Rewiring ERBB Signaling in Cancer Cells

描述（由申请人提供）：在一些实体瘤中，尤其是在乳腺癌中，ERBB2的过表达导致更积极的生长，增加转移潜力和出现耐药性的可能性。这种反应的一个重要方面是ERBB2和ERBB3之间的相互作用。同时，ERBB3磷酸化水平升高（在许多情况下受体水平升高）导致抗凋亡信号传导增强，因为几个PI3K结合位点位于激酶缺陷的ERBB3中。除非ERBB2抑制是定量的（目前还不可行），在激酶抑制剂治疗后，通过稳定残余活化的ERBB3，磷酸化ERBB3的稳态水平迅速恢复到抑制前水平，而不管ERBB2是否持续和实质性的抑制。这严重削弱了ERBB2定向治疗，但它也反映了ERBB2- erbb3信号传导的内在方面。ERBB2显然已经进化到避免稳定的自结合，并在非常短暂的相互作用中被本构激活。我们的初步数据表明，ERBB2具有内在的控制机制，可以进一步抑制这种瞬态复合物的“意外激活”。ERBB2过表达导致ERBB2高水平激活，但处于空间分离和低比例激活状态。我们认为这反映了癌细胞为确保激活受体的低空间密度所做的努力，因为在稳定的ERBB2二聚体中，空间密度和ERBB3的排除将有利于细胞毒性信号转导的结果。我们通过使用嵌合受体结构来研究这一点，嵌合受体结构具有改变的内在控制和关联行为，以及配体变体，这些配体变体对ERBB3簇的破坏程度不同。与ERBB2相反，ERBB3在激活过程中似乎需要簇的自结合和有序解离。扰乱该系统会导致细胞死亡，其方式似乎与细胞粘附特性的变化有关，可能是因为细胞向更倾向转移的途径发展，没有足够的信号来“支撑”细胞凋亡等疾病的发生。避免ERBB2聚集和维持ERBB3聚集对于获得平衡的信号反应非常重要，并且是故意系统扰动的非常容易达到的目标。我们将在不同的细胞环境中评估细胞死亡的性质，采用现有的和新的方法来直接评估受体聚类和激活受体的空间邻近性，以评估它们对信号转导结果的贡献，并确定由环境外和细胞死亡促进激活产生的信号转导的性质。我们的长期目标是利用这一认识，通过工程疗法（抗体或适体）来加强ERBB2的体外激活。这里提出的研究也可能阐明目前使用的治疗方法（如赫赛汀）的机制，尽管没有机制基础，但可能已经利用了这种反应的一部分。我们也希望更好地理解为什么以前的尝试没有更成功地加强ERBB2介导的细胞凋亡。

DESCRIPTION (provided by applicant): In several solid tumors, but especially in breast cancers, overexpression of ERBB2 results in more aggressive growth, and enhanced metastatic potential and probability of emergence of drug resistance. An important aspect in this response is the interaction between ERBB2 and ERBB3. Simultaneously elevated levels of ERBB3 phosphorylation (and in many cases receptor levels) result in enhanced anti-apoptotic signaling since several PI3K binding sites reside in the kinase-deficient ERBB3. Unless ERBB2 inhibition is quantitative (which is currently not feasible), steady state levels of phospho-ERBB3 rapidly return to pre-inhibition levels after kinase inhibitor treatment through stabilization of the residual activated ERBB3, regardless of sustained and substantial inhibition of ERBB2. This seriously undercuts ERBB2 directed therapy, but it also reflects on intrinsic aspects of ERBB2-ERBB3 signaling. ERBB2 has apparently evolved to avoid stable self-association and is constitutively activated in very transient interactions. Our preliminary data suggest that ERBB2 has built-in control mechanisms to further suppress "accidental activation" in such transient complexes. ERBB2 overexpression results in high levels of activated ERBB2 but at a state of spatial separation and low fraction of activation. We propose that this reflects efforts by the cancer cell to ensure a low spatial density of activated receptors, because the combination of spatial density and exclusion of ERBB3 in stable ERBB2 dimers would favor a cytotoxic signaling outcome. We have investigated this by using chimeric receptor constructs with altered intrinsic control and association behavior as well as ligand variants that differ in their degree of disruption of ERBB3 clusters. In contrast to ERBB2, self-association and orderly dissociation of clusters during activation appears to be required for ERBB3. Perturbing this system causes cell death in a manner that appears to be related to changes in cell adhesion characteristics, possibly because the cells progress towards a more pro-metastatic path without sufficient signaling to "brace" against the onset of anoikis like apoptosis. The avoidance of ERBB2 clustering and maintenance of ERBB3 clusters are important in getting a balanced signaling response and are very accessible targets for deliberate system perturbation. We will evaluate the nature of cell death in different cellular contexts, adopt existing and novel methodology for direct assessment of receptor clustering and spatial proximity of activated receptors to evaluate their contribution to signaling outcomes, and determine the nature of signaling that emanates from out-of-context and cell-death promoting activation. It is our long range goal to capitalize on this understanding to enforce out-of-context activation of ERBB2 by engineered therapeutics (antibodies or aptamers). The studies proposed here are also likely to shed light on the mechanisms of currently used therapeutics (such as Herceptin) which may, albeit without mechanistic foundation, already utilize part of this response. We also expect to better understand why previous attempts to actively enforce ERBB2 mediated apoptosis have not been more successful.