Hirofumi Kawakubo

Loss of B-cell translocation gene-2 in estrogen receptor-positive breast carcinoma is associated with tumor grade and overexpression of cyclin d1 protein.

The B-cell translocation gene-2 (BTG2) is present in the nuclei of epithelial cells in many tissues, including the mammary gland where its expression is regulated during glandular proliferation and differentiation in pregnancy. In immortalized mammary epithelial cells and breast cancer cells, BTG2 protein localized predominantly to the nucleus and cytoplasm, respectively. The highly conserved domains (BTG boxes A, B, and C) were required for regulating localization, suppression of cyclin D1 and growth inhibitory function of BTG2. Expression analysis of BTG2 protein in human breast carcinoma (n = 148) revealed the loss of nuclear expression in 46% of tumors, whereas it was readily detectable in the nuclei of adjacent normal glands. Loss of nuclear BTG2 expression in estrogen receptor-alpha (ERalpha)-positive breast tumors correlated significantly with increased histologic grade and tumor size. Consistent with its ability to suppress cyclin D1 transcription, loss of nuclear BTG2 expression in ER-positive breast carcinomas showed a significant correlation with cyclin D1 protein overexpression, suggesting that loss of BTG2 may be a factor involved in deregulating cyclin D1 expression in human breast cancer.

Expression of the NF-κB-responsive gene BTG2 is aberrantly regulated in breast cancer

BTG2, a p53-inducible antiproliferative gene, is stimulated in breast cancer cells by activation of nuclear factor kappa B (NF-κB). In rat mammary glands, BTG2 is expressed in epithelial cells and levels decreased during pregnancy and lactation but recovered during involution. Estrogen and progestin suppress BTG2 expression, suggesting that these steroids, which stimulate proliferation and lobuloalveolar development of mammary epithelial cells, may downregulate BTG2 in the mammary gland during pregnancy. Consistent with the report that BTG2 inhibits cyclin D1 expression, suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and progestin, correlated with stimulation of cyclin D1. Ectopic expression of BTG2 inhibited breast cancer cell growth by arresting cells in the G1 phase, an effect reversed by cyclin D1. BTG2 expression was very low or undetectable in human breast cancer cell lines compared with nontumorigenic mammary epithelial cells, and nuclear expression of BTG2 was absent in 65% of human breast tumors compared with adjacent matched normal glands. Spontaneous mammary tumors arising in a mouse model with targeted expression of the early region of the SV40 large tumor Ag demonstrated loss of BTG2 protein very early during the tumorigenic process. Thus deregulation of BTG2 may be an important step in the development of mammary tumors.

Mullerian Inhibiting Substance Promotes Interferon γ-induced Gene Expression and Apoptosis in Breast Cancer Cells

This report demonstrates that in addition to interferons and cytokines, members of the TGFβ superfamily such as Mullerian inhibiting substance (MIS) and activin A also regulate IRF-1 expression. MIS induced IRF-1 expression in the mammary glands of mice in vivo and in breast cancer cells in vitro and stimulation of IRF-1 by MIS was dependent on activation of the NFκB pathway. In the rat mammary gland, IRF-1 expression gradually decreased during pregnancy and lactation but increased at involution. In breast cancer, the IRF-1 protein was absent in 13% of tumors tested compared with matched normal glands. Consistent with its growth suppressive activity, expression of IRF-1 in breast cancer cells induced apoptosis. Treatment of breast cancer cells with MIS and interferon γ (IFN-γ) co-stimulated IRF-1 and CEACAM1 expression and synergistic induction of CEACAM1 by a combination of MIS and IFN-γ was impaired by antisense IRF-1 expression. Furthermore, a combination of IFN-γ and MIS inhibited the growth of breast cancer cells to a greater extent than either one alone. Both reagents alone significantly decreased the fraction of cells in the S-phase of the cell cycle, an effect not enhanced when they were used in combination. However, MIS promoted IFN-γ-induced apoptosis demonstrating a functional interaction between these two classes of signaling molecules in regulation of breast cancer cell growth.

Transforming Growth Factor-beta superfamily: evaluation as breast cancer biomarkers and preventive agents

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Mullerian-inhibiting substance induces Gro-β expression in breast cancer cells through a nuclear factor-κB-dependent and Smad1-dependent mechanism

Mullerian-inhibiting substance (MIS), a transforming growth factor-β family member, activates the nuclear factor-κB (NF-κB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-β. Inhibiting NF-κB activation with a phosphorylation-deficient IκBα mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-β promoter-driven reporter expression and Gro-β mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-β mRNA, suggesting that regulation of Gro-β expression by MIS was dependent on activation of NF-κB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of κB-binding sequences within Gro-β, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-κB complex bound to the κB site of the Gro-β promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-β promoter in vivo , whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-κB signaling in which enhancement of NF-κB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the κB consensus site within the promoter. [Cancer Res 2007;67(6):2747–56]

Correlation between change of FA score and postoperative recurrence in esophageal cancer patients who received neoadjuvant treatment.

41 Background: We previously reported that fibrinogen and albumin score (FA score), which was consisted of plasma fibrinogen level (FNG) and serum albumin level (Alb), was shown to predict postoperative survival in esophageal cancer patients who underwent transthoracic esophagectomy. In this study, in patients who received neoadjuvant chemotherapy (NAC), change of FA score during NAC was reviewed and the correlation with recurrence free survival (RFS) was investigated. Methods: We retrospectively reviewed 125 patients who received neoadjuvant chemotherapy and underwent transthoracic esophagectomy in our institution between 2001 and 2012. FNG and Alb before (preTx) and after (preope) NAC were confirmed in 92 patients. Based on our previous reports, patients with elevated fibrinogen ( > 350 mg/dL) and decreased albumin ( < 3.8 g/dl) levels were allocated a FA score of 2, those with only one of these abnormalities were allocated a FA score of 1, and those with neither of these abnormalities were allocated a ...

The influence of perioperative decreasing total psoas area on prognosis of esophagieal cancer patients.

38 Background: Depression of skeletal muscle mass (sarcopenia) has been linked to postoperative mortality in several carcinomas and various types of surgeries. Total psoas muscle area (TPA) has received attention as one of the indicators of systemic total skeletal muscle volume. The objective of this study is to determine the correlation between the patients who decreased TPA during pre-post operation and prognosis of esophageal cancer treated by surgical resection. Methods: We enrolled 40 esophageal cancer patients who underwent esophagectomy between April 2008 and May 2009. TPA was estimated by measuring the cross-sectional area of the psoas major muscle at the level of the third lumbar vertebra using an image-analysing software, identified on a preoperative CT scan, and a postoperative CT scan, which was taken on post-operative day 6. We divided the patients who decreased TPA between pre-post operation, into “decreasing group”, and the patients who preserved or increased TPA, into “non-decreasing group...

Phase I study of combination chemotherapy consisting of capecitabine plus fractional cisplatin for unresectable gastric cancer in an outpatient setting.

173 Background: The combination chemotherapy of capecitabine and cisplatin is one of the standard regimen for unresectable gastric cancer. However, the high dose of cisplatin could cause the nephrotoxicity and require the adequate hydration to avoid the damage of renal during the administration. For that reason, the hospitalization is recommended for the patients to receive the chemotherapy including high dose of cisplatin. The present study aimed to establish the proper dose of fractional cisplatin in the combination with capecitabine in an outpatient setting. Methods: Capecitabine was administered orally every day from the evening of day 1 to the morning of 15. Cisplatin were infused on days 1, 8 and 15 for 60 minutes without hydration. The starting dose of cisplatin was 15 mg/m2 as level 1. The following cisplatin dose could be given to subsequent cohorts of patients depending on safety findings observed in the previous cohort: level 2, 20 mg/m2 and level 3, 25 mg/m2, respectively. Serum concentration ...

Abstract 433: Role of IL-8/CXCR2 network in the tumor microenvironment of esophageal squamous cell carcinoma

Background: IL-8, a pro-inflammatory cytokine, and its receptor, CXCR2, are expressed in various cancer cells. The IL-8/CXCR2 network is believed to be involved in angiogenesis, tumor cell proliferation and invasion. However, its role in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we investigated the role of the IL-8/CXCR2 network in the tumor microenvironment of ESCC. Methods: CXCR2 expression was investigated immunohistochemically in 82 primary ESCCs with R0 resection but without any preoperative treatment. We evaluated the intensity of the staining in four grades; grades 3 (strong) and 2 (medium) were deemed “positive”, and 1 (weak) and 0 (negative) as “negative”. The concentration of IL-8 and CXCR2 were investigated using the human ESCC cell lines TE1, 4, 5, 6, 8, 9, 10, 11, 14 and 15. IL-8/CXCR2 signaling was stimulated by IL-8 addition, and suppressed by SB225002 (Cayman Chemical) treatment, a selective antagonist of CXCR2. Stable IL-8 over-expressing TE4 cells were established, and cell proliferation assays were performed. To evaluate tumor growth in vivo, we subcutaneously inoculated 1.0×106 TE4 cells or transfectant into nude mice, and half of the transfectant-inoculated mice received 1mg/kg of SB225002 by intraperitoneal injection thrice weekly. The size and incidence of subcutaneous tumors were recorded. Result: Thirty-two (38.6%) patients were positive for CXCR2 immunostaining and 50 (61.4%) patients were negative. CXCR2 expression was related to poor survival in ESCC patients (P = 0.063). IL-8 concentration was high in TE4, 5, 10 and 14 supernatant, and low in that of TE1, 6, 8, 9, 11 and 15. CXCR2 expression was high in TE1, 4, 5, and 14 cells, and low in TE6, 8, 9, 10, 11 and 15. We chose TE1 and TE4 cells for further investigation. Stimulation or inhibition of the IL-8/CXCR2 network resulted in significant enhancement or suppression of cell proliferation in both cell lines (P Conclusion: Our results demonstrated that stimulation of the IL-8/CXCR2 network clearly enhanced ESCC cell proliferation, while its inhibition obviously suppressed ESCC cell proliferation in vitro. These results indicate that control of IL-8/CXCR2 network signaling may be a new therapeutic strategy for ESCC. Citation Format: Masazumi Inoue, Hiroya Takeuchi, Sachiko Matsuda, Tomohiko Nishi, Kazumasa Fukuda, Rieko Nakamura, Tsunehiro Takahashi, Norihito Wada, Hirofumi Kawakubo, Yoshiro Saikawa, Yuko Kitagawa. Role of IL-8/CXCR2 network in the tumor microenvironment of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2015-433