Heterodimerization of CXCR4 and CB2 Inhibits Prostate Cancer Cell Movement

描述(申请人提供)：当被基质细胞衍生因子1α(SDF1α)激活时，G蛋白偶联趋化因子受体CXCR4产生信号，最终导致原发肿瘤细胞在远端器官中的转移扩散和存活。事实上，CXCR4蛋白在前列腺组织中的高表达与转移和总体预后不良有关。CXCR4可以与不相关的受体形成同源二聚体或异源二聚体，如增量阿片受体(DOR)。同时用适当的受体激动剂处理异源二聚化的单个受体，如CXCR4/DOR异二聚体，会导致一个不能发出信号的二聚体，尽管每个受体都可以结合它各自的配体。因此，异源二聚可以通过这种受体复合体导致信号减少，代表功能脱敏。在癌症治疗的背景下，CXCR4信号和后续功能可以通过与其他受体的异源二聚体结合而被脱敏而沉默，从而抑制CXCR4产生的信号，否则会导致转移。因此，通过异源二聚化来拮抗CXCR4的功能可能是预防和治疗转移性前列腺癌的一种合理的方法，也可能是一种有效的替代目前使用的中和抗体或CXCR4拮抗剂的治疗方法，这两种方法都会产生不良的后果。研究报道，CXCR4和大麻素受体2(CB2R)激动剂同时治疗可减少CB2R诱导的镇痛，这表明CB2R具有功能性脱敏作用，尽管没有分析二聚化。在这种情况下，CXCR4/CB2R的异源二聚体可能会减弱CXCR4或CB2R单独触发的反应，而不会出现使用受体抗体或拮抗剂所产生的副作用，特别是在两种受体都在同一肿瘤细胞上表达的情况下。我们将证明，受体异二聚化可以形成分子基础，以减少CXCR4介导的信号，从而减少转移。 假设：CXCR4的功能可通过CXCR4与CB2R的同时配体依赖的异二聚化而被取消，从而导致CXCR4信号的减少、细胞转移和整个肿瘤的发展。 具体目标：(1)：确定CXCR4和CB2R在体外是否存在物理联系；(2)：确定CXCR4与CB2R异源二聚导致哪些信号通路被调制；(3)：确定CXCR4与CB2R异源二聚是否拮抗CXCR4介导的完整动物的转移。 研究设计：我们的策略将包括：(I)通过FRET分析确认CXCR4和CB2R二聚体；(Ii)确定CB2R是否需要作为二聚体伙伴通过CB2R的shRNA来消除CXCR4的信号和已知功能；以及(Iii)检测CXCR4和CB2R的异源二聚是否会抑制肿瘤生长和前列腺癌细胞在体内的骨转移。

DESCRIPTION (provided by applicant): Upon activation by stromal cell derived factor 1 alpha (SDF1 alpha), the G-protein-coupled chemokine receptor, CXCR4, generates signals that eventually lead to the metastatic spread and survival of primary tumor cells in distal organs. Indeed, elevated expression of CXCR4 protein in prostate tissues correlates with metastasis and overall prediction of poor survival. CXCR4 can form homodimers or can heterodimerize with unrelated receptors, such as the delta opioid receptor (DOR). Simultaneous treatment with appropriate agonists for the individual receptors that are heterodimerizing, such as the CXCR4/DOR heterodimer, results in a dimer that is unable to signal, although each receptor can bind its respective ligand. Therefore, heterodimerization can cause decreased signaling through such receptor complexes, representing functional desensitization. In the context of cancer treatment, CXCR4 signaling and subsequent functions can be silenced by desensitization through heterodimeric association with other receptors, thereby inhibiting CXCR4-generated signals that would otherwise lead to metastasis. Thus, antagonizing the function of CXCR4 through heterodimerization could be a rational approach to the prevention and management of metastatic prostate cancer, and could be an effective alternative to current therapeutics involving neutralizing antibodies or antagonists against CXCR4, both of which have undesirable consequences. Studies reported that simultaneous treatment with CXCR4 and cannabinoid receptor 2 (CB2R) agonists caused a reduction in CB2R-induced analgesia, suggesting a functional desensitization of CB2R, although dimerization was not analyzed. Given this scenario, a heterodimer of CXCR4/CB2R could potentially attenuate responses triggered individually by CXCR4 or CB2R, without the side effects experienced with the use of receptor antibodies or antagonists, especially in situations where both receptors are expressed on the same tumor cells. We will demonstrate that receptor heterodimerization could form the molecular basis for decreasing CXCR4-mediated signaling, and therefore, metastasis. Hypothesis: CXCR4 function can be abrogated by simultaneous ligand-dependent heterodimerization of CXCR4 with CB2R, resulting in decreased CXCR4 signaling, cell metastasis and overall tumor development. Specific aims: (1): To determine whether CXCR4 and CB2R physically associate in vitro; (2): To determine which signaling pathways are modulated as a result of heterodimerization of CXCR4 with CB2R; and (3): To determine whether heterodimerization of CXCR4 with CB2R antagonizes CXCR4-mediated metastasis in intact animals. Research design: Our strategy will include: (i) confirming that CXCR4 and CB2R dimerize by FRET analysis; (ii) determining whether CB2R is required as a dimer partner to abrogate signaling and known functions of CXCR4 by shRNA for CB2R; and (iii) examining whether heterodimerization of CXCR4 with CB2R will inhibit tumor growth and metastasis of prostate cancer cells to the bone in vivo.