Mechanisms and therapeutic targeting of colon cancer stem cell plasticity

摘要： 肿瘤起始细胞或癌症干细胞（CSC）与耐药性、转移和复发有关， 使得CSC成为有效治疗癌症的主要障碍。最近的研究明确地表明， 确定了成体干细胞标记物LGR 5（富含亮氨酸的重复序列，G蛋白偶联受体5） 在原发性结肠肿瘤中高度表达，只有LGR 5阳性的结肠CSC能够驱动肿瘤 生长和转移。然而，LGR 5在CSC中的功能和机制仍然相对未知。我们 产生的掺入毒素单甲基-奥瑞他汀E的LGR 5靶向抗体-药物缀合物（ADC） 并表明它们可以破坏结肠癌细胞和异种移植物，但肿瘤最终复发。 此外，LGR 5阳性结肠CSC的选择性基因消融未能完全根除肿瘤。 由于LGR 5阴性癌细胞能够在治疗后迅速再生LGR 5阳性细胞， 终止，表明高度的癌细胞可塑性。因此，要开发出完全 为了根除原发性结肠肿瘤和转移性疾病，关键是鉴定LGR 5介导的功能， 参与CSC可塑性的信号通路。我们报道了LGR 5结合R-spondin生长因子， 其功能是增强Wnt/β-连环蛋白信号传导，这是CSC中激活的一种重要途径。最近， 我们已经发现LGR 5与支架蛋白IQGAP 1（含有GT3活化的IQ基序）相互作用， 蛋白1）增加结肠癌细胞中的细胞-细胞粘附，并且LGR 5的敲低显著增强 耐药性和与存活信号相关的蛋白质的活化。LGR 5诱导表达的丧失 粘附G蛋白偶联受体GPR 56，在结肠肿瘤中高度上调。在目标1中，我们 确定LGR 5在控制CSCs的干细胞性和耐药性中的作用和机制。我们将 研究LGR 5敲除（KO）与消除LGR 5阳性CSC相比如何影响肿瘤生长， 使用患者来源的异种移植物（PDX）类器官模型的转移、耐药性和癌细胞信号传导 离体和体内。LGR 5-IQGAP 1相互作用在调节定义免疫缺陷的不同性质中的作用 还将审查CSC。在目标2中，我们将开发独特的双特异性ADC以靶向CSC可塑性。我们将 测试安全性、有效性并建立双靶向治疗同时破坏 LGR 5阳性CSC和LGR 5阴性癌细胞。在目标3中，我们将描述和验证GPR 56 通过使用GPR 56描述其在肿瘤生长和耐药性中的作用， KO结肠癌细胞系和PDX模型。本研究将揭示LGR 5在肿瘤细胞中的功能和作用机制， CSC和产生创新的治疗导致目标干细胞和可塑性的治疗， 根除结肠癌。

ABSTRACT: Tumor-initiating or cancer stem cells (CSCs) have been implicated in drug resistance, metastasis, and relapse, making CSCs a major impediment for the effective treatment of cancer. Recent studies have unequivocally established that the adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein-coupled receptor 5) is highly expressed in primary colon tumors and only LGR5-positive colon CSCs are capable of driving tumor growth and metastasis. However, the function and mechanism of LGR5 in CSCs is still relatively unknown. We generated LGR5-targeted antibody-drug conjugates (ADCs) incorporating the toxin monomethyl-auristatin E and showed they could destroy colon cancer cells and xenografts, yet tumors eventually recurred. Furthermore, selective genetic ablation of LGR5-positive colon CSCs failed to completely eradicate tumors since LGR5-negative cancer cells were able to rapidly regenerate LGR5-positive cells after treatment was terminated, indicating a high degree of cancer cell plasticity. Therefore, to develop therapies that completely eradicate primary colon tumors and metastatic disease, it is critical to identify LGR5-mediated functions and signaling pathways involved in CSC plasticity. We reported that LGR5 binds R-spondin growth factors and functions to potentiate Wnt/beta-catenin signaling, an essential pathway that is activated in CSCs. Recently, we have found that LGR5 interacts with the scaffold protein IQGAP1 (IQ Motif Containing GTPase Activating Protein 1) to increase cell-cell adhesion in colon cancer cells and knockdown of LGR5 significantly enhanced drug resistance and activation of proteins associated with survival signaling. Loss of LGR5 induced expression of adhesion G-protein-coupled receptor GPR56, which is highly upregulated in colon tumors. In Aim 1, we will determine the roles and mechanisms of LGR5 in the control of stemness and drug resistance of CSCs. We will investigate how LGR5 knockout (KO) compared to elimination of LGR5-positive CSCs affects tumor growth, metastasis, drug resistance, and cancer cell signaling using patient-derived xenograft (PDX) organoids models ex vivo and in vivo. The role of LGR5-IQGAP1 interaction in regulating the different properties that define CSCs will also be examined. In Aim 2, we will develop unique bispecific ADCs to target CSC plasticity. We will test safety, efficacy and establish proof-of-concept for dual-targeted therapeutics to simultaneously destroy both LGR5-positive CSCs and LGR5-negative cancer cells. In Aim 3, we will characterize and validate GPR56 as a novel target for colon cancer by delineating its roles in tumor growth and drug resistance using GPR56 KO colon cancer cell lines and PDX models. This study will uncover the function and mechanism of LGR5 in CSCs and generate innovative therapeutic leads to target stemness and plasticity for the treatment and eradication of colon cancer.