Investigating the mechanisms of CD44s splice isoform in breast cancer metastasis

描述(申请人提供)：肿瘤转移是包括乳腺癌在内的大多数人类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背后的选择性剪接的分子机制。在这个项目中，我们建议研究涉及CD44s剪接异构体和Akt激活的正反馈环路的机制，该环路与乳腺癌转移有关。细胞表面分子CD44由一系列通过选择性剪接产生的蛋白质组成。包含不同组合的可变外显子会产生CD44v。相反，排除所有可变外显子会产生CD44s。CD44可以被视为细胞外信号的传感器。CD44通过与受体酪氨酸激酶(RTK)及其生长因子形成共受体复合体，增强生长因子刺激的RTK信号传导。我们以前的研究表明，CD44v和CD44s作用于不同的信号级联：CD44s激活Akt信号，而Akt信号对促进细胞存活至关重要，而CD44v则促进RAS/MAPK信号，导致细胞增殖状态。我们最近报道，CD44s亚型在上皮-间充质转化(EMT)中起重要作用，EMT是一种在肿瘤转移中异常激活的发育过程。我们还发现，通过shRNA去除CD44可以抑制动物乳腺肿瘤的转移，并且CD44s在高级别患者的乳腺肿瘤标本中表达上调。这些结果表明CD44s在乳腺癌转移中起关键作用。在机制上，我们已经证明CD44s增强Akt的激活并促进细胞存活。我们还发现依赖CD44s的Akt信号上调透明质酸合成酶2(HAS2)的表达。重要的是，HAS2的产物透明质酸(HA)是一种与CD44结合的配体，促进CD44s介导的Akt激活。这些观察结果使我们假设，CD44s和Akt信号之间存在正反馈环路，导致Akt持续激活，促进乳腺癌转移。为了验证我们的假设，我们制定了以下具体目标：目标1，确定CD44s激活Akt信号的分子机制。目的研究CD44s依赖的Akt激活如何促进乳腺癌细胞HAS2的表达。目的研究HAS2的产物HA是否促进CD44s依赖的Akt活化，并探讨其在临床乳腺肿瘤转移中的正反馈回路。该项目的成功完成将定义一种促进乳腺肿瘤转移的正反馈回路的新机制。干预这一正反馈回路可能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.