Tomokazu Tanaka

Methylation-mediated gene silencing as biomarkers of gastric cancer: A review

Despite a decline in the overall incidence of gastric cancer (GC), the disease remains the second most common cause of cancer-related death worldwide and is thus a significant global health problem. The best means of improving the survival of GC patients is to screen for and treat early lesions. However, GC is often diagnosed at an advanced stage and is associated with a poor prognosis. Current diagnostic and therapeutic strategies have not been successful in decreasing the global burden of the disease; therefore, the identification of reliable biomarkers for an early diagnosis, predictive markers of recurrence and survival and markers of drug sensitivity and/or resistance is urgently needed. The initiation and progression of GC depends not only on genetic alterations but also epigenetic changes, such as DNA methylation and histone modification. Aberrant DNA methylation is the most well-defined epigenetic change in human cancers and is associated with inappropriate gene silencing. Therefore, an increasing number of genes methylated at the promoter region have been targeted as possible biomarkers for different purposes, including early detection, classification, the assessment of the tumor prognosis, the development of therapeutic strategies and patient follow-up. This review article summarizes the current understanding and recent evidence regarding DNA methylation markers in GC with a focus on the clinical potential of these markers.

HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1α in the development of peritoneal dissemination of gastric cancer. HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1α exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1α was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1α played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1α-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1α activates tumor angiogenesis.

Loss of trefoil factor 1 is regulated by DNA methylation and is an independent predictive factor for poor survival in advanced gastric cancer

Trefoil factor 1 (TFF1) is considered to be a tumor suppressor gene in gastric cancer. However, the role of TFF1 expression and its regulation in gastric cancer patients remain unclear. The aims of this study were to clarify the clinical significance of TFF1 and to determine its regulatory mechanisms. We assessed the immunohistochemical expression of TFF1 in 182 gastric cancer patients and examined whether or not TFF1 is associated with the clinicopathological factors and patient survival. In vitro study using TFF1 knockdown gastric cancer cells evaluated the role of TFF1 in cancer invasion. Bisulfite sequencing was performed to assess DNA methylation of TFF1 in cells and resected tissues. Patients with low expression of TFF1 showed a significantly deeper invasion of the tumor than those with high expression (p=0.037). Low expression of TFF1 was also associated with a poor survival (p=0.029) in 108 patients who were treated by surgery alone. Both TFF1 expression and lymph node metastasis are independent predictive factors for disease-specific survival in a multivariate analysis. In an in vitro study, invasive power of the cells was significantly increased in the TFF1‑deficient cells compared with the control cells. Bisulfate sequencing showed that TFF1 expression is strongly dependent on DNA methylation in both gastric cancer cells and tissues. Interestingly, methylation status of two specific CpG sites, which are located close to a TATA box and hypoxia response element (HRE), determined the TFF1 expression in the resected tissues. TFF1 expression is silenced by DNA methylation and is associated with tumor invasion and a poor survival in gastric cancer patients. The expression and̸or methylation status of TFF1 may, therefore, serve as a useful biomarker for predicting survival in patients with advanced gastric cancer.

The Apoptotic Effect of HIF-1α Inhibition Combined with Glucose plus Insulin Treatment on Gastric Cancer under Hypoxic Conditions.

Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O2) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.

The value of trefoil factor 3 expression in predicting the long‑term outcome and early recurrence of colorectal cancer

The trefoil factor (TFF) family comprises three thermo-stable and protease-resistant proteins (TFF1, TFF2 and TFF3) and plays an essential role in gastrointestinal mucosa protection and regeneration, and TFFs have recently been found to be involved in the development and progression of various types of cancer. However, the clinical significance of TFFs in colorectal cancer (CRC) patients remains unclear. The present study determined the relationship between TFF expression and clinicopathological findings, as well as long-term outcome in CRC patients. The mRNA expression levels of TFFs were examined in the excised CRC specimens obtained from 154 consecutive CRC patients who underwent surgical resection between 2005 and 2007 at our institution. TFF3 expression was significantly associated with the presence of distant metastasis (p=0.017), although neither TFF1 nor TFF2 expression was associated with the clinicopathological features. Survival rate of the patients with positive TFF3 was significantly worse compared to those with negative TFF3 (p=0.011). A multivariate analysis revealed that the expression of TFF3, lymph node metastasis, and vascular invasion were independent prognostic factors for disease-specific survival. Furthermore, among 134 patients with no clinical findings of metastasis at surgery, the patients with positive TFF3 experienced recurrence within one year more frequently than those with negative TFF3 (p=0.039). In conclusion, TFF3 is not only a useful biomarker for a long-term surgical result in CRC patient, but also may be a risk factor of early recurrence.

Hypoxia-induced ANGPTL4 sustains tumour growth and anoikis resistance through different mechanisms in scirrhous gastric cancer cell lines

Patients with scirrhous gastric cancer (SGC) frequently develop peritoneal dissemination, which leads to poor prognosis. The secreted protein angiopoietin-like-4 (ANGPTL4), which is induced by hypoxia, exerts diverse effects on cancer progression. Here, we aimed to determine the biological function of ANGPTL4 in SGC cells under hypoxia. ANGPTL4 levels were higher in SGC cells under hypoxia than in other types of gastric cancer cells. Hypoxia-induced ANGPTL4 mRNA expression was regulated by hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, monolayer cultures of ANGPTL4 knockdown (KD) 58As9 SGC (58As9-KD) cells were arrested in the G1 phase of the cell cycle through downregulation of c-Myc and upregulation of p27, in contrast to control 58As9-SC cells. Moreover, the ability of 58As9-KD xenografts to form tumours in nude mice was strongly suppressed. When 58As9-KD cells were cultured in suspension, hypoxia strongly increased their susceptibility to anoikis through suppression of the FAK/Src/PI3K-Akt/ERK pro-survival pathway, followed by activation of the apoptotic factors caspases-3, -8 and -9. The development of peritoneal dissemination by 58As9-KD cells was completely inhibited compared with that by 58As9-SC cells. In conclusion, ANGPTL4 is uniquely induced by hypoxia in cultured SGC cells and is essential for tumour growth and resistance to anoikis through different mechanisms.

Promising immunotherapies for esophageal cancer

ABSTRACT Introduction: Esophageal cancer (EC) is the eighth most common cancer in the world, and the prognosis of EC is still poor. Although immunotherapy has been developed in melanoma and lung cancer, it is also expected to show efficacy in EC. Currently, several clinical trials are ongoing to evaluate the safety and efficacy of immunotherapies, immune checkpoint inhibitors, adoptive T cell transfer, and therapeutic cancer vaccines in EC. Areas covered: This review provides an overview and the status of immunotherapy in EC. Clinical significance of molecules related immune checkpoints, especially PD-1 and PD-L1 is presented and the designs, results and future directions of clinical trials using immunotherapy in EC are provided. Expert opinion: To bring immunotherapy to the forefront of treatment for EC, it is necessary to select patients who can obtain a high efficacy of immunotherapy and to also elucidate the correct timing for administration. Moreover, combination therapies of immunotherapy with existing chemotherapy or radiation or other immunotherapy with different mechanisms of action must be evaluated to achieve excellent outcomes in patients with EC.

Abstract 2096: HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background The molecular mechanisms of peritoneal dissemination in gastric cancer still remain unclear. This study was designed to clarify the role of HIF-1α in the development of peritoneal dissemination of gastric cancer. Methods HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9 by siRNA transfection. In western blot analysis, HIF-1α expression was assessed using KD and the control transfectant (SC) cells under normoxia (20%O2) as well as hypoxia (1%O2). A series of in vitro analysis were conducted and compared between the 2 transfectants. Using nude mice, peritoneal dissemination was evaluated by orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. Finally, tumor angiogenesis of venous and lymph vessels was immunohistochemically analyzed. Results Deficient HIF-1α expression was observed in KD, but not SC cells under normoxia and hypoxia. In vitro proliferation and invasion was significantly reduced in KD compared with SC cells. On contrary, HIF-1α exerts unfavorable effects on anoikis resistance and adhesion ability. In o.i. model, stomach tumor was highly developed in both of KD (86.7%) and SC mice(100%). On the other hand, peritoneal dissemination was more frequently observed in the SC mice (93%) than in the KD mice (13%) (p < 0.001). In the i.p. model, the dissemination highly occurred in both mice (each 100%), while greater number of nodules was observed in the KD mice (p = 0.017). Angiogenesis with vascular invasion were more highly developed in the SC stomach tumors (venous vessels: p=0.001, lymph vessels: p=0.043). In the disseminated nodules, the vascular invasion was developed at intratumoral regions in the SC o.i. mice, however such appearance was not found in i.p. mice. Conclusions This study clearly showed that HIF-1α is crucial for the development of peritoneal dissemination in the spontaneous metastatic (o.i.) model. Moreover, in i.p. model, larger number of dissemination was found in KD than SC mice, indicating that the dissemination by o.i. would not be developed via seeding mechanism. Analysis of tumor angiogenesis in o.i. model demonstrated that HIF-1α activated venous as well as lymph angiogenesis in the stomach tumor. In peritoneal disseminated nodules, vascular invasion was observed at intratumoral area in o.i. SC mice, but not in the i.p. mice. This unique feature further suggests that the dissemination in o.i. mice might be developed by extravasation of cancer cells, which are micro-connected to the stomach tumor via trans-vessel route. Citation Format: Shuusuke Miyake, Yoshihiko Kitajima, Jun Nakamura, Keita Kai, Kazuyoshi Yanagihara, Tomokazu Tanaka, Masatsugu Hiraki, Kohji Miyazaki, Hirokazu Noshiro. HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2014-2096