Hyaluronan-mediated CD 44 Interaction with p 300 and SIRT 1 Regulates-Catenin Signaling and NF B-specific Transcription Activity Leading to MDR 1 and Bcl-xL Gene Expression and Chemoresistance in Breast Tumor Cells *-猫眼课题宝

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2009

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作者：

L. Bourguignon;W. Xia;G. Wong

在这项研究中，我们研究了人乳腺肿瘤细胞（MCF-7 细胞）中透明质酸 (HA) 介导的 CD44（anHA 受体）与 p300（一种组蛋白乙酰转移酶）和 SIRT1（一种组蛋白脱乙酰酶）的相互作用。具体而言，我们的结果表明，HA 与 CD44 结合上调 p300 表达及其乙酰转移酶活性，进而促进 β 连环蛋白和 NF B-p65 的乙酰化，分别导致 β 连环蛋白相关 T 细胞因子/淋巴细胞增强因子转录共激活和 NF B 特异性转录上调。这些变化随后导致 MDR1（P-糖蛋白/P-gp）基因和抗凋亡基因 Bcl-xL 的表达，从而导致 MCF-7 细胞出现化疗耐药。我们的数据还表明，MCF-7细胞中p300、β-连环蛋白或NF B-p65的下调（通过用p300-、-连环蛋白-或NF B-p65特异性小干扰RNA转染细胞）会抑制HA/CD44介导的-连环蛋白/NF B-p65乙酰化并消除上述转录活性。随后，乳腺肿瘤细胞中 MDR1 和 Bcl-xL 基因表达显着降低，caspase-3 活性和化疗敏感性增强。进一步分析表明，白藜芦醇（一种天然抗氧化剂）激活SIRT1（脱乙酰酶）会诱导SIRT1-p300关联和乙酰转移酶失活，导致HA/CD44诱导的β-catenin和NF B-p65脱乙酰化，抑制β-catenin-T细胞因子/淋巴细胞增强因子和NF B特异性转录激活，以及MDR1和Bcl-xL基因表达受损。所有这些多重作用都会导致 caspase-3 的激活和化疗耐药性的降低。总之，这些发现表明，HA/CD44 刺激的 p300（乙酰转移酶）和白藜芦醇激活的 SIRT1（脱乙酰酶）之间的相互作用在调节乳腺肿瘤细胞的细胞存活与凋亡、多药耐药性与敏感性之间的平衡中发挥着关键作用。多药耐药性和疾病复发是乳腺癌治疗中具有挑战性的临床问题 (1-3)。由于对乳腺肿瘤细胞信号传导和化疗反应的分子基础知之甚少，因此识别可用于预测乳腺癌来源的癌细胞的致癌潜力和可能的化疗耐药性的分子非常重要。在寻找与乳腺肿瘤细胞功能和可能的化疗耐药相关的细胞调节因子的过程中，透明质酸 (HA)2（大多数哺乳动物组织细胞外基质的主要成分）被确定为主要候选者 (4, 5)。 HA 是一种非硫酸化、无支链糖胺聚糖，由重复二糖单元、D-葡萄糖醛酸和 N-乙酰基-D-葡萄糖胺组成 (6, 7)。 HA 由特定的 HA 合酶合成 (7, 8)，并被透明质酸酶消化成各种较小的分子 (9)。 HA 在干细胞微环境和乳腺肿瘤中明显富集 (10, 11)。在乳腺癌患者中，恶性肿瘤中的 HA 浓度通常高于良性或正常组织，并且在某些肿瘤类型中，HA 水平可预测恶性肿瘤 (11)。此外，在乳腺癌患者的血清中发现 HA 水平升高 (12)。 CD44 是细胞表面糖蛋白家族，在多种细胞和组织中表达，包括乳腺肿瘤细胞和癌组织 (5, 13–16)。核苷酸序列分析表明，存在多种 CD44 亚型（由选择性剪接机制衍生），所有这些亚型都是标准形式 CD44 的变体 (17, 18)。所有 CD44 同工型的胞外域均含有 HA 结合位点，因此可作为 HA 的主要细胞表面受体 (19)。乳腺癌干细胞中表达的 CD44 同工型显示出启动肿瘤细胞特异性特性的独特能力 (20-22)。最近的研究表明，乳腺癌肿瘤含有高致瘤性癌症干细胞亚群，其特征是高 CD44 表达和低（或无）CD24 表达（CD44 CD24 /低）(20-22)。纯化的 CD44 CD24 /低乳腺肿瘤细胞能够产生表型不同的细胞，从而在免疫缺陷小鼠中产生异质性肿瘤 (20-22)。这些发现表明 * 这项工作全部或部分得到了美国国立卫生研究院拨款 R01 CA66163、R01 CA 78633 和 P01 AR39448 (USPHS) 的支持。这项工作还得到了退伍军人事务部功绩审查拨款和国防部拨款的支持。这篇文章的出版费用部分是通过支付版面费来支付的。因此，根据 18 U.S.C. 规定，本文必须特此标记为“广告”。第1734章只是为了表明这个事实。 1 退伍军人事务研究职业科学家。信件和重印请求的收件人：内分泌科 (111N)，医学系，加州大学旧金山分校和退伍军人事务医疗中心，4150 Clement St., San Francisco, CA 94121。电话：415-2214810，分机号：115-2214810。第3321章传真：415-383-1638；电子邮件：lilly.bourguignon@ucsf.edu。 2 使用的缩写为：HA、透明质酸； HAT，组蛋白乙酰转移酶； HDAC，组蛋白脱乙酰酶； TCF/LEF、T细胞因子/淋巴细胞增强因子； siRNA，小干扰RNA； Z，苄氧基羰基； FMK，氟甲基酮； Q-PC，定量PCR； IKK，B激酶抑制剂； MDR，多重耐药性。生物化学杂志卷。 284，没有。 5，第 2657–2671 页，2009 年 1 月 30 日美国印刷

In this study we have investigated hyaluronan (HA)-mediated CD44 (anHA receptor) interactionswith p300 (a histone acetyltransferase) and SIRT1 (a histone deacetylase) in human breast tumor cells (MCF-7 cells). Specifically, our results indicate that HA binding to CD44 up-regulates p300 expression and its acetyltransferase activity that, in turn, promotes acetylation of -catenin and NF B-p65 leading to activation of -catenin-associated T-cell factor/lymphocyte enhancer factor transcriptional co-activation andNF B-specific transcriptional up-regulation, respectively. These changes then cause the expression of the MDR1 (P-glycoprotein/P-gp) gene and the anti-apoptotic gene Bcl-xL resulting in chemoresistance in MCF-7 cells. Our data also show that down-regulation of p300, -catenin, or NF B-p65 in MCF-7 cells (by transfecting cells with p300-, -catenin-, orNF B-p65-specific small interfering RNA) inhibits the HA/CD44-mediated -catenin/NF B-p65 acetylation and abrogates the aforementioned transcriptional activities. Subsequently, there is a significant decrease in bothMDR1 and Bcl-xL gene expression and an enhancement in caspase-3 activity and chemosensitivity in the breast tumor cells. Further analyses indicate that activation of SIRT1 (deacetylase) by resveratrol (a natural antioxidant) induces SIRT1-p300 association and acetyltransferase inactivation, leading to deacetylation of HA/CD44-induced -catenin and NF B-p65, inhibition of -catenin-T-cell factor/lymphocyte enhancer factor andNF Bspecific transcriptional activation, and the impairment of MDR1 and Bcl-xL gene expression. All these multiple effects lead to an activation of caspase-3 and a reduction of chemoresistance. Together, these findings suggest that the interactions betweenHA/CD44-stimulated p300 (acetyltransferase) and resveratrol-activated SIRT1 (deacetylase) play pivotal roles in regulating the balance between cell survival versus apoptosis, and multidrug resistance versus sensitivity in breast tumor cells. Multidrug resistance and disease relapse are challenging clinical problems in the treatment of breast cancers (1–3). Because little is known regarding the molecular basis of breast tumor cell signaling and chemotherapeutic responses, it is important to identifymolecule(s) that can be used to predict the oncogenic potential and possible chemoresistance of breast carcinoma-derived cancer cells. During the search for cellular regulators that correlate with breast tumor cell functions and possible chemoresistance, hyaluronan (HA)2 (a major component in the extracellular matrix of most mammalian tissues) was identified as a prime candidate (4, 5). HA is a nonsulfated, unbranched glycosaminoglycan consisting of repeating disaccharide units, D-glucuronic acid and N-acetyl-D-glucosamine (6, 7). HA is synthesized by specific HA synthases (7, 8) and digested into various smaller molecules by hyaluronidases (9). HA is clearly enriched in stem cell niches and in breast tumors (10, 11). In breast cancer patients, HA concentrations are often higher in malignant tumors than in benign or normal tissues, and in some tumor types the level of HA is predictive of malignancy (11). Furthermore, elevatedHA levels have been found in the serum of breast cancer patients (12). CD44 denotes a family of cell-surface glycoproteins that are expressed in a variety of cells and tissues, including breast tumor cells and carcinoma tissues (5, 13–16). Nucleotide sequence analyses reveal that there exist numerous CD44 isoforms (derived by alternative splicingmechanisms), all of which are variants of the standard form, CD44s (17, 18). All CD44 isoforms contain an HA-binding site in their extracellular domain and thereby serve as major cell surface receptors for HA (19). The CD44 isoforms expressed in breast cancer stem cells display a unique ability to initiate tumor cell-specific properties (20–22). Recent studies indicate that breast cancer tumors contain a subpopulation of highly tumorigenic cancer stem cells characterized by high CD44 expression and low (or no) CD24 expression (CD44 CD24 /low) (20–22). Purified CD44 CD24 /low breast tumor cells are capable of generating phenotypically distinct cells resulting in heterogeneous tumors in immunodeficient mice (20–22). These findings indicate that * This work was supported, in whole or in part, by National Institutes of Health Grants R01 CA66163, R01 CA 78633, and P01 AR39448 (USPHS). This work was also supported by a Veterans Affairs merit review grant and a Department of Defense grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Veterans Affairs Research Career Scientist. To whom correspondence and reprint requests should be addressed: Endocrine Unit (111N), Dept. of Medicine, University of California at San Francisco and Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-2214810, ext. 3321; Fax: 415-383-1638; E-mail: lilly.bourguignon@ucsf.edu. 2 The abbreviations used are: HA, hyaluronan; HAT, histone acetyltransferase; HDAC, histone deacetylase; TCF/LEF, T-cell factor/lymphocyte enhancer factor; siRNA, small interfering RNA; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; Q-PC, quantitative PCR; IKK, inhibitor of B kinase; MDR, multidrug resistance. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 5, pp. 2657–2671, January 30, 2009 Printed in the U.S.A.

Hyaluronan-mediated CD 44 Interaction with p 300 and SIRT 1 Regulates-Catenin Signaling and NF B-specific Transcription Activity Leading to MDR 1 and Bcl-xL Gene Expression and Chemoresistance in Breast Tumor Cells *

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