MUC1在HER2阳性乳腺癌发病中的作用及机制研究

doi:10.3969/j.issn.1674-8115.2021.07.001

摘要/Abstract

摘要：

目的·研究黏蛋白1（mucin 1，MUC1）在人类表皮生长因子受体2 （human epidermal growth factor receptor 2，HER2）阳性乳腺癌发病中的作用及其调控的分子机制。方法·使用病毒感染技术构建MT2/MUC1诱导表达细胞株和MT2/Vec及MT2/CD过表达细胞株；采用蛋白质印迹法（Western blotting）检测过表达MUC1和MUC1-CD后6-磷酸葡萄糖脱氢酶（glucose-6-phosphate 1-dehydrogenase，G6PD）的蛋白质表达水平；通过细胞计数试剂盒8（cell counting kit-8，CCK-8）法、平板克隆形成实验、细胞划痕实验、Transwell迁移实验和肿瘤细胞成球实验，研究MUC1对HER2阳性乳腺癌细胞MT2增殖、克隆形成、迁移和成球的影响；使用GP6D的抑制剂6-AN抑制酶活性，CCK-8检测乳腺癌细胞增殖；采用GEPIA和Kaplan-Meier Plotter数据库分析HER2、MUC1和G6PD在乳腺癌中的表达及其对乳腺癌患者生存期的影响。结果·过表达MUC1或MUC1-CD促进HER2阳性乳腺癌细胞MT2的增殖、克隆形成、迁移和成球，并且提高G6PD的蛋白质表达水平。抑制G6PD活性显著降低MUC1和MUC1-CD诱导的细胞增殖。G6PD蛋白在乳腺癌组织中表达上调且与乳腺癌患者生存期缩短相关。结论·MUC1可能通过调控G6PD的表达促进HER2阳性乳腺癌的进程，提示抑制磷酸戊糖途径可能成为HER2阳性乳腺癌治疗的靶点。

关键词: 乳腺癌, 黏蛋白1, 磷酸戊糖途径, 6-磷酸葡萄糖脱氢酶

Abstract:

Objective·To study the role and mechanism of mucin 1 (MUC1) in the pathogenesis of HER2-positive breast cancer.

Methods·Virus infection technology was employed to construct MT2/MUC1 and MT2/Vec/MUC1-CD overexpression cell lines; Western blotting was used to detect the expression level of relative proteins; the ability of cell proliferation, migration and sphere formation were detected by using cell counting kit-8 (CCK-8), colony formation, wound healing, transwell and sphere formation experiments respectively; inhibitor 6-AN was used to inhibit glucose-6-phosphate 1-dehydrogenase (G6PD) activity, and the proliferation of breast cancer cells was detected by CCK-8. GEPIA and Kaplan-Meier Plotter database were analyzed to figure out the relationship of HER2, MUC1 and G6PD expression with the survival of breast cancer patients.

Results·Overexpression of MUC1 or MUC1-CD promoted the proliferation, clone formation, migration and sphere formation of MT2 HER2-positive breast cancer cells, as well as elevating the protein level of G6PD. Inhibition of G6PD activity significantly reduced cell proliferation induced by MUC1 and MUC1-CD. High level of G6PD protein in breast cancer tissues is associated with significantly lower survival of breast cancer patients.

Conclusion·MUC1 may promote the progression of HER2-positive breast cancer by up-regulating the expression of G6PD, suggesting that inhibition of the pentose phosphate pathway may be a target for the treatment of HER2-positive breast cancer.

Key words: breast cancer, mucin 1 (MUC1), pentose phosphate pathway (PPP), glucose-6-phosphate 1-dehydrogenase (G6PD)

中图分类号:

R737.9

王成志, 邓华云, 庞智, 黄雷. MUC1在HER2阳性乳腺癌发病中的作用及机制研究[J]. 上海交通大学学报(医学版), 2021, 41(7): 839-848.

Cheng-zhi WANG, Hua-yun DENG, Zhi PANG, Lei HUANG. Mechanism of MUC1 in the pathogenesis of HER2-positive breast cancer[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 839-848.

图/表 6

图1 Western blotting检测MUC1的表达Note： A. The MT2/MUC1 cells were treated with DOX (1 μg·mL-1) for 48 h, and then collected for Western blotting to detect MUC1 expression. B. The expression of MUC1 was detected by Western blotting in MT2/CD cells.

Fig 1 Expression of MUC1 was detected by Western blotting

图2 MUC1促进乳腺癌细胞增殖和克隆形成能力Note： A/B. 5 000 cells were incubated per well, and the proliferation ability was detected by CCK-8 for MT2/MUC1 cells (A) and MT2/CD cells (B). C/D. 1 000 cells were incubated per well and cultured for 10 d, and colony formation experiment was performed with MT2/MUC1 cells (C) and MT2/CD cells (D).

Fig 2 MUC1 promotes breast cancer cell proliferation and colony formation ability

图3 MUC1促进乳腺癌细胞迁移Note： A/B. Wound healing experiment was performed to detect the migration ability of MT2/MUC1 cells (A) and MT2/CD cells (B). C/D. Transwell was used to detect the migration of MT2/MUC1 cells (C) MT2/CD cells (D).

Fig 3 MUC1 promotes breast cancer cell migration

图4 MUC1增加乳腺癌细胞的成球能力Note： A. Sphere formation of MT2/MUC1 cells. B. Sphere formation of MT2/MUC1-CD cells.

Fig 4 MUC1 increases the sphere formation of breast cancer cells

图5 G6PD参与MUC1诱导的乳腺癌细胞增殖作用Note： A. The correlation between HER2 (ERBB2) and MUC1 in breast cancer was analyzed through GEPIA database. B. The correlation between HER2 (ERBB2) and G6PD in breast cancer was analyzed by searching GEPIA database. C. The expression of G6PD was detected in MT2/MUC1 cells with DOX (1 μg·mL-1) induction for 48 h. D. The expression of G6PD was detected in MT2/CD cells. E/F. 5 000 cells were incubated per well and treated with or without 6-AN (50 μmol), and the cell proliferation was detected by CCK-8 in MT2/MUC1 cells (E) and MT2/CD cells (F).

Fig 5 G6PD is involved in the proliferation of breast cancer cells induced by MUC1

图6 利用数据库分析G6PD表达与生存期的关系Note： A/B. HER2 (A) and MUC1 (B) expression levels were analyzed in GEPIA database. C. G6PD expression level were analyzed in TCGA database. D. 5-year OS of breast cancer was analyzed in Kaplan-Meier plotter with high or low level of G6PD expression. E. RFS was analyzed in Kaplan-Meier plotter with high or low level of G6PD expression. ①P<0.05. TPM—transcript per million.

Fig 6 Database analysis of the expression of G6PD and survival

参考文献 27

1	Chen W, Sun K, Zheng R, et al. Cancer incidence and mortality in China, 2014[J]. Chin J Cancer Res, 2018, 30(1): 1-12.
2	Harbeck N, Gnant M. Breast cancer[J]. Lancet, 2017, 389(10074): 1134-1150.
3	Mitri Z, Constantine T, O′Regan R. The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy[J]. Chemother Res Pract, 2012, 2012: 743193.
4	Pernas S, Tolaney SM. HER2-positive breast cancer: new therapeutic frontiers and overcoming resistance[J]. Ther Adv Med Oncol, 2019, 11: 1758835919833519.
5	Loibl S, Gianni L. HER2-positive breast cancer[J]. Lancet, 2017, 389(10087): 2415-2429.
6	von Minckwitz G, Procter M, de Azambuja E, et al. Adjuvant pertuzumab and trastuzumab in early HER2-positive breast cancer[J]. N Engl J Med, 2017, 377(2): 122-131.
7	Nemeth BT, Varga ZV, Wu WJ, et al. Trastuzumab cardiotoxicity: from clinical trials to experimental studies[J]. Br J Pharmacol, 2017, 174(21): 3727-3748.
8	Nishimura R, Toh U, Tanaka M, et al. Role of HER2-related biomarkers (HER2, p95HER2, HER3, PTEN, and PIK3CA) in the efficacy of lapatinib plus capecitabine in HER2-positive advanced breast cancer refractory to trastuzumab[J]. Oncology, 2017, 93(1): 51-61.
9	Vu T, Claret FX. Trastuzumab: updated mechanisms of action and resistance in breast cancer[J]. Front Oncol, 2012, 2: 62.
10	McAuley JL, Linden SK, Png CW, et al. MUC1 cell surface mucin is a critical element of the mucosal barrier to infection[J]. J Clin Invest, 2007, 117(8): 2313-2324.
11	Li X, Xu Y, Zhang L. Serum CA153 as biomarker for cancer and noncancer diseases[J]. Prog Mol Biol Transl Sci, 2019, 162: 265-276.
12	Jiang XT, Tao HQ, Zou SC. Detection of serum tumor markers in the diagnosis and treatment of patients with pancreatic cancer[J]. Hepatobiliary Pancreat Dis Int, 2004, 3(3): 464-468.
13	Nath S, Mukherjee P. MUC1: a multifaceted oncoprotein with a key role in cancer progression[J]. Trends Mol Med, 2014, 20(6): 332-342.
14	Ahmad R, Raina D, Joshi MD, et al. MUC1-C oncoprotein functions as a direct activator of the nuclear factor-κB p65 transcription factor[J]. Cancer Res, 2009, 69(17): 7013-7021.
15	Ahmad R, Rajabi H, Kosugi M, et al. MUC1-C oncoprotein promotes STAT3 activation in an autoinductive regulatory loop[J]. Sci Signal, 2011, 4(160): ra9.
16	Khodarev N, Ahmad R, Rajabi H, et al. Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor prognosis human breast cancer[J]. Oncogene, 2010, 29(6): 920-929.
17	Raina D, Uchida Y, Kharbanda A, et al. Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells[J]. Oncogene, 2014, 33(26): 3422-3431.
18	Kharbanda A, Rajabi H, Jin C, et al. Oncogenic MUC1-C promotes tamoxifen resistance in human breast cancer[J]. Mol Cancer Res, 2013, 11(7): 714-723.
19	Kufe DW. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches[J]. Oncogene, 2013, 32(9): 1073-1081.
20	Ren J, Raina D, Chen W, et al. MUC1 oncoprotein functions in activation of fibroblast growth factor receptor signaling[J]. Mol Cancer Res, 2006, 4(11): 873-883.
21	Park J, Rho HK, Kim KH, et al. Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity[J]. Mol Cell Biol, 2005, 25(12): 5146-5157.
22	Zhang HS, Zhang ZG, Du GY, et al. Nrf2 promotes breast cancer cell migration via up-regulation of G6PD/HIF-1α/Notch1 axis[J]. J Cell Mol Med, 2019, 23(5): 3451-3463.
23	Pan S, World CJ, Kovacs CJ, et al. Glucose 6-phosphate dehydrogenase is regulated through c-Src-mediated tyrosine phosphorylation in endothelial cells[J]. Arterioscler Thromb Vasc Biol, 2009, 29(6): 895-901.
24	Au SW, Gover S, Lam VM, et al. Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP⁺ molecule and provides insights into enzyme deficiency[J]. Structure, 2000, 8(3): 293-303.
25	Ju HQ, Lu YX, Wu QN, et al. Disrupting G6PD-mediated redox homeostasis enhances chemosensitivity in colorectal cancer[J]. Oncogene, 2017, 36(45): 6282-6292.
26	Gunda V, Souchek J, Abrego J, et al. MUC1-Mediated metabolic alterations regulate response to radiotherapy in pancreatic cancer[J]. Clin Cancer Res, 2017, 23(19): 5881-5891.
27	Jing X, Liang H, Hao C, et al. Overexpression of MUC1 predicts poor prognosis in patients with breast cancer[J]. Oncol Rep, 2019, 41(2): 801-810.

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