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 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.

PTPN3 Could Βe a Therapeutic Target of Pancreatic Cancer.

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.

Cancer therapy that targets the Hedgehog signaling pathway considering the cancer microenvironment (Review).

Recently, the cancer microenvironment (CME) has received significant attention. At the local site of the tumor, cancer progression is affected by secreted cytokines and conditions derived from the CME and stimulation by cancer­induced cytokines in an autocrine manner. The CME is characterized by various types of conditions, such as hypoxia, inflammation stimulation, and angiogenesis, and contains various components, such as reactive oxygen species, cancer­associated fibroblasts, infiltrated immune cells, exosomes, and cancer stem cells (CSCs). These conditions and components complicate the progression of cancer. The Hedgehog (HH) signaling pathway is a morphogenesis signaling pathway that is reactivated in some cancers. In these cancers, reactivated HH signaling is involved in the induction of the malignant phenotype. HH signaling is also activated under hypoxic conditions and is considered to be strongly correlated with the CME, including the induction of cancer fibrosis and maintenance of CSCs. The aim of the present review was to elucidate a cancer therapy that targets HH signaling by considering the CME, particularly focusing on hypoxia.

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.

PTPN3 is a potential target for a new cancer immunotherapy that has a dual effect of T cell activation and direct cancer inhibition in lung neuroendocrine tumor.

In our previous study, we found that inhibition of protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is expressed in lymphocytes, enhances lymphocyte activation, suggesting PTPN3 may act as an immune checkpoint molecule. However, PTPN3 is also expressed in various cancers, and the biological significance of PTPN3 in cancer cells is still not well understood, especially for lung neuroendocrine tumor (NET).Therefore, we analyzed the biological significance of PTPN3 in small cell lung cancer and examined the potential for PTPN3 inhibitory treatment as a cancer treatment approach in lung NET including small cell lung cancer (SCLC) and large cell neuroendocrine cancer (LCNEC). Experiments in a mouse xenograft model using allo lymphocytes showed that PTPN3 inhibition in SCLC cells enhanced the anti-tumor effect of PTPN3-suppressed activated lymphocytes. In addition, PTPN3 was associated with increased vascularization, decreased CD8/FOXP3 ratio and cellular immunosuppression in SCLC clinical specimens. Experiments in a mouse xenograft model using autocrine lymphocytes also showed that PTPN3 inhibition in LCNEC cells augmented the anti-tumor effect of PTPN3-suppressed activated lymphocytes. In vitro experiments showed that PTPN3 is involved in the induction of malignant traits such as proliferation, invasion and migration. Signaling from PTPN3 is mediated by MAPK and PI3K signals via tyrosine kinase phosphorylation through CACNA1G calcium channel. Our results show that PTPN3 suppression is associated with lymphocyte activation and cancer suppression in lung NET. These results suggest that PTPN3 suppression could be a new method of cancer treatment and a major step in the development of new cancer immunotherapies.

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.

NFκB and TGFß contribute to the expression of PTPN3 in activated human lymphocytes.

We previously reported that protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is upregulated in activated lymphocytes, acts as an immune checkpoint. However, the mechanism by which PTPN3 expression is enhanced in activated lymphocytes is unknown. In this study, we analyzed the mechanism of PTPN3 expression in activated lymphocytes with a view for developing a novel immune checkpoint inhibitor that suppresses PTPN3. Through the activation process, lymphocytes showed enhanced NFκB activation as well as increased PTPN3 expression. NFκB enhanced proliferation, migration, and cytotoxicity of lymphocytes. Furthermore, NFκB enhanced PTPN3 expression and tyrosine kinase activation. TGFß reduced PTPN3 expression and NFκB activation in the cancer microenvironment, and suppressed the biological activity of lymphocytes. The results of this study are expected to provide significant implications for improving existing immunotherapy and developing novel immunotherapy.

FAM115C could be a novel tumor suppressor associated with prolonged survival in pancreatic cancer patients.

Hypoxia is a characteristic feature of the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC). We have recently explored new targeting molecules and pathways in PDAC cells under hypoxic conditions. In this study, we performed a microarray experiment to analyze the genes up-regulated in PDAC cell lines under hypoxia compared to normoxia, and identified human family with sequence similarity 115, member C (FAM115C) as a candidate gene for further study. Our data showed that FAM115C was overexpressed in PDAC cell lines under hypoxia, and FAM115C inhibition promoted PDAC cell migration and invasion in vitro. FAM115C inhibition did not affect tumor cell proliferation in PDAC. Immunohistochemically, FAM115C expression was observed ubiquitously in normal pancreas, pancreatic intraepithelial neoplasia (PanIN) and PDAC tissue, and it was located mainly in the nucleus but also in the cytoplasm of cells. In qPCR analysis, high expression of FAM115C was correlated with better prognosis in patients with PDAC. Our findings suggest that FAM115C could be a novel tumor suppressor associated with prolonged survival in patients with PDAC.

Patched 1-interacting Peptide Represses Fibrosis in Pancreatic Cancer to Augment the Effectiveness of Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to immunotherapy. As a factor of resistance, the dense fibrosis of this cancer acts as a barrier to inhibit immune cell infiltration into a tumor. We examined the influence of a Hedgehog signal inhibitor, Patched 1-interacting peptide, on fibrosis, infiltration of immune cells, and immunotherapeutic effects on PDAC. We found that this peptide inhibited proliferation and migration of cancer-associated fibroblasts and cancer cells. Furthermore, this peptide reduced the production of extracellular matrix and transforming growth factor ß1 in cancer-associated fibroblasts and induced expression of HLA-ABC in PDAC cells and interferon-γ in lymphocytes. In vivo, the peptide suppressed fibrosis of PDAC and increased immune cell infiltration into tumors. The combination of this peptide and an anti-programmed death-1 antibody augmented the antitumor effect, and this combination showed the same effect in experiments using cancer cells and autologous lymphocytes. These results indicate that, in addition to the direct effect of tumor suppression, the Patched 1-interacting peptide increases the infiltration of immune cells by reducing fibrosis of PDAC and consequently enhances the effect of immunotherapy. Therefore, treatment with this peptide may be a novel therapy with 2 different mechanisms: direct tumor suppression and enhancing the immune response against PDAC.

PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor.

It has been shown that protein tyrosine phosphatase non-receptor type (PTPN) 3 inhibits T-cell activation. However, there is no definitive conclusion about how the inhibition of PTPN3 in lymphocytes affects immune functions in human lymphocytes. In the present study, we showed that PTPN3 inhibition significantly contributes to the enhanced activation of activated human lymphocytes. The PTPN3 expression of lymphocytes was significantly increased through the activation process using IL-2 and anti-CD3 mAb. Interestingly, inhibiting the PTPN3 expression in activated lymphocytes significantly augmented the proliferation, migration, and cytotoxicity through the phosphorylation of zeta-chain-associated protein kinase 70 (ZAP-70), lymphocyte-specific protein tyrosine kinase (LCK), and extracellular signal-regulated kinases (ERK). Lymphocyte activation by PTPN3 inhibition was observed only in activated CD3+ T cells and not in NK cells or resting T cells. In therapy experiments using autologous tumors and lymphocytes, PTPN3 inhibition significantly augmented the number of tumor-infiltrated lymphocytes and the cytotoxicity of activated lymphocytes. Our results strongly imply that PTPN3 acts as an immune checkpoint in activated lymphocytes and that PTPN3 inhibitor may be a new non-antibody-type immune checkpoint inhibitor for cancer therapy.

Liprin-α4 as a Possible New Therapeutic Target for Pancreatic Cancer.

BACKGROUND/AIM: In pancreatic cancer, where the microenvironment is extremely hypoxic, analyzing signal transduction under hypoxia is thought to be significantly important. By investigating microarray analysis of pancreatic cancer cells cultured under both normoxia and hypoxia, we found that the expression of leukocyte common antigen-related (LAR)-interacting protein (liprin)-α4 was extremely increased under hypoxia compared to under normoxia. MATERIALS AND METHODS: In the present study, the biological significance of liprin-α4 in pancreatic cancer was investigated and whether liprin-α4 has potential as a therapeutic target for pancreatic cancer was estimated. RESULTS: Suppression of liprin-α4 reduced proliferation of pancreatic cancer cells both in vitro and in vivo. Inhibition of liprin-α4 also reduced invasiveness through the suppression of endothelial-mesenchymal transition. Stimulation by liprin-α4 was through phosphoinositide 3-kinase and mitogen-activated protein kinase signaling pathways. CONCLUSION: Liprin-α4 plays a pivotal role in inducing malignant phenotypes such as increased proliferation and invasion in pancreatic cancer, and that liprin-α4 could be a new effective therapeutic target for pancreatic cancer.

Long-term effect of cinacalcet hydrochloride on abdominal aortic calcification in patients on hemodialysis with secondary hyperparathyroidism.

BACKGROUND: Secondary hyperparathyroidism (SHPT) is one of the common complications in dialysis patients, and is associated with increased risk of vascular calcification. The effects of cinacalcet hydrochloride treatment on bone and mineral metabolism have been previously reported, but the benefit of cinacalcet on vascular calcification remains uncertain. The aim of this study was to evaluate the impact of cinacalcet on abdominal aortic calcification in dialysis patients. SUBJECTS AND METHODS: Patients were on maintenance hemodialysis with insufficiently controlled SHPT (intact parathyroid hormone [PTH] >180 pg/mL) by conventional therapies. All subjects were initially administered 25 mg cinacalcet daily, with concomitant use of calcitriol analogs. Abdominal aortic calcification was annually evaluated by calculating aortic calcification area index (ACAI) using multidetector computed tomography (MDCT), from 12 months before to 36 months after the initiation of cinacalcet therapy. RESULTS: Twenty-three patients were analyzed in this study. The mean age was 59.0±8.7 years, 34.8% were women, and the mean dialysis duration was 163.0±76.0 months. After administration of cinacalcet, serum levels of intact PTH, phosphorus, and calcium significantly decreased, and mean Ca × P values significantly decreased from 67.4±7.9 mg(2)/dL(2) to 52±7.7 mg(2)/dL(2). Although the ACAI value did not decrease during the observation period, the increase in ACAI between 24 months and 36 months after cinacalcet administration was significantly suppressed. CONCLUSION: Long-term administration of cinacalcet was associated with reduced progression of abdominal aortic calcification, and achieving appropriate calcium and phosphorus levels may reduce the rates of cardiovascular events and mortality in patients on hemodialysis.