PI3K PROTAC协同拉帕替尼在PIK3CA基因突变乳腺癌HER2靶向治疗耐药的功能与机制研究

【作者】 张红艳；

【导师】 陈策实；

【作者基本信息】 昆明理工大学 ， 生物学， 2024， 博士

【摘要】 乳腺癌是一类异质性很强的癌种,具有不同的病理组织学类型、分子分型、遗传易感基因的突变或体细胞重编程基因突变等特征,这些不同的特征都可以影响乳腺癌患者对抗肿瘤治疗的应答,从而导致不同的治疗反应及疾病预后的迥异。另一方面,乳腺癌的发病率逐年高升,是女性发病率最高的恶性肿瘤,威胁女性的身心健康。尽管近年来关于乳腺癌的治疗有许多的新的突破和进展,然而研究显示乳腺癌患者对治疗产生抵是导致疾病进展、不良预后的关键性因素。HER2阳性乳腺癌主要是指通过IHC染色鉴定ERBB2/HER2 3+或FISH检测鉴定ERBB2/HER2扩增的一类乳腺癌分子亚型,在乳腺癌人群中大约占比15%。HER2阳性乳腺癌被广泛认为是最具有侵袭性临床特征的乳腺癌类型之一,其疾病侵袭转移率高、生存预后差。这与ERBB2/HER2基因扩增导致促生长信号的转化具有密切的相关性。因而靶向ERBB2/HER2的治疗药物应运而生,显著改善了疾病的控制率和患者的生存率。尽管抗HER2治疗药物蓬勃发展,但抗HER2药物治疗天然及获得性耐药并不罕见,是HER2阳性乳腺癌患者疾病进展、转移和预后恶化的主要原因。临床上急需要新的策略来克服抗HER2治疗的耐药问题。目前的研究认为PI3K-AKT信号通路过度激活是诱导HER2靶向治疗耐药的重要机制。而PIK3CA基因突变可以诱导该信号通路过度活化。研究发现PIK3CA基因突变在乳腺癌群体中具有高达36%的发生频率,而在HER2阳性乳腺癌亚型中其突变频率更是高达～42%。临床前研究显示曲妥珠单抗或拉帕替尼联合PI3K-AKT-mTOR(PAM)信号通路抑制剂可以逆转抗HER2治疗耐药。蛋白降解靶向嵌合体(Proteolysis-targeting chimeras,PROTACs)具有选择性、特异性降解癌基因蛋白的功能,从而降低了传统分子抑制剂的毒副作用,是一种极具前景的开发抗癌药物的新模式。我们的合作团队清华大学药学院饶燏教授团队设计并合成了靶向PI3K的PROTAC分子,他们的研究报道该PI3KPROTAC分子在多株乳腺癌细胞系中具有良好的抑癌活性。而在本研究中,我们着重关注PI3KPROTAC分子在HER2靶向治疗耐药中的功能与机制的研究。通过在多种乳腺癌细胞系中进行抗癌疗效的初筛,我们发现PI3K-PROTAC分子对正常细胞无明显杀伤效果,而对携有PIK3CA基因突变的乳腺癌细胞系具有更敏感的抑癌活性。随后我们选择了两株HER2耐药细胞株作为研究模型,进行深入的研究。首先我们发现PI3K PROTAC分子在这两株细胞系中具有较低的IC50(<100 nM)、DC50(<6 nM)及T 1/2(<12h)。进一步的研究发现,低浓度的PI3K PROTAC分子可协同拉帕替尼,增加耐药细胞株G1期细胞阻滞比例,恢复拉帕替尼等HER2靶向治疗药物对HER2阳性耐药细胞株的抑癌活性,同时还可增强拉帕替尼对HER2敏感细胞株的抑癌活性,以及浓度依赖地抑制耐药细胞克隆形成的数量。与此同时,我们还与临床上选择性PI3K-p110α激酶抑制剂A1pelisib进行疗效的对比,分别从细胞层面、动物层面以及人源乳腺癌类器官层面都进行平行比较。发现不论是体内还是体外,单用或是联用拉帕替尼,PI3KPROTAC分子的抗肿瘤活性都要显著优于Alpelisib。这提示PI3K PROTAC分子是靶向PAM信号通路可供选择的强有力的先导化合物。综上所述,本研究阐述了新型靶向p110α的PI3K PROTAC分子,通过选择性、高效地靶向p110α,使其通过蛋白酶体降解,抑制PAM信号通路的活化,从而可以逆转PIK3CA基因突变导致的HER2靶向治疗药物拉帕替尼耐药。PI3K PROTAC分子是一个有潜力的先导化合物,不论是单用还是联用,都可以有效克服临床上HER2靶向治疗耐药的难题。更多还原

【Abstract】 Breast cancer is a highly heterogeneous carcinoma.The influences of the response of breast cancer patients to anti-tumor therapy including different histopathological types,molecular types,genetic susceptibility gene mutations or somatic reprogramming gene mutations,eventually resulting in different therapeutic reactivity and disease prognosis.The increasing of the incidence of breast cancer year by year,ranking at the highest incidence malignant tumors of female,significantly threatening the physical and mental health of women seriously.Although plenty of new breakthroughs and advances in the treatment of breast cancer in recent years,the therapeutic resistance of breast cancer patients is a crucial factor leading to disease progression and poor prognosis.HER2-positive breast tumors are characterized by HER2 3+staining in immunohistochemistry(IHC)or HER2 amplification identified through fluorescence in situ hybridization(FISH).This subtype accounts for approximately 15%of all breast cancers.HER2positive breast cancer generally regarded as the most aggressive breast cancer types with high invasion and metastasis rate,patient usually face a worse survival prognosis.This phenotype associated with the transformation of growth promoting signal caused by ERBB2/HER2 gene amplification.However,the advent of anti-HER2 targeted therapy has significantly improved the outcome for individuals with this subtype,including disease control rate and the survival rate of patients.Despite the booming development of anti-HER2 therapies,innate and acquired resistance to anti-HER2 therapies is not uncommon and is a major cause of disease progression,metastasis,leading worse prognosis in patients with HER2+breast cancer.New strategies are urgently needed to overcome resistance to anti-HER2 therapy.Current studies suggest that hyper-activation of the PI3K-AKT signaling pathway is an important mechanism leading to drug resistance in HER2targeted therapy.PIK3CA gene mutation can induce hyper-activation of this signaling pathway.The studies found that PIK3CA gene mutations have a frequency of up to 36%in breast cancer populations,and mutations in HER2+breast cancer subtypes are as high as 42%.Preclinical studies have shown that trastuzumab or lapatinib combined with PI3K-AKT-mTOR(PAM)signaling pathway inhibitors can reverse resistance to HER2 therapy.Proteolysis-targeting chimeras(PROTACs)characterize to selectively and specifically degrade oncogene proteins,thereby reducing the toxic side effects of traditional molecular inhibitors,which is a promising new model for the development of anticancer drugs.Our partner Prof.Rao Yu,School of Pharmacy,Tsinghua University,designed and synthesized a PROTAC molecule targeting PI3K.Their research has reported that the PI3K PROTAC molecule exhibited high anti-cancer efficacy in multiple breast cancer cell lines.In this study,we focused on the function and mechanism of PI3K PROTAC molecule in HER2-targeted therapy resistance.Through preliminary screening of anti-cancer efficacy in a variety of breast cancer cell lines,we found that PI3K-PROTAC molecule has high tolerant on normal cells and is susceptible sensitive to breast cancer cell lines carrying PIK3CA gene mutation.Subsequently,we selected two HER2-resistant cell lines as research models and conducted further investigations.First,we found that the PI3K PROTAC molecule had a low IC50(<100 nM),DC50(<6 nM),and T 1/2(<12 h)in these two cell lines.Further studies found that PI3K PROTAC could cooperate with lapatinib at low concentrations,resulted increased the proportion of G1 phase cell arrest of drug-resistant cell lines,restored the proliferation inhibition efficacy of lapatinib on HER2+resistant cell lines,and inhibited the number of clonal formation of drug-resistant cell lines in a concentration-dependent manner.Meanwhile,we also compared the efficacy of PI3K PROTAC to Alpelisib,a selective PI3K-p110α kinase inhibitor in clinical,from the models of breast cancer cells,xenograft animals,and patient derived human breast cancer organoids.It was found that the efficacy of antitumor activity of PI3K PROTAC was significantly superior to Alpelisib either in vivo or in vitro,regardless of administration alone or combined with lapatinib.The superior antitumor activity of PI3K PROTAC has also been demonstrated in animal xenograft tumor models and patient-derived breast cancer organoids.In summary,this study demonstrates a novel PI3K PROTAC molecule targeting p110α,with selectively and efficiently degraded p110α through proteasome,thus efficiently inhibited the activation of PAM signaling pathway,reversing lapatinib resistance caused by PIK3CA gene mutation.PI3K PROTAC molecule is a promising pre-compound of anti-cancer drug,which can effectively overcome the problem of drug resistance in clinical HER2-targeted therapy,whether it is used alone or in combination.更多还原