PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer

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Enhanced PEAK1 expression facilitates tumor cell survival, invasion, metastasis and chemoresistance. However, the role of PEAK1 in breast cancer is not clear. Here, we investigated the PEAK1 expression in breast cancer and analyzed its relation with clinicopathological status and chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated the role of PEAK1 on breast cancer cells in vitro and in vivo . Methods Immunohistochemistry (IHC) was performed in 112 surgical resected breast cancer tissues. The associations between clinicopathological status, multi-drug resistance and PEAK1 expression were determined. Effect of PEAK1 overexpression or down-expression on proliferation, colony formation, invasion, migration, metastasis and Doxorubicin sensitivity in the MCF-7 cells in vitro and in vivo was detected. Results PEAK1 was overexpressed in breast cancer tissues and NAC -resistant breast cancer tissues. High PEAK1 expression was related with tumor size, high tumor grade, T stage, LN metastasis, recurrence, Ki-67 expression, Her-2 expression and multi-drug resistance. Targeting PEAK1 inhibited cell growth, invasion, metastasis and reversed chemoresistance to Doxorubicin in breast cancer cells in vitro and in vivo. Conclusion High PEAK1 expression was associated with invasion, metastasis and chemoresistance of breast cancers. Furthermore, targeting PEAK1 could inhibit cell growth and metastasis, and reverse chemoresistance in breast cancer cells, which provides an effective treatment strategies for breast cancer. Clinical Pharmacology Surgery General Surgery Breast cancer multi-drug resistance metastasis Pseudopodium-enriched atypical kinase 1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Breast cancer metastasis results in poor prognosis and increased mortality, the mechanisms of which are yet to be fully resolved. Tumour resection combined with radiotherapy, endocrine-therapy, and/or chemotherapy is the main way to treat breast cancer, but the development of chemoresistance limits the effectiveness of chemotherapy [ 1 ]. Therefore, identifying the molecular mechanisms contributing to breast cancer progression and chemoresistance would produce new biomarkers for the precise prediction of patient prognosis and for molecular targeted-therapy. Overexpression of PEAK1 (pseudopodium enriched atypical kinase 1, Sgk269), which was discovered in the pseudopodia of migrating cells [ 2 ], is also found in colorectal cancer [ 3 ] and pancreatic cancer [ 4 ]. PEAK1 expression is associated with metastasis and proliferation in many cancer cells, such as colorectal cancer [ 3 , 5 ], lung cancer [ 6 ] and pancreatic cancer [ 4 ]. Enforced PEAK1 expression can cause cell cycle deregulation and resistance to chemotherapy in pancreatic cancer cells [ 4 ]. Altering PEAK1 expression can interfere tumor formation and metastasis in pancreatic cancer cells in vivo, indicating that PEAK1 plays an important role in pancreatic cancer growth and metastasis. In breast cancer cells in vivo, enforced PEAK1 expression could induce new blood vessel formation by upregulation of vascular endothelial growth factor receptor-2, which facilitates cell movement and growth [ 7 ]. It has recently found that PEAK1 overexpression was significantly associated with advanced clinical stage and poor prognosis in colon cancer [ 3 ] and pancreatic cancer [ 4 ]. In breast cancer, PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse [ 8 ]. Croucher et al. [ 9 ] reported that PEAK1 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. However, the relation of PEAK1 expression and clinicopathological status and the relation of PEAK1 expression with chemosensitvity in breast cancer is currently unknown. In the present study, we examined the PEAK1 protein expression in human breast cancer tissues and explored the relationship between PEAK1 expression and clinical characteristics. Furthermore, we investigated the effect of PEAK1 on breast cancer cell growth, invasion, migration, metastasis and Doxorubicin sensitivity in vitro and in vivo . Materials And Methods Cell culture The human breast cancer MCF-7 cells were purchased from Institute of Cell Research, Chinese Academy of Sciences (Beijing, China). Doxorubicin (DOX)-resistant MCF-7 DOX cells were established by induction with gradient concentrations (0.1-2 μg/mL) of ADR in vitro . Cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco), 100 units/ml of penicillin, 100 μg/ml, streptomycin in a 5% CO 2 at 37°C. MCF-7 DOX cells were cultured at a final concentration of 2.0 μg/mL. Patient samples Breast cancer samples were obtained from Breast disease Center, the affiliated Hospital of Qingdao University. In all, 112 surgical resected tumors from Feb. 2012 to 2018. Pathological diagnosis was verified by two pathologists independently. All human samples were collected with informed consents from the donors according to the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The study was performed after approval by the institutional review board (IRB) of the affiliated Hospital of Qingdao University. Written informed consent was obtained from each individual or patient. Immunohistochemistry of PEAK1 Formalin-fixed paraffin-embedded tissue sections from excised specimens were processed according to standard procedures. Specific primary antibodies against PEAK1 (1:50) was purchased from Sigma-Aldrich (Shanghai, China). IHC staining for PEAK1 was performed as the manufacture’s instruction . The expression of PEAK1 was positive when 10% of tumor cells showed PEAK1 immunopositivity, and negative when less than 10% of tumor cells showed PEAK1 immunopositivity. PEAK1 shRNA vector construction and transfection The short-hairpin RNA direct against human PEAK1 gene (PEAK1 shRNA) was synthesized and constructed into the pcDNA3.1 expression vector (Shanghai, China) as the manufacture’s instruction. The constructed vectors (PEAK1 shRNA or NC shRNA) were transfected into the MCF-7 cells using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) as the manufacturer's instruction. The PEAK1 shRNA or NC shRNA transfected MCF-7 cells were selected by puromycin (10 mg/ml) for 5 days. The puromycin-resistant colonies were then picked and expanded. The relative protein was detected by Western blot assay. Plasmid constructs and transfection The full-length human PEAK1 adenovirus was constructed as to the manufacturer’s instructions using the AdEasy Adenoviral Vector System. Viral particles were produced by GenScript Biotechnology, China. The virus particles containing PEAK1 or control vector were used to infect MCF-7 cells. Transfected cells were selected with G418 (600 μg/ml, Gibco) for 10-12 days. The expression of PEAK1 in stable PEAK1 transfected colonies was detected by western blot analysis. In vitro doxorubicin sensitivity by MTT assay MCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in a 96-well plate in triplicate for 24 h, and the cells (300 cells / well) were then exposed to a concentration of 2.0 μg/mL Doxorubicin for 72 h. Subsequently, 20 μL MTT (Sigma-Aldrich) was added to each well and then incubated for 4 hours at 37°C and 5% CO2 humidified atmosphere. The optical density (OD) at 450 nm was measured and considered an indirect index of relative cell viability. In vitro doxorubicin sensitivity by colony formation assay MCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in triplicate at 1000 cells per well in 6-well plates with or without 2.0 μg/mL Doxorubicin and cultured for 12 days, then methanol-fixed and Giemsa-stained (GS, Sigma-Aldrich), which was followed by colony counting. Matrigel invasion assay. Cell migration and invasion was determined using a Transwell Chamber assay (BD Biosciences) according to the manufacturer's instructions. MCF-7 cells (1 × 10 5 cells) after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 24 h were added to the upper compartment (in triplicate), and DMEM plus 10% FCS was added to the lower compartment. Bestatin (Sigma) was added to both compartments. The cells were incubated for 24 h, and the total number of invaded cells was calculated as the manufacture’s instruction. Western Blotting Cells and tissues were lysed and protein concentration was measured with BCA protein assay reagents (Pierce). The proteins were then resolved on SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore) and probed with the primary antibodies: PEAK1 and a-Tubulin. Band densitometry analysis was performed using ImageJ software (NCI). In vivo metastasis assay Female BALB/c nude mice (5-6 weeks of age, 16-18 g) were obtained from National Rodent Shanghai Experimental Branch Center, Chinese Academy of Sciences (Shanghai, China). All experimental procedures involving animals were conducted in accordance with the institutional guidelines by the Affiliated hospital of Qingdao University. The stable Lv-PEAK1 or PEAK1 shRNA or its control transfected MCF-7 cells were cultured to log phase. 2 × 10 6 cells (n = 6 per group) were injected into mice via tail veins. After 5 weeks, the whole lung tissues were removaled and the numbers of visible nodules on the lung surface were numbered. H&E staining was used to evaluate the tumor metastasis. In vivo growth and doxorubicin sensitivity assay A total of stable PEAK1 shRNA transfected MCF-7 DOX or stable Lv-PEAK1 transfected MCF-7 cells (5 × 10 6 ) were injected s.c. to the left front flank of mice on day 0. From day 3 to day 9, DOX was administered to all groups of mice by i.v. injection (0.1 mL, 10 mg/kg) for a total of 4 times at 2 day intervals. Tumor dimensions were measured in 2 dimensions with microcalipers every other day and tumor volume was calculated by the following formula: tumor volume = (length × width 2 )/2. Non-retrospective ethical approval obtained for the animal experiments conducted in the study. Statistical analysis Data were shown as mean ± SEM; Data were analyzed using Student t test and pearson χ 2 test. Value of p <0.05 were considered significant. Results PEAK1 is overexpressed in breast cancer tissues Using immunohistochemical methods, we detected the expression of PEAK1 in 112 cases of breast cancer tissues and 34 cases of corresponding adjacent tissues (Fig. 1). PEAK1 expression is significantly upregulated in breast cancer tissues 61.6% (69/112) in comparison with adjacent normal tissues 26.4% (9/34) (P=0.033). PEAK1 overexpression is associated with high tumor size, tumor grade, T stage, advanced nodal status, regional recurrence, HER2, Ki-67 and chemotherapy status (Table 1). PEAK1 promotes cell growth ,invasion and migration in vitro The Lv-PEAK1 or empty vector was transfected into MCF-7 cells. PEAK1 expression was significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells (Fig. 2A). Cell growth was significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells by MTT assay (Fig. 2B). To confirm the MTT data, we carried out in vitro colony formation assays. Representative pictures and their quantification confirm that PEAK1 overexpression significantly promoted MCF-7 cells growth (Fig. 2C). The cell migration and invasion of transfected cells in vitro was observed by transwell assays. As shown in Fig. 2D, the invasive and migrative cells were significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells. Targeting PEAK1 inhibits cell growth ,invasion and migration in vitro The Lv-PEAK1 shRNA or empty vector was transfected into MCF-7 cells. PEAK1 expression was significantly decreased in MCF-7/ PEAK1 shRNA cells compared to the MCF-7/ control shRNA cells (Fig.2A). Cell growth was significantly decreased with PEAK1 depletion in the MCF-7/ PEAK1 shRNA cells by MTT assay (Fig. 3A) and colony formation assays (Fig. 3B). As shown in Fig. 3C, the invasive and migrative cells were significantly decreased in MCF-7/ PEAK1 shRNA cells compared to the MCF-7/control shRNA cells. PEAK1 regulated doxorubicin sensitivity in MCF-7 cells The Lv-PEAK1 or empty vector was transfected into MCF-7 cells, then treated with 2.0 μg/mL doxorubicin for 72 h. The results confirmed that PEAK1 overexpression reduced doxorubicin-induced cytotoxicity ( Fig. 4A-4B). Similarly, the MCF-7 DOX cells were transfected with PEAK1 shRNA, then treated with 2.0 μg/mL doxorubicin for 72 h. The results confirmed that PEAK1 depletionincreased doxorubicin-induced cytotoxicity ( Fig. 4A-4B). These data suggest that targeting PEAK1 reverses doxorubicin resistance in doxorubicin-resistant breast cancer cells. PEAK1 depelation increases the sensitivity of MCF-7 cells to doxorubicin in vivo To determine whether the effect of PEAK1 on chemosensitivity in vitro also extended to tumors growing in vivo, we injected female BALB/c mice with 5 × 10 5 MCF-7 DOX or MCF-7 cells and then evaluated their response to doxorubicin treatment. The results showed that MCF-7 DOX or MCF-7cells with PEAK1 knockdown had a markedly reduced growth rate following doxorubicin treatment (Fig. 5A-5B). These data suggest that targeting PEAK1 inhibited tumor growth in vito and sensitized MGF-7 cells to doxorubicin treatment. PEAK1 depelation inhibited lung metastasis of MCF-7 cells in vivo Finally, we investigated the role of PEAK1 in mediating breast cancer cells metastasis in vivo . The stable PEAK1 shRNA or Lv-PEAK1 transfected MCF-7 cells were injected into the female nude mice by the tail vein. After 8 weeks, the whole lung tissues were removaled and the numbers of visible nodules on the lung surface were numbered. H&E staining was used to evaluate the tumor metastasis. The results showed that the PEAK1 shRNA transfected MCF-7 cells has fewer tumor nodes (Fig. 5C) and the Lv-PEAK1 transfected MCF-7 cells has more tumor nodes (Fig. 5C). These data suggest that targeting PEAK1 inhibited lung metastasis in vivo . Discussion Breast cancer is the commonest female cancer worldwide and its propensity to metastasize negatively impacts on therapeutic outcome. Several clinicopathological parameters with prognostic/predictive significance have been associated with metastatic suppressor expression levels. PEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. It has reported to be upregulated in human malignancies and related with poor prognosis [ 3 , 4 ]. In the present study, we performed immunohistochemistry (IHC) of PEAK1 in 112 surgical resected breast cancer tissues and 43 adjacent non-tumor breast tissues. PEAK1 is localized in the cytoplasm, membrane and nuclear, and predominately cytoplasm staining. These observations were consistent with the IHC staining results in colorectal cancer [ 3 ] and pancreatic cancer [ 4 ]. We also found that PEAK1 is overexpressed in breast cancer tissues at significantly higher levels than adjacent non-tumor breast tissues. PEAK1 is upregulated in multiple human malignancies and has been associated with tumor invasion and metastasis [ 3 , 4 ]. However, PEAK1 was downexpressed in gastric cancers, higher PEAK1 expression was related with non-lymph node metastases and good prognosis [ 10 ]. Here we found that PEAK1 overexpression was related with larger tumor size, higher tumor stage, tumor grade, T stage, LN metastasis and recurrence, suggesting that PEAK1 overexpression might promote the malignant potential of breast cancer. Therefore, PEAK1 might act as a valuable marker for prediction of breast cancer invasion, and also play an important role in prognosis prediction. Ki-67 expression was reported to be positively correlated with a higher incidence of lymphovascular invasion and lymph node metastasis in breast cancer [ 11 , 12 ]. In the study, PEAK1 expression was correlated with lymph node metastasis and Ki-67 expression, further confirming that PEAK1 may be used as a diagnostic marker for breast cancer invasion and prognosis. In clinical settings, low-proliferative tumors are less sensitive to chemotherapy [ 13 ]. Nowadays immunohistochemistry for Ki-67 assessment is used for estimation of cell proliferation and guiding the decision on adjuvant treatment choice and predicting of the neoadjuvant treatment response in breast cancer [ 14 ]. Immunohistochemistry for ER, PR and HER2 assessment is also used to predict sensitivity to drugs and to determine the application and types of systemic therapy [ 15 ]. In the present study, PEAK1 expression was related with both Ki-67 expression and HER2 expression in the breast cancer, suggesting that PEAK1 expression may predict chemosensitivity in breast cancer. In addition, PEAK1 was overexpressed in 112 cases of breast cancer patients accepting chemotherapy. However, the difference in PEAK1 expression in groups of radiation and hormonal treatments and without radiation and hormonal treatments did not show significance. To confirm our observations, we tested PEAK1 expression in 53 cases of breast cancer patients accepting neoadjuvant chemotherapy (NAC) treatment, and found that PEAK1 expression was significantly enhanced in chemoresistant breast cancer. In the laboratory, targeting PEAK1 expression inhibited cell proliferation and metastasis in vitro and in vivo , and vice verse [ 2 – 5 , 7 , 16 ]. Croucher et al. [ 9 ] reported that PEAK1 was overexpressed in luminal, HER2, primary basal breast cancers and cell lines by western blot assay. Furthermore, enhanced PEAK1 promoted acinar growth and cell invasion in vitro. Abu-Thuraia et al.[ 17 ] reported that PEAK1 is required for both tumor growth and metastasis in a TNBC cellular model. In the present study, the blockade of PEAK1 expression inhibited cell growth, invasion and migration in vitro , and inhibited tumor growth and lung metastasis in vivo , suggesting that PEAK1 is the target gene for breast cancer gene therapy. To further confirm whether PEAK1 expression regulates the chemosensitivity of breast cancer cells to therapeutic drug, the PEAK1 knockdown and overexpresssing MCF-7 cells were treated with doxorubicin. As might be predicted from prior studies of doxorubicin, a remarkably decreased cell or tumor growth was observed in MCF-7 cells with suppressed PEAK1 expression, whereas more cell growth were detected in MCF-7 cells overexpressing PEAK1 in vitro and in vivo . These data indicated that enhanced PEAK1 expression promoted the development of chemoresistance, and vice versa. Conclusion In conclusion, PEAK1 is overexpressed in breast cancer and chemoresistant breast cancers. PEAK1 overexpression is related with clinicopathological parameters. Monitoring PEAK1 level and PEAK1 depletion may be utilized to predict and reverse the chemoresistance of breast cancers. PEAK1 may be as a useful prognostic biomarker and a potential therapeutic target for patients with breast cancer. Declarations Competing interests The authors declare that they have no competing interests. Data availability statement Data sharing is not applicable to this article as no new data were created or analyzed in this study. Acknowledgements : We would like to thank all of the participants for their excellent submissions and thoughtful commentary. Funding No Funding in the manuscript was not funded. Author Contribution Yu Wang and Xingang Wang designed the experiments, analysed the data and prepared the manuscript. Yan Zheng selected the materials. All authors read and approved the final manuscript. References Saha S, Mukherjee S, Khan P, Kajal K, Mazumdar M, Manna A. Aspirin Suppresses the Acquisition of Chemoresistance in Breast Cancer by Disrupting an NFkappaB-IL6 Signaling Axis responsible for the Generation of Cancer Stem Cells. Cancer Res. 2016;76:2000–2012 Wang Y, Kelber JA, Tran Cao HS, Cantin GT, Lin R, Wang W, Kaushal S, Bristow JM, Edgington TS, Hoffman RM, Bouvet M, Yates JR 3rd, Klemke RL. Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression. Proc Natl Acad Sci U S A. 2010;107(24):10920-5. Huang L, Wen C, Yang X, Lou Q, Wang X, Che J, Chen J, Yang Z, Wu X, Huang M, Lan P, Wang L, Iwamoto A, Wang J, Liu H. PEAK1, acting as a tumor promoter in colorectal cancer, is regulated by the EGFR/KRas signaling axis and miR-181d. Cell Death Dis. 2018; 9(3):271. Strnadel J, Choi S, Fujimura K, Wang H, Zhang W, Wyse M, Wright T, Gross E, Peinado C, Park HW, Bui J, Kelber J, Bouvet M, Guan KL, Klemke RL. eIF5A-PEAK1 Signaling Regulates YAP1/TAZ Protein Expression and Pancreatic Cancer Cell Growth. Cancer Res. 2017;77(8):1997-2007. Ding C, Tang W, Wu H, Fan X, Luo J, Feng J, Wen K, Wu G. The PEAK1-PPP1R12B axis inhibits tumor growth and metastasis by regulating Grb2/PI3K/Akt signalling in colorectal cancer. Cancer Lett. 2019;442:383-395. Ding C, Tang W, Fan X, Wang X, Wu H, Xu H, Xu W, Gao W, Wu G. Overexpression of PEAK1contributes to epithelial-mesenchymal transition and tumor metastasis in lung cancer through modulating ERK1/2 and JAK2 signaling. Cell Death Dis. 2018;9(8):802. Wang H, Lapek J, Fujimura K, Strnadel J, Liu B, Gonzalez DJ, Zhang W, Watson F, Yu V, Liu C, Melo CM, Miller YI, Elliott KC, Cheresh DA, Klemke RL. Pseudopodium-enriched atypical kinase 1 mediates angiogenesis by modulating GATA2-dependent VEGFR2 transcription. Cell Discov. 2018;4:26. Agajanian M, Campeau A, Hoover M, Hou A, Brambilla D, Kim SL, Klemke RL, Kelber JA. PEAK1 Acts as a Molecular Switch to Regulate Context-Dependent TGFbeta Responses in Breast Cancer. PLoS One. 2015;10(8):e0135748. Croucher DR, Hochgräfe F, Zhang L, Liu L, Lyons RJ, Rickwood D, Tactacan CM, Browne BC, Ali N, Chan H, Shearer R, Gallego-Ortega D, Saunders DN, Swarbrick A, Daly RJ. Involvement of Lyn and the atypical kinase SgK269/PEAK1 in a basal breast cancer signaling pathway. Cancer Res. 2013;73(6):1969-80. Guo Q, Qin W, Li B, Yang H, Guan J, Liu Z, Li S. Analysis of a cytoskeleton-associated kinase PEAK1 and E-cadherin in gastric cancer. Pathol Res Pract. 2014;210(12):793-8. Talaat IM, Hamoudi RA, Yakout NM, Oweiss NY, Omar AM. Correlation of securin and Ki67 in invasive breast Histol Histopathol. 2019;34(6):697-709. Robertson S, Stålhammar G, Darai-Ramqvist E, Rantalainen M, Tobin NP, Bergh J, Hartman J. Prognostic value of Ki67 analysed by cytology or histology in primary breast cancer . J Clin Pathol. 2018;71(9):787-794. Mu K, Li L, Yang Q, Yun H, Kharaziha P, Ye DW, Auer G, Lagercrantz SB, Zetterberg A. A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer. Ann Diagn Pathol. 2015;19(4):243-8. Penault-Llorca F, Radosevic-Robin N. Ki67 assessment in breast cancer: an update. Pathology. 2017; 49(2): 166 -171. Coates AS, Winer EP, Goldhirsch A et al. Tailoring therapies-improving the management of early breast cancer: St Gallen international expert consensus on the primary therapy of early breast cancer 2015. Ann Oncol 2015; 26: 1533-46. Kelber JA, et al. KRas induces a Src/PEAK1/ErbB2 kinase amplification loop that drives metastatic growth and therapy resistance in pancreatic cancer. Cancer Res. 2012;72:2554-2564. Abu-Thuraia A, Goyette MA, Boulais J, Delliaux C, Apcher C, Schott C, Chidiac R, Bagci H, Thibault MP, Davidson D, Ferron M, Veillette A, Daly RJ, Gingras AC, Gratton JP, Côté JF. AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network. Nat Commun. 2020;11(1):3586. Tables Table 1. PEAK1 expression and clinicopathological status in 112 patients with breast cancer PEAK1 expression Variable Low expression(n=43) High expression(n=69) p-value Age(year) 0.547 ≤50 15 28 >50 28 41 Tumor size (cm) 0.032 ≤2 27 29 >2 16 40 Tumor grade 0.049 1 6 11 2 26 26 3 11 32 T stage 0.012 T1 26 24 T2 17 40 T3 0 5 LN metastasis 0.036 Negative 12 33 Positive 31 36 Distant metastasis 0.073 No 30 58 Yes 13 11 ER 0.074 positive 28 33 negative 15 36 PR 0.262 positive 24 31 negative 19 38 HER-2 0.0218 positive 9 29 negative 34 40 Triple-negative 0.180 positive 9 8 negative 34 61 Chemotherapy 0.0135 No 29 30 Yes 14 39 Radiation therapy 0.208 No 34 47 Yes 9 22 Endocrine therapy 0.269 No 30 41 Yes 13 28 Local recurrence 0.243 No 28 52 Yes 15 17 Regional recurrence 0.0473 No 37 48 Yes 6 21 Ki-67 0.0366 positive 25 53 negative 18 16 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 09 Jul, 2021 Reviews received at journal 26 May, 2021 Reviewers invited by journal 22 May, 2021 Editor assigned by journal 22 May, 2021 First submitted to journal 21 May, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-549884","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":28686807,"identity":"2c32f925-63cb-47cb-9795-b4d4e2c53568","order_by":0,"name":"xingang wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYDACZgST/cfHPzZybOzNB4jWwiA5syHNmI/nWALxNkrzNhxKnCeRo4BXlcFx3sMvfu6oTeyf3X7BcOaOA+ltDDkMDD8qtuHUItnMl2bZe+Z44ow7ZwoSPp65k9vGcPYAY8+Z2zi18DPzmBnwth3LbbiRk3BwBtuz3DbGvgRmxjbcWtiAWgz/ArXMv5GT2MzDdjgdKGKAVwvQFuPHvG01uRtupB9m5m07nMDGRkCLZDOPGbNs24H6jTdy2BhnnEkzbONhSziIzy8G588Yf3zbVmcsdyP9GcOHCht5+fmPDz74UYFbC8g7EgwMh4E0jwFc6AA+9UDA/IGBoQ5Isz8goHAUjIJRMApGKgAARW5ffuAfeZ8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2407-5053","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"xingang","middleName":"","lastName":"wang","suffix":""},{"id":28686808,"identity":"868358c7-b4da-42bc-8c39-35524630a42f","order_by":1,"name":"YAN ZHENG","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"YAN","middleName":"","lastName":"ZHENG","suffix":""},{"id":28686809,"identity":"9037d6c9-54a5-406e-8262-313948fb2a43","order_by":2,"name":"YU WANG","email":"","orcid":"https://orcid.org/0000-0003-1379-7736","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"YU","middleName":"","lastName":"WANG","suffix":""}],"badges":[],"createdAt":"2021-05-21 21:35:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-549884/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-549884/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9557733,"identity":"9660f89c-7eda-4a1f-b2c9-71c65f6c9c99","added_by":"auto","created_at":"2021-05-25 14:51:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":507717,"visible":true,"origin":"","legend":"PEAK1 expression in breast cancer tissues and adjacent tissues by immunohistochemistry. A, High PEAK1 expression in breast cancer tissues; B, Low PEAK1 expression in the adjacent tissues of A.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/77997395e31615a58513d67f.jpg"},{"id":9557731,"identity":"24d991fc-82c9-4e39-8ace-8eb92ceecabd","added_by":"auto","created_at":"2021-05-25 14:51:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332536,"visible":true,"origin":"","legend":"Effect of PEAK1 overexpression on cell growth, invasion and migration in vitro. A, PEAK1 expression was detected in MCF-7/Lv-PEAK1 or MCF-7/Lv-PEAK1 shRNA and MCF-7/ empty vector cells by western blot assay. B, cell growth was detected by MTT assay in MCF-7/Lv-PEAK1 and MCF-7/ empty vector cells. C, Cell growth was detected by colony formation assay in MCF-7/Lv-PEAK1 and MCF-7/ empty vector cells. D, The MCF-7 cells invasion and migration abilities were measured by transwell after PEAK1 overexpression. VS control, *p\u003c0.01.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/e98b6a6cda5877b1264cc1fd.jpg"},{"id":9558116,"identity":"e8354297-9038-4723-95f4-066b383bb142","added_by":"auto","created_at":"2021-05-25 14:54:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281244,"visible":true,"origin":"","legend":"Effect of PEAK1 depletion on cell growth, invasion and migration in vitro. A, cell growth was detected by MTT assay in MCF-7/Lv-PEAK1 shRNA and MCF-7/empty vector cells. B, Cell growth was detected by colony formation assay in MCF-7/Lv-PEAK1 shRNA and MCF-7/ empty vector cells. C, The MCF-7 cells invasion and migration abilities were measured by transwell after PEAK1 depletion. VS control, *p\u003c0.01.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/d4c8aa4ba11402d96e2858a5.jpg"},{"id":9557735,"identity":"6e6df4a4-5ecd-4631-b16d-3b567b3081e8","added_by":"auto","created_at":"2021-05-25 14:51:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215901,"visible":true,"origin":"","legend":"Effect of PEAK1 depletion or overexpression on doxorubicin sensitivity in MCF-7 cells.\nMCF-7/Lv-PEAK1 and MCF-7/ empty vector cells were treated with 2.0 μg/mL Doxorubicin for 72 h, MCF-7 DOX/Lv-PEAK1 shRNA and MCF-7 DOX / empty vector cells were treated with 2.0 μg/mL Doxorubicin for 72 h. Cell growth was detected by MTT assay (A) and colony formation assay(B). *p\u003c0.01.\n","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/161fbddb30096d66b042ee0a.jpg"},{"id":9558115,"identity":"66dd8c9f-e63a-4493-bc74-b8d977e65afd","added_by":"auto","created_at":"2021-05-25 14:54:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176879,"visible":true,"origin":"","legend":"PEAK1 expression influences metastasis and chemosensitivity of MCF-7 cells in vivo. \n MCF-7DOX cells (A) or MCF-7 cells (B) with varying PEAK1 status, were injected s.c. in the left front flank of female BALB/c mice (n = 4/group) on day 0. All mice were treated with DOX by i.v. injection (day 3-9). When tumor growth became visible, tumor volume was monitored and results were graphically displayed. MCF-7 / PEAK1 or NC cells，MCF-7 /Lv-NC shRNA or MCF-7/Lv-PEAK1 shRNA cells were inoculated subcutaneously into nude mice for 35 days. Lung metastatic nodes were counted in the lungs (C).*, P \u003c 0.05, versus control group.\n","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/6f2f1373268bfc44ee02a413.jpg"},{"id":13694852,"identity":"1a4d9635-fd38-4e9d-9c45-f0c0d8ac04df","added_by":"auto","created_at":"2021-09-17 12:54:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1039302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-549884/v1/29937788-c091-4288-a835-ae5590c4eb9e.pdf"}],"financialInterests":"","formattedTitle":"PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer","fulltext":[{"header":"Introduction","content":" \u003cp\u003eBreast cancer metastasis results in poor prognosis and increased mortality, the mechanisms of which are yet to be fully resolved. Tumour resection combined with radiotherapy, endocrine-therapy, and/or chemotherapy is the main way to treat breast cancer, but the development of chemoresistance limits the effectiveness of chemotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, identifying the molecular mechanisms contributing to breast cancer progression and\u003c/p\u003e \u003cp\u003echemoresistance would produce new biomarkers for the precise prediction of patient prognosis and for molecular targeted-therapy.\u003c/p\u003e \u003cp\u003eOverexpression of PEAK1 (pseudopodium enriched atypical kinase 1, Sgk269), which was discovered in the pseudopodia of migrating cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], is also found in colorectal cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and pancreatic cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PEAK1 expression is associated with metastasis and proliferation in many cancer cells, such as colorectal cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], lung cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and pancreatic cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Enforced PEAK1 expression can cause cell cycle deregulation and resistance to chemotherapy in pancreatic cancer cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Altering PEAK1 expression can interfere tumor formation and metastasis in pancreatic cancer cells in vivo, indicating that PEAK1 plays an important role in pancreatic cancer growth and metastasis. In breast cancer cells in vivo, enforced PEAK1 expression could induce new blood vessel formation by upregulation of vascular endothelial growth factor receptor-2, which facilitates cell movement and growth [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has recently found that PEAK1 overexpression was significantly associated with advanced clinical stage and poor prognosis in colon cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and pancreatic cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In breast cancer, PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Croucher et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] reported that PEAK1 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. However, the relation of PEAK1 expression and clinicopathological status and the relation of PEAK1 expression with chemosensitvity in breast cancer is currently unknown.\u003c/p\u003e \u003cp\u003eIn the present study, we examined the PEAK1 protein expression in human breast cancer tissues and explored the relationship between PEAK1 expression and clinical characteristics. Furthermore, we investigated the effect of PEAK1 on breast cancer cell growth, invasion, migration, metastasis and Doxorubicin sensitivity \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human breast cancer MCF-7 cells were purchased from Institute of Cell Research, Chinese Academy of Sciences (Beijing, China). Doxorubicin (DOX)-resistant MCF-7\u003csup\u003eDOX\u003c/sup\u003e cells were established by induction with gradient concentrations (0.1-2\u0026nbsp;\u0026mu;g/mL) of ADR \u003cem\u003ein vitro\u003c/em\u003e. Cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco), 100 units/ml of penicillin, 100 \u0026mu;g/ml, streptomycin in a 5% CO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;at 37\u0026deg;C. MCF-7\u003csup\u003eDOX\u003c/sup\u003e cells were cultured at a final concentration of 2.0 \u0026mu;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBreast cancer samples were obtained from Breast disease Center, the affiliated Hospital of Qingdao University. In all, 112 surgical resected tumors from Feb. 2012 to 2018. Pathological diagnosis was verified by two pathologists independently. All human samples were collected with informed consents from the donors according to the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The study was performed after approval by the institutional review board (IRB) of the affiliated Hospital of Qingdao University. Written informed consent was obtained from each individual or patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry of PEAK1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded tissue sections from excised specimens were processed according to standard procedures. Specific primary antibodies against PEAK1 (1:50) was purchased from Sigma-Aldrich (Shanghai, China). IHC staining for PEAK1 was performed as the manufacture\u0026rsquo;s instruction\u003cstrong\u003e. \u003c/strong\u003eThe expression of PEAK1 was positive when 10% of tumor cells showed PEAK1 immunopositivity, and negative when less than 10% of tumor cells showed PEAK1 immunopositivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePEAK1 shRNA vector construction and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe short-hairpin RNA direct against human PEAK1 gene (PEAK1 shRNA) was synthesized and constructed into the pcDNA3.1 expression vector (Shanghai, China) as the manufacture\u0026rsquo;s instruction.\u0026nbsp;The constructed vectors (PEAK1 shRNA or NC shRNA) were transfected into the MCF-7 cells using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) as the manufacturer's instruction. The PEAK1 shRNA or NC shRNA transfected MCF-7 cells were selected by puromycin (10\u0026nbsp;mg/ml) for 5\u0026nbsp;days. The puromycin-resistant colonies were then picked and expanded. The relative protein was detected by Western blot assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid constructs and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length human PEAK1 adenovirus was constructed as to the manufacturer\u0026rsquo;s instructions using the AdEasy Adenoviral Vector System. Viral particles were produced by GenScript Biotechnology, China. The virus particles containing PEAK1 or control vector were used to infect MCF-7 cells. Transfected cells were selected with G418 (600\u0026nbsp;\u0026mu;g/ml, Gibco) for 10-12 days. The expression of PEAK1 in stable PEAK1 transfected colonies was detected by western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edoxorubicin sensitivity by MTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in a 96-well plate in triplicate for 24 h, and the cells (300 cells / well) were then exposed to a concentration of 2.0 \u0026mu;g/mL Doxorubicin for 72 h. Subsequently, 20 \u0026mu;L MTT (Sigma-Aldrich) was added to each well and then incubated for 4 hours at 37\u0026deg;C and 5% CO2\u0026nbsp;humidified atmosphere.\u0026nbsp;The optical density (OD) at 450 nm was measured and considered an indirect index of relative cell viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edoxorubicin sensitivity by colony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCF-7 cells after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 48 h were plated in triplicate at 1000 cells per well in 6-well plates with or without 2.0 \u0026mu;g/mL Doxorubicin and cultured for 12 days, then methanol-fixed and Giemsa-stained (GS, Sigma-Aldrich), which was followed by colony counting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMatrigel invasion assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell migration and invasion was determined using a Transwell Chamber assay (BD Biosciences) according to the manufacturer's instructions. MCF-7 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;cells) after transfection with Lv-PEAK1 or PEAK1 shRNA or its control for 24 h were added to the upper compartment (in triplicate), and DMEM plus 10% FCS was added to the lower compartment. Bestatin (Sigma) was added to both compartments. The cells were incubated for 24 h, and the total number of invaded cells was calculated as the manufacture\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells and tissues were lysed and protein concentration was measured with BCA protein assay reagents (Pierce). The proteins were then resolved on SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore) and probed with the primary antibodies: PEAK1 and a-Tubulin. Band densitometry analysis was performed using ImageJ software (NCI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo metastasis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale BALB/c nude mice (5-6 weeks of age, 16-18\u0026thinsp;g) were obtained from National Rodent Shanghai Experimental Branch Center, Chinese Academy of Sciences (Shanghai, China). All experimental procedures involving animals were conducted in accordance with the institutional guidelines by the Affiliated hospital of Qingdao University. The stable Lv-PEAK1 or PEAK1 shRNA or its control transfected MCF-7 cells were cultured to log phase. 2\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;cells (n\u0026thinsp;=\u0026thinsp;6 per group) were injected into mice via tail veins. After 5 weeks, the whole lung tissues were removaled and the numbers of visible nodules on the lung surface were numbered. H\u0026amp;E staining was used to evaluate the tumor metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u0026nbsp;growth and \u003c/strong\u003e\u003cstrong\u003edoxorubicin sensitivity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of stable PEAK1 shRNA transfected MCF-7\u003csup\u003eDOX\u003c/sup\u003e or stable Lv-PEAK1 transfected MCF-7 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;) were injected s.c. to the left front flank of mice on day 0. From day 3 to day 9, DOX was administered to all groups of mice by i.v. injection (0.1\u0026thinsp;mL, 10\u0026thinsp;mg/kg) for a total of 4 times at 2 day intervals. Tumor dimensions were measured in 2 dimensions with microcalipers every other day and tumor volume was calculated by the following formula:\u0026nbsp;tumor volume\u0026thinsp;=\u0026thinsp;(length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. Non-retrospective ethical approval obtained for the animal experiments conducted in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were shown as mean \u0026plusmn; SEM; Data were analyzed using Student\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test and pearson \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026nbsp;test. Value of p \u0026lt;0.05 were considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePEAK1 is overexpressed in breast cancer tissues \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing immunohistochemical methods, we detected the expression of PEAK1 in 112 cases of breast cancer tissues and 34 cases of corresponding adjacent tissues (Fig. 1). PEAK1 expression is significantly upregulated in breast cancer tissues 61.6% (69/112) in comparison with adjacent normal tissues 26.4% (9/34) (P=0.033). PEAK1 overexpression is associated with high tumor size, tumor grade, T stage, advanced nodal status, regional recurrence, HER2, Ki-67 and chemotherapy status (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePEAK1 promotes cell growth ,invasion and migration\u003cem\u003e in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Lv-PEAK1 or empty vector was transfected into MCF-7 cells. PEAK1 expression was significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells (Fig. 2A). Cell growth was significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells by MTT assay (Fig. 2B). To confirm the MTT data, we carried out in vitro colony formation assays. Representative pictures and their quantification confirm that PEAK1 overexpression significantly promoted MCF-7 cells growth (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eThe cell migration and invasion of transfected cells in vitro was observed by transwell assays. As shown in Fig. 2D, the invasive and migrative cells were significantly increased in MCF-7/Lv-PEAK1 cells compared to the MCF-7/ empty vector cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeting PEAK1 inhibits cell growth ,invasion and migration\u003cem\u003e in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Lv-PEAK1 shRNA or empty vector was transfected into MCF-7 cells. PEAK1 expression was significantly decreased in MCF-7/ PEAK1 shRNA cells compared to the MCF-7/ control shRNA cells (Fig.2A). Cell growth was significantly decreased with PEAK1 depletion in the MCF-7/ PEAK1 shRNA cells by MTT assay (Fig. 3A) and colony formation assays (Fig. 3B). As shown in Fig. 3C, the invasive and migrative cells were significantly decreased in MCF-7/ PEAK1 shRNA cells compared to the MCF-7/control shRNA cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePEAK1 regulated\u003c/strong\u003e\u003cstrong\u003e doxorubicin sensitivity in MCF-7 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Lv-PEAK1 or empty vector was transfected into MCF-7 cells, then treated with 2.0 \u0026mu;g/mL doxorubicin for 72 h. The results confirmed that PEAK1 overexpression reduced doxorubicin-induced cytotoxicity\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cstrong\u003eFig. 4A-4B).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly, the MCF-7\u003csup\u003eDOX\u003c/sup\u003e cells were transfected with PEAK1 shRNA, then treated with 2.0 \u0026mu;g/mL doxorubicin for 72 h. The results confirmed that PEAK1 depletionincreased doxorubicin-induced cytotoxicity\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cstrong\u003eFig. 4A-4B).\u003c/strong\u003e These data suggest that targeting PEAK1 reverses \u003cstrong\u003edoxorubicin\u003c/strong\u003e resistance in doxorubicin-resistant breast cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePEAK1 depelation increases the sensitivity of MCF-7\u003c/strong\u003e\u003cstrong\u003ecells to \u003c/strong\u003e\u003cstrong\u003edoxorubicin \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the effect of PEAK1 on chemosensitivity\u0026nbsp;in vitro\u0026nbsp;also extended to tumors growing\u0026nbsp;in vivo, we injected female BALB/c mice with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;MCF-7\u003csup\u003eDOX\u003c/sup\u003e or MCF-7 cells and then evaluated their response to doxorubicin treatment. The results showed that MCF-7\u003csup\u003eDOX\u003c/sup\u003e or MCF-7cells with PEAK1 knockdown had a markedly reduced growth rate following doxorubicin treatment (Fig. 5A-5B). These data suggest that targeting PEAK1 inhibited tumor growth \u003cem\u003ein vito\u003c/em\u003e and sensitized MGF-7 cells to doxorubicin treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePEAK1 depelation inhibited lung metastasis of MCF-7 cells\u003cem\u003e in vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we investigated the role of PEAK1 in mediating breast cancer cells metastasis \u003cem\u003ein vivo\u003c/em\u003e. The stable PEAK1 shRNA or Lv-PEAK1 transfected MCF-7 cells were injected into the female nude mice by the tail vein. After 8 weeks, the whole lung tissues were removaled and the numbers of visible nodules on the lung surface were numbered. H\u0026amp;E staining was used to evaluate the tumor metastasis. The results showed that the PEAK1 shRNA transfected MCF-7 cells has fewer tumor nodes (Fig. 5C) and the Lv-PEAK1 transfected MCF-7 cells has more tumor nodes (Fig. 5C). These data suggest that targeting PEAK1 inhibited lung metastasis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eBreast cancer is the commonest female cancer worldwide and its propensity to metastasize negatively impacts on therapeutic outcome. Several clinicopathological parameters with prognostic/predictive significance have been associated with metastatic suppressor expression levels.\u003c/p\u003e \u003cp\u003ePEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. It has reported to be upregulated in human malignancies and related with poor prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the present study, we performed immunohistochemistry (IHC) of PEAK1 in 112 surgical resected breast cancer tissues and 43 adjacent non-tumor breast tissues. PEAK1 is localized in the cytoplasm, membrane and nuclear, and predominately cytoplasm staining. These observations were consistent with the IHC staining results in colorectal cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and pancreatic cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We also found that PEAK1 is overexpressed in breast cancer tissues at significantly higher levels than adjacent non-tumor breast tissues.\u003c/p\u003e \u003cp\u003ePEAK1 is upregulated in multiple human malignancies and has been associated with tumor invasion and metastasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, PEAK1 was downexpressed in gastric cancers, higher PEAK1 expression was related with non-lymph node metastases and good prognosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Here we found that PEAK1 overexpression was related with larger tumor size, higher tumor stage, tumor grade, T stage, LN metastasis and recurrence, suggesting that PEAK1 overexpression might promote the malignant potential of breast cancer. Therefore, PEAK1 might act as a valuable marker for prediction of breast cancer invasion, and also play an important role in prognosis prediction. Ki-67 expression was reported to be positively correlated with a higher incidence of lymphovascular invasion and lymph node metastasis in breast cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the study, PEAK1 expression was correlated with lymph node metastasis and Ki-67 expression, further confirming that PEAK1 may be used as a diagnostic marker for breast cancer invasion and prognosis. In clinical settings, low-proliferative tumors are less sensitive to chemotherapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nowadays immunohistochemistry for Ki-67 assessment is used for estimation of cell proliferation and guiding the decision on adjuvant treatment choice and predicting of the neoadjuvant treatment response in breast cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Immunohistochemistry for ER, PR and HER2 assessment is also used to predict sensitivity to drugs and to determine the application and types of systemic therapy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the present study, PEAK1 expression was related with both Ki-67 expression and HER2 expression in the breast cancer, suggesting that PEAK1 expression may predict chemosensitivity in breast cancer. In addition, PEAK1 was overexpressed in 112 cases of breast cancer patients accepting chemotherapy. However, the difference in PEAK1 expression in groups of radiation and hormonal treatments and without radiation and hormonal treatments did not show significance. To confirm our observations, we tested PEAK1 expression in 53 cases of breast cancer patients accepting neoadjuvant chemotherapy (NAC) treatment, and found that PEAK1 expression was significantly enhanced in chemoresistant breast cancer.\u003c/p\u003e \u003cp\u003eIn the laboratory, targeting PEAK1 expression inhibited cell proliferation and metastasis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and vice verse [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Croucher et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] reported that PEAK1 was overexpressed in luminal, HER2, primary basal breast cancers and cell lines by western blot assay. Furthermore, enhanced PEAK1 promoted acinar growth and cell invasion in vitro. Abu-Thuraia et al.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reported that PEAK1 is required for both tumor growth and metastasis in a TNBC cellular model. In the present study, the blockade of PEAK1 expression inhibited cell growth, invasion and migration \u003cem\u003ein vitro\u003c/em\u003e, and inhibited tumor growth and lung metastasis \u003cem\u003ein vivo\u003c/em\u003e, suggesting that PEAK1 is the target gene for breast cancer gene therapy.\u003c/p\u003e \u003cp\u003eTo further confirm whether PEAK1 expression regulates the chemosensitivity of breast cancer cells to therapeutic drug, the PEAK1 knockdown and overexpresssing MCF-7 cells were treated with doxorubicin. As might be predicted from prior studies of doxorubicin, a remarkably decreased cell or tumor growth was observed in MCF-7 cells with suppressed PEAK1 expression, whereas more cell growth were detected in MCF-7 cells overexpressing PEAK1 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These data indicated that enhanced PEAK1 expression promoted the development of chemoresistance, and vice versa.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn conclusion, PEAK1 is overexpressed in breast cancer and chemoresistant breast cancers. PEAK1 overexpression is related with clinicopathological parameters. Monitoring PEAK1 level and PEAK1 depletion may be utilized to predict and reverse the chemoresistance of breast cancers. PEAK1 may be as a useful prognostic biomarker and a potential therapeutic target for patients with breast cancer.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no new data were created or analyzed in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: We would like to thank all of the participants for their excellent submissions and thoughtful commentary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo Funding in the manuscript was not funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Yu Wang and Xingang Wang designed the experiments, analysed the data and prepared the manuscript. Yan Zheng selected the materials. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSaha S, Mukherjee S, Khan P, Kajal K, Mazumdar M, Manna A. Aspirin Suppresses the Acquisition of Chemoresistance in Breast Cancer by Disrupting an NFkappaB-IL6 Signaling Axis responsible for the Generation of Cancer Stem Cells.\u0026nbsp;Cancer Res.\u0026nbsp;2016;76:2000\u0026ndash;2012\u003c/li\u003e\n\u003cli\u003eWang Y, Kelber JA, Tran Cao HS, Cantin GT, Lin R, Wang W, Kaushal S, Bristow JM, Edgington TS, Hoffman RM, Bouvet M, Yates JR 3rd, Klemke RL. Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression. Proc Natl Acad Sci U S A. 2010;107(24):10920-5.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eHuang L, Wen C, Yang X, Lou Q, Wang X, Che J, Chen J, Yang Z, Wu X, Huang M, Lan P, Wang L, Iwamoto A, Wang J, Liu H. PEAK1, acting as a tumor promoter in colorectal cancer, is regulated by the EGFR/KRas signaling axis and miR-181d. Cell Death Dis. 2018; 9(3):271.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eStrnadel J, Choi S, Fujimura K, Wang H, Zhang W, Wyse M, Wright T, Gross E, Peinado C, Park HW, Bui J, Kelber J, Bouvet M, Guan KL, Klemke RL. eIF5A-PEAK1\u0026nbsp;Signaling Regulates YAP1/TAZ Protein Expression and Pancreatic Cancer Cell Growth. Cancer Res. 2017;77(8):1997-2007.\u003c/li\u003e\n\u003cli\u003eDing C, Tang W, Wu H, Fan X, Luo J, Feng J, Wen K, Wu G. The\u0026nbsp;PEAK1-PPP1R12B axis inhibits tumor growth and metastasis by regulating Grb2/PI3K/Akt signalling in colorectal cancer. Cancer Lett. 2019;442:383-395.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eDing C, Tang W, Fan X, Wang X, Wu H, Xu H, Xu W, Gao W, Wu G. Overexpression of\u0026nbsp;PEAK1contributes to epithelial-mesenchymal transition and tumor metastasis in lung cancer through modulating ERK1/2 and JAK2 signaling. Cell Death Dis. 2018;9(8):802.\u003c/li\u003e\n\u003cli\u003eWang H, Lapek J, Fujimura K, Strnadel J, Liu B, Gonzalez DJ, Zhang W, Watson F, Yu V, Liu C, Melo CM, Miller YI, Elliott KC, Cheresh DA, Klemke RL. Pseudopodium-enriched atypical kinase 1 mediates angiogenesis by modulating GATA2-dependent VEGFR2 transcription. Cell Discov. 2018;4:26.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eAgajanian M, Campeau A, Hoover M, Hou A, Brambilla D, Kim SL, Klemke RL, Kelber JA. PEAK1\u0026nbsp;Acts as a Molecular Switch to Regulate Context-Dependent TGFbeta Responses in\u0026nbsp;Breast\u0026nbsp;Cancer. PLoS One. 2015;10(8):e0135748.\u003c/li\u003e\n\u003cli\u003eCroucher DR, Hochgr\u0026auml;fe F, Zhang L, Liu L, Lyons RJ, Rickwood D, Tactacan CM, Browne BC, Ali N, Chan H, Shearer R, Gallego-Ortega D, Saunders DN, Swarbrick A, Daly RJ. Involvement of Lyn and the atypical kinase SgK269/PEAK1\u0026nbsp;in a basal breast cancer signaling pathway. Cancer Res. 2013;73(6):1969-80.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eGuo Q, Qin W, Li B, Yang H, Guan J, Liu Z, Li S. Analysis of a cytoskeleton-associated kinase PEAK1 and E-cadherin in gastric cancer. Pathol Res Pract. 2014;210(12):793-8.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eTalaat IM, Hamoudi RA, Yakout NM, Oweiss NY, Omar AM. Correlation of securin and Ki67 in invasive\u0026nbsp;\u003cstrong\u003ebreast\u003c/strong\u003e Histol Histopathol. 2019;34(6):697-709.\u003c/li\u003e\n\u003cli\u003eRobertson S, St\u0026aring;lhammar G, Darai-Ramqvist E, Rantalainen M, Tobin NP, Bergh J, Hartman J. Prognostic value of Ki67 analysed by cytology or histology in primary\u0026nbsp;\u003cstrong\u003ebreast\u003c/strong\u003e\u003cstrong\u003ecancer\u003c/strong\u003e. J Clin Pathol. 2018;71(9):787-794.\u0026nbsp;\u003c/li\u003e\n\u003cli\u003eMu K, Li L, Yang Q, Yun H, Kharaziha P, Ye DW, Auer G, Lagercrantz SB, Zetterberg A. A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer. Ann Diagn Pathol. 2015;19(4):243-8.\u003c/li\u003e\n\u003cli\u003ePenault-Llorca F, Radosevic-Robin N. Ki67 assessment in\u0026nbsp;breast\u0026nbsp;cancer: an update. Pathology. 2017; 49(2): 166 -171.\u003c/li\u003e\n\u003cli\u003eCoates AS, Winer EP, Goldhirsch A et al. Tailoring therapies-improving the management of early breast cancer: St Gallen international expert consensus on the primary therapy of early breast cancer 2015. Ann Oncol 2015; 26: 1533-46.\u003c/li\u003e\n\u003cli\u003eKelber JA, et al. KRas induces a Src/PEAK1/ErbB2 kinase amplification loop that drives metastatic growth and therapy resistance in pancreatic cancer.\u0026nbsp;Cancer Res.\u0026nbsp;2012;72:2554-2564.\u003c/li\u003e\n\u003cli\u003eAbu-Thuraia A, Goyette MA, Boulais J, Delliaux C, Apcher C, Schott C, Chidiac R, Bagci H, Thibault MP, Davidson D, Ferron M, Veillette A, Daly RJ, Gingras AC, Gratton JP, C\u0026ocirc;t\u0026eacute; JF. AXL confers cell migration and invasion by hijacking a\u0026nbsp;PEAK1-regulated focal adhesion protein network. Nat Commun. 2020;11(1):3586.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable style=\"width: 578px;\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 551px;\" colspan=\"4\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. PEAK1 expression and clinicopathological status in 112 patients with breast cancer\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 292.656px;\" colspan=\"2\"\u003e\n\u003cp\u003ePEAK1 expression\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003eLow expression(n=43)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003eHigh expression(n=69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003ep-value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eAge(year)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026le;50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026gt;50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eTumor size (cm)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.032\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026le;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026gt;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eTumor grade\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.049\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eT stage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eT2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eT3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eLN metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.036\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eDistant metastasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.073\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eER\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.074\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003epositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003enegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003ePR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.262\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003epositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003enegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eHER-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.0218\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003epositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003enegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eTriple-negative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.180\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003epositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003enegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eChemotherapy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.0135\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eRadiation therapy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.208\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eEndocrine therapy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.269\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eLocal recurrence\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.243\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eRegional recurrence\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.0473\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003e\u0026nbsp; Ki-67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e0.0366\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003epositive\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 187.344px;\"\u003e\n\u003cp\u003enegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 141.656px;\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 151px;\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Breast cancer, multi-drug resistance, metastasis, Pseudopodium-enriched atypical kinase 1","lastPublishedDoi":"10.21203/rs.3.rs-549884/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-549884/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Aims\u003c/strong\u003e\u003c/p\u003e\u003cp\u003ePseudopodium-enriched atypical kinase 1 (PEAK1) has reported to be upregulated in human malignancies and related with poor prognosis. Enhanced PEAK1 expression facilitates tumor cell survival, invasion, metastasis and chemoresistance. However, the role of PEAK1 in breast cancer is not clear. Here, we investigated the PEAK1 expression in breast cancer and analyzed its relation with clinicopathological status and chemotherapy resistance to the neoadjuvant chemotherapy (NAC). We also investigated the role of PEAK1 on breast cancer cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemistry (IHC) was performed in 112 surgical resected breast cancer tissues. The associations between clinicopathological status, multi-drug resistance and PEAK1 expression were determined. Effect of PEAK1 overexpression or down-expression on proliferation, colony formation, invasion, migration, metastasis and Doxorubicin sensitivity in the MCF-7 cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e was detected. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003ePEAK1 was overexpressed in breast cancer tissues and NAC -resistant breast cancer tissues. High PEAK1 expression was related with tumor size, high tumor grade, T stage, LN metastasis, recurrence, Ki-67 expression, Her-2 expression and multi-drug resistance. Targeting PEAK1 inhibited cell growth, invasion, metastasis and reversed chemoresistance to Doxorubicin in breast cancer cells in vitro and in vivo. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eHigh PEAK1 expression was associated with invasion, metastasis and chemoresistance of breast cancers. Furthermore, targeting PEAK1 could inhibit cell growth and metastasis, and reverse chemoresistance in breast cancer cells, which provides an effective treatment strategies for breast cancer.\u003c/p\u003e","manuscriptTitle":"PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-25 14:51:03","doi":"10.21203/rs.3.rs-549884/v1","editorialEvents":[{"type":"communityComments","content":1},{"type":"decision","content":"Major revisions","date":"2021-07-09T05:04:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-05-27T01:33:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-22T12:23:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-22T08:41:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2021-05-21T06:58:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3bcd50e2-170b-4625-850c-5ee3e1f717f2","owner":[],"postedDate":"May 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4548666,"name":"Clinical Pharmacology"},{"id":4548667,"name":"Surgery"},{"id":4548668,"name":"General Surgery"}],"tags":[],"updatedAt":"2021-09-04T02:13:21+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-25 14:51:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-549884","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-549884","identity":"rs-549884","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}