition of PTPN3 expression activated the mitogen-activated protein kinase pathway in DCs. CONCLUSION: Based on our findings, inhibition of PTPN3 expression could contribute to the development of novel cancer immunotherapies that activate not only lymphocytes but also DCs.

MAML3 Contributes to Induction of Malignant Phenotype of Gallbladder Cancer Through Morphogenesis Signalling Under Hypoxia.

BACKGROUND/AIM: Hedgehog (HH) signalling is a potential therapeutic target for gallbladder cancer (GBC), and Mastermind-like 3 (MAML3) is involved in the transcription of Smoothened (SMO), which is a key protein of HH signalling during hypoxia in the cancer microenvironment. MAML3 is a NOTCH signalling activator, and HH and NOTCH are involved in morphogenesis signalling. However, the association between MAML3-NOTCH and HH signalling and its role in regulating GBC cells remain unknown. This study aimed to determine whether NOTCH signalling affects tumour aggressiveness in GBC under hypoxic conditions and if MAML3 could be a new comprehensive therapeutic target that regulates morphogenesis signalling, HH, and NOTCH in GBC. MATERIALS AND METHODS: We used three cell lines (NOZ, TYGBK1, and TGBC2TKB) and 58 resected specimens. These samples were subjected to cell proliferation, RNA interference, invasion, western blot, and immunohistochemical analyses. RESULTS: MAML3 expression was higher under hypoxic conditions than under normoxic conditions and was involved in the activation of HH and NOTCH signalling. It contributed to the proliferation, migration, and invasion of GBC cells through the NOTCH signalling pathway and enhanced gemcitabine sensitivity. Immunohistochemical analysis showed that MAML3 expression was related to lymphatic invasion, lymph node metastasis, stage category, and a poor prognosis. CONCLUSION: MAML3 contributes to the induction of the malignant phenotype of GBC under hypoxia through the HH and NOTCH signalling pathways and may be a comprehensive therapeutic target of morphogenesis signalling in GBC.

C4orf47 Contributes to the Induction of Stem-like Properties in Gallbladder Cancer Under Hypoxia.

BACKGROUND/AIM: Gallbladder cancer (GBC) is a refractory cancer with poor prognosis. Recently, therapy targeting the tumor microenvironment (TME) has gained attention. Cancer hypoxia is a significant factor in the tumor microenvironment (TME). Our research has shown that hypoxia activates several molecules and signaling pathways that contribute to the development of various types of cancer. Our analysis indicated that C4orf47 expression was up-regulated in a hypoxic environment and had a role in the dormancy of pancreatic cancer. There are no other reports on the biological significance of C4orf47 in cancer and its mechanism is still unknown. This study analyzed how C4orf47 affects refractory GBC to develop a new effective therapy for GBC. MATERIALS AND METHODS: Two human gallbladder carcinomas were used to examine how C4orf47 affects proliferation, migration, and invasion. C4orf47 was silenced using C4orf47 siRNA. RESULTS: C4orf47 was over-expressed in gallbladder carcinomas under hypoxic conditions. C4orf47 inhibition increased the anchor-dependent proliferation and decreased the anchor-independent colony formation of GBC cells. C4orf47 inhibition reduced epithelial-mesenchymal transition and suppressed migration and invasiveness of GBC cells. C4orf47 inhibition decreased CD44, Fbxw-7, and p27 expression and increased C-myc expression. CONCLUSION: C4orf47 enhanced invasiveness and CD44 expression, and reduced anchor-independent colony formation, suggesting that C4orf47 is involved in plasticity and the acquisition of the stem-like phenotype of GBC. This information is useful for the development of new therapeutic strategies for GBC.

C4orf47 contributes to the dormancy of pancreatic cancer under hypoxic conditions.

In our comprehensive analysis of pancreatic cancer pathology, we found that the C4orf47 molecule was upregulated in hypoxic environments. C4orf47 is reported to be a centrosome-associated protein, but its biological significance in cancer is completely unknown; therefore, we assessed its role in pancreatic cancer. We found that C4orf47 was a direct target of HIF-1α and is upregulated in hypoxic conditions, in which it suppressed the cell cycle and inhibits cell proliferation through up-regulation of the cell cycle repressors Fbxw-7, P27, and p57; and the down-regulation of the cell cycle promoters c-myc, cyclinD1, and cyclinC. Furthermore, C4orf47 induced epithelial-mesenchymal transition and enhanced their cell plasticity and invasiveness. In addition, the p-Erk/p-p38 ratio was significantly enhanced and down-regulated CD44 expression by C4orf47 suppression, suggesting that C4orf47 is involved in pancreatic cancer dormancy under hypoxic conditions. Furthermore, the potential of C4orf47 expression was a good prognostic biomarker for pancreatic cancer. These results contribute to the elucidation of the pathology of refractory pancreatic cancer and the development of novel therapeutic strategies.

TrkB/BDNF Signaling Could Be a New Therapeutic Target for Pancreatic Cancer.

BACKGROUND/AIM: Tropomyosin-related kinase B (TrkB)/brain-derived neurotrophic factor (BDNF) signaling plays a role in inducing malignant phenotypes in several aggressive types of cancers. To create a conclusive therapy targeting TrkB/BDNF signaling in solid refractory cancers, the biological significance of TrkB/BDNF signaling was analyzed in pancreatic ductal adenocarcinoma (PDAC) cells. MATERIALS AND METHODS: Three PDAC cell lines were used as target cells to investigate proliferation and invasiveness. Small interfering RNA (siRNA) and the TrkB tyrosine kinase inhibitor k252a were used as TrkB/BDNF signaling inhibitors. RESULTS: All PDAC cell lines expressed TrkB and BDNF. When TrkB and BDNF were inhibited by siRNA or k252a, the invasiveness of PANC-1 and SUIT-2 cells significantly decreased. When TrkB was inhibited by siRNA or k252a, proliferation was significantly inhibited in PDAC cells. CONCLUSION: TrkB/BDNF signaling may be a new therapeutic target for PDAC. Therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy for refractory solid cancer.

GLI2 but not GLI1/GLI3 plays a central role in the induction of malignant phenotype of gallbladder cancer.

We previously reported that Hedgehog (Hh) signal was enhanced in gallbladder cancer (GBC) and was involved in the induction of malignant phenotype of GBC. In recent years, therapeutics that target Hh signaling have focused on molecules downstream of smoothened (SMO). The three transcription factors in the Hh signal pathway, glioma­associated oncogene homolog 1 (GLI1), GLI2, and GLI3, function downstream of SMO, but their biological role in GBC remains unclear. In the present study, the biological significance of GLI1, GLI2, and GLI3 were analyzed with the aim of developing novel treatments for GBC. It was revealed that GLI2, but not GLI1 or GLI3, was involved in the cell cycle­mediated proliferative capacity in GBC and that GLI2, but not GLI1 or GLI3, was involved in the enhanced invasive capacity through epithelial­mesenchymal transition. Further analyses revealed that GLI2 may function in mediating gemcitabine sensitivity and that GLI2 was involved in the promotion of fibrosis in a mouse xenograft model. Immunohistochemical staining of 66 surgically resected GBC tissues revealed that GLI2­high expression patients had fewer numbers of CD3+ and CD8+ tumor­infiltrating lymphocytes (TILs) and increased programmed cell death ligand 1 (PD­L1) expression in cancer cells. These results suggest that GLI2, but not GLI1 or GLI3, is involved in proliferation, invasion, fibrosis, PD­L1 expression, and TILs in GBC and could be a novel therapeutic target. The results of this study provide a significant contribution to the development of a new treatment for refractory GBC, which has few therapeutic options.