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FZR1 As a Novel Biomarker for Breast Cancer Neoadjuvant Chemotherapy

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FZR1 As a Novel Biomarker for Breast Cancer Neoadjuvant Chemotherapy Liu et al. Cell Death and Disease (2020) 11:804 https://doi.org/10.1038/s41419-020-03004-9 Cell Death & Disease ARTICLE Open Access FZR1 as a novel biomarker for breast cancer neoadjuvant chemotherapy prediction Shuo Liu1,HaobinWang2,JunLi1,JianhuiZhang3,JianWu2,YiLi4, Yongjun Piao1,LeitingPan5,RongXiang 1,6 and Shijing Yue 1 Abstract The concept of breast-conserving surgery is a remarkable achievement of breast cancer therapy. Neoadjuvant chemotherapy is being used increasingly to shrink the tumor prior to surgery. Neoadjuvant chemotherapy is reducing the tumor size to make the surgery with less damaging to surrounding tissue and downstage locally inoperable disease to operable. However, non-effective neoadjuvant chemotherapy could increase the risks of delaying surgery, develop unresectable disease and metastatic tumor spread. The biomarkers for predicting the neoadjuvant chemotherapy effect are scarce in breast cancer treatment. In this study, we identified that FZR1 can be a novel biomarker for breast cancer neoadjuvant chemotherapy according to clinical patient cohort evaluation and molecular mechanism investigation. Transcriptomic data analysis indicated that the expression of FZR1 is correlated with the effect of neoadjuvant chemotherapy. Mechanistically, we demonstrate that FZR1 is pivotal to the chemotherapy drugs induced apoptosis and cell cycle arrest. FZR1 is involved in the stability of p53 by impairing the phosphorylation at ser15 site. We demonstrate that the expression of FZR1 detected by quantification of IHC can be an effective predictor of neoadjuvant chemotherapy in animal experiment and clinical patient cohort. To obtain more benefit for breast cancer patient, we propose that the FZR1 IHC score using at the clinical to predict the effect of neoadjuvant chemotherapy. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction remarkable achievements of cancer therapy. Currently, To date, breast cancer is considered as a heterogeneous Neoadjuvant chemotherapy (NACT) contributes to the disease with distinct subtypes based on the gene expres- idea of breast-conserving cancer therapy that che- sion profiling. In the field of breast cancer treatment, a motherapy drugs are delivered before surgery to shrink comprehensive treatment strategy is developed that the tumor. NACT is reducing the tumor size prior to included surgery, chemotherapy, radiotherapy, endocrine surgery makes the surgeon with less damage to sur- therapy and molecular targeted therapy1,2. Surgical rounding tissue and enables the surgeon to better differ- treatment is still the most important approach for breast entiate the edge of the tumor from healthy tissue3. NACT cancer. The concept of breast-conserving and the devel- also reduces the inflammation of neighboring metastasis opment of breast-conserving surgery is one of the most tissue and allows more of the healthy tissue to remain. Overall, NACT improves the outcome of breast cancer treated with surgery4,5. It is suppose that the combination Correspondence: Rong Xiang ([email protected])or of NACT with traditional treatment will bring the best Shijing Yue ([email protected]) benefits to patients in the research field6. 1 School of Medicine, Nankai University, Tianjin, China NACT is widely used in breast cancer to downstage 2Department of Breast & Thyroid Surgery, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, The Second locally advanced (inoperable) disease and make it oper- Chengdu Hospital Affiliated to Chongqing Medical University, Chengdu, able, particularly for large tumors7,8. There is a number of Sichuan, China potential advantages of NACT, including reduction of Full list of author information is available at the end of the article These authors contributed equally: Shuo Liu, Haobin Wang tumor burden to facilitate surgical resection, insights into Edited by S. Tait © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Liu et al. Cell Death and Disease (2020) 11:804 Page 2 of 14 the chemotherapy drugs resistance and response, impor- Materials and methods tant information of prognostic, toxicity, and survival9,10. Cell lines and culture Studies of preclinical models suggest that NACT might Human breast cancer cells T-47D and MDA-MB-231 provide a survival benefit over adjuvant therapy in the were purchased from ATCC. T-47D cells were cultivated context of treatment with an immune checkpoint block- in RPMI-1640 medium supplemented with 10% fetal ade11,12. Although the listed potential benefits of NACT, bovine serum (FBS) and 1% penicillin/streptomycin. the risks of delaying surgery with poor treatment response MDA-MB-231 cells were maintained in Dulbecco’s could develop unresectable disease and increased chances modified Eagle’s medium (DMEM) supplemented with for metastatic tumor spread. In addition, the toxic effects 10% fetal bovine serum (FBS) and 1% penicillin/strepto- of NACT might aggravate morbidity or further delay mycin. HEK-293T cells were purchased from ATCC and surgical resection, and increasing surgical risk13,14. maintained in Dulbecco’s modified Eagle’s medium Thereby, it is greatly need to develop a specific biomarker (DMEM) supplemented with 10% FBS and 1% penicillin/ signature to define the ideal patient population for NACT. streptomycin. However, the molecular biomarkers are scarcity for the pathological response of NACT in breast cancer. Patient derived samples Recent studies demonstrate that the efficacy of NACT Breast cancer clinical samples were obtained from The differs significantly among breast cancer subtypes and the Third People’s Hospital of Chengdu and Sichuan Cancer NACT ability to increase the risk of cancer progression. Center. This study was approved by the institutional The increasing evidence concluded that NACT elevated ethics committees of The Third People’s Hospital of the local recurrence rate, despite reduced tumor size9,15. Chengdu. All patients signed an informed consent. An unfavorable effect of chemotherapy is associated with Patient’s information is reported in Table 1. the selection of chemoresistant clones and the conditions for cancer progression16. It is believed that NACT pro- Plasmids and transfections motes metastasis via the induction of cellular stress and The human FZR1 and CHK2 cDNA derived from the – remodeling tumor microenvironment17 20. According to breast cancer cell line T-47D fused with Flag, His or Myc- the current knowledge, we need to establish a consensus peptide at the C-terminal was cloned into the lentiviral and recommendations for the NACT in breast cancer. expression vector pLV-EF1α-MCS-IRES-Bsd/puro (Bio- Investigations to identify the biomarker or the methods settia, San Diego, CA, USA). The TP53 shRNAs were for the efficacy of NACT have been executed in breast cloned into the pLV-RNAi-Vector (Biosettia, San Diego, – cancer21 23. Although these biomarkers are considered to CA, USA). The FZR1 sgRNA was cloned into the lenti- be the predictor of pathologic complete responses to CRISPRv2 vector (Addgene). All constructs were verified NACT in breast cancer, there is a large limitation for the by DNA sequencing. The primers used for the generation clinical application. The existing biomarkers for NACT of gene overexpression, shRNAs for gene knockdown and have been reported including the lncRNA profile, the sgRNA for gene knockout cell lines are listed in Supple- change of axillary lymph nodes, and the recurrence scores mentary Table 1. with NACT. The lncRNA profiling has identified several lncRNAs associated with the pathological complete Transduction of lentivirus response, which is limited for clinical application. The Stable breast cancer cell lines expressing FZR1-Flag, change of axillary lymph nodes and the recurrence scores sgRNA or shRNA were established using a lentiviral- can be explored during or proceed the NACT that are not based delivery system. For the generation of lentiviral suitable for clinical application. particles, 4 × 106 HEK293T cells were seeded in a 10 cm In this study, we identified that FZR1 as a biomarker for culture dish and cultivated overnight. Next day cells were breast cancer NACT based on the transcriptomic data co-transfected with 9 µg of empty or FZR1-containing analysis and the molecular mechanism investigation. pLV-EF1α-MCS-IRES-Bsd/puro vector, or shRNA- FZR1 is involved in the regulation of the stability and containing pLV-RNAi-Vector, or sgRNA-containing len- transcriptional activity of tumor suppressor p53. The tiCRISPRv2 plasmids together with packaging and molecular function of FZR1 to modulate the cell apoptosis envelope vectors (4.5 µg pMDLg/pRRE, 1.8
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