Darpp-32 and its truncated variant t-Darpp have antagonistic effects on breast cancer cell growth and herceptin resistance.
Darpp-32 and its truncated variant t-Darpp have antagonistic effects on breast cancer cell growth and herceptin resistance.
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DOI：
10.1371/journal.pone.0006220
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发表时间：
2009-07-13
期刊：
影响因子：
3.7
通讯作者：
Kane SE
中科院分区：
文献类型：
--
作者：
Gu L;Waliany S;Kane SE
赫赛汀（曲妥珠单抗）是一种人源化单克隆抗体，被批准用于治疗肿瘤过表达Her2 （erbB2/neu）的转移性乳腺癌患者。高达70%的her2阳性乳腺癌对基于赫赛汀的治疗有反应，但在最初的反应后一年内几乎不可避免地出现耐药性。为了帮助理解赫赛汀耐药机制，我们分离了her2阳性BT474人乳腺癌细胞（BT/HerR）的克隆变体，这些克隆变体对赫赛汀具有高度耐药性。这些细胞系表现出持续的PI3K/Akt信号传导，这是herceptin耐药增殖的重要组成部分。蛋白激酶A （PKA）信号网络中的几个基因在BT/HerR细胞中表达改变，包括PPP1R1B，它编码一种32 kDa的蛋白，称为Darpp-32及其氨基末端截断变体t-Darpp。当前工作的目的是确定Darpp-32和t-Darpp在赫赛汀耐药中的作用。我们检测了对赫赛汀耐药的BT/HerR细胞中Darpp-32和t-Darpp的表达。随后，将编码Darpp-32两种亚型的cdna分别或一起转染到her2阳性的SK-Br-3乳腺癌细胞中。检测转染后的细胞对赫赛汀和赫赛汀介导的Akt去磷酸化的耐药性。同时测定了cAMP反应元件结合蛋白（CREB）的DNA结合活性。我们发现BT/HerR细胞过表达t-Darpp，但不表达Darpp-32。此外，SK-Br-3细胞中的t-Darpp过表达足以赋予赫赛汀抗性和赫赛汀介导的Akt去磷酸化。Darpp-32共表达逆转了t-Darpp对赫赛汀耐药和Akt磷酸化的影响。t-Darpp过表达导致CREB结合活性增加，这也是由Darpp-32可逆的。t-Darpp和Darpp-32似乎对赫赛汀耐药具有拮抗作用。我们提出了一个统一的模型，通过该模型，这些影响可能通过PKA调控网络介导。
Herceptin (trastuzumab) is a humanized monoclonal antibody that is approved for the treatment of metastatic breast cancer patients whose tumors overexpress Her2 (erbB2/neu). Up to 70% of Her2-positive breast cancers demonstrate a response to Herceptin-based therapies, but resistance almost inevitably arises within a year of the initial response. To help understand the mechanism of Herceptin resistance, we isolated clonal variants of Her2-positive BT474 human breast cancer cells (BT/HerR) that are highly resistant to Herceptin. These cell lines exhibit sustained PI3K/Akt signaling as an essential component of Herceptin-resistant proliferation. Several genes in the protein kinase A (PKA) signaling network have altered expression in BT/HerR cells, including PPP1R1B, which encodes a 32 kDa protein known as Darpp-32 and its amino-terminal truncated variant, t-Darpp. The purpose of the current work was to determine the role of Darpp-32 and t-Darpp in Herceptin resistance. We determined expression of Darpp-32 and t-Darpp in BT/HerR cells selected for resistance to Herceptin. Subsequently, cDNAs encoding the two isoforms of Darpp-32 were transfected, separately and together, into Her2-positive SK-Br-3 breast cancer cells. Transfected cells were tested for resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. DNA binding activity by the cAMP response element binding protein (CREB) was also measured. We found that BT/HerR cells overexpressed t-Darpp but not Darpp-32. Moreover, t-Darpp overexpression in SK-Br-3 cells was sufficient for conferring resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. Darpp-32 co-expression reversed t-Darpp's effects on Herceptin resistance and Akt phosphorylation. t-Darpp overexpression led to increased CREB binding activity, which was also reversible by Darpp-32. t-Darpp and Darpp-32 appear to have antagonistic effects on Herceptin resistance. We present a unified model by which these effects might be mediated via the PKA regulatory network.
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