Investigating the mechanisms of CD44s splice isoform in breast cancer metastasis

描述（由申请人提供）：肿瘤转移是大多数类型的人类癌症（包括乳腺癌）中癌症相关死亡的主要原因。我们研究的长期目标是更好地了解乳腺癌转移的选择性剪接的分子机制。在这个项目中，我们建议调查涉及CD 44剪接异构体和Akt激活的正反馈环的机制，这是负责乳腺肿瘤转移。细胞表面分子CD 44由通过选择性剪接产生的蛋白质家族组成。包含可变外显子的不同组合产生⑶ 44 v。相反，排除所有可变外显子产生CD 44。CD 44可以被视为细胞外信号的传感器。通过与受体酪氨酸激酶（RTK）及其生长因子形成共受体复合物，CD 44增强生长因子刺激的RTK信号传导。我们以前的研究表明，CD 44 v和CD 44 s作用于不同的信号级联：CD 44 s激活Akt信号，这对促进细胞存活至关重要，而CD 44 v，另一方面，促进Ras/MAPK信号，导致细胞增殖状态。我们最近报道了CD 44 s亚型在上皮-间质转化（EMT）中起着重要作用，EMT是一个在肿瘤转移中异常激活的发育过程。我们还发现，CD 44的消减shRNA抑制乳腺肿瘤转移的动物和CD 44 s的表达上调，在高级别患者乳腺肿瘤标本。这些结果表明CD 44在乳腺癌转移中起关键作用。从机制上讲，我们已经表明，CD 44 s增强Akt激活和促进细胞存活。我们还发现，CD 44 s依赖性Akt信号上调透明质酸合成酶2（HAS 2）的表达。重要的是，HAS 2产物透明质酸（HA）是结合CD 44并促进CD 44 ε介导的Akt活化的配体。这些观察使我们假设正反馈环耦合CD 44和Akt信号，导致持续的Akt激活并促进乳腺癌转移。为了验证我们的假设，我们开发了以下具体目标：目标1，确定CD 44激活Akt信号的分子机制。目的2：研究CD 44 s依赖性Akt激活对乳腺癌细胞HAS 2表达的影响。目的3、探讨HAS 2的产物HA是否促进CD 44 s依赖的Akt活化，并探讨其在乳腺癌转移中的正反馈环。成功完成该项目将定义一种促进乳腺肿瘤转移的正反馈回路的新机制。干预这种正反馈回路可以为转移性乳腺癌的治疗提供一种令人兴奋的新治疗方法。

DESCRIPTION (provided by applicant): Tumor metastasis is the major cause of cancer-related death in most types of human cancers including breast cancer. The long-term goal of our research is to better understand molecular mechanisms of alternative splicing underlying breast cancer metastasis. In this project we propose to investigate the mechanisms of a positive feedback loop involving the CD44s splice isoform and Akt activation that is responsible for breast tumor metastasis. The cell surface molecule CD44 is comprised of a family of proteins that are generated by alternative splicing. Inclusion of different combinations of variable exons generates CD44v. Conversely, exclusion of all of the variable exons produces CD44s. CD44 can be viewed as a sensor for extracellular cues. By forming co-receptor complexes with receptor tyrosine kinases (RTKs) and their growth factors, CD44 augments growth factor-stimulated RTK signaling. Our previous studies showed that CD44v and CD44s act on different signaling cascades: CD44s activates Akt signaling that is critical for promoting cell survival, while CD44v, on the other hand, promotes Ras/MAPK signaling resulting in a cell proliferative state. We recently reported that the CD44s isoform plays an essential role in epithelial-mesenchymal transition (EMT), a developmental process that is abnormally activated in tumor metastasis. We also found that depletion of CD44 by shRNA inhibits breast tumor metastasis in animals and that CD44s expression is upregulated in high-grade patient breast tumor specimens. These results suggest a critical role for CD44s in breast cancer metastasis. Mechanistically, we have shown that CD44s potentiates Akt activation and promotes cell survival. We also found that CD44s-dependent Akt signaling upregulates hyaluronic acid synthase 2 (HAS2) expression. Importantly, the HAS2 product, hyaluronic acid (HA), is a ligand that binds to CD44 and facilitates CD44s- mediated Akt activation. These observations led us to hypothesize that a positive feedback loop couples CD44s and Akt signaling, resulting in sustained Akt activation and promoting breast cancer metastasis. To test our hypothesis we have developed the following specific aims: Aim 1, Determine the molecular mechanism by which CD44s activates Akt signaling. Aim 2, Examine how CD44s-dependent Akt activation promotes HAS2 expression in breast cancer cells. Aim 3, Investigate whether HA, product of HAS2, promotes CD44s-dependent Akt activation and examine the positive-feedback loop in clinical breast tumor metastasis. Successfully accomplishing this project will define a novel mechanism of a positive feedback loop that promotes breast tumor metastasis. Intervening this positive feedback loop could offer an exciting new therapeutic approach for the treatment of metastatic breast cancer.