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Rationale and Objectives: To evaluate whether ultrasound-based radiomics features can effectively predict HER2-low expression in patients with breast cancer (BC)., Material and Methods: Between January 2021 and June 2023, patients who received US scans with pathologically confirmed BC in this multicenter study were included. In total, 383 patients from institution 1 were comprised of training set, 233 patients from institution 2 were comprised of validation set and 149 patients from institution 3 were comprised of external validation set. Radiomics features were derived from conventional ultrasound (US) images. The minimum redundancy and maximum relevancy and the least absolute shrinkage and selector operation algorithm were used to generate an US-based radiomics score (RS). Multivariable logistic regression analysis was used to select variables associated with HER2 expressions. The diagnostic performance of the RS was evaluated through the area under the receiver operating characteristic curve (AUC)., Results: In the training set, the RS yield an AUC of 0.81 (95%CI: 0.76-0.84) for differentiation HER2-zero from HER2-low and -positive cases, and performed well in validation set (AUC 0.84, 95%CI: 0.78-0.88) and external validation set (AUC 0.82, 95%CI: 0.73-0.90). In the subgroups analysis, the RS showed good performance in distinguishing HER2-zero from HER2 1 + , HER2 2 + and HER2-low tumors (AUC range, 0.79-0.87)., Conclusion: The RS based on conventional US is proven effective for predicting HER2-low expression in BC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Background: Breast cancer-related lymphedema (BCRL) may benefit from acupuncture as a therapeutic. However, the findings of systematic reviews (SRs) and meta-analyses (MAs) are inconsistent and their quality needs to be evaluated critically. We aimed to provide an overview of the methodological quality, risk of bias, quality of reporting, and quality of evidence for SRs/MAs of acupuncture for BCRL., Methods: Publications were retrieved from four Chinese databases and four English databases. The methodological quality, risk of bias, reporting quality, and evidence quality of the included SRs/MAs were assessed by two independent researchers using the A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR-2), Risk of Bias in Systematic Reviews (ROBIS), Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), and Grading of Recommendations, Assessment, Development and Evaluation (GRADE), respectively., Results: There were a total of 8 SRs/MAs included. By AMSTAR-2, all SRs/MAs were graded as having low or very low methodological quality. By ROBIS, all SRs/MAs in phase 1, domain 1, and domain 4 of phase 2 were at low risk, while in domain 2 were at high risk. By PRISMA, reporting weaknesses in protocol and registration, as well as search method, were identified. By GRADE, the level of evidence quality was "low" to "very low", and the most commonly downgraded factor was the risk of bias., Conclusions: Acupuncture may be beneficial in improving BCRL. However, due to the identified limitations and conflicting findings, further more prescriptive and rigorous SRs/MAs are required to give strong evidence for final judgments., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This review pooled data from the literature to examine the association between preeclampsia (PE) and subsequent risk of breast cancer in women. Cohort studies published in the databases of PubMed, Embase, Scopus, and Web of Science up to 18 July 2023 were searched. Adjusted data were pooled to obtain the risk ratio (RR). Eleven studies with 15 cohorts and a cumulative sample size of 7,838,693 women were included. Meta-analysis of all studies demonstrated a reduced risk of breast cancer in women with PE as compared to those without PE (RR: 0.89 95% CI: 0.83, 0.95 p  < 0.001 I 2  = 50%). Follow-up ranged from 8 to 29.2 years. Results did not change during sensitivity analysis. Outcomes varied on subgroup analysis based on location, study type, data extraction method, incidence of breast cancer, and follow-up. To conclude, women with PE may have a reduced risk of breast cancer later in life. However, the risk reduction is minimal and may not have much clinical significance. The evidence is also limited by high inter-study heterogeneity and lack of adjustment of all possible confounders.

Objectives: This study aimed to determine whether post-neoadjuvant therapy (NAT) axillary ultrasound (AUS) could reduce the false-negative rate (FNR) of sentinel lymph node biopsy (SLNB). We also performed subgroup analyses to identify the appropriate patient for SLNB., Methods: A total of 220 patients with cytologically proven axillary node-positive breast cancer who underwent both SLNB and axillary lymph node dissection (ALND) after NAT were included. We calculated the FNR of SLNB. In the case of post-NAT AUS results available, AUS was classified as negative or positive. Then the FNR of post-NAT AUS combined with SLNB was evaluated. Subgroup analyses based on the number of sentinel lymph nodes removed, molecular subtypes, and the clinical N stage were also performed., Results: The overall axillary lymph node pathological complete response rate was 45.5% (100/220). The FNR of SLNB alone was 15.8% (95%CI: 9.2 to 22.5%). Post-NAT AUS results were available for 181 patients. When combined negative post-NAT AUS results and SLNB, the FNR was reduced to 7.5% (95%CI: 2.4 to 12.7%). Subgroup analyses of the FNR for SLNB alone and negative post-NAT AUS combined with SLNB were shown as follows: in cases patients with less than three sentinel lymph nodes (SLNs) and at least three SLNs removed, the FNR was decreased from 24.5 to 13.2%, and 9.0 to 5.0%, respectively. The FNR was decreased from 20.8 to 10.5% in HR+/HER2+subgroup, 21.4 to 16.7% in HR-/HER2+subgroup, 15.9 to 7.0% in HR+/HER2- subgroup, and 0% in HR-/HER2- subgroup, respectively. For cN1 patients, the FNR was decreased from 18.1 to 12.1% while 17.1 to 3.6% for cN2 patients and 0% for cN3 patients., Conclusion: Using negative post-NAT AUS may help to decrease the FNR and improve patient selection for SLNB., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: The purpose of our study is to present a method combining radiomics with deep learning and clinical data for improved differential diagnosis of sclerosing adenosis (SA)and breast cancer (BC)., Methods: A total of 97 patients with SA and 100 patients with BC were included in this study. The best model for classification was selected from among four different convolutional neural network (CNN) models, including Vgg16, Resnet18, Resnet50, and Desenet121. The intra-/inter-class correlation coefficient and least absolute shrinkage and selection operator method were used for radiomics feature selection. The clinical features selected were patient age and nodule size. The overall accuracy, sensitivity, specificity, Youden index, positive predictive value, negative predictive value, and area under curve (AUC) value were calculated for comparison of diagnostic efficacy., Results: All the CNN models combined with radiomics and clinical data were significantly superior to CNN models only. The Desenet121+radiomics+clinical data model showed the best classification performance with an accuracy of 86.80%, sensitivity of 87.60%, specificity of 86.20% and AUC of 0.915, which was better than that of the CNN model only, which had an accuracy of 85.23%, sensitivity of 85.48%, specificity of 85.02%, and AUC of 0.870. In comparison, the diagnostic accuracy, sensitivity, specificity, and AUC value for breast radiologists were 72.08%, 100%, 43.30%, and 0.716, respectively., Conclusions: A combination of the CNN-radiomics model and clinical data could be a helpful auxiliary diagnostic tool for distinguishing between SA and BC.

Introduction: During breast cancer chemotherapy, the chemoresistance that frequently accompanies the treatment has become a big challenge. Long noncoding RNAs (LncRNAs) have been related to the development of chemoresistance in multiple cancer types. LncRNA DDX11-AS1 has shown a carcinogenic role in lung and colorectal cancer and was reported to enhance oxaliplatin resistance in gastric cancer and Taxol insensitivity in esophageal cancer. But its role in breast cancer chemotherapy drug resistance remains unknown. This study aimed to investigate the function and mechanism of lncRNA DDX11-AS1 in breast cancer chemoresistance., Methods: The relationship between DDX11-AS1 and adriamycin (ADR) resistance was confirmed by qPCR, cell viability tests, and survival analysis. Then, RNA immunoprecipitation was conducted to evaluate the interaction between DDX11-AS1 and RNA-binding protein LIN28A. The regulation effect of LIN28A on autophagy-related genes ATG7 or ATG12 was detected by RNA stability assay and Western blot. Their correlation analysis was evaluated in GEO datasets and further validated by immunohistochemical results. The clinical significance of DDX11-AS1, ATG7, or ATG12 was evaluated by Kaplan-Meier Plotter analysis., Results: Here, we reported DDX11-AS1 was significantly upregulated in chemoresistant breast cancer cells and overexpression of DDX11-AS1 promoted ADR resistance in breast cancer. LIN28A could interact with DDX11-AS1 and was involved in DDX11-AS1-mediated ADR resistance. Interfering with LIN28A reversed DDX11-AS1-induced ADR resistance. LIN28A could increase the protein level of ATG7 and ATG12 by increasing their mRNA stability. Survival analysis showed that ATG12 expression level was negatively correlated with the prognosis of breast cancer patients., Conclusion: This study clarifies the role of DDX11-AS1 in breast cancer chemoresistance and revealed a new mechanism, that is, interacting with LIN28A to stabilize ATG7 and ATG12 and jointly promote chemorefractory. These findings warrant further in vivo investigations to study DDX11-AS1 as a potential target to overcome chemoresistance., (© 2022 S. Karger AG, Basel.)

A carbon nanotube (CNT) supported single-site Fe-N-C catalyst (CNTs/Fe-N-C) exhibited attractive properties in peroxidase (POD)-like activity and photothermal effect. Herein, we designed a therapeutic platform by wrapping doxorubicin (DOX) in mesoporous CNTs/Fe-N-C with the cell membrane (CM) of breast cancer. The ultimate nanoagent (CNTs/Fe-N-C/DOX/CM) exhibited high antitumor activity on account of its efficient catalytic ability, increased drug release rates, and significant photothermal effect. Due to the POD-like activity, CNTs/Fe-N-C could effectively catalyze hydrogen peroxide (H 2 O 2 ) into cytotoxic hydroxyl radicals ( • OH) for chemodynamic therapy (CDT) of the tumor. Besides, the released DOX not only merely induced the diagnosis of the tumor cells for chemotherapy (CT) but also generated H 2 O 2 to promote CDT. Moreover, the photothermal effect of the nanoagent could use for photothermal therapy (PTT). The increasing temperature was conducive to the release of DOX from micropore into the cell, which indirectly enhanced CT and CDT effects. As an intelligent and multifunctional drug delivery platform, the present CNTs/Fe-N-C/DOX/CM nanoagent could be engineered with synergistic treatments and favorable biosafety, which provides a promising paradigm in site-specific antitumor treatment and biomedicine.

Rationale: The decreased HER2-accessibility by epitope masking is a primary trastuzumab-resistance mechanism. In this study, we developed a HER2-targeted dual radiotracer approach to predict the HER2-trastuzumab engagement noninvasively. Methods: Two novel HER2-specific VHHs, MIRC208 and MIRC213, were acquired by immunizing alpaca with human HER2 protein, and were site-specifically labeled with 99m Tc. Biodistribution and SPECT/CT imaging studies were performed in mice bearing HER2-positive and HER2-negative tumors. The HER2 binding sites of 99m Tc-MIRC208 and 99m Tc-MIRC213 were investigated by cell binding and SPECT/CT imaging studies. We evaluated the therapeutic predictive ability of our dual-radiotracer imaging approach for trastuzumab treatment in mice bearing MUC4-positive tumors (trastuzumab-resistant JIMT-1 and 87MUC4) and MUC4-negative tumors (trastuzumab-sensitive 7HER2 and NCI-N87). The preliminary clinical studies of 99m Tc-MIRC208 were performed in two patients with HER2-positive breast tumors. Results: 99m Tc-MIRC208 and 99m Tc-MIRC213 clearly visualized HER2-positive tumors, but not HER2-negative tumors. 99m Tc-MIRC208 competes with trastuzumab for HER2-binding while 99m Tc-MIRC213 recognizes HER2 on an epitope that is not masked by MUC4. The SPECT/CT studies with 99m Tc-MIRC208 and 99m Tc-MIRC213 clearly showed that the MUC4-negative and trastuzumab-sensitive 7HER2 and NCI-N87 tumors had very similar tumor uptake with the SUV 208 /SUV 213 (2 h) ratios of 1.11 ± 0.17 in 7HER2 and 1.25 ± 0.22 in NCI-N87. However, the MUC4-positive JIMT-1 tumors showed the decreased SUV 208 /SUV 213 (2 h) ratio (0.63 ± 0.07), which correlated well with the low response rate to trastuzumab therapy. The SUV 208 /SUV 213 (2 h) ratio was reduced to 0.72 ± 0.02 in MUC4-expressing NCI-N87 cells, and resulting in the decreased trastuzumab sensitivity, further supporting the correlation between the SUV 208 /SUV 213 (2 h) ratio and trastuzumab-sensitivity. The primary and metastatic HER2-positive lesions of patients were clearly visualized by 99m Tc-MIRC208 SPECT at 2 h post injection. Conclusion: Overall, we demonstrated that the dual radiotracer imaging strategy is a valid noninvasive approach for the cancer patient selection before trastuzumab therapy. 99m Tc-MIRC213 SPECT is utilized to quantify the tumor HER2 expression and screen HER2-positive cancer patients, while 99m Tc-MIRC208 SPECT is used to determine the HER2-accessibility of trastuzumab. The SUV 208 /SUV 213 (2 h) ratio is an important biomarker to determine the responsiveness of trastuzumab therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objective: To explore the value of ultrasonic multimodality imaging for characterizing nonpuerperal mastitis (NPM) lesions and feasibility of distinguishing different subtypes., Methods: Thirty-eight NPM lesions were assessed using conventional ultrasonography (US), strain elastography (SE), and contrast-enhanced ultrasound (CEUS). The lesions were confirmed pathologically and classified as granulomatous lobular mastitis (GLM), plasma cell mastitis (PCM), or nonspecific mastitis (NSM). Furthermore, diagnostic indicators were evaluated. The diagnostic performances of the modalities were compared using the area under the receiver operating characteristic curve (AUC)., Results: The overall morphological features on US differed significantly between the GLM and PCM groups (p = 0.002). Lesion size (≤10 mm) (p = 0.003) and mean SE score (p = 0.001) differed significantly between the PCM and NSM groups. The frequent NPM characteristic on CEUS was hyperenhancement with (or without) increased lesion size; intergroup differences were not significant. Breast Imaging Reporting and Data System > 3 was considered to indicate malignancy; accordingly, the accuracy of US alone, US with CEUS, and US with SE was 10.5%, 21.1%, and 65.8%, respectively. Moreover, the AUC for US with SE for classifying GLM and PCM was 0.616., Conclusion: CEUS cannot accurately classify NPM subtypes, while US and SE are valuable for classification., (© 2022 Wiley Periodicals LLC.)

This study was conducted to investigate the expression of the spindle assembly checkpoint kinase tyrosine/threonine kinase (TTK) in triple positive breast cancer (TPBC) and its effect on TPBC cells. We analyzed the status of TTK in 69 TPBC samples using immunohistochemistry. The correlation between TTK and clinicopathological parameters was analyzed using a chi-squared test. The prognostic value of TTK was evaluated using Kaplan-Meier survival curves. We analyzed the role of TTK in the invasion and proliferation of TPBC cells in vitro and in vivo. The mean age of the 69 patients with TPBC enrolled in this study was 53 years (range: 29-86 years). TTK expression was positively correlated with tumor size (p=0.034), p53 status (p=0.023), TNM stage ([p=0.023), and Ki-67 index (p<0.001). The Kaplan-Meier curves revealed that TTK expression was correlated with poor disease-free survival (p=0.001) and overall survival (p=0.050). Multivariate proportional hazard regression analyses showed that TTK and TNM staging were significant independent predictors of disease-free survival (p=0.007 and p=0.034, respectively). Additionally, TTK knockdown inhibited the invasion and proliferation of the BT474 TPBC cell line. The findings of this study indicate that TTK overexpression is associated with cancer progression and prognosis in patients with TPBC, whereas TTK knockdown inhibits the invasion and proliferation of TPBC cells. Thus, TTK might serve as a prognostic marker for TPBC.

MicroRNAs (miRNA) have been shown to be associated with tumor diagnosis, prognosis, and therapeutic response. MiR-328-3p plays a significant role in breast cancer growth; however, its actual function and how it modulates specific biological functions is poorly understood. Here, miR-328-3p was significantly downregulated in breast cancer, especially in patients with metastasis. Mitochondrial carnitine palmitoyl transferase 1a (CPT1A) is a downstream target gene in the miR-328-3p-regulated pathway. Furthermore, the miR-328-3p/CPT1A/fatty acid β-oxidation/stemness axis was shown responsible for breast cancer metastasis. Collectively, this study revealed that miR-328-3p is a potential therapeutic target for the treatment of breast cancer patients with metastasis, and also a model for the miRNA-fatty acid β-oxidation-stemness axis, which may assist inunderstanding the cancer stem cell signaling functions of miRNA., (© 2021. The Author(s).)

Background: Continual expression of PD-L1 in tumor cells is critical for tumor immune escape and host T cell exhaustion, however, knowledge on its clinical benefits through inhibition is limited in breast cancer. N 6 -methyladenosine (m 6 A) plays a crucial role in multiple biological activities. Our study aimed to investigate the regulatory role of the m 6 A modification in PD-L1 expression and immune surveillance in breast cancer., Methods: MeRIP-seq and epitranscriptomic microarray identified that PD-L1 is the downstream target of METTL3. MeRIP-qPCR, absolute quantification of m 6 A modification assay, and RIP-qPCR were used to examine the molecular mechanism underlying METTL3/m 6 A/IGF2BP3 signaling axis in PD-L1 expression. B-NDG and BALB/c mice were used to construct xenograft tumor models to verify the phenotypes upon METTL3 and IGF2BP3 silencing. In addition, breast cancer tissue microarray was used to analyze the correlation between PD-L1 and METTL3 or IGF2BP3 expression., Results: We identified that PD-L1 was a downstream target of METTL3-mediated m 6 A modification in breast cancer cells. METTL3 knockdown significantly abolished m 6 A modification and reduced stabilization of PD-L1 mRNA. Additionally, METTL3-mediated PD-L1 mRNA activation was m 6 A-IGF2BP3-dependent. Moreover, inhibition of METTL3 or IGF2BP3 enhanced anti-tumor immunity through PD-L1-mediated T cell activation, exhaustion, and infiltration both in vitro and in vivo. PD-L1 expression was also positively correlated with METTL3 and IGF2BP3 expression in breast cancer tissues., Conclusion: Our study suggested that METTL3 could post-transcriptionally upregulate PD-L1 expression in an m 6 A-IGF2BP3-dependent manner to further promote stabilization of PD-L1 mRNA, which may have important implications for new and efficient therapeutic strategies in the tumor immunotherapy., (© 2022. The Author(s).)

Objective: This study is to explore the correlation between the contrast-enhanced ultrasound (CEUS) characteristics of breast cancer and the epithelial-mesenchyme transformation (EMT)., Methods: Totally 119 patients of breast cancer underwent CEUS. Tissues in the active area were collected and subjected to the immunohistochemical detection, PT-PCR and Western blot. Correlation analysis was conducted between the clinical pathological parameters and the CEUS indicators., Results: The expression levels of CD44, N-cadherin, and β-catenin in breast cancer tissues were higher than those in adjacent tissues (P<0.05). However, the expression levels of CD24 and E-cadherin in breast cancer tissues were lower than those in adjacent tissues (P<0.05). There was no significant difference in E-cadherin mRNA and Vimentin levels between cancer and adjacent tissues (P>0.05). The expressions were up-regulated in the CSCs, with higher histological grade, lymph node metastasis, and negative estrogen receptor (ER) expression. Smaller breast tumors, with no lymph node metastasis, lower clinical stage, and positive ER expression, tended to exhibit the up-regulated epithelial phenotype. Breast tumors, with high histological grade, lymph node metastasis, high clinical staging grade, and negative ER expression, tended to exhibit the up-regulated interstitial phenotype. The peak intensity of the time-intensity curve (TIC) for the CEUS was positively correlated with the CSC marker CD44 and the interstitial phenotype marker N-cadherin. The starting time of enhancement was negatively correlated with the N-cadherin. Area under the curve was positively correlated with the expression of CD44 and N-cadherin, while negatively correlated with the epithelial phenotype marker β-catenin. The time to peak was negatively correlated with the interstitial phenotypes Vimentin and N-cadherin, with no correlation with the E-cadherin or β-catenin., Conclusion: Breast cancers show the enlarged lesions after enlargement and perfusion defect for the CEUS. The fast-in pattern, high enhancement, and high perfusion in the TIC are correlated with the CSCs and EMT expressions, suggesting poor disease prognosis., Competing Interests: The authors have declared that no competing interests exist.

Circular RNA (circRNA) is closely related to tumorigenesis and cancer progression. Yet, the roles of cancer-specific circRNAs in the circRNA-related ceRNA network of breast cancer (BRCA) remain unclear. The aim of this study was to construct a ceRNA network associated with circRNA and to explore new therapeutic and prognostic targets and biomarkers for breast cancer. We downloaded the circRNA expression profile of BRCA from Gene Expression Omnibus (GEO) microarray datasets and downloaded the miRNA and mRNA expression profiles of BRCA from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNAs (DEmRNAs), differentially expressed miRNAs (DEmiRNAs), and differentially expressed circRNAs (DEcircRNAs) were identified, and a competitive endogenous RNA (ceRNA) regulatory network was constructed based on circRNA-miRNA pairs and miRNA-mRNA pairs. Gene ontology and pathway enrichment analyses were performed on mRNAs regulated by circRNAs in ceRNA networks. Survival analysis and correlation analysis of all mRNAs and miRNAs in the ceRNA network were performed. A total of 72 DEcircRNAs, 158 DEmiRNAs, and 2762 DE mRNAs were identified. The constructed ceRNA network contains 60 circRNA-miRNA pairs and 140 miRNA-mRNA pairs, including 40 circRNAs, 30 miRNAs, and 100 mRNAs. Functional enrichment indicated that DEmRNAs regulated by DEcircRNAs in ceRNA networks were significantly enriched in the PI3K-Akt signaling pathway, microRNAs in cancer, and proteoglycans in cancer. Survival analysis and correlation analysis of all mRNAs and miRNAs in the ceRNA network showed that 13 mRNAs and 6 miRNAs were significantly associated with overall survival, and 48 miRNA-mRNA interaction pairs had a significant negative correlation. A PPI network was established, and 21 hub genes were determined from the network. This study provides an effective bioinformatics basis for further understanding of the molecular mechanisms and predictions of breast cancer. A better understanding of the circRNA-related ceRNA network in BRCA will help identify potential biomarkers for diagnosis and prognosis., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Han Sheng et al.)

Objectives: Clarifying the expression of breast cancer receptor is the key to clinical treatment for breast cancer. This study aims to explore the correlation between X-ray and clinical characteristics of 4 molecular subtypes and their receptor types of breast cancer., Methods: A total of 439 breast cancer patients who confirmed by pathology and performed X-ray examination were enrolled. The X-ray and clinical characteristics of 4 molecular subtypes and the expression of their receptors were analyzed., Results: Luminal A type showed the highest proportion of spiculate masses, and the lowest calcification score, showing significant difference with other 3 subtypes (all P <0.001). The age in the human epidermal growth factor 2 (HER2) overexpression type group was older, the proportions of menopause, the calcification score, and the calcification score with 9-12 were higher, the sizes of the tumor were greater in the HER2 overexpression type group than those in the other 3 molecular subtype groups (age P <0.05, the rest P <0.01). The proportions of regular shape, edge indistinct, and high-grade invasive ductal carcinoma in the triple-negative type group were higher than those in the other 3 molecular subtype groups (all P <0.001). The proportions of non-menopausal patients and spiculate tumors in the estrogen receptor (ER) positive and/or progesterone receptor (PR) positive groups were higher than those in both ER and PR negative group ( P <0.001 and P =0.001, respectively). The proportions of calcification fraction and high-grade invasive ductal carcinoma were higher, tumor sizes were greater in the HER2 positive group, Ki-67≥20% group than those in the HER2 negative group, Ki-67<20% group, respectively ( P <0.001 or P <0.05, respectively)., Conclusions: Four molecular subtypes of breast cancer and their receptor expressions are correlated with X-ray and clinical characteristics, which can provide a basis for clinical diagnosis and treatment.

Objective To investigate the effect of progranulin (PGRN) on the invasion and migration of mouse breast cancer 4T1 cells and its mechanism. Methods After treated with PGRN (1 μg/mL) for 24 hours, the invasion ability of breast cancer 4T1 cells was detected by Transwell TM invasion assay, the migration ability was detected by scratch test, and the epithelial cadherin (E-cadherin), vimentin mRNA expression was detected by real-time fluorescent quantitative PCR. Western blot assay was used to detect the expression of E-cadherin, vimentin, extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2). After treated with 1 μg/mL PGRN and ERK1/2 signaling pathway inhibitor U0126 (10 μmol/L) simultaneously, the migration and invasion ability of 4T1 cells and the changes in the expression of E-cadherin, vimentin and p-ERK proteins were detected again. Results After treated with PGRN, the migration and invasion capabilities of breast cancer 4T1 cells were significantly enhanced; E-cadherin expression decreased; vimentin and p-ERK1/2 expression increased. After treated with ERK1/2 signaling pathway inhibitor, the ability of PGRN to promote breast cancer 4T1 cell migration, invasion and epithelial-mesenchymal transition (EMT) was significantly inhibited. Conclusion PGRN can promote the migration and invasion of breast cancer 4T1 cells by promoting EMT and activating the ERK1/2 pathway.

Curcumin has a variety of anticancer properties, but low bioavailability prevents its use in chemotherapeutic applications. To address this problem, we tested the efficacy of the synthetic curcumin analog B14 in breast cancer cells and explored the mechanism by which B14 inhibits proliferation and metastasis of breast cancer cells. We used the breast cancer cell line MCF-7, MDA-MB-231 to study the anticancer effects of B14 and assessed cell viability, cell migration and invasion, cell cycle, and apoptosis, in addition, the antitumor effect of B14 in vivo was examined in mice bearing MDA-MB-231 cells. We found that, as the concentration of B14 increased, cell viability decreased in a dose-dependent manner. Compound B14 exerted the best antitumor activity and selectivity for MCF-7 and MDA-M-231 cells (IC 50  = 8.84 μmol/L and 8.33 μmol/L, respectively), while its IC 50 value for MCF-10A breast epithelial cells was 34.96 μmol/L. B14 has been shown to be a multi-targeted drug that alters the expression of cyclin D1, cyclin E1, and cyclin-dependent kinase 2 (CDK2), and ultimately induces G1 phase cell cycle arrest. At the same time, B14 activates the mitochondrial apoptosis pathway in breast cancer cells. Furthermore, B14 was more effective than curcumin in inhibiting cell migration, invasion, and colony formation. In tumor-bearing mice, analog B14 significantly reduced tumor growth and inhibited cell proliferation and angiogenesis. The pharmacokinetic test found that B14 was more stable than curcumin in vivo. Our data reveal the therapeutic potential of the curcumin analog B14 and the underlying mechanisms to fight breast cancer cells., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Progranulin (PGRN), as a multifunctional growth factor, is overexpressed in multiple tumors, but the role of PGRN on tumor immunity is still unclear. Here, we studied the effect of PGRN on breast cancer tumor immunity and its possible molecular mechanism., Methods: The changes of macrophage phenotypes after PGRN treatment were detected by western blot, quantitative polymerase chain reaction (PCR) and flow cytometry. Western blot was used to study the signal molecular mechanism of PGRN regulating this process. The number and localization of immune cells in Wild-type (WT) and PGRN -/- breast cancer tissues were analyzed by immunohistochemical staining and immunofluorescence techniques. The activation and proliferation of CD8 + T cells were measured by flow cytometry., Results: After being treated with PGRN, the expressions of M2 markers and programmed death ligand 1 (PD-L1) on macrophages increased significantly. Signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitor Stattic significantly inhibited the expression of PD-L1 and M2 related markers induced by PGRN. In WT group, CD8 were co-localized with macrophages and PD-L1, but not tumor cells. The number of immune cells in PGRN -/- breast cancer tissue increased, and their infiltration into tumor parenchyma was also enhanced. Moreover, in the co-culture system, WT peritoneal macrophages not only reduced the ratio of activated CD8 + T cells but also reduced the proportion of proliferating CD8 + T cells. The addition of programmed death receptor 1 (PD-1) and PD-L1 neutralizing antibodies effectively reversed this effect and restored the immune function of CD8 + T cells., Conclusion: These results demonstrate that PGRN promotes M2 polarization and PD-L1 expression by activating the STAT3 signaling pathway. Furthermore, through PD-1/PD-L1 interaction, PGRN can promote the breast tumor immune escape. Our research may provide new ideas and targets for clinical breast cancer immunotherapy.

Breast cancer is one of the most malignant diseases world-wide and ranks the first among female cancers. Progranulin (PGRN) plays a carcinogenic role in breast cancer, but its mechanisms are not clear. In addition, there are few reports on the relationship between PGRN and tumor-associated macrophages (TAMs)., Aims: To investigate the effects of exosomes derived from PGRN -/- TAMs on invasion and migration of breast cancer cells., Main Methods: Mouse breast cancer xenograft model was constructed to explore the effect of PGRN -/- tumor environment (TME) on breast cancer. Flow cytometry was used to compare TAMs of wild type (WT) and PGRN -/- tumor tissue. Transwell assay, wound healing assay and western blot were used to explore the effect of WT and PGRN -/- TAMs and their exosomes on invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer cells. MicroRNA (miRNA) assay was used to find out the differentially expressed miRNA of negative control (NC) and siPGRN-TAMs exosomes. Quantitative PCR and luciferase report assay were used to explore the target gene., Key Findings: The lung metastasis of breast cancer of PGRN -/- mice was inhibited. PGRN -/- TAMs inhibited invasion, migration and EMT of breast cancer cells through their exosomes. MiR-5100 of PGRN -/- TAMs-derived exosomes was up-regulated, which might regulate expression of CXCL12, thereby inhibiting the CXCL12/CXCR4 axis, and ultimately inhibiting the invasion, migration and EMT of breast cancer cells., Significance: Our study elucidates a new molecular mechanism of lung metastasis of breast cancer, so it may contribute to efficient prevention and therapeutic strategies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: In recent years, the regulatory activities of long noncoding RNAs have received increasing attention as an important research focus. This study aimed to characterize the expression and detailed roles of TTC39A antisense RNA 1 (TTC39A-AS1) in breast cancer (BC), in addition to concentrating on its downstream mechanisms., Methods: Quantitative RT-PCR was performed to determine the expression levels of TTC39A-AS1, microRNA-483-3p (miR-483-3p), and metastasis-associated gene 2 (MTA2). Further, the detailed functions of TTC39A-AS1 in BC cells were confirmed using the Cell Counting Kit 8 assay, flow cytometric analysis, and Transwell cell migration and invasion assays. The targeting relationship between TTC39A-AS1, miR-483-3p, and MTA2 in BC was predicted via bioinformatics analysis and further confirmed by performing the luciferase reporter assay and RNA immunoprecipitation., Results: TTC39A-AS1 was present in high levels in BC; this result was confirmed in our sample cohort and The Cancer Genome Atlas database. Patients with BC with a high level of TTC39A-AS1 had a shorter overall survival than those with a low level of TTC39A-AS1. Functionally, the absence of TTC39A-AS1 accelerated cell apo-ptosis but retained cell proliferation, migration, and invasion. Mechanistically, TTC39A-AS1 functioned as a competing endogenous RNA in BC by sponging miR-483-3p and thereby indirectly increasing MTA2 expression. Finally, rescue experiments revealed that the tumor-inhibiting actions of TTC39A-AS1 knockdown on the malignant characteristics of BC cells could be reversed by inhibiting miR-483-3p or upregulating MTA2., Conclusion: The newly identified TTC39A-AS1/miR-483-3p/MTA2 pathway was revealed to be a critical regulator in the tumorigenicity of BC, possibly offering a novel therapeutic direction for the anticancer treatment of BC., (© 2021 S. Karger AG, Basel.)

Aims: It is well known that pyruvate dehydrogenase kinase 1 (PDK1) is highly expressed in breast cancer (BC) tissues and promotes tumor growth, but the underlying mechanisms of this process are unclear. Here, we investigated the effects of nuclear PDK1 on growth, migration and invasion in human BC cells., Main Methods: The sub-cellular localization of PDK1 in BC cells was performed with subcellular fractionation followed by Western blot and immunofluorescence. The localization of PDK1 in breast normal tissue and breast duct carcinoma was detected by Immunohistochemistry. Then the protein-protein interaction between PDK1 and Importin β was verified by co-immunoprecipitation assay. Finally, the effects of nuclear PDK1 on cell proliferation, apoptosis, migration and invasion of BC cells were assessed., Key Findings: In addition to its well-known sub-cellular localization, PDK1 was present in the nucleus of BC cells, and EGF treatment increased nucleus distribution of PDK1. Moreover, the level of nuclear PDK1 accumulation facilitated the growth of BC cells. We also found that the entry of PDK1 into nucleus mainly relied on the nuclear localization signal (NLS), and NLS mutation inhibited the entry of PDK1 into nucleus; as a result, the migration and invasion abilities of BC cells were impaired, and the number of apoptotic cells was significantly increased., Significance: Our findings provided a new supplement to the sub-cellular localization of PDK1 in BC cells and uncovered the function of nuclear PDK1 in facilitating BC cells growth, migration and invasion., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: This study aimed to compare the diagnostic performance of contrast-enhanced ultrasound (CEUS), MRI, and the combined use of the two modalities for differentiating breast lesions of different sizes., Methods: A total of 406 patients with 406 solid breast masses detected by conventional ultrasound underwent both CEUS and MRI scans. Histological results were used as reference standards. The lesions were categorized into three groups according to size (Group 1, ≤ 20 mm; Group 2, > 20 mm, Group 3: total lesions). Sensitivity, specificity, accuracy, and receiver operating characteristic (ROC) curve analysis were used to assess the diagnostic performance of these imaging methods for breast lesions., Results: There were 194 benign and 212 malignant breast lesions according to the histological diagnosis. Compared with MRI, CEUS demonstrated similar sensitivity in detecting breast cancer ( p = 1.0000 for all) in all the three groups. With regard to specificity, accuracy, and the area under the ROC curve (Az) values, MRI showed a better performance than that shown by CEUS (p ＜0.05 for all), and the combination of the two modalities improved the diagnostic performance of CEUS alone significantly (p ＜0.05 for all) in all the three groups. However, the diagnostic specificity and accuracy of the combined method was not superior to that of MRI alone except for Group 2., Conclusion: CEUS demonstrated good sensitivity in detecting breast cancer, and the combined use with MRI can optimize the diagnostic specificity and accuracy in breast cancer prediction., Advances in Knowledge: Few studies have compared the diagnostic efficacy of CEUS and MRI, and this study is the first attempt to seek out the diagnostic values for breast lesions of variable sizes (lesions with ≤20 mm and >20 mm).

Breast cancer gene 1 and 2 ( BRCA1 and BRCA2 ) are human tumor suppressor genes. BRCA mutations increase the risk for breast, ovarian, and gastric cancer. However, double heterozygosity for BRCA1 and BRCA2 mutations in gastric cancer have not been reported and their clinical significance is unclear. In this study, a 52-year-old Chinese male patient with gastric cancer was chosen for analysis. A tumor tissue biopsy and blood sample were collected, and next-generation sequencing-based deep panel sequencing was performed on the IlluminaNextSeq-500 platform. Comprehensive genomic alterations of 450 cancer-related genes and 47 tumor susceptibility genes were analyzed. Here, we report for the first time a case of gastric cancer that carried both BRCA1 S1841Vfs*2 and BRCA2 Q1886* heterozygous mutations. Unfortunately, the patient was resistant to olaparib treatment. Further RNA analysis revealed that only the wild-type alleles of BRCA1 and BRCA2 were expressed, although genetic BRCA1 and BRCA2 mutations were present in the patient. This genetic finding may explain the mechanism for primary resistance to olaparib observed in the BRCA1/2 -mutated patient.

Introduction: We evaluated the current status of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) detection in invasive breast carcinoma (IBC) in various laboratories across China., Materials and Methods: The Breast Pathology Study Group of the Chinese Society of Pathologists collected HR and HER2 data from 12,467 IBC cases from 19 representative clinical centers in China. The data from every center were compared with the pooled data from the other centers., Results: The assessment of HR status showed that the overall positive estrogen receptor (ER) and progesterone receptor (PR) rates were 71.7% and 63.7% (range, 60.9%-87.9% and 43.9%-84.8%), respectively. The ER results in 3 centers and the PR results in 6 centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). Of the 12,467 cases, 62.4% were ER + /PR + , 9.3% were ER + /PR - , 1.3% were ER - /PR + , and 27.0% were ER - /PR - . The assessment of HER2 status showed that the overall positive rate of HER2 (with a definition of immunohistochemistry [IHC] 3+ or IHC2+/ in situ hybridization-positive) was 24.7% (range, 13.7%-35.7%) in each center. Three centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). The proportion of HER2 IHC3+ was 21.1%. The positive rates of HER2 gene amplification in IHC 0/1+, 2+, 3+ cases were 2.0%, 17.6%, and 85.9%, respectively., Conclusions: As the largest study of HR and HER2 status in Chinese patients with IBC, the data from the present study have indicated that the overall rates of HR and HER2 were comparable to those reported in previous studies. However, the rates varied among the laboratories. Individual centers had not met the target values, and they need to improve the detection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To evaluate the efficacies of conventional ultrasound (US), US elasticity imaging (EI), and acoustic radiation force impulse (ARFI) elastography in breast malignancy diagnosis., Methods: We included 315 women (mean age, 44 years; range, 18-81 years) with 336 pathologically proven breast lesions in this retrospective study. All lesions underwent conventional US, EI, and ARFI (including virtual touch tissue imaging [VTI], virtual touch tissue quantification [VTQ], and virtual touch tissue imaging and quantification [VTIQ]) elastography. Multivariate logistic regression analysis was performed to assess 12 independent variables for malignancy prediction. Diagnostic performance was evaluated with receiver operating characteristic (ROC) curve analysis., Results: Irregular lesion shape was the strongest independent predictor for breast malignancy, followed by poorly defined margins, taller than wide dimensions, posterior echo attention, VTIQ, and VTI boundaries (P < 0.05). Area under the ROC curve (AUC) for VTIQ was higher than other significant independent variables. With the best cut-off value of 3.74 m/s, the AUC, sensitivity, and specificity were 0.93 (95% CI: 0.90, 0.96), 90.1%, and 91.1%, respectively., Conclusions: ARFI elastography is a promising method in breast malignancy prediction, with good diagnostic performance. For patients requiring surgery, the combination of various methods can provide better diagnostic results and may help to reduce unnecessary biopsy or surgery.

Background: Epithelioid sarcoma (ES) is a rare malignant soft tissue tumor, commonly occurring in distal extremities, such as fingers, hands and wrists. For oncologists and surgeons, a female patient with enlarged axillary lymph node on one side only is easily diagnosed with an occult breast carcinoma rather than ES. Besides, whole breast metastasis of ES has not been reported yet., Case Presentation: A 47-year-old Chinese woman came to the outpatient clinic of First Affiliated Hospital of Zhejiang University (FAHZU) with a complaint of an asymptomatic right axillary mass for 3 months. Then she received surgical resection of the right axillary lymph nodes and right supraclavicular lymph nodes. According to the clinical tumor site and routine immunohistochemistry (IHC), suspicion of metastatic epithelial sarcoma and metastatic breast cancer could not be ruled out. Subsequently, with more detailed medical history review and physical examination, a mass on the right forearm was found, which was considered as the primary lesion. Further IHC and Molecular Genetics revealed that all the neoplastic cells exhibited loss of INI1 protein and were negative for ERG gene rearrangement yet positive for epithelial membrane antigen (EMA), cytokeratin (CK) 8, CK19, Vimentin, CD34. The final diagnosis was ES. She received postoperative chemotherapy, without radiotherapy. Unexpectedly, an ipsilateral breast metastasis was developed at ten months after surgery. Regrettably, there was no positive result of the metastatic breast sample, based on a genome sequencing by a 381-cancer-gene panel in a lab. Therefore, she went through another round of chemotherapy and took Apatinib for maintenance treatment. During the last follow-up (26 months after diagnosis), the disease was under control., Conclusion: This rare but interesting case enables breast surgeons and pathologists to accumulate more experience of differential diagnosis of axillary mass for personalized treatment in clinical practice. Meanwhile, ipsilateral breast metastasis of ES we reported in the case urges that clinicians attach greater importance to the tumor metastasis mechanism.

Aim: To identify novel competing endogenous RNA (ceRNA) network related to patients prognosis in breast cancer. Materials & methods: Dysregulated mRNA based on intersection of three Gene Expression Omnibus and The Cancer Genome Atlas datasets were analyzed by bioinformatics. Results: In total 72 upregulated and 208 downregulated genes were identified. Functional analysis showed that some pathways related to cancer were significantly enriched. By means of stepwise reverse prediction and validation from mRNA to lncRNA, 19 hub genes, nine key miRNA and four key lncRNAs were identified by expression and survival analysis. Ultimately, the coexpression analysis identified RRM2 -let-7a-5p- SNHG16 / MAL2 as key ceRNA subnetwork associated with prognosis of breast cancer. Conclusion: We successfully constructed a novel ceRNA network, among which each component was significantly associated with breast cancer prognosis.

Intracellular Ca 2+ signals play many important cellular functions such as migration, proliferation and differentiation. Store-operated Ca 2+ entry (SOCE) is a major route of Ca 2+ entry in nonexcitable cells. The activation of SOCE requires engagement between stromal interaction molecule 1 (STIM1) molecules on the endoplasmic reticulum and Ca 2+ release-activated Ca 2+ (CRAC) channel Orais (Orai1-3) on the plasma membrane. Accumulating evidence indicates that SOCE plays critical roles in cancer cell proliferation, invasion and metastasis. Here, we used the synthetic intracellular peptides derived from the C-termini of Orai channels to treat the breast cancer cells. We have found that Orai3-CT peptide exhibits stronger binding to STIM1 than Orai1-CT, and Orai3-CT peptide acts in a dominant negative fashion, blocking the STIM1-Orai1 interaction and reducing the Ca 2+ entry and proliferation of breast cancer cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective To explore the functions and mechanisms of macrophages derived from PGRN gene knockout (PGRN -/- ) C57BL/6 mice in the invasion and migration of breast cancer cells. Methods Breast cancer cells were cultured in conditioned medium of macrophages derived from WT and PGRN -/- mice. Transwell TM assay and scratch assay were used to detect the invasion and migration ability of cancer cells. Western blot analysis was used to detect the expression of E-cadherin and N-cadherin in cancer cells. Cytokine array, real-time quantitative PCR and ELISA were performed to investigate the differences of cytokines secreted by macrophages derived from WT and PGRN -/- mice. Breast cancer cells were treated by the differentially expressed cytokine interleukin-6 (IL-6), and then the above methods were used to investigate its effect on cancer cells. Western blot analysis was used to verify the roles of NF-κB and JAK/STAT3 signaling pathways. Results The macrophages derived from PGRN -/- mice blocked NF-κB signaling pathway, reduced IL-6 secretion, and inhibited the invasion and migration of breast cancer cells. IL-6 activated JAK/STAT3 signaling pathway to promote the invasion and migration of breast cancer cells. Conclusion The macrophages derived from PGRN -/- mice can block the NF-κB and JAK/STAT3 signaling pathways, down-regulate IL-6 expression, and inhibit the invasion and migration of breast cancer cells.

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2 . CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. IMPLICATIONS: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells., (©2018 American Association for Cancer Research.)

Breast cancer (BrCa) is the malignant tumor that most seriously threatens female health; however, the molecular mechanism underlying its progression remains unclear. Here, we found that conditional deletion of hypermethylated in cancer 1 (HIC1) in the mouse mammary gland might contribute to premalignant transformation in the early stage of tumor formation. Moreover, the chemokine (C-X-C motif) ligand 14 (CXCL14) secreted by HIC1-deleted BrCa cells bound to its cognate receptor GPR85 on mammary fibroblasts in the microenvironment and was responsible for activating these fibroblasts via the ERK1/2, Akt, and neddylation pathways, whereas the activated fibroblasts promoted BrCa progression via the induction of epithelial-mesenchymal transition (EMT) by the C-C chemokine ligand 17 (CCL17)/CC chemokine receptor 4 (CCR4) axis. Finally, we confirmed that the HIC1-CXCL14-CCL17 loop was associated with the malignant progression of BrCa. Therefore, the crosstalk between HIC1-deleted BrCa cells and mammary fibroblasts might play a critical role in BrCa development. Exploring the progression of BrCa from the perspective of microenvironment will be beneficial for identifying the potential prognostic markers of breast tumor and providing more effective treatment strategies.

Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers.

Though most patients with breast cancer are cured, there are still many patients progressed to metastatic breast cancer (MBC) and some are diagnosed as MBC. Human epidermal growth factor receptor 2 (HER2) is positive in about 20% all breast cancer patients and considered as a poor prognostic factor. The advent of ant-HER2 therapy has prominently prolonged the time of disease progression and survival for HER2-positive MBC patients. This review summarizes the advance in anti-HER2 therapy in HER2-positive MBC according to the phase III clinical trials, and briefly discusses some new agents and anti-HER2 therapy for HER2 low-or non-expression breast cancer patients.

Ductal carcinoma in situ (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2 + macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2 + fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients. Implications: This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. Mol Cancer Res; 16(2); 296-308. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Chemoresistance often leads to the failure of breast cancer treatment. MicroRNAs (miRNAs) play an important role in the progression and chemoresistance of cancer. However, because of the complexity of the mechanisms of chemoresistance and the specificity of miRNA regulation in different cell types, the function of miR-20a in breast cancer chemoresistance is still unclear. Here, by using miRNA microarray and high-content screening techniques, we found that miR-20a/b were significantly downregulated in breast cancer tissues compared with normal breast tissues, and low miR-20a/b expression was correlated with poor survival in breast cancer patients. Ectopic overexpression of miR-20a sensitized breast cancer cells to a broad spectrum of chemotherapy drugs and suppress their proliferation both in vitro and in vivo. Further study demonstrated that miR-20a directly targeted the 3'untranslated region of MAPK1, and thus downregulated the expression of P-gp and c-Myc by inhibiting the MAPK/ERK signaling pathway, whereas c-Myc can bind to the promoter region of the miR-20a gene to promote the expression of miR-20a. Together, our study identified a novel miR-20a/MAPK1/c-Myc feedback loop that regulates breast cancer growth and chemoresistance. These findings suggest that miR-20a synergizing with anticancer drugs will be a promising treatment strategy, especially for chemoresistant patients.

The transcriptional co-activator YES-associated protein (YAP) has been reported to act as both an oncogene and tumor suppressor in breast cancers. In this study, we evaluated YAP expression immunohistochemically in 324 breast cancer tissues and correlated the expression with clinicopathological findings and patient survival data. Additionally, we reviewed the literature to clarify the role of YAP in breast cancer. We detected YAP, estrogen receptor, progesterone receptor (PR), and human epidermal growth receptor-2 (HER2) expression and a Ki67 labeling index >20% in 53.4%, 49.0%, 45.0%, 28.3%, and 57.4% of invasive ductal carcinoma tissues, respectively. YAP is mainly localized within the tumor cell nuclei, and its expression was associated with the PR status and luminal A subtype. YAP expression also inversely correlated with the HER2 and Ki67 levels and lymph node metastasis. Kaplan-Meier curves revealed associations of YAP expression with favorable disease-free survival (DFS) and overall survival in patients with luminal A breast cancer and with favorable DFS association among patients with invasive ductal carcinoma, luminal B (HER2-), and luminal B (HER2+) breast cancers. A multivariate Cox analysis revealed that YAP expression and PR status were independent favorable predictors of DFS and overall survival, respectively, among patients with breast cancer, whereas tumor-node-metastasis stage and an old age were independent predictors of a poor DFS. Our results, together with the literature review findings, suggest that YAP could be a prognostic marker in patients with breast cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To provide novel insight into the development of new therapeutic strategies to combat breast cancer using trivalent arsenic (AsIII)-based combination therapy, the cytotoxicity of a combination of AsIII and tetrandrine (Tetra), a Chinese plant-derived alkaloid, was investigated in the human breast cancer cell line MCF-7. Cytotoxicity was evaluated using cell viability, colony formation, wound healing, lactate dehydrogenase leakage and cell cycle assay. Alterations of genes associated with cell proliferation and death were analyzed using real-time PCR and western blotting. Intracellular arsenic accumulation (As[i]) was also determined. Tetra significantly enhanced the cytotoxicity of AsIII in MCF-7 cells in a synergistic manner. The combined treatment upregulated the expression level of FOXO3a, and subsequently resulted in a concomitant increase in the expression levels of p21, p27, and decrease of cycline D1, which occurred in parallel with G0/G1 phase arrest. Autophagy induction was also observed in the combination treatment. Importantly, combining AsIII with Tetra exhibited a synergistic inhibitory effect on the expression level of survivin. Furthermore, enhanced As[i] along with synergistic cytotoxicity was observed in MCF-7 cells treated with AsIII combined with Tetra or Ko134, an inhibitor of breast cancer resistance protein (BCRP), suggesting that Tetra or the BCRP inhibitor probably intervened in the occurrence of resistance to arsenic therapy by enhancing the As[i] via modulation of multidrug efflux transporters. These results may provide a rational molecular basis for the combination regimen of AsIII plus Tetra, facilitating the development of AsIII-based anticancer strategies and combination therapies for patients with solid tumors, especially breast cancer.

Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2-, ER- HER2-, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2- and ER- HER2- primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2- and ER- HER2- patients, showing that these events were truly subtype specific.

Objective: To explore the expression and clinicopathological significance of Ser10 phosphorylated p27(kip1)(P-p27Ser10) protein in human breast carcinoma tissue. Methods: Tissue specimens were obtained from 111 cases with breast carcinoma from January 2004 to December 2005. And the expression of P-p27Ser10 protein for each specimen was detected by immunohistochemical (EnVision) analysis. Six representative paired samples of cancerous and paired adjacent normal tissues were collected and detected by Western blot. The relationships between the expression levels of P-p27Ser10 protein and its clinicopathological characteristics in primary breast carcinoma were retrospectively analyzed.The association of P-p27Ser10 and Jab1 protein was detected by co-immunoprecipitation method. Results: A high-level expression of P-p27Ser10 was present in cancerous tissues but not in paired adjacent normal tissues. The positive expression rate of P-p27Ser10 protein was as high as 88.3% (98/111) and 7.2% (8/111) in cancerous and paired adjacent normal tissues respectively ( P <0.05). P-p27Ser10 expression was correlated with tumor size, lymph node metastasis and TNM stage (all P <0.05). P-p27Ser10 expression was significantly correlated with Ki-67 ( r =0.582, P <0.05), and inversely correlated with p27 ( r =-0.426, P <0.05). Co-immunoprecipitation revealed that the association of P-p27Ser10 and Jab1 protein was increased in breast carcinoma. Conclusion: Over-expression of P-p27Ser10 protein might play an important role in the pathogenesis of breast carcinoma. P-p27Ser10 protein therefore represents a novel diagnostic marker and therapeutic target in patients with breast carcinoma.

NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DLs). NKG2DLs are expressed on malignant cells and sensitize them to early elimination by cytotoxic lymphocytes. We investigated the clinical importance of NKG2DLs and the mechanism of NKG2DL regulation in breast cancer (BC). Among the NKG2DLs MICA/B and ULBP1/2/3, the expression levels of MICA/B in BC tissues were inversely associated with the Tumor Node Metastasis stage. We first found that the high expression of MICB, but not MICA, was an independent prognostic factor for overall survival in patients with BC. Investigation into the mechanism revealed that a group of microRNAs (miRNAs) belonging to the miR-17-92 cluster, especially miR-20a, decreased the expression of ULBP2 and MICA/B. These miRNAs downregulated the expression of MICA/B by targeting the MICA/B 3'-untranslated region and downregulated ULBP2 by inhibiting the MAPK/ERK signaling pathway. Functional analysis showed that the silencing of NKG2DL-targeting miRNAs in BC cells increased NK cell-mediated cytotoxicity in vitro and inhibited immune escape in vivo. In addition, histone deacetylase inhibitors (HDACis) increased NKG2DL expression in BC cells by inhibiting members of the miR-17-92 cluster. Thus, targeting miRNAs with antisense inhibitors or HDACis may represent a novel approach for increasing the immunogenicity of BC.

Despite advances in treatment, up to 30% of breast cancer patients experience disease recurrence accompanied by more aggressive disease and poorer prognosis. Treatment of breast cancer is complicated by the presence of multiple breast cancer subtypes, including: luminal, Her2 overexpressing, and aggressive basal-like breast cancers. Identifying new biomarkers specific to breast cancer subtypes could enhance the prediction of patient prognosis and contribute to improved treatment strategies. The microenvironment influences breast cancer progression through expression of growth factors, angiogenic factors and other soluble proteins. In particular, chemokine C-C ligand 2 (CCL2) regulates macrophage recruitment to primary tumors and signals to cancer cells to promote breast tumor progression. Here we employed a software-based approach to evaluate the prognostic significance of CCL2 protein expression in breast cancer subtypes in relation to its expression in the epithelium or stroma or in relation to fibroblast-specific protein 1 (Fsp1), a mesenchymal marker. Immunohistochemistry analysis of tissue microarrays revealed that CCL2 significantly correlated with Fsp1 expression in the stroma and tumor epithelium of invasive ductal carcinoma. In the overall cohort of invasive ductal carcinomas (n=427), CCL2 and Fsp1 expression in whole tissues, stroma and epithelium were inversely associated with cancer stage and tumor size. When factoring in molecular subtype, stromal CCL2 was observed to be most highly expressed in basal-like breast cancers. By Cox regression modeling, stromal CCL2, but not epithelial CCL2, expression was significantly associated with decreased recurrence-free survival. Furthermore, stromal CCL2 (HR=7.51 P=0.007) was associated with a greater hazard than cancer stage (HR=2.45, P=0.048) in multivariate analysis. These studies indicate that stromal CCL2 is associated with decreased recurrence-free survival in patients with basal-like breast cancer, with important implications on the use of stromal markers for predicting patient prognosis.

Chemoresistance in breast cancer has been of great interest in past studies. However, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge in clinical oncology. By integrating data from global differences of gene expression and phospho-receptor tyrosine kinases between sensitive parental cells (MCF-7) and doxorubicin-resistant cells (MCF-7/ADR), we identified Axl as a potential target for chemoresistance and metastasis in multidrug resistant breast cancer cells. We analyzed Axl expression in 57 breast cancer cell lines and detected a dramatic increase in its expression level in mesenchymal breast cancer cell lines. Axl silencing suppressed invasive and metastatic potentials of chemoresistant breast cancer cells as well as increased elimination of cancer cells when combined with doxorubicin. Furthermore, in preclinical assays, an Axl inhibitor R428 showed increased cell death upon doxorubicin treatment. Additionally, using phospho-kinase array based proteomic analysis, we identified that Akt/GSK-3β/β-catenin cascade was responsible for Axl-induced cell invasion. Nuclear translocation of β-catenin then induced transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and doxorubicin-resistance in breast cancer cells. Most importantly, Axl was correlated with its downstream targets in tumor samples and was associated with poor prognosis in breast cancer patients. These results demonstrate that Gas6/Axl axis confers aggressiveness in breast cancer and may represent a therapeutic target for chemoresistance and metastasis.

Purpose: Cancer stem cell-tumor microenvironment ecosystem is proposed to drive tumor heterogeneity. Tumor-infiltrating lymphocytes (TILs) in breast cancer ecosystem were demonstrated to indicate better prognosis and benefit from chemotherapy. This study sought to detect the association between breast cancer stem cells and TILs., Methods: 92 patients with breast cancer were enrolled. Matched cancerous and paracancerous tissues were assembled in a tissue microarray and immunohistochemistry was employed to test expression of breast cancer stem cells (BCSCs) markers. TILs counts were estimated with global hematoxylin-eosin staining. The association between TILs and BCSCs phenotypes was analysed by multivariate analysis., Results: Although it was unable to find direct significant association between BCSCs phenotypes and TILs, the BCSCs phenotype with CD44(+)CD24(-)ALDH1A1(+)EpCAM(+)CD49f(+) was proved to be associated with worse DFS and OS (P=0.037 and 0.001). This result was confirmed by cox proportional-hazards regression model (for DFS and OS respectively, HR=2.438 and 3.383, P=0.019 [95%CI 1.418-3.457] and 0.025 [95%CI 1.162-9.843]). Additionally, in results of TILs, plasma cell-predominant breast cancer (PPBC) was unexpectedly found to indicate worse OS and HR was 2.686 (P=0.038 [95%CI 1.582-3.789])., Conclusions: The BCSCs phenotype and PPBC may be helpful stratified factors in future clinical trials. The underlying mechanism needs further research., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Currently drug resistance has remained a major challenge in successful breast cancer therapy. Wogonin, one of the active components of scutellaria baicalensis, has shown anticarcinogenic, chemopreventive, and immunoregulatory functions. The present study aimed to explore whether wogonin exerted synergistic cytotoxicity with doxorubicin in breast cancer. Our data indicated that wogonin inhibited the proliferation of breast cancer cells in a dose- and time-dependent manner. Combined treatment with wogonin increased the doxorubicin sensitivity in breast cancer cells. Moreover, administration with wogonin alone or in combination with doxorubicin suppressed the expression of insulin like growth factor 1 receptor (IGF-1R) in Bcap-37 and MCF-7 cells. Incubation with insulin like growth factor (IGF) I or IGF-II promoted cell growth, which was reversed by wogonin co-administration. Mechanically, we found that down-regulation of IGF-1R diminished the chemosensitization role of wogonin in breast cancer. In addition, wogonin suppressed the phosphorylation levels of AKT and addition of AKT inhibitor abolished the synergistic cytotoxicity of wogonin and doxorubicin. Taken together, combined treatment with wogonin increased the doxorubicin sensitivity in breast cancer cells through regulation of IGF-1R/AKT signaling pathway. Therefore, these findings demonstrated that combination therapy with wogonin led to better therapeutic effects via regulating IGF-1R/AKT signaling pathway in doxorubicin-based chemotherapy for breast cancer.

Background: The eIF5A2 gene (encoding the eukaryotic initiation factor 5A2) located at 3q26 is a putative oncogene that is overexpressed in colon and rectal carcinomas, lung cancer and hepatocellular carcinoma. EIF5A2 overexpression correlates significantly with tumor metastasis and is an adverse prognostic marker. However, eIF-5A2 overexpression in breast cancer and its effect on chemotherapy are unknown., Methods: We measured eIF-5A2 expression and doxorubicin sensitivity in different human breast cancer cell lines (Bcap-1937, HCC1937, and MCF-7). To investigate a role for eIF-5A2 in chemoresistance, cells were treated with eIF-5A2-siRNA, exposed to various concentrations of doxorubicin, and toxicity was assayed by CCK-8 (cell counting kit)., Results: The eIF-5A2 expression levels varied among breast cancer cells. Higher expression levels correlated with decreased doxorubicin sensitivity. Silencing of eIF-5A2 significantly improved doxorubicin toxicity in all three breast cancer cell lines., Conclusion: This study shows that eIF-5A2 plays an important role in doxorubicin chemoresistance in breast cancer cells.

Luminal A and B breast cancers are the most prevalent forms of breast cancer diagnosed in women. Compared to luminal A breast cancer patients, patients with luminal B breast cancers experience increased disease recurrence and lower overall survival. The mechanisms that regulate the luminal B subtype remain poorly understood. The chemokine CCL2 is overexpressed in breast cancer, correlating with poor patient prognosis. The purpose of this study was to determine the role of CCL2 expression in luminal B breast cancer cells. Breast tissues, MMTV-PyVmT and MMTV-Neu transgenic mammary tumors forming luminal B-like lesions, were immunostained for CCL2 expression. To determine the role of CCL2 in breast cancer cells, CCL2 gene expression was silenced in mammary tumor tissues and cells using TAT cell-penetrating peptides non-covalently cross linked to siRNAs (Ca-TAT/siRNA). CCL2 expression was examined by ELISA and flow cytometry. Cell growth and survival were analyzed by flow cytometry, immunocytochemistry, and fluorescence microscopy. CCL2 expression was significantly increased in luminal B breast tumors, MMTV- PyVmT and MMTV-Neu mammary tumors, compared or normal breast tissue or luminal A breast tumors. Ca-TAT delivery of CCL2 siRNAs significantly reduced CCL2 expression in PyVmT mammary tumors, and decreased cell proliferation and survival. CCL2 gene silencing in PyVmT carcinoma cells or BT474 luminal B breast cancer cells decreased cell growth and viability associated with increased necrosis and autophagy. CCL2 expression is overexpressed in luminal B breast cancer cells and is important for regulating cell growth and survival by inhibiting necrosis and autophagy.