AFAP-110 as a cSrc activator in breast cancer

AFAP-110 作为乳腺癌的 cSrc 激活剂

• 批准号: 8288607
• 负责人: Jeffrey T Holt
• 金额: $ 22.78万
• 依托单位: GEISINGER COMMONWEALTH SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288607
• 关键词: Address; Adhesions; Affect; Binding; Binding Sites; Breast; Breast Cancer Cell; Breast Carcinoma; Cell Adhesion; Cells; Data; Docking; Drug Delivery Systems; Fibronectins; Filament; Fostering; Generations; Health; Integrins; Link; Malignant neoplasm of ovary; Mammary Gland Parenchyma; Mammary gland; Membrane; Microfilaments; Molecular Conformation; Mus; Mutate; Mutation; Neoplasm Metastasis; Ovarian Carcinoma; Pharmaceutical Preparations; Phosphatidic Acid; Phosphatidylinositols; Phospholipids; Phosphorylation; Process; Protein Tyrosine Kinase; Proteins; Role; Signal Transduction; Site; Stress; Structure; Time; Tissues; Transgenic Mice; Variant; Vesicle; Work; crosslink; malignant breast neoplasm; neoplastic cell; overexpression; platelet protein P47; response; restoration; tumor

DESCRIPTION (provided by applicant): As breast and ovarian carcinoma progress the tumor cells develop invasive structures, which provide the cells with a mechanism to cross tissue barriers and metastasize. Activation of the tyrosine kinase cSrc is known to occur in breast and ovarian cancers, will stimulate the formation of invasive structures, and promote invasion. Although cSrc is activated in these tumors, it is not mutated. Rather, cSrc is activated in response to input signals. One example of a cSrc-activating protein is AFAP-110, which has two known functions ? it cross-links actin filaments and can bind to and activate cSrc. AFAP-110 is a substrate of PKCa. Phosphorylation by PKCa results in a conformational change in AFAP-110 that releases 'autoinhibition' and enables it to efficiently cross-link stress filaments and direct cSrc activation. This signaling cascade results in the formation of invasive structures. The question to be addressed in this application is, 'can increased expression of AFAP-110 in mouse breast promote cSrc activation, and if so, how?' Our preliminary data indicate that knockdown of AFAP-110 in breast cancer cells results in reduced cell adhesion. This effect is linked with reduced stress filament cross-linking and decreased invasive potential. Upon MDA-231 cell adhesion to fibronectin, AFAP-110 becomes phosphorylated on Tyr94, a Src target. Thus, in Aim 1, we hypothesize that AFAP-110 promotes cell adhesion by activating cSrc. We also find that the ability of AFAP-110 to activate cSrc requires that the PH1 domain bind to phosphatidic acid (PA). There are two grooves in the PH1 domain that can bind phospholipids. In Aim 2, we predict that binding of PA and PtdIns4P are required for AFAP-110 to activate cSrc and that they bind to separate grooves and work cooperatively to dock AFAP-110 on membranes and subsequently activated cSrc. Interestingly, we identified a polymorphic variant of AFAP-110 that has a nonsynomous SNP in the carboxy terminal pleckstrin homology (PH2) domain. This Ser403?Cys403 change enables AFAP-110403C to independently activate cSrc, but only under conditions of overexpression. We hypothesize that the Cys403 SNP may result in a less efficiently stabilized AFAP-110 multimer, which could weaken autoinhibition and result in cSrc activation. In Aim 3, we will determine how the PH2 domain fosters self-association and if mutations in the opposing PH2 binding site within AFAP-110 also destabilize the multimer and enable cSrc activation. Lastly, we find that AFAP-110 and cSrc are overexpressed together in the same cells in breast and ovarian cancer tissues. We hypothesize that AFAP-110 has the capacity to activate cSrc in breast tissue. In Aim 4, we will create a transgenic mouse and determine if, under conditions of overexpression in mouse mammary tissue, AFAP-110 has the potential to activate cSrc. The significance of these studies is that AFAP-110 may be one protein responsible for activating cSrc. Thus, it may be a legitimate drug target, with a 'drug-able' site in the PH1 domain that could be targeted to disrupt cSrc activation and invasive potential. PUBLIC HEALTH RELEVANCE: This project is relevant to breast cancer in that it will address a mechanism by which cSrc becomes activated in breast cancer. This will be the first time anyone has attempted to determine if cSrc activating proteins (AFAP-110) can direct cSrc activation in mouse breast tissues. As AFAP-110 has a phospholipid binding pocket that appears to be crucial for this function, it may be a drug-able target.

描述（由申请人提供）：随着乳腺癌和卵巢癌的进展，肿瘤细胞形成侵袭性结构，这为细胞提供了穿过组织屏障和转移的机制。 已知酪氨酸激酶cSrc的激活发生在乳腺癌和卵巢癌中，将刺激侵袭性结构的形成，并促进侵袭。 虽然cSrc在这些肿瘤中被激活，但它没有突变。 相反，cSrc响应于输入信号而被激活。 cSrc激活蛋白的一个例子是AFAP-110，它有两个已知的功能？它交联肌动蛋白丝并能结合和激活cSrc。 AFAP-110是PKCa的底物。 通过PKCa的磷酸化导致AFAP-110的构象变化，释放“自抑制”，使其能够有效地交联应力丝和直接cSrc激活。 这种信号级联导致侵入性结构的形成。 本申请中要解决的问题是，小鼠乳腺中AFAP-110的表达增加是否能促进cSrc活化，如果是，如何促进？“我们的初步数据表明，乳腺癌细胞中AFAP-110的敲低导致细胞粘附力降低。 这种效应与应力丝交联减少和侵袭潜力降低有关。 后 MDA-231细胞粘附于纤连蛋白，AFAP-110在Src靶Tyr 94上磷酸化。 因此，在目的1中，我们假设AFAP-110通过激活cSrc促进细胞粘附。 我们还发现AFAP-110激活cSrc的能力需要PH 1结构域与磷脂酸（PA）结合。 在PH 1结构域中有两个可以结合磷脂的凹槽。 在目标2中，我们预测PA和PtdIns 4P的结合是AFAP-110激活cSrc所需的，并且它们结合到单独的凹槽并协同工作以将AFAP-110停靠在膜上并随后激活cSrc。 有趣的是，我们鉴定了AFAP-110的多态性变体，其在羧基末端普列克底物蛋白同源性（PH 2）结构域中具有非synomous SNP。 这是Ser 403吗Cys 403变化使 AFAP-110403 C独立激活cSrc，但仅在过表达的条件下。 我们假设Cys 403 SNP可能导致AFAP-110多聚体的稳定性降低，这可能会削弱自身抑制并导致cSrc激活。 在目标3中，我们将确定PH 2结构域如何促进自缔合，以及AFAP-110内相对的PH 2结合位点中的突变是否也使多聚体不稳定并使cSrc活化。 最后，我们发现AFAP-110和cSrc在乳腺癌和卵巢癌组织中的相同细胞中一起过表达。 我们假设AFAP-110具有激活乳腺组织中cSrc的能力。 在目标4中，我们将创建转基因小鼠，并确定在小鼠乳腺组织中过表达的条件下，AFAP-110是否具有激活cSrc的潜力。 这些研究的意义在于AFAP-110可能是负责激活cSrc的一种蛋白。 因此，它可能是一个合法的药物靶点，在PH 1结构域中有一个“药物活性”位点，可以靶向破坏cSrc激活和侵袭潜力。 公共卫生关系：该项目与乳腺癌相关，因为它将解决cSrc在乳腺癌中被激活的机制。 这将是第一次有人试图确定cSrc激活蛋白（AFAP-110）是否可以指导小鼠乳腺组织中的cSrc激活。 由于AFAP-110有一个磷脂结合口袋，这似乎是至关重要的这一功能，它可能是一个药物的目标。

项目成果

AFAP120 regulates actin organization during neuronal differentiation.

• DOI: 10.1016/j.diff.2008.09.006
• 发表时间: 2009-01
• 期刊: DIFFERENTIATION
• 影响因子: 2.9
• 作者: Xu, Xiaohua;Harder, Jennifer;Flynn, Daniel C.;Lanier, Lorene M.
• 通讯作者: Lanier, Lorene M.

Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland.

• DOI: 10.1038/onc.2014.205
• 发表时间: 2015-05-14
• 期刊: Oncogene
• 影响因子: 8

Confirmation of gene expression-based prediction of survival in non-small cell lung cancer.

• DOI: 10.1158/1078-0432.ccr-08-0095
• 发表时间: 2008-12-15
• 期刊: CLINICAL CANCER RESEARCH
• 影响因子: 11.5
• 作者: Guo, Nancy L.;Wan, Ying-Wooi;Tosun, Kursad;Lin, Hong;Msiska, Zola;Flynn, Daniel C.;Remick, Scot C.;Vallyathan, Val;Dowlati, Afshin;Shi, Xianglin;Castranova, Vincent;Beer, David G.;Qian, Yong
• 通讯作者: Qian, Yong

AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes.

• DOI: 10.1016/j.ejcb.2010.11.016
• 发表时间: 2011-05
• 期刊: European journal of cell biology
• 影响因子: 6.6
• 作者: Snyder BN;Cho Y;Qian Y;Coad JE;Flynn DC;Cunnick JM
• 通讯作者: Cunnick JM