DataSheet_1_Modulated TRPC1 Expression Predicts Sensitivity of Breast Cancer to Doxorubicin and Magnetic Field Therapy: Segue Towards a Precision Medicine Approach.docx

Chemotherapy is the mainstream treatment modality for invasive breast cancer. Unfortunately, chemotherapy-associated adverse events can result in early termination of treatment. Paradoxical effects of chemotherapy are also sometimes observed, whereby prolonged exposure to high doses of chemotherapeutic agents results in malignant states resistant to chemotherapy. In this study, potential synergism between doxorubicin (DOX) and pulsed electromagnetic field (PEMF) therapy was investigated in: 1) MCF-7 and MDA-MB-231 cells in vitro; 2) MCF-7 tumors implanted onto a chicken chorioallantoic membrane (CAM) and; 3) human patient-derived and MCF-7 and MDA-MB-231 breast cancer xenografts implanted into NOD-SCID gamma (NSG) mice. In vivo, synergism was observed in patient-derived and breast cancer cell line xenograft mouse models, wherein PEMF exposure and DOX administration individually reduced tumor size and increased apoptosis and could be augmented by combined treatments. In the CAM xenograft model, DOX and PEMF exposure also synergistically reduced tumor size as well as reduced Transient Receptor Potential Canonical 1 (TRPC1) channel expression. In vitro, PEMF exposure alone impaired the survival of MCF-7 and MDA-MB-231 cells, but not that of non-malignant MCF10A breast cells; the selective vulnerability of breast cancer cells to PEMF exposure was corroborated in human tumor biopsy samples. Stable overexpression of TRPC1 enhanced the vulnerability of MCF-7 cells to both DOX and PEMF exposure and promoted proliferation, whereas TRPC1 genetic silencing reduced sensitivity to both DOX and PEMF treatments and mitigated proliferation. Chronic exposure to DOX depressed TRPC1 expression, proliferation, and responses to both PEMF exposure and DOX in a manner that was reversible upon removal of DOX. TRPC1 channel overexpression and silencing positively correlated with markers of epithelial-mesenchymal transition (EMT), including SLUG, SNAIL, VIMENTIN, and E-CADHERIN, indicating increased and decreased EMT, respectively. Finally, PEMF exposure was shown to attenuate the invasiveness of MCF-7 cells in correlation with TRPC1 expression. We thus demonstrate that the expression levels of TRPC1 consistently predicted breast cancer sensitivity to DOX and PEMF interventions and positively correlated to EMT status, providing an initial rationale for the use of PEMF-based therapies as an adjuvant to DOX chemotherapy for the treatment of breast cancers characterized by elevated TRPC1 expression levels.

化疗是侵袭性乳腺癌的主流治疗手段。但遗憾的是，化疗相关不良反应可导致治疗提前终止。此外，化疗有时还会表现出矛盾效应：长期暴露于高剂量化疗药物后，反而会产生化疗耐药的恶性表型。本研究探讨了阿霉素（doxorubicin, DOX）与脉冲电磁场（pulsed electromagnetic field, PEMF）治疗的潜在协同效应，实验体系包括：1）体外培养的MCF-7与MDA-MB-231细胞；2）接种于鸡绒毛尿囊膜（chicken chorioallantoic membrane, CAM）的MCF-7肿瘤；3）植入NOD-SCID gamma (NSG)小鼠体内的人源乳腺癌异种移植物以及MCF-7、MDA-MB-231乳腺癌细胞系异种移植物。体内实验中，在人源乳腺癌异种移植瘤及细胞系异种移植瘤小鼠模型中均观察到协同效应：单独使用PEMF辐照或DOX给药均可缩小肿瘤体积、促进细胞凋亡，联合治疗可进一步增强这一效果。在CAM异种移植瘤模型中，DOX与PEMF辐照联合也可协同缩小肿瘤体积，并降低瞬时受体电位阳离子通道蛋白1（Transient Receptor Potential Canonical 1, TRPC1）的表达水平。体外实验中，单独PEMF辐照可降低MCF-7与MDA-MB-231细胞的存活率，但对非恶性乳腺细胞MCF10A无此影响；这一乳腺癌细胞对PEMF辐照的选择性易感性在人类肿瘤活检样本中得到了验证。TRPC1稳定过表达可增强MCF-7细胞对DOX与PEMF辐照的易感性，并促进细胞增殖；而TRPC1基因沉默则会降低细胞对两种治疗的敏感性，并抑制细胞增殖。长期DOX暴露会下调TRPC1的表达、抑制细胞增殖，并降低细胞对PEMF辐照与DOX的响应能力，这一效应在撤除DOX后可恢复。TRPC1通道的过表达与基因沉默分别与上皮间质转化（epithelial-mesenchymal transition, EMT）标志物（包括SLUG、SNAIL、波形蛋白（VIMENTIN）、E-钙粘蛋白（E-CADHERIN））的表达呈正相关，分别对应EMT进程的增强与减弱。最后，研究证实PEMF辐照可减弱MCF-7细胞的侵袭能力，且这一效应与TRPC1的表达水平相关。综上，本研究证实TRPC1的表达水平可稳定预测乳腺癌对DOX与PEMF干预的敏感性，且与EMT状态呈正相关，这为以TRPC1高表达为特征的乳腺癌采用PEMF疗法作为DOX化疗的辅助治疗手段提供了初步理论依据。