中国科学院昆明动物研究所机构知识库

乳腺癌是女性中发病率最高的一种癌症。尽管预防、诊断以及治疗上都有了极大的提高，但在女性肿瘤死亡率中仍排在第二位。目前临床上，乳腺癌根据雌激素受体 (ER)、孕激素 (PR) 和人表皮生长因子受体2 (HER2) 表达情况，分成：Luminal A，Luminal B，HER2和三阴性乳腺癌（Triple Negative Breast Cancer, TNBC）等四种亚型，其中三阴性乳腺癌占15%左右。三阴性乳腺癌由于其具有很强的侵袭性和缺少可以靶向的药物，且具有非常高的异质性，是所有乳腺癌类型中预后最差的一种。肿瘤干细胞也被称作肿瘤起始细胞，是肿瘤中一群具有自我更新能力并能产生异质性肿瘤细胞的一类细胞群。目前临床上常用的治疗方式对肿瘤干细胞不仅不能起到很好的杀伤效果，反而富集肿瘤干细胞，进而造成耐药和复发。三阴性乳腺癌中富含肿瘤干细胞, 因此对三阴性乳腺癌干细胞进行深入研究，发现有效的靶向药物，才能很好地控制三阴性乳腺癌的发生发展。本项研究，我们利用三阴性乳腺癌HCC1806和HCC1937细胞系，以及三阴性乳腺癌患者来源的肿瘤异种移植模型（PDX），发现二甲双胍可以降低乳腺癌干细胞标志物ALDH阳性细胞群以及抑制微球体形成能力。继续通过肿瘤干细胞检测金标准，有限稀释成瘤实验发现，二甲双胍可以显著降低三阴性乳腺癌肿瘤形成和增殖能力。说明二甲双胍可以抑制三阴性乳腺癌干细胞。我们在三阴性乳腺癌细胞中发现，二甲双胍可以显著下调KLF5表达，而过表达KLF5，可以部分挽救二甲双胍对三阴性乳腺癌干细胞的降低。说明二甲双胍部分通过下调KLF5来抑制三阴性乳腺癌干细胞。同时发现二甲双胍主要通过降低KLF5蛋白稳定性来下调KLF5表达。根据目前报道，二甲双胍主要通过AKT、mTOR、AMPK和Stat3等信号通路来发挥其抑癌作用。而我们的研究发现，二甲双胍在三阴性乳腺癌中对KLF5的调控不依赖于AKT、mTOR、AMPK和Stat3等信号通路。进一步实验发现，在三阴性乳腺癌中二甲双胍可以抑制PKA活性，PKA抑制剂下调KLF5、激活剂则可以上调KLF5、敲低PKA能降低KLF5蛋白的稳定性，说明二甲双胍可能是通过抑制PKA来下调KLF5表达。GSK3β可以直接磷酸化KLF5，造成KLF5被泛素-蛋白酶体系统降解。本研究发现，二甲双胍可以抑制PKA活性的同时会降低GSK3β的磷酸化而激活GSK3β，siRNA干扰GSK3β表达可以阻止二甲双胍对KLF5的下调。继续用二甲双胍处理细胞不同时间发现，PKA活性的抑制和GSK3β活性的激活先于KLF5发生磷酸化，随后KLF5发生降解。同时，PKA激活剂也可以抑制KLF5发生磷酸化，用siRNA干扰GSK3β的表达可以阻止PKA激活剂对KLF5的上调。因此我们证明了在三阴性乳腺癌中二甲双胍和PKA是通过GSK3β来调控KLF5。我们进一步在三阴性乳腺癌临床样本中，利用免疫组化染色的方法发现PKA高度活化（通过染色p-CREB）以及KLF5高表达，统计结果揭示PKA活化程度与KLF5表达水平呈显著正相关性。综合以上研究，我们可以得出结论：在三阴性乳腺癌中，二甲双胍通过抑制PKA活性，进而激活GSK3β，而活化的GSK3β直接磷酸化KLF5，磷酸化的KLF5进而被泛素-蛋白酶体系统降解，即二甲双胍通过PKA - GSK3β - KLF5这一新的信号通路抑制三阴性乳腺癌干细胞。进一步阐明了二甲双胍治疗癌症的机制，为临床对二甲双胍在治疗三阴性乳腺癌方面提供了理论依据，为三阴性乳腺癌靶向药物的研发提供了新的靶点。

Breast cancer is the most commonly diagnosed cancer and the second cause of cancer death in women, despite substantial progress in its prevention, diagnosis, and treatment. According to the expression level of ERa, PR, or HER2, the classification involves four subtypes: Luminal A, Luminal B, HER2, and Triple Negative Breast Cancer (TNBC). TNBC refers to breast cancer that does not express ERa, PR, or Her-2. TNBC accounts for around 15% of all breast cancer cases. TNBC is an aggressive and heterogeneous disease with poor prognosis and the shortest survival rate because of no targeted therapies.Cancer stem cells, also known as tumor initiating cells, are a small cell population with unique characteristic, such as self-renewal, and the ability to generate heterogenic lineage of cancer cells. The conventional cancer treatments fail to eliminate the cancer stem cells due to their high chemoresistance, and recurrence. TNBC is enriched of cancer stem cells. Therefore, it is urgent to develop more effective therapies to target cancer stem cells to cure breast cancer patients, especially for triple negative breast cancer patients.In this study, we found that metformin significantly decreases the percentage of cancer stem cells in TNBC. The TNBC cell line HCC1806 and HCC1937 as well as patient derived exnografts treated with metformin significantly decrease Aldehyde dehydrogenase (ALDH) positive cells and their mammoshpere formation ability. We further found that metformin inhibits TNBC cell growth and tumorigenesis.Metformin suppresses TNBC stem cells partially through the inhibiton of KLF5. Metformin downregulated the expression of KLF5 through reducing its protein stability. This phenotype was partially rescued by KLF5 overexpression. Metformin has been reported to function through regulating the AKT, mTOR, AMPK and Stat3 signaling pathways. In this study, we found that Metformin suppresses TNBC stem cells independent of these signaling pathways.Metformin downregulates KLF5 expression level through the inhibition of PKA. PKA activators and inhibitors increased and decreased the KLF5 expression levels respectively. We further found metformin and PKA regulate KLF5 protein stability through GSK3β. Metformin decreased the activity of PKA and increased the activity of GSK3β, which can directly phosphorylate KLF5 and target it for degradation by proteasome. Additionally, PKA activators decreased KLF5 phosphorylation level. The PKA activity, as measured by the p-CREB level, positively correlates with the KLF5 expression level in human TNBC samples.In summary, we demonstrated that metformin significantly suppresses TNBC stem cells and revealed that metformin acts through the PKA - GSK3β - KLF5 pathway. Additionally, our results indicate that PKA and KLF5 are potential therapeutic targets in TNBC and support the idea that metformin is a promising agent for the treatment of patients with TNBC. Our findings may provide a new therapeutic strategy for TNBC patients.