Naoki Aomatsu

Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.

Advantages of adjuvant chemotherapy for patients with triple-negative breast cancer at Stage II: usefulness of prognostic markers E-cadherin and Ki67

IntroductionTriple-negative breast cancer (TNBC), which is characterized by negativity for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2), is a high risk breast cancer that lacks specific targets for treatment selection. Chemotherapy is, therefore, the primary systemic modality used in the treatment of this disease, but reliable parameters to predict the chemosensitivity of TNBC have not been clinically available.MethodsA total of 190 TNBC patients who had undergone a curative resection of a primary breast cancer were enrolled. The adjuvant chemotherapy was performed for 138 (73%) of 190 TNBC cases; 60 cases had an anthracyclin-based regimen and 78 a 5-fluorouracil-based regimen. The prognostic value of E-cadherin, Ki67 and p53 expression in the outcome of TNBC patients with adjuvant chemotherapy was evaluated by immunohistochemistry.ResultsThe adjuvant therapy group, especially those with Stage II TNBC, had a more favorable prognosis than the surgery only group (P = 0.0043), while there was no significant difference in prognosis between the anthracyclin-based regimen and 5-fluorouracil-based regimen. Patients with E-cadherin-negative and Ki67-positive expression showed significantly worse overall survival time than those with either E-cadherin-positive or Ki67-negative expression (P < 0.001). Multivariate analysis showed that the combination of E-cadherin-negative and Ki67-positive expression was strongly predictive of poor overall survival (P = 0.004) in TNBC patients receiving adjuvant chemotherapy. In contrast, p53 status was not a specific prognostic factor.ConclusionsAdjuvant therapy is beneficial for Stage II TNBC patients. The combination of E-cadherin and Ki67 status might be a useful prognostic marker indicating the need for adjuvant chemotherapy in Stage II TNBC patients.

CD133 Is a Useful Surrogate Marker for Predicting Chemosensitivity to Neoadjuvant Chemotherapy in Breast Cancer

Background Neoadjuvant chemotherapy (NAC) is a standard care regimen for patients with breast cancer. However, the pathologic complete response (pCR) rate remains at 30%. We hypothesized that a cancer stem cell marker may identify NAC-resistant patients, and evaluated CD133 and ALDH1 as a potential surrogate marker for breast cancer. The aim of this study was to find a surrogate maker to predict chemosensitivity of NAC for breast cancer. Methodology/Findings A total of 102 patients with breast cancer were treated with NAC consisting of epirubicin followed by paclitaxel. Core needle biopsy (CNB) specimens and resected tumors were obtained from all patients before and after NAC, respectively. Chemosensitivity and prognostic potential of CD133 or ALDH1 expression was evaluated by immunohistochemistry. Clinical CR (cCR) and pCR rates were 18% (18/102) and 29% (30/102), respectively. Forty-seven (46%) patients had CD133-positive tumors before NAC, and CD133 expression was significantly associated with a low pCR rate (p = 0.035) and clinical non-responders. Multivariate analysis revealed that CD133 expression was significantly (p = 0.03) related to pCR. Recurrence was more frequent in patients with CD133-positive tumors (21/47, 45%) than that in patients with CD133-negative tumors (7/55, 13%). The number of patients with CD133-positive tumors (62%) after NAC was higher than that (46%) before NAC. Furthermore, most patients with CD133-positive tumors before NAC maintained the same status after NAC. Conclusion/Significance CD133 before NAC might be a useful marker for predicting the effectiveness of NAC and recurrence of breast cancer after NAC.

c‐Kit expression as a prognostic molecular marker in patients with basal‐like breast cancer

As patients with basal‐like breast cancer (BLBC) have a poor prognosis and there is no specifically tailored therapy, molecular biological characterization of BLBC is necessary. c‐Kit is a transmembrane receptor tyrosine kinase known to play important roles in various solid cancers. This study classified BLBCs from patients with breast carcinoma, and addressed the significance of c‐Kit expression in these tumours.

Carbonic anhydrase 9 is associated with chemosensitivity and prognosis in breast cancer patients treated with taxane and anthracycline

BackgroundNeoadjuvant chemotherapy (NAC) is one of the standard care regimens for patients with resectable early-stage breast cancer. It would be advantageous to determine the chemosensitivity of tumors before initiating NAC. One of the parameters potentially compromising such chemosensitivity would be a hypoxic microenvironment of cancer cells. The aim of this study was thus to clarify the correlation between expression of the hypoxic marker carbonic anhydrase-9 (CA9) and chemosensitivity to NAC as well as prognosis of breast cancer patients.MethodsA total of 102 patients with resectable early-stage breast cancer was treated with NAC consisting of FEC (5-fluorouracil, epirubicin, and cyclophosphamide) followed by weekly paclitaxel before surgery. Core needle biopsy (CNB) specimens and resected tumors were obtained from all patients before and after NAC, respectively. Chemosensitivity to NAC and the prognostic potential of CA9 expression were evaluated by immunohistochemistry.ResultsCA9 positivity was detected in the CNB specimens from 47 (46%) of 102 patients. The CA9 expression in CNB specimens was significantly correlated with pathological response, lymph node metastasis, and lymph-vascular invasion. Multivariate analysis revealed that the CA9 expression in CNB specimens was an independent predictive factor for pathological response. The Kaplan-Meier survival curve revealed a significant negative correlation (p = 0.013) between the disease-free survival (DFS) and the CA 9 expression in resected tissues after NAC. Multivariate regression analyses indicated that the CA9 expression in resected tissues was an independent prognostic factor for DFS.ConclusionsCA9 expression in CNB specimens is a useful marker for predicting chemosensitivity, and CA9 expression in resected tissue is prognostic of DFS in patients with resectable early-stage breast cancer treated by sequential FEC and weekly paclitaxel prior to resection.

Abstract 3337: TGF-β from niche fibroblasts increases the stemness cancer stem cell-like side population cells in gastric cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Cancer Stem Cells (CSCs) are thought to possess tumor initiation and self-renewal and are associated with tumor progression. The niche cells of microenvironment, such as fibroblast might play an important role for the progression of cancer cells. The aim of this study is to examine the effect of cancer-associated fibroblasts on the stemness of CSCs. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblast, CaF-37, were used. SP cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechest33342, and were named as OCUM-12/SP. The percentage of SP cells was evaluated by flowcytometry, after it was cultured with condition medium from CaF-37 for three days. SP fraction was evaluated by flowcytometry, in the presence or absence of TGF-β receptor inhibitor, FGF receptor inhibitor, and c-Met Receptor inhibitor. In vivo, tumorigenicity of OCUM-12/SP cells were evaluated by subcutaneous inoculation in the presence or abcence of CaF-37. Results: The inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells with 5x105 CaF-37 fibroblasts resulted in tumor formation in all of mice, respectively. However, the inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells alone results in poor tumor formation at 3(60%) of 5 mice, 0(0%) of 5 mice and 0(0%) of 5 mice, respectively. The percentage of SP fraction of OCUM-12/SP was significantly increased in the presence of condition medium from CaF-37. The increase of SP fraction of OCUM-12/SP cells by condition medium from CaF-37 was inhibited by TGF-β receptor inhibitor, but not FGF receptor inhibitor and c-Met inhibitor. Conclusion: TGF-β from the niche, fibroblasts, might sustain the stemness of CSCs and might be associated with the progression of Cancer Cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3337. doi:1538-7445.AM2012-3337

Cytomegalovirus colitis followed by ischemic colitis in a non-immunocompromised adult:A case report

We report a rare case of cytomegalovirus (CMV) colitis followed by severe ischemic colitis in a non-immunocompromised patient. An 86-year-old woman was admitted after experiencing episodes of vomiting and diarrhea. The next day, hematochezia was detected without abdominal pain. The initial diagnosis of ischemic colitis was based on colonoscopy and histological findings. The follow-up colonoscopy revealed a prolonged colitis. Immunohistochemical staining detected CMV-positive cells following conservative therapy. Intravenous ganciclovir therapy led to successful healing of ulcers and disappearance of CMV-positive cells. The prevalence of CMV infection is common in adults. CMV colitis is relatively common in immunocompromised patients; however, it is rare in immunocompetent patients. In our case, CMV infection was allowed to be established due to the disruption of the colonic mucosa by the prior severe ischemic colitis. Our experience suggests that biopsies may be necessary to detect CMV and the prompt management of CMV colitis should be instituted when intractable ischemic colitis is observed.

Prognostic significance of pathological complete response following neoadjuvant chemotherapy for operable breast cancer

The aim of the present retrospective study was to ascertain the significance of pathological complete response (pCR) on overall survival (OS) and disease-free survival (DFS) in each disease subtype of operable breast cancer. Using a single-institution database, 90 patients were identified, who received neoadjuvant chemotherapy (NAC) for operable breast cancer and were eligible for the analysis. In total, 10 patients (11.1%) had succumbed to their diseases and 20 (22.2%) had succumbed to their diseases or exhibited recurrences. The OS of patients with triple-negative (TN) tumors was significantly lower than that of patients with other disease subtypes (P=0.016). The DFS of patients with luminal tumors was higher than that of patients with other subtypes. Survival was improved with pCR following NAC (P=0.044). Across all subtypes, patients who achieved pCR exhibited a higher DFS than patients who did not, but not significantly. pCR only improved OS and DFS in the TN disease subtype (P=0.022 and P=0.048, respectively). pCR following NAC may have prognostic value in TN breast cancer.

Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ.

The antitumor activity of prostaglandin (PG) D2 has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD2 is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD synthase (PGDS), on gastric cancer cells. The effects of PGD2 and PGDS on the proliferation of gastric cancer cells were examined in vivo and in vitro. The expression levels of PGD2 receptors and peroxisome proliferator‐activated receptor γ (PPARγ) were evaluated by RT‐PCR. The effects of a PPARγ antagonist or siPPARγ on the proliferation of cancer cells and the c‐myc and cyclin D1 expression were examined in the presence or absence of PGD2 or PGDS. PPARγ was expressed in gastric cancer cell lines, but PGD2 receptors were not. PGD2 and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD2 production of gastric cancer cells. A PPARγ antagonist and siPPARγ transfection significantly suppressed the growth‐inhibitory effects of PGD2 and PGDS. Expression of c‐myc and cyclin D1 was significantly decreased by PGD2; this inhibitory effect was suppressed by PPARγ antagonist. Both PGD2 and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than PGD2 treatment. These findings suggest that PGDS and PGD2 decrease the proliferation of gastric cancer cells through PPARγ signaling. PGDS is a potentially promising therapeutic agent for gastric cancers that express PPARγ.

Abstract 3995: Antitumor effect of prostaglandin D2 by peroxisome proliferator-activated receptor gamma (PPARγ)-dependent pathway in gastric carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Prostaglandin D2 (PGD2) has been demonstrated to have not only physiological responses but also anti-tumor effects against some types of cancer cells such as a lung cancer. PGD2 act through two major receptors, DP1 and DP2 of chemoattractant receptor-like molecule on the Th2 cell. Recently, some papers reported that PGD2 may act through PPARγ, suggesting that the effects of PGD2 are involved in either PGD2 dependent or independent pathway. PGD2 metabolite, 15-deoxy-Δ12 14-PGJ2, activates peroxisome proliferator-activated receptor γ (PPARγ) which induces growth inhibition of various cells. PPARγ is expressed in various types of cancers including gastric cancer. Since the role of PGD2 on gastric cancer cells is still unknown, this study is aimed to investigate the effect of PGD2 signalings on the proliferation of gastric cancer cells. Materials and Methods: Three human gastric cancer cell lines, OCUM-2M, OCUM-12, and MKN-74, were used. Effect of PGD2 or PPARγ antagonist on the proliferation of cancer cells was examined by MTT assay. The expression level of PGD2 receptor, DP1 and DP2, and PPARγ of gastric cancer cells was examined by RT-PCR. The effect of PGD2 on cell cycle was examined with PI staining by flowcytometry. Results: PGD2 significantly decreased the proliferation of OCUM-2M and MKN-74 cells at a dose-dependent manner, but not that of OCUM-12. PGD2 receptors, DP1 and DP2, were not expressed in 3 gastric cancer cell lines while PPARγ was expressed in OCUM-2M and MKN-74. BADGE, a PPARγ antagonist, significantly suppressed the growth-inhibitory effects of PGD2 on OCUM-2M and MKN-74 cells. PGD2 increased G0/G1 phase of cancer and decereased G2/M phase. Conclusion: PGD2 decreased the proliferation of gastric cancer cells through PPARγ dependent pathway. PGD2 might be a promising therapeutic agent for gastric cancer with PPARγ expression. Citation Format: Tatsunari Fukuoka, Masakazu Yashiro, Hiroshi Takeda, Takayuki Maruyama, Haruhito Kinoshita, Tamammi Morisaki, Tsuyoshi Hasegawa, Toshiki Hirakawa, Naoki Aomatsu, Katsunobu Sakurai, Kenjiro Kimura, Hisashi Nagahara, Takahiro Toyokawa, Ryosuke Amano, Eiji Noda, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Antitumor effect of prostaglandin D2 by peroxisome proliferator-activated receptor gamma (PPARγ)-dependent pathway in gastric carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2013-3995