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Supplementary Materials and Methods, Supplementary Figure Legends, Supplementary Table S1 Alteration of SSBP1 protein expression levels in relation to clinicopathological characteristics in breast cancer patients. Supplementary Table S2 Antibodies used in this study. Supplementary Table S3 Primers for plasmid construction. Supplementary Table S4 Primers for ChIP. Supplementary Table S5 iTRAQ: Dysregulated proteins with gene symbol.

(A) The relative mtDNA content was analyzed by real-time PCR amplification of mtDNA encoded COX I and nuclear encoded GAPDH. (B) Statistical data of mitochondrial ROS generation in the indicated cells. (C) Western blot analysis of p-SMAD3 and SMAD3 expression in the indicated cells. (D) The migration abilities of indicated cell line were evaluated by Transwell assays in vitro. *** P < 0.005 compared with control. (E) Left: Control and CCCP-treated (20 μM for 2 h) MDA-MB-231 and MDA-MB-231shSSBP1 cells were incubated with or without added FK506 (10 nM). Total cellular protein fractions were subjected to western blot analysis using indicated antibodies. Right: Total cellular protein fractions of MDA-MB-231 cells transfected with CnA and treated with or without FK506 analyzed by western blot analysis using indicated antibodies. (F) Control and CCCP-treated (20 μM for 2 h) MDA-MB-231 and MDA-MB-231shSSBP1 cells were incubated with or without added FK506 (10 nM). Nuclear protein fractions were subjected to western blot analysis using indicated antibodies. (G) Western blot analysis of E-cadherin and Fibronectin expression in the indicated cells.

Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Mitochondrial function is dysregulated frequently in cancer cells to allow for adaptation to a harsh tumor microenvironment. Targeting mitochondrial biogenesis and bioenergetics is, therefore, an attractive therapeutic strategy. In this study, we performed quantitative proteomic analyses in human parental and metastatic breast cancer cell lines to identify mitochondrial proteins involved in TNBC metastasis. We found that single-strand DNA-binding protein 1 (SSBP1) was downregulated in highly metastatic breast cancer cells. Moreover, SSBP1 downregulation promoted TNBC cell metastasis in vitro and in vivo. Mechanistically, SSBP1 loss decreased mitochondrial DNA copy number, thereby potentiating calcineurin-mediated mitochondrial retrograde signaling that induced c-Rel/p50 nuclear localization, activated TGFβ promoter activity, and TGFβ-driven epithelial-to-mesenchymal transition. Low SSBP1 expression correlated with tumor progression and poor prognosis in patients. Collectively, our findings identified SSBP1 as a novel metastasis suppressor and elucidated the mechanisms by which dysregulated mitochondrial signaling contributes to metastatic potential, providing potential new prognostic indicators for patients with TNBC. Cancer Res; 76(4); 952–64. ©2015 AACR.

The objective of this study was to investigate the clinicopathological characteristics and survival outcomes of Paget disease (PD), Paget disease concomitant infiltrating duct carcinoma (PD‐IDC), and Paget disease concomitant intraductal carcinoma (PD‐DCIS). We identified 501,631 female patients from 2000 to 2013 in the Surveillance, Epidemiology, and End Results (SEER) database. These identified patients included patients with PD (n = 469), patients with PD‐IDC (n = 1832), and patients with PD‐DCIS (n = 1130) and infiltrating ductal carcinoma (IDC) (n = 498,076). Then, we compared the clinical characteristics of these patients with those who were diagnosed with IDC during the same period. The outcomes of these subtypes of breast carcinoma were different. Based on the overall survival, the patients with PD‐IDC had the worst prognosis (5‐year survival rate = 84.1%). The PD‐DCIS had the best prognosis (5‐year survival rate = 97.5%). Besides, among patients with Paget disease, the one who was married had a better prognosis than who were not. And, according to our research, the marital status was associated with the hormone receptor status in patients with PD‐IDC. Among three subtypes of Paget disease, patients with PD‐IDC had the worst prognosis. Besides, patients who were unmarried had worse outcomes. And the marital status of patients with PD‐IDC is associated with hormone status. The observation underscores the importance of individualized treatment.

// He-Fen Sun 1, 2 , Yang Zhao 1, 2 , Shui-Ping Gao 1, 2 , Liang-Dong Li 1, 2 , Wen-Yan Fu 1, 2 , Hong-lin Jiang 3 , Meng-Ting Chen 1, 2 , Li-Peng Yang 4 and Wei Jin 1, 2 1 Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Collaborative Innovation Center of Cancer Medicine, Fudan University Shanghai Cancer Center, Shanghai, 200030, China 2 Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200030, China 3 Division of Molecular Medicine and Genetic, Department of Internal Medicine and Life Sciences Insititute, University of Michigan, Ann Abor, Michgan 48109, USA 4 Department of pathology, School of Basic Medical Sciences, Fudan University, Shanghai, 200030, China Correspondence to: Wei Jin, email: jinwei7207@163.com Keywords: male breast cancer, race, overall survival, SEER Received: December 30, 2016 Accepted: May 15, 2017 Published: May 26, 2017 ABSTRACT To investigate the clinicopathological characteristics and survival outcomes of breast cancer in the male population, 8,607 cases of patients were identified in the Surveillance, Epidemiology, and End Results (SEER) database, including white males ( n = 7122), black males ( n = 1111), and other males (American Indian/AK Native, Asian/Pacific Islander) ( n = 374). Black male breast cancer patients were more likely to be in stages II–IV and have more advanced tumors. The rate of lymph node (LN) involvement at diagnosis was higher in black men than in whites and others. The ER- and PR-positive rates were lower in black men than in whites and others. The distant metastasis rate was higher in blacks than in whites and others. Furthermore, the overall survival (OR) rates and breast cancer-specific survival rates were significantly poorer in blacks than in whites and others (χ 2 = 29.974, P < 0.001; χ 2 = 7.285, P = 0.026, respectively). In a multivariate analysis, the results showed that race could also be a prognostic indicator ( P < 0.001). Moreover, significant differences were also observed in OS among 1:1:1 matched white, black, and other groups ( P < 0.001). Differences in outcomes may be partially explained by differences in tumor grades, LN status, and ER and PR status between the 3 groups. This study might provide insights into a better understanding of male breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Mitochondrial function is dysregulated frequently in cancer cells to allow for adaptation to a harsh tumor microenvironment. Targeting mitochondrial biogenesis and bioenergetics is, therefore, an attractive therapeutic strategy. In this study, we performed quantitative proteomic analyses in human parental and metastatic breast cancer cell lines to identify mitochondrial proteins involved in TNBC metastasis. We found that single-strand DNA-binding protein 1 (SSBP1) was downregulated in highly metastatic breast cancer cells. Moreover, SSBP1 downregulation promoted TNBC cell metastasis in vitro and in vivo. Mechanistically, SSBP1 loss decreased mitochondrial DNA copy number, thereby potentiating calcineurin-mediated mitochondrial retrograde signaling that induced c-Rel/p50 nuclear localization, activated TGFβ promoter activity, and TGFβ-driven epithelial-to-mesenchymal transition. Low SSBP1 expression correlated with tumor progression and poor prognosis in patients. Collectively, our findings identified SSBP1 as a novel metastasis suppressor and elucidated the mechanisms by which dysregulated mitochondrial signaling contributes to metastatic potential, providing potential new prognostic indicators for patients with TNBC. Cancer Res; 76(4); 952–64. ©2015 AACR.

Cancer metastasis and relapse are the primary cause of mortality for patients with breast cancer. The present study performed quantitative proteomic analysis on the differentially expressed proteins between highly metastatic breast cancer cells and parental cells. It was revealed that forkhead box P2 (FOXP2), a transcription factor in neural development, may become a potential inhibitor of breast cancer metastasis. The results demonstrated that patients with a lower level of FOXP2 expression had significantly poorer relapse-free survival (P=0.0047). The transcription of FOXP2 was also significantly downregulated in breast cancer tissue compared with normal breast tissue (P=0.0005). In addition, FOXP2 may inhibit breast cancer cell migration and invasion in vitro. It was also revealed that the underlying mechanism may include the epithelial-mesenchymal transition process driven by the tumor growth factor β/SMAD signaling pathway. In conclusion, the present study identified FOXP2 as a novel suppressor and prognostic marker of breast cancer metastasis. These results may provide further insight into breast cancer prevention and the development of novel treatments.

TIM50 is an essential component of TIM23 complex and involved in protein translocating into the inner mitochondrial membrane. Here, we found that TIM50 was increased in breast cancer cells by SILAC. However, its biological functions and molecular mechanisms in breast cancer are poorly understood. To gain insight into the functions of TIM50 in breast cancer, we constructed two stably transfected cell lines and examined TIM50 expression in tissue samples. Our data showed that TIM50 expression was increased in breast cancer. The stable suppression of TIM50 expression through lentivirus-mediated shRNA was shown to inhibit the abilities of cancer cell proliferation and induce apoptosis. What is more, depletion of TIM50 could decrease mitochondrial membrane potential, which may be associated with cell viability. Taken together, our findings reveal a new role for TIM50 in regulating cell proliferation and apoptosis through decreasing mitochondrial membrane potential in breast cancer cell and suggest that TIM50 might be a potential target for controlling breast cancer progression.

// Wenyan Fu 1, 2, * , Hefen Sun 1, 2, * , Yang Zhao 1, 2 , Mengting Chen 1, 2 , Lipeng Yang 3 , Shuiping Gao 1, 2 , Liangdong Li 1, 2 and Wei Jin 1, 2 1 Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Collaborative Innovation Center of Cancer Medicine, Fudan University Shanghai Cancer Center, Shanghai 200030, China 2 Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200030, China 3 Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai 200030, China * These authors contributed equally to this work Correspondence to: Wei Jin, email: jinwei7207@163.com Keywords: breast cancer; radiotherapy; radiation sequence with surgery; survival rate; SEER Received: August 20, 2017 Accepted: December 04, 2017 Published: May 11, 2018 ABSTRACT Background: Although neoadjuvant treatment has become the established approach for women with large primary tumors or locally advanced breast cancer for which immediate surgery is not the best approach, it may also stimulate cancer stem cell self-renewal and facilitate recurrence. We sought to determine the survival outcomes of preoperative radiotherapy (PRRT) compared with postoperative radiotherapy (PORT). Materials and Methods: The Surveillance, Epidemiology, and End Results (SEER) registry was queried for patients who were diagnosed with breast cancer and underwent cancer-directed surgery. Survival analyses were performed with Cox proportional hazard regression for both overall survival (OS) and disease-specific survival (DSS), and 1:1 propensity score (PS) matching-adjusted competing risk analyses were conducted for DSS. Results: We first identified 1,111,218 eligible patients in 18 registries from 1973 to 2013 and found that, outside of the Utah registry, sequence patterns other than PORT were rarely used. Thus, we next identified eligible patients registered in Utah ( n = 7,042) from 1988 to 2007. The treatment trends shifted abruptly in 1988. Compared with the PORT group, the PRRT group showed significantly higher risks of overall mortality (absolute difference, 22.4%; P < 0.001), breast cancer-specific mortality (absolute difference, 8.6%; P < 0.001), and cardiovascular disease-specific mortality (absolute difference, 11.5%; P = 0.021). Survival differences in treatment sequences were correlated with stage. Conclusions: Substantial shifts in treatment patterns for malignant breast cancer were identified in Utah. Compared with PORT, PRRT showed significantly worse outcomes. These results could inform future standardized options for radiation sequence with surgery and further clinical trials.

Background: Cytochrome c oxidase subunit VB (COX5B), a subunit of mammalian COX, takes roles in COX assembling and functions. Online database predicts high COX5B transcription may be associated with worse disease-free survival (DFS). However, the clinical implications of COX5B in breast cancer remain unclear. Methods: We carried out immunohistochemistry on tissue microarrays of 244 patients with invasive ductal breast carcinoma to detected COX5B expression. Results: Our results suggest that COX5B protein level might be associated with tumor size. COX5B overexpression indicated a worse DFS (p < 0.05) in breast cancer. Furthermore, high COX5B expression may act as an independent factor for worse DFS in breast cancer. Conclusions: Cumulatively, our findings suggest that COX5B might serve as an important prognostic factor for breast cancer.

UHRF1 is an epigenetic regulator and perform pivotal functions in cell tumorigenesis. We found UHRF1 is increased in breast cancer and patients with high UHRF1 levels have poorer prognoses than those with low UHRF1 levels. However, the underlying mechanisms remain largely unknown. Here, we found overexpression UHRF1 indeed promoted cell proliferation and migration, whereas its downregulation had the opposite functions. In vivo, UHRF1 also accelerated tumor growth. Mechanistically, microarrays were performed in MDA-MB-231 sh-UHRF1 and NC cells and KLF17, with rich CpG islands on its promoter region, finally caused our attention. Then, the expression of UHRF1 and KLF17 was testified negatively correlated in breast cancer cell lines and tissues. Additionally, the inhibition of cell proliferation and migration by UHRF1 depletion can be rescued by KLF17 silencing, suggesting KLF17 is downstream gene of UHRF1. The potential mechanism is that overexpression UHRF1 increased methylation of CpG nucleotides on KLF17 promoter, while UHRF1 silence decreased methylation. Collectively, our results demonstrated that increased UHRF1 can promote breast cancer cell proliferation and migration via silencing of KLF17 expression through CpG island methylation on its promoter.

The last step of ethylene biosynthesis in higher plants is to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). In our investigation, a cross-introns genomic DNA gene encoding ACC oxidase, PsgACO, was isolated from Paeonia suffruticosa. It revealed that PsgACO (FJ855434) has 1281 bases, containing four exons and three introns, encoding 312 amino acids. The four exons stretched from 1 to 105, 217 to 434, 592 to 925 and 1000 to 1281 bases. A splicing junction sequence of all the exon-introns conformed to the GT-AG rule in the cross-intron genomic DNA sequence of the ACO gene. PsgACO showed high homology to many characterized ACC oxidases both at the nucleic acid and amino acid levels. As well, twelve amino acid residues were conserved among many ACOs from other species. A phylogenetic tree analysis indicated that the amino acid of ACOs is quite conserved among the different eudicots. The phylogenetic tree showed that both the tree peony and herbaceous peony are quite isolated taxa. Bioinformatic analysis showed that the molecular weight of ACO is 35.29 kD, with a theoretical pI of 5.25. It is a non-secrete, stable hydrophilic protein, located in the cytoplasm.

Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank (AF248988), a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter (AF248988) was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vector pAtChs::GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.

COX5B, a peripheral subunit of the cytochrome c oxidase complex, has previously been reported to maintain the stability of this complex. However, its functions and mechanisms involved in breast cancer progression remain unclear. Here, by performing SILAC assays in breast cancer cell models and detecting COX5B expression in tissues, we found that COX5B expression was elevated in breast cancer. Down-regulation of COX5B in breast cancer cell lines can suppress cell proliferation and induced cell senescence which was accompanied by elevating production of IL-8 and other cytokines. Interestingly, conditioned medium from COX5B knockdown cells could promote breast cancer cell migration. Mechanistic studies reveal that COX5B silence induces an increase in production of ROS, depolarization of MMP and a decrease in ATP. What's more, silence of COX5B leads to metabolic disorders, such as increased glucose uptake and decreased lactate secretion. Collectively, our study shows that loss of COX5B induces mitochondrial dysfunction and subsequently leads to cell growth suppression and cell senescence. Cytokines such as IL-8 secreted by senescent cells may in turn alter the microenvironment which could enhance cell migration. These findings may provide a novel paradigm for the treatment which combined anti-cancer drugs with particular cytokine inhibitors such as IL-8 blockers.

Belonging to MADS-Box gene family, APETALA3 ( AP3 ) gene of Arabidopsis floral organ class B is expressed in petals and stamens specifically. AP3 gene which encodes a transcription factor controls the development of petals and stamens of dicotyledons by cooperation with class A and class C genes, respectively. The results showed that the promoter of AP3 was the flower-specific expression promoter. Therefore, the cloning and functional identification of AP3 gene promoter from Arabidopsis thaliana will play significant effects on the oriented improvement of the commercial traits related to the flowers of ornamental plants. Here, a pair of specific PCR primers was designed according to the promoter sequence of AP3 gene of Arabidopsis thaliana ecotype (Ler) reported in GenBank (U30729). A length of 1 767 bp of AP3 gene promoter was obtained from Arabidopsis thaliana ecotype (Col) by using PCR technique with high-fidelity DNA polymerase KOD-plus, named as pAtAP3 , the GenBank accession No. is FJ619533. Online Bl2seq analysis indicated that the sequence similarity between pAtAP3 and U30729 was 98%. It also showed that the sequence similarity between pAtAP3 and the BAC clone T12E18 (AL132971) of Arabidopsis thaliana ecotype (Col) from 9 264 to 11 030 was up to 100%, and its downstream sequence encoded AP3 protein (CAB81799), which approved that the sequence we cloned was the promoter of AP3 gene of Arabidopsis thaliana ecotype (Col). Online PLACE analysis displayed that pAtAP3 contained the basic cis -elements of promoter such as TATA-box and CAAT-box. It also comprised several cis -elements related to flower-specific expression, such as CArG1, CArG2, CArG3 and anther-box. Moreover, plant expression vector of pAtAP3 :: GUS was successfully constructed in this research, which would lay the foundation for functional identification of the AP3 promoter of Arabidopsis thaliana ecotype (Col).