Expression Of Anx A4 Research Articles

Abstract 3029: Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer

Abstract [Introduction] Ovarian cancer (OvCa) is one of the main causes of gynecologic cancer death in many countries and the number of patients is increasing. The standard treatment for advanced OvCa is cytoreductive surgery and adjuvant chemotherapy. The key drug for OvCa chemotherapy is platinum. However more than 70% of advanced stage OvCa relapse within 5 years, and the major issue to treat them is platinum resistance. We previously reported that Annexin (Anx) A4 protein was associating with chemoresistance to platinum-based cancer drugs. Nucleic acid therapeutics is promising therapy to target molecule existing intra cell membrane. The aim of this study is to evaluate the usefulness of antisense oligonucleotides (ASOs) and toovercome the platinum chemoresistance with ASOs by suppressing the expression of Anx A4 in cancer cells. [Methods] Cell line: To assess AnxA4 ASOs, two human ovarian cancer cell lines, RMG-1 and OVISE, which have platinum resistance and strongly express Anx A4, were used in each experiment. Anx A4 ASOs: We selected targeting Annexin A4 mRNA sequence in silico and made 16 ASOs. To enhance the stability, 2’, 4’-bridged nucleic acid were inserted in both 3’and 5’ends. And we analyzed suppression of Annexin A4 in ovarian clear cell cancer cell lines in vitro using real time PCR and western blotting. Platinum resistance verification: In vitro, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50values) were calculated. In vivo, we used ICRnu/nu mice xenografted subcutaneously with RMG-I and OVISE cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. [Result] Of the 16 types of antisense oligonucleotides, we focused on 2 type of ASO, #7, #9 which had stronger knockdown effect for Anx A4 expression. In RMG-I cell, IC50value of Anx A4 ASO transfected cells were 6.42 µM in compared with 11.12 µM in control ASO transfected cells. (p=0.012) In OVISE cells, IC50value was also significantly higher in transfected Anx A4 ASO cells. (10.30 µM, 4.66 µM, p<0.0001) Significantly more amount of platinum had accumulated in Anx A4 ASO transfected cells in compared with control ASO transfected cells and no treatment cells.In vivo, tumor growth of mice treated with cisplatin+Anx A4 ASOs were significantly inhibited compared to cisplatin + control ASOs in both the RMG-1 and OVISE cell lines xenograft mice. [Conclusion] By transfection of ANXA4 ASO, platinum resistance has been improved both in vitro and in vivo. Targeting ANX A4 with nucleic therapeutics could be an option for platinum resistant ovarian cancer. Citation Format: Satoshi Nakagawa, Reisa Kakubari, Shinya Matsuzaki, Yutaka Ueda, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Tadashi Kimura, Tetsuji Naka. Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3029.

Quantitative proteomic analysis for novel biomarkers of buccal squamous cell carcinoma arising in background of oral submucous fibrosis.

BackgroundIn South and Southeast Asian, the majority of buccal squamous cell carcinoma (BSCC) can arise from oral submucous fibrosis (OSF). BSCCs develop in OSF that are often not completely resected, causing local relapse. The aim of our study was to find candidate protein biomarkers to detect OSF and predict prognosis in BSCCs by quantitative proteomics approaches.MethodsWe compared normal oral mucosa (NBM) and paired biopsies of BSCC and OSF by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) to discover proteins with differential expression. Gene Ontology and KEGG networks were analyzed. The prognostic value of biomarkers was evaluated in 94 BSCCs accompanied with OSF. Significant associations were assessed by Kaplan-Meier survival and Cox-proportional hazards analysis.ResultsIn total 30 proteins were identified with significantly different expression (false discovery rate < 0.05) among three tissues. Two consistently upregulated proteins, ANXA4 and FLNA, were validated. The disease-free survival was negatively associated with the expression of ANXA4 (hazard ratio, 3.4; P = 0.000), FLNA (hazard ratio, 2.1; P = 0.000) and their combination (hazard ratio, 8.8; P = 0.002) in BSCCs.ConclusionThe present study indicates that iTRAQ quantitative proteomics analysis for tissues of BSCC and OSF is a reliable strategy. A significantly up-regulated ANXA4 and FLNA could be not only candidate biomarkers for BSCC prognosis but also potential targets for its therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2650-1) contains supplementary material, which is available to authorized users.

Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A

Although platinum drugs are often used for the chemotherapy of human cancers, platinum resistance is a major issue and may preclude their use in some cases. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to carboplatin through increased extracellular efflux of the drug. However, the precise mechanisms underlying that chemoresistance and the relationship of Anx A4 to platinum resistance in vivo remain unclear. In this report, the in vitro mechanism of platinum resistance induced by Anx A4 was investigated in endometrial carcinoma cells (HEC1 cells) with low expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, HEC1 control cells were compared with Anx A4-overexpressing HEC1 cells in xenografted mice. Significantly greater chemoresistance to cisplatin was observed in vivo in Anx A4-overexpressing xenografted mice. Immunofluorescence analysis revealed that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became colocalized with ATP7A, a copper transporter also well known as a mechanism of platinum efflux. ATP7A expression suppressed by small interfering RNA had no effect on HEC1 control cells in terms of chemosensitivity to platinum drugs. However, suppression of ATP7A in Anx A4-overexpressing platinum-resistant cells improved chemosensitivity to platinum drugs (but not to 5-fluorouracil) to a level comparable to that of control cells. These results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A.

Abstract 4059: Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A.

Abstract Because platinum drugs are often used for the chemotherapy of human cancers, when platinum resistance occurs it is a major issue. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to Carboplatin via increased extracellular efflux of the drug. However, the precise mechanisms of that chemoresistance, and the relationship of Anx A4 to platinum resistance in vivo, remain unclear. In this report we investigate in vitro the mechanism of platinum resistance induced by Anx A4 in endometrial carcinoma cells (HEC1 cells) which normally have a low level of expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, we compared HEC1 control cells with Anx A4-overexpressing HEC1 cells when both were xenografted to mice, Anx A4-overexpressing xenografted mice presented with significantly greater chemoresistance to Cisplatin in vivo. By immunofluorescence analysis we found that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became co-localized with ATP7A, a copper transporter also well known to be a platinum effluxer. When the expression of ATP7A was suppressed by small interfering RNA in HEC1 control cells, they showed no change of chemosensitivity to platinum drugs. However, suppressed ATP7A in Anx A4 overexpressing platinum-resistant cells, they showed improved chemosensitivity to platinum drugs, to a level comparable of that of control cells. Our results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A. Citation Format: Shinya Matsuzaki, Akiko Morimoto, Satoshi Serada, Takuhei Yokoyama, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takayuki Enomoto, Tetsuji Naka, Tadashi Kimura. Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A. [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 4059. doi:10.1158/1538-7445.AM2013-4059

Abstract 4057: Functional domain of Annexin A4: which domain is indispensable for chemoresistance to platinum drugs.

Abstract Background: Platinum drugs are often used for the postoperative adjuvant therapy or treatment in advanced and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtype of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. Purpose: To identify the domain of Anx A4 relevant to chemoresistance. Materials and methods: Anx A4 possesses the NH2-terminal domain and four annexin repeat domains with one Ca2+-binding site in each domain. Deleting annexin repeat domains one by one from C-terminal site, we generated four deletion mutants of Anx A4 named R1, 2, 3, and 4 (arabic figure shows the number of annexin repeat domains). Stable cell lines overexpressing each deletion mutants were established in NUGC3 cells. Chemosensitivity to platinum drugs was evaluated using the MTT assay. Results. Compared with mock transfected cells, all the four deletion mutants induced chemoresistance to both cisplatin and carboplatin. Conclusions. The deletion derivative covering only one annexin repeat domain can induce chemoresistance to platinum drugs. Further study is underway to determine the specific site for chemoresistance of Anx A4. Citation Format: Akiko Morimoto, Takayuki Enomoto, Shinya Matsuzaki, Kousuke Hiramatsu, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takuhei Yokoyama, Satoshi Serada, Tetsuji Naka, Tadashi Kimura. Functional domain of Annexin A4: which domain is indispensable for chemoresistance to platinum drugs. [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 4057. doi:10.1158/1538-7445.AM2013-4057

Abstract 4225: Annexin A4 induces chemoresistance for multiple drugs in ovarian clear cell carcinoma

Abstract BACKGROUND: Platinum drugs play the key role for postoperative adjuvant therapy and treatment in progress and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtypes of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. However, it remains unclear whether targeting Anx A4 is effective for improving the sensitization against chemotherapy, like platinum drugs. The aim of this study was to evaluate the role of Anx A4 in chemoresistance in ovarian CCC. MATERIAL AND METHODS: RMG-I cell lines were used to establish the stable knockdown of Anx A4. Plasmids encoding short hairpin RNAs (shRNAs) targeting Anx A4 were transfected. Chemoresistance was assessed by IC50 value in various drugs. RESULTS: Compared with control cells, knockdown of Anx A4 gene expression in RMG-I cells induced chemosentisitzation around 2-fold to cisplatin (IC50: 11.2 μM to 5.8 μM, P&amp;lt;0.01) and Carboplatin (IC50: 130.4 μM to 63.3 μM, P&amp;lt;0.01). Unexpectedly, knockdown of Anx A4 also improved the chemosentisitzation of epirubicin (IC50: 256.7 nM to 180.3 nM, P&amp;lt;0.01) and Paclitaxel (IC50: 5.5 nM to 2.9 nM, P&amp;lt;0.01). In consistent with the improved chemosensitivity, drugs induced p53 was markedly increased by knockdown of Anx A4 in RMG-I cells. In xenograft models, significant chemosensitization toward cisplatin was demonstrated by knockdown of Anx A4 in RMG-I cells. CONCLUSIONS: Inhibition of Anx A4 induced chemosensitization in ovarian CCC cells. Anx A4 blockade might be a novel therapeutic target of chemoresistance in patients with ovarian CCC. 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 4225. doi:1538-7445.AM2012-4225

Enhanced expression of Annexin A4 in clear cell carcinoma of the ovary and its association with chemoresistance to carboplatin

Clear cell carcinoma (CCC) of the ovary is known to be highly resistant to platinum-based chemotherapy. The purpose of our study was to identify a candidate protein that is associated with chemoresistance of CCC and to investigate the specific mechanism of chemoresistance conferred by the identified protein. Enhanced expression of Annexin A4 (Anx A4) was identified in ovarian CCC cells using 2-D differential gel electrophoresis (2D-DIGE) and mass spectrometry. Anx A4 levels were elevated in CCC cells compared with non-CCC cells as determined by real-time RT-PCR and Western blot analysis. Immunohistochemical analysis of Anx A4 was performed in 126 epithelial ovarian cancer tissue samples and demonstrated significantly elevated levels of Anx A4 protein levels in ovarian CCC tumors compared with ovarian serous and endometrioid tumors (p < 0.01). Anx A4-transfected ovarian non-CCC cells were more resistant to carboplatin (IC50 = 42 microM) compared with control cells (IC50 = 23 microM) as determined by modified MTT assay. Intracellular platinum levels were significantly lower in Anx A4-transfected cells compared with control cells after carboplatin treatment (p = 0.0020) and after an additional 360 min of carboplatin-free incubation (p = 0.0004), as measured by atomic absorption spectrophotometry. Expression of Anx A4 is elevated in ovarian CCC tumors and is associated with chemoresistance in cultured ovarian cancer cells. These results demonstrate that Anx A4 confers chemoresistance in ovarian cancer cells in part by enhancing drug efflux. Thus, Anx A4 may represent a novel therapeutic target of chemoresistance in patients with ovarian CCC.