Carcinogenesis Page 12 of 210 1 2 3 Full title: 4 5 6 p62/SQSTM1 interacts with vimentin to enhance breast cancer metastasis 7 8 Authors：：： 9 10 1# 2# 1# 3# 1 1 11 Si-Si Li , Ling-Zhi Xu , Wei Zhou , Shang Yao , Chun-Li Wang , Jiang-Long Xia , 12 13 He-Fei Wang1, Muhammad Kamran1, Xiao-Yuan Xue1, Lin Dong1, Jing Wang4, Xu-Dong 14 15 5 3 3 1 1 6 16 Ding , Laura Bella , Laurence Bugeon , Jie Xu , Fei-Meng Zheng , Margaret J. Dallman , 17 3 *1 18 Eric W.F. Lam , QuentinFor Liu Peer Review 19 20 Affiliations: 21 22 1 23 Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian, China; 24 25 State Key Laboratory of Oncology in South China, Collaborative Innovation Center for 26 27 Cancer Medicine, Sun Yat-sen University, Guangzhou, China 28 29 2 30 Department of Oncology, the Second Affiliated Hospital of Dalian Medical University, 31 32 Dalian, China 33 34 3Department of Surgery and Cancer, Imperial College London, W12 0NN, UK 35 36 4 37 Department of Oncology, the First Affiliated Hospital of Dalian Medical University, 38 39 Dalian, China 40 41 5 42 Department of Pathology, the Second Affiliated Hospital of Dalian Medical University, 43 44 Dalian, China 45 46 6Department of Life Sciences, Imperial College London, SW7 2AZ, UK 47 48 49 50 51 #These authors contributed equally to this work. 52 53 *Corresponding author: 54 55 56 Quentin Liu, Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Section, 57 58 59 60 1 Page 13 of 210 Carcinogenesis 1 2 3 Lvshun South Street, Lvshunkou District, Dalian 116044, PR China. Phone: 4 5 6 +86-411-86110508; Fax: +86-411-86110508; Email: [email protected]. 7 8 9 10 11 Key words: 12 13 Breast cancer; metastasis; p62; vimentin; protein interaction 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Carcinogenesis Page 14 of 210 1 2 3 4 5 6 Abstract 7 8 The signalling adaptor p62 is frequently overexpressed in numerous cancer types. Here, 9 10 11 we found that p62 expression was elevated in metastatic breast cancer and its 12 13 overexpression correlated with reduced metastasis-free and relapse-free survival times. 14 15 Analysis of p62 expression in breast cancer cell lines demonstrated that high p62 16 17 18 expression was associatedFor withPeer the invasive Review phenotypes of breast cancer. Indeed, 19 20 silencing p62 expression attenuated the invasive phenotypes of highly metastatic cells, 21 22 whereas overexpressing p62 promoted the invasion of non-metastatic cells in in vitro 23 24 25 microfluidic model. Moreover, MDA-MB-231 cells with p62 depletion which were 26 27 grown in a three-dimensional culture system exhibited a loss of invasive protrusions. 28 29 Consistently, genetic ablation of p62 suppressed breast cancer metastasis in both 30 31 32 zebrafish embryo and immunodeficient mouse models, as well as decreased 33 34 tumorigenicity in vivo. To explore the molecular mechanism by which p62 promotes 35 36 37 breast cancer invasion, we performed a co-immunoprecipitation (co-IP)-MS analysis and 38 39 revealed that p62 interacted with vimentin, which mediated the function of p62 in 40 41 promoting breast cancer invasion. Vimentin protein expression was downregulated upon 42 43 44 p62 suppression and upregulated with p62 overexpression in breast cancer cells. Linear 45 46 regression analysis of clinical breast cancer specimens showed a positive correlation 47 48 between p62 and vimentin protein expression. Together, our findings provide strong 49 50 51 evidence that p62 functions as a tumour metastasis promoter by binding vimentin and 52 53 promoting its expression. This finding might help to develop novel molecular therapeutic 54 55 strategies for breast cancer metastasis treatment. 56 57 58 59 60 3 Page 15 of 210 Carcinogenesis 1 2 3 Summary: p62 functions as a tumor metastasis promoter through its interaction with vimentin. This 4 5 6 finding might help to develop novel molecular therapeutic strategies for breast cancer metastasis 7 8 treatment. 9 10 11 12 Introduction 13 14 15 Cancer metastasis is a complex multi-step cell biological process termed the 16 17 invasion-metastasis cascade, by which primary tumour cells acquire the invasive ability 18 For Peer Review 19 and then spread to distant organs [1-3]. Metastases are responsible for more than 90% of 20 21 22 cancer-related deaths in various solid malignancies, including breast cancer [4, 5]. 23 24 Despite the early diagnostic methods, surgical resection and adjuvant therapy have 25 26 improved, metastatic disease is largely incurable [6]. Recent studies have shown that the 27 28 29 primary cause for cancer cells to drive the invasion-metastasis process is the acquisition 30 31 of the genetic or epigenetic alterations [7, 8]. Thus, the exploration of key molecules and 32 33 34 mechanisms underlying breast cancer metastasis regulation is urgently needed. 35 36 The adaptor protein p62 (also known as SQSTM1) is initially identified as a 37 38 cytosolic 62kDa protein which can bind to the isolated src homology 2 (SH2) domain of 39 40 lck 41 p56 [9]. This multidomain protein interacts selectively with different signalling 42 43 intermediaries, such as Raptor [10], Nrf2-binding site on Keap1 [11], ubiquitin and LC3 44 45 [12, 13], to regulate metabolic reprogramming, antioxidant response and selective 46 47 48 autophagy, respectively. Abnormal p62 overexpression has been documented in various 49 50 neoplasms [14-19], especially in breast cancer [20-22]. For example, the scaffold p62 51 52 53 protein cooperates with the Wnt/PCP protein VANGL2 and recruits JNK to VANGL2, 54 55 which is required for tumour growth in breast cancer [23]. In addition, high p62 56 57 expression is associated with breast tumours exhibiting the clinicopathological features of 58 59 60 4 Carcinogenesis Page 16 of 210 1 2 3 aggressive disease, as well as overexpression of EGF receptor (EGFR), HER2, HER3 and 4 5 6 HER4 [21]. In triple-negative breast cancers, patients with p62 accumulation exhibit a 7 8 higher risk of positive lymph node and lymphovascular invasion [22]. These findings 9 10 11 highlight the potential of p62 as a therapeutic target during cancer progression. However, 12 13 the detailed mechanism by which p62-mediates cancer cell invasion and metastasis 14 15 remains largely unknown. 16 17 18 Studies of epithelialFor malignancies Peer indicate Review that the acquisition of invasion and 19 20 metastasis potential by the incipient cancer cell may depend on the transition of a 21 22 mesenchymal phenotype [24]. Vimentin, a Type Ⅲ intermediate filament, serves as a 23 24 25 classical mesenchymal phenotype biomarker [25, 26]. Furthermore, vimentin protein 26 27 overexpression positively correlates with cell motility, induction of EMT, metastatic 28 29 30 disease and poor prognosis [27-31]. Vimentin-deficient (-/-) mice reveal weakened wound 31 32 healing ability in all stages of growth as a result of the seriously impaired fibroblasts in 33 34 their capacity to migrate [32, 33]. In this study, we found that p62 binds to vimentin and 35 36 37 regulates vimentin protein level, which in turn contributes to cancer cell invasion and 38 39 metastasis. Thus, this p62-vimentin interaction may be a promising target and provide 40 41 42 new opportunities for therapeutic intervention. 43 44 45 46 Materials and methods 47 48 49 Clinical samples, cell culture and reagents 50 51 All breast cancer specimens and adjacent normal tissues were obtained from clinical 52 53 diagnosed patients with prior patient consent and the approval of the Institutional Clinical 54 55 st 56 Ethics Review Board in the 2 Affiliated Hospital of Dalian Medical University. Samples 57 58 59 60 5 Page 17 of 210 Carcinogenesis 1 2 3 were kept in liquid nitrogen for protein extraction. 4 5 6 The human breast cancer cell lines (MDA-MB-231, SKBR-3, BT549 and MCF7), 7 8 the immortalized human breast epithelial cell MCF-10A and human embryonic kidney 9 10 11 HEK293T cell line were obtained from the American Type Culture Collection (ATCC). 12 13 The SUM149 cell line was kindly provided by Prof. Zhi-Min Shao (Deparment of 14 15 Medical Oncology, Cancer Hospital of Fudan University, Shanghai Medical College, 16 17 18 Shanghai, China). ForAll the cell Peer lines were tested Review and authenticated by the standard short 19 20 tandem repeat DNA typing methodology before used in this study [46]. Each cell line 21 22 was cultured in its standard medium as recommended by ATCC. SUM149 cells were 23 24 25 cultured in F-12 Hams (Gibco) supplemented with 5% FBS (Hyclone), 5 µg/ml insulin 26 27 (Sigma-Aldrich), and 1 µg/ml hydrocortisone (Sigma-Aldrich). The cells were last tested 28 29 in July 07th, 2015. 30 31 32 Plasmid construction 33 34 Full-length of p62 fragment was cloned from human genome cDNA and ligated into 35 36 37 pLVX-DsRed-Monomer-N1 vector (Clontech). The primers were as followed: p62: 5’ 38 39 Xho I, 5’-CCGCTCGAGCGGGCCACCATGGCGTCGCTCACCGTGAAGGCC; 3’ 40 41 EcoR I, 5’-CCGGAATTCCGTCACAACGGCGGGGGATGCTTTGA. Flag-p62 was a 42 43 44 gift from Prof. Song-Shu Meng (Department of Cancer Biology, Dalian Medical 45 46 University, Dalian, China). The short hairpin RNA (shRNA) targeting p62 (1#, 2#) and 47 48 the nontarget shRNA (SHC002) were kindly provided by Dr.