Rewiring ERBB Signaling in Cancer Cells

描述(申请人提供)：在一些实体肿瘤中，尤其是在乳腺癌中，ERBB2的过度表达会导致更具侵袭性的生长，并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磷酸化水平的升高(在许多情况下也是受体水平)导致了抗凋亡信号的增强，因为几个PI3K结合位点驻留在激酶缺陷的ERBB3中。除非ERBB2的抑制是定量的(目前还不可行)，否则在通过稳定剩余的激活的ERBB3而进行激酶抑制剂治疗后，稳态的磷酸化ERBB3水平迅速恢复到抑制前的水平，而不管ERBB2的持续和实质性抑制。这严重削弱了ERBB2的定向治疗，但它也反映了ERBB2-ERBB3信号的内在方面。ERBB2的进化显然是为了避免稳定的自结合，并且在非常短暂的相互作用中被结构性地激活。我们的初步数据表明，ERBB2具有内置的控制机制，可以进一步抑制这种瞬时复合体中的“意外激活”。ERBB2过表达导致高水平的激活ERBB2，但处于空间分离状态和低激活分数。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，因为空间密度和稳定的ERBB2二聚体中ERBB3的排除将有利于细胞毒信号转导结果。我们通过使用具有改变的内在控制和结合行为的嵌合受体结构以及不同程度破坏ERBB3簇的配体变体来研究这一点。与ERBB2相反，ERBB3似乎需要在激活过程中自结合和有序解离簇。干扰这一系统会导致细胞死亡，其方式似乎与细胞黏附特性的改变有关，可能是因为细胞朝着更有利于转移的路径前进，而没有足够的信号来对抗类失巢凋亡的开始。避免ERBB2集群和维护ERBB3集群对于获得平衡的信令响应是重要的，并且是非常容易受到故意系统扰动的目标。我们将评估不同细胞环境下细胞死亡的性质，采用现有的和新的方法直接评估受体聚集和激活受体的空间邻近程度，以评估它们对信号转导结果的贡献，并确定来自环境外和促进细胞死亡激活的信号的性质。我们的长期目标是利用这一理解，通过工程疗法(抗体或适配子)实施ERBB2的脱离上下文的激活。这里提出的研究也可能阐明目前使用的疗法(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。我们还希望更好地理解为什么以前积极执行ERBB2介导的细胞凋亡的尝试没有更成功。

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.