Pitavastatin induces autophagy-dependent ferroptosis in MDA-MB-231 cells via the mevalonate pathway.

Triple-negative breast cancer (TNBC) is more prone to recurrence and metastasis relative to other subtypes of breast cancer, leading to an extremely poor prognosis. The increasing potential chemoresistance of TNBC patients is mainly due to that tumor cells escape from apoptosis. In recent years, statins have demonstrated extensive anti-tumor effects. It is worth noting that statins have more effective anti-tumor effects on TNBC cells and drug-resistant breast cancer cells. Therefore, this study examines the superior cytotoxic effects of statins on TNBC cell lines and further explores their potential therapeutic mechanisms. We detected different cell phenotypes and found that statins significantly reduced the cell viability of TNBC cells. Specifically, pitavastatin showed an obvious induction in cell death, cell cycle arrest and oxidative stress in TNBC MDA-MB-231 cells. The reversal effect of iron chelator desferrioxamine (DFO) on the morphological and molecular biological changes induced by pitavastatin has revealed a new mode of cell death induced by pitavastatin: ferroptosis. This ferroptotic effect was strengthened by the decreased expression of glutathione peroxidase 4 (GPx4) as well as newly discovered ferroptosis suppressor protein 1 (FSP1). The data showed that ferroptotic death of MDA-MB-231 cells is autophagy-dependent and mediated by the mevalonate pathway. Finally, we found that therapeutic oral doses of statins can inhibit the growth of transplanted tumors, which establishes statins as a potential treatment for TNBC patients. In conclusion, we found pitavastatin could induce autophagy-dependent ferroptosis in TNBC cells via the mevalonate pathway which may become a potential adjuvant treatment option for TNBC patients.

Integration of bioinformatics and machine learning strategies identifies APM-related gene signatures to predict clinical outcomes and therapeutic responses for breast cancer patients.

BACKGROUND: Tumor antigenicity and efficiency of antigen presentation jointly influence tumor immunogenicity, which largely determines the effectiveness of immune checkpoint blockade (ICB). However, the role of altered antigen processing and presentation machinery (APM) in breast cancer (BRCA) has not been fully elucidated. METHODS: A series of bioinformatic analyses and machine learning strategies were performed to construct APM-related gene signatures to guide personalized treatment for BRCA patients. A single-sample gene set enrichment analysis (ssGSEA) algorithm and weighted gene co-expression network analysis (WGCNA) were combined to screen for BRCA-specific APM-related genes. The non-negative matrix factorization (NMF) algorithm was used to divide the cohort into different clusters and the fgsea algorithm was applied to investigate the altered signaling pathways. Random survival forest (RSF) and the least absolute shrinkage and selection operator (Lasso) Cox regression analysis were combined to construct an APM-related risk score (APMrs) signature to predict overall survival. Furthermore, a nomogram and decision tree were generated to improve predictive accuracy and risk stratification for individual patients. Based on Tumor Immune Dysfunction and Exclusion (TIDE) method, random forest (RF) and Lasso logistic regression model were combined to establish an APM-related immunotherapeutic response score (APMis). Finally, immune infiltration, immunomodulators, mutational patterns, and potentially applicable drugs were comprehensively analyzed in different APM-related risk groups. IHC staining was used to assess the expression of APM-related genes in clinical samples. RESULTS: In this study, APMrs and APMis showed favorable performances in risk stratification and therapeutic prediction for BRCA patients. APMrs exhibited more powerful prognostic capacity and accurate survival prediction compared to conventional clinicopathological features. APMrs was closely associated with distinct mutational patterns, immune cell infiltration and immunomodulators expression. Furthermore, the two APM-related gene signatures were independently validated in external cohorts with prognosis or immunotherapeutic responses. Potential applicable drugs and targets were mined in the APMrs-high group. APM-related genes were further validated in our in-house samples. CONCLUSION: The APM-related gene signatures established in our study could improve the personalized assessment of survival risk and guide ICB decision-making for BRCA patients.

Tumor-derived small extracellular vesicles promote breast cancer progression by upregulating PD-L1 expression in macrophages.

BACKGROUND: The metastasis of breast cancer (BC) is a complex multi-step pathological process, strictly dependent on the intrinsic characteristics of BC cells and promoted by a predisposing microenvironment. Although immunotherapy has made important progress in metastasis BC, the heterogeneity of PD-L1 in tumor associated macrophages (TAMs) in BC and the underlying mechanisms in the metastasis development of BC are still not completely elucidated. Small extracellular vesicles (sEVs) represent essential interaction mediators between BC cells and TAMs. It is worth noting to explore the underlying mechanisms typical of sEVs and their role in the metastasis development of BC. METHODS: The structure of sEVs was identified by TEM, while the particle size and amounts of sEVs were detected by BCA and NTA analysis. The specific PD-L1 + CD163 + TAM subpopulation in metastasis BC was identified by scRNA-seq data of GEO datasets and verified by IHC and IF. The function of TAMs and sEVs in metastasis BC was explored by RT-qPCR, WB, IF, flow cytometry and in vivo experiment. The expression profiles of plasma sEVs-miRNA in relation to BC metastasis was analyzed using next-generation sequencing. Further detailed mechanisms of sEVs in the metastasis development of BC were explored by bioinformatics analysis, RT-qPCR, WB and luciferase reporter assay. RESULTS: In this study, we identified that the immunosuppressive molecule PD-L1 was more abundant in TAMs than in BC cells, and a specific PD-L1 + CD163 + TAM subpopulation was found to be associated with metastasis BC. Additionally, we found that BC cells-derived sEVs can upregulate the PD-L1 expression and induce the M2 polarization, enhancing the metastasis development both in vitro and in vivo. Also, Clinical data showed that sEV-miR-106b-5p and sEV-miR-18a-5p was in relation to BC metastasis development and poor prognosis of BC patients. Further mechanistic experiments revealed that BC-derived sEV-miR-106b-5p and sEV-miR-18a-5p could synergistically promoted the PD-L1 expression in M2 TAMs by modulating the PTEN/AKT and PIAS3/STAT3 pathways, resulting in the enhancement of the BC cells invasion and metastasis. CONCLUSIONS: Our study demonstrated that BC-derived sEVs can induce metastasis in BC through miR-106b-5p/PTEN/AKT/PD-L1 and miR-18a-5p/PIAS3/STAT3/PD-L1 pathways in TAMs. Therefore, the inhibition of these specific interactions of signaling pathways would represent a promising target for future therapeutic strategies for treatment of BC.

Hyperthermia promotes M1 polarization of macrophages via exosome-mediated HSPB8 transfer in triple negative breast cancer.

PURPOSE: To investigate the mechanism underlying the modulation of M1 macrophage polarization by exosomes released from hyperthermia-treated triple-negative breast cancer (TNBC) cells. MATERIALS AND METHODS: In this study, the effects of hyperthermia on TNBC cells were examined using cell counting kit-8, apoptosis, and cell cycle assays. Transmission electron microscopy was used to identify the structure of exosomes, while bicinchoninic acid and nanoparticle tracking analysis were used to detect particle size and amounts of exosomes released after hyperthermia. The polarization of macrophages incubated with exosomes derived by hyperthermia-pretreated TNBC cells were assessed by RT-qPCR and flow cytometry analysis. Next, RNA sequencing was performed to determine the targeting molecules changed in hyperthermia-treated TNBC cells in vitro. Finally, the mechanism underlying the modulation of macrophage polarization by exosomes derived from hyperthermia-treated TNBC cells was examined by using RT-qPCR, immunofluorescence and flow cytometry analysis. RESULTS: Hyperthermia markedly reduced cell viability in TNBC cells and promoted the secretion of TNBC cell-derived exosomes. The hub genes of hyperthermia-treated TNBC cells were significantly correlated with macrophage infiltration. Additionally, hyperthermia-treated TNBC cell-derived exosomes promoted M1 macrophage polarization. Furthermore, the expression levels of heat shock proteins, including HSPA1A, HSPA1B, HSPA6, and HSPB8, were significantly upregulated upon hyperthermia treatment, with HSPB8 exhibiting the highest upregulation. Moreover, hyperthermia can induce M1 macrophage polarization by promoting exosome-mediated HSPB8 transfer. CONCLUSION: This study demonstrated a novel mechanism that hyperthermia can induce M1 polarization of macrophages via exosome-mediated HSPB8 transfer. These results will help with future development of an optimized hyperthermia treatment regime for clinical application, especially for combination treatment with immunotherapy.

GATA3 mediates doxorubicin resistance by inhibiting CYB5R2-catalyzed iron reduction in breast cancer cells.

AIMS: Neoadjuvant chemotherapy (NAC) is the primary preoperative therapy for breast cancer. The luminal subtype of breast cancer shows less NAC response than the basal subtype, with an inefficient NAC treatment effect. Understanding of the molecular and cellular mechanisms responsible for this chemoresistance is an important issue when determining optimal treatment. METHODS: Doxorubicin-induced apoptosis and ferroptosis was investigated using cytotoxicity, western blotting, and flow cytometry assays. The role of GATA3 in modulating doxorubicin-induced cell death was investigated both in vitro and in vivo. RNA-seq, qPCR, ChIP, and luciferase assay and association analyses were performed to investigate the regulation of CYB5R2 by GATA3. The function of GATA3 and CYB5R2 in regulating doxorubicin-induced ferroptosis was evaluated with iron, ROS, and lipid peroxidation detection assays. Immunohistochemistry was performed for results validation. RESULTS: Doxorubicin-induced basal breast cancer cell death is dependent on iron-mediated ferroptosis. Overexpression of the luminal signature transcriptional factor GATA3 mediates doxorubicin resistance. GATA3 promotes cell viability by decreasing ferroptosis-related gene CYB5R2 expression and by maintaining iron homeostasis. Analyzing data from the public and our cohorts demonstrates that GATA3 and CYB5R2 are associated with NAC response. CONCLUSIONS: GATA3 promotes doxorubicin resistance by inhibiting CYB5R2-mediated iron metabolism and ferroptosis. Therefore, patients with breast cancer who display high GATA3 expression do not benefit from doxorubicin-based NAC regimens.

Construction of an m6A-related lncRNA model for predicting prognosis and immunotherapy in patients with lung adenocarcinoma.

N6-methyladenosine (m6A)-related lncRNAs could be involved in the development of multiple tumors with an unknown role in lung adenocarcinoma (LUAD). Hence, gene expression data and clinical data of LUAD patients were acquired from The Cancer Genome Atlas Database. The prognostic m6A-related lncRNAs were identified through differential lncRNA expression analysis and Spearman's correlation analysis. The least absolute shrinkage and selection operator regression was used to establish the prognostic risk model, so as to evaluate and validate the predictive performance with survival analysis and receiver operating characteristic curve analysis. The expression of immune checkpoints, immune cell infiltration and drug sensitivity of patients in different risk groups were analyzed separately. A total of 19 prognostic m6A-related lncRNAs were identified to set up the prognostic risk model. The patients were divided into high- and low-risk groups based on the median value of the risk scores. Compared with the patients in the low-risk group, the prognosis of the patients in the high-risk group was relatively worse. The receiver operating characteristic curves indicated that this model had excellent sensitivity and specificity. Multivariate Cox regression analysis demonstrated that the risk score could be supposed as an independent prognostic risk factor. We highlighted that the risk scores were correlated with immune cell infiltration and drug sensitivity for constructing a prognostic risk model in LUAD patients based on m6A-related lncRNAs.

CBX3 promotes breast cancer progression and high level of CBX3 predicts poor prognosis in patients.

Breast cancer is one of the leading cancer deaths around the world. Targeted drugs have greatly increased the survival rate of breast cancer patients in recent years. But in some patients, the current regimen is still ineffective. Therefore, more therapeutic targets for treating breast cancer are demanding. The core heterochromatin-related genes of breast cancer were identified by utilizing prognostic survival analysis and multivariate Cox hazard proportional regression analysis. Both breast cancer and adjacent normal tissue were collected and analyzed with western blot and immunohistochemistry. Colony formation assay, CCK-8 assay, and EdU assay were used to measure the effect of CBX3 on breast cancer cell growth, wound-healing assay and Transwell assay were used to analyze the effect of CBX3 on breast cancer cell migration and invasion. Flow cytometry assay and western blot were used to study the molecular mechanism of CBX3 in breast cancer. High expression of heterochromatin-related proteins CBX3, H2AFY, and SULF1 showed a poor prognosis in patients in both TCGA dataset and GEO datasets. Western blot demonstrated that the expression level of CBX3 was significantly higher in breast cancer than that in adjacent normal tissues. Colony formation assay, CCK-8 assay, and EdU assay showed that the knockdown of CBX3 could significantly inhibit breast cancer cell growth, and the overexpression of CBX3 could promote the growth of breast cancer cells. Transwell assay and wound healing assay showed that knockdown of CBX3 inhibited breast cancer cell migration and invasion, and the overexpression of CBX3 promoted breast cancer cell migration and invasion. Western blot showed that CBX3 might promote breast cancer cell proliferation, invasion, and migration in breast cancer by modulating the ERK1/2 signaling pathway and epithelial-mesenchymal transition (EMT)-related genes. CBX3 was a biomarker of poor prognosis in breast cancer patients. CBX3 promoted the proliferation of breast cancer cells through the ERK signaling pathway, and migration and invasion of breast cancer cells through EMT-related genes. The CBX3/p-ERK1/2 signaling axis might provide a new therapeutic method against breast cancer.

Glucose metabolism and lncRNAs in breast cancer: Sworn friend.

BACKGROUND: Glucose metabolism disorder is a common feature in cancer. Cancer cells generate much energy through anaerobic glycolysis, which promote the development of tumors. However, long non-coding RNA may play an important role in this process. Our aim is to explore a prognostic risk model based on the glucose metabolism-related lncRNAs which provides clues that lncRNAs predict a clinical outcome through glucose metabolism in breast cancer. METHODS: 1222 RNA-seq were extracted from the TCGA database, and 74 glucose metabolism-related genes were loaded from the GSEA website. Then, 7 glucose metabolism-related lncRNAs risk score model was developed by univariate, Lasso, and multivariate regression analysis. The lncRNA risk model showed that high-risk patients predict a poor clinical outcome with high reliability (P=2.838×10-6). Univariate and multivariate independent prognostic analysis and ROC curve analysis proved that the risk score was an independent prognostic factor in breast cancer with an AUC value of 0.652. Finally, Gene set enrichment analysis showed that cell cycle-related pathways were significantly enriched in a high-risk group. RESULTS: Our results showed that glucose metabolism-related lncRNAs can affect breast cancer progression. 7 glucose metabolism-related lncRNAs prognostic signature was established to evaluate the OS of patients with breast cancer. PICSAR, LINC00839, AP001505.1, LINC00393 were risk factors and expressed highly in the high-risk group. A Nomogram was made based on this signature to judge patients' living conditions and prognosis. CONCLUSION: 7 glucose metabolism-related lncRNAs risk score model had a high prognostic value in breast cancer. PICSAR, LINC00839, AP001505.1, LINC00393 were risk factors. AP001505.1 expression was increased in most triple-negative breast cancer cells treated with high glucose, which may also take part in breast cancer progression and potential therapeutic targets.

Vaspin accelerates the proliferation, invasion and metastasis of Triple-Negative breast cancer through MiR-33a-5p/ABHD2.

OBJECTIVE: To explore the influence and the underlying mechanism of vaspin (visceral adipose tissue-derived serpin) on the development of triple-negative breast malignancy. METHODS: First, we analyzed medical records and screened out 22 breast cancer patients with different BMI according to inclusion and exclusion criterion, and measured serum vaspin of those patients. Then we studied the effects of vaspin on TNBC cell lines by using EdU assay, colony formation, transwell and wound-healing assay. Later, we used bioinformatics analysis to identify downstream effectors and verify with qRT-PCR, luciferase assay, western blot, etc. RESULTS: We found the vaspin level was positively correlated with BMI in breast malignant patients and vaspin could significantly enhance the proliferation, infiltration and transferring of triple-negative breast cancer cells by restraining the expression of miR-33a-5p. By using bioinformatic analysis and luciferase assay, we identified miR-33a-5p directly regulating ABHD2. CONCLUSION: Vaspin, as a cancer-promoting cytokine, may inhibit miR-33a-5p thus increasing the level of ABHD2 to promote the development of the triple-negative breast cancer.

Dietary patterns, nutritional status, and mortality risks among the elderly.

Introduction: While most epidemiological studies have focused on the effects of individual dietary patterns and nutritional status on health, the relationships between the combinations of these factors and patient prognosis requires further investigation. Objective: This study explored mortality risk in individuals with different combinations of dietary patterns or nutritional status. Methods: Unsupervised K-means clustering was used to classify populations. The analyses included Cox proportional risk and competing risk models. Results: After considering a complex sampling design, the results showed that among 12,724 participants aged >60 years, 6.99% died from cancer and 10.47% from cardiovascular and cerebrovascular disease (CCVD). After correcting for participant baseline information and chronic conditions, the geriatric nutritional risk index and healthy eating index (HEI) were negatively associated with the risk of all-cause and cause-specific mortality. The opposite was true for the dietary inflammatory index (DII). After sorting the population three clusters based on study scores showed higher risks of all-cause mortality and cancer-related death in Cluster 2 and 3. Discussion: These results suggest that different nutritional status and dietary patterns are associated with the risk of all-cause mortality and death from cancer and CCVD in people aged >60 years in the United States. Dietary patterns with high HEI and low DII were beneficial to health, whereas nutritional status needs to be maintained at a level that is not too low.

PUS1 is a novel biomarker for predicting poor outcomes and triple-negative status in breast cancer.

Breast cancer patients' outcomes have improved dramatically in recent years, but relapses and poor prognosis remain common due to its aggressiveness and heterogeneity. The development of reliable biomarkers is still needed for predicting prognosis and treatment effectiveness. Recently, a growing body of research suggests that pseudouridine synthases contribute to the development of many cancers, but their contribution to breast cancer remains largely unknown. Using an integrative analysis, we selected pseudouridine synthase1(PUS1) as the candidate biomarker. A tissue microarray of 131 breast cancer patients was then utilized to determine the clinical significance and prognostic value of PUS1. RNA sequencing analysis was conducted to identify downstream genes that differ between control and PUS1 knockdown cells. The effect of PUS1 on phenotypes of cells was assessed using cell proliferation, colony formation, and transwell invasion assays. We found that breast tumors overexpressed PUS1 compared with paired normal tissues. PUS1 expression was positively correlated with triple-negative breast cancer (TNBC) status (P= 0.020) and tumor grade (P <0.0001), but not with age (P= 0.736), tumor size (P= 0.608), lymph node (P= 0.742), oestrogen receptor (ER) (P= 0.162), progesterone receptor (PR) (P= 0.901), human epidermal growth factor receptor 2 (HER2) (P= 0.608) or tumor stage (P= 0.411). Comparatively, patients with high PUS1 levels had shorter overall survival time (P=0.0001) and relapse-free survival time (P = 0.0093). A univariate and multivariate survival analysis suggested that the overall survival of patients was independently influenced by the PUS1 score (Univariate Cox P <0.0001, HR=5.176, 95% CI =2.420-11.07; Multivariate Cox P = 0.001, HR = 5.291, 95% CI =1.893-14.78). RNA sequencing data revealed the PUS1 knockdown significantly affects a series of cancer related biological process such as regulation of cell proliferation and cell migration, as well as KEGG pathways including Mitophagy and PI3K-Akt signaling. In vitro, knockdown of PUS1 significantly suppressed the proliferation and colony formation abilities of MDA-MB-231 cells and BT-549 cells. Additionally, the ability of tumor cells to invade was remarkably attenuated in low PUS1 expression groups compared with the corresponding control groups. Our results suggested that PUS1 is a novel biomarker that predicts poor outcomes in patients with breast cancer and may prove to be a promising treatment target.

Endoplasmic reticulum stress targeted therapy for breast cancer.

Recurrence, metastasis, and drug resistance are still big challenges in breast cancer therapy. Internal and external stresses have been proven to substantially facilitate breast cancer progression through molecular and systemic mechanisms. For example, endoplasmic reticulum stress (ERS) results in activation of the unfolded protein response (UPR), which are considered an important cellular stress response. More and more reports indicate its key role in protein homeostasis and other diverse functions involved in the process of breast cancer progression. Therefore, therapies targeting the activation of ERS and its downstream signaling pathways are potentially helpful and novel tools to counteract and fight breast cancer. However, recent advances in our understanding of ERS are focused on characterizing and modulating ERS between healthy and disease states, and so little attention has been paid to studying the role and clinical application of targeting ERS in a certain cancer. In this review, we summarize the function and main mechanisms of ERS in different molecular types of breast cancer, and focus on the development of agents targeting ERS to provide new treatment strategies for breast cancer. Video Abstract.

Identification of TIMP2 as a Prognostic Biomarker and Its Correlation with Tumor Immune Microenvironment: A Comprehensive Pan-Cancer Analysis.

Background: Tissue inhibitor of metalloproteinase-2 (TIMP2), an endogenous inhibitor of matrix metalloproteinases, has been disclosed to participate in the development and carcinogenesis of multiple malignancies. However, the prognosis of TIMP2 in different cancers and its correlation with tumor microenvironment and immunity have not been clarified. Methods: In this study, we conducted a comprehensive bioinformatics analysis to evaluate the prognostic and therapeutic value of TIMP2 in cancer patients by utilizing a series of databases, including Oncomine, GEPIA, cBioPortal, GeneMANIA, Metascape, and Sangerbox online tool. The expression of TIMP2 in different cancers was analyzed by Oncomine, TCGA, and GTEx databases, and mutation status of TIMP2 in cancers was then verified using the cBioPortal database. The protein-protein interaction (PPI) network of the TIMP family was exhibited by GeneMANIA. The prognosis of TIMP2 in cancers was performed though the GEPIA database and Cox regression. Additionally, the correlations between TIMP2 expression and immunity (immune cells, gene markers of immune cells, TMB, MSI, and neoantigen) were explored using Sangerbox online tool. Results: The transcriptional level of TIMP2 in most cancerous tissues was significantly elevated. Survival analysis revealed that an elevated expression of TIMP2 is associated with unfavorable survival outcome in multiple cancers. Enrichment analysis demonstrated the possible mechanisms of TIMPs and their associated genes mainly involved in pathways including extracellular matrix (ECM) regulators, degradation of ECM and ECM disassembly, and several other signaling pathways. Conclusions: Our findings systematically dissected that TIMP2 is a potential prognostic maker in various cancers and use the inhibitor of TIMP2, which may be an effective strategy for cancer therapy to improve the poor cancer survival and prognostic accuracy, but concrete mechanisms need to be validated by subsequent experiments.

Potential values of circulating tumor cell for detection of recurrence in patients of thyroid cancer: a diagnostic meta-analysis.

BACKGROUND: Several studies have reported that circulating tumor cells (CTCs) are a promising marker for the diagnosis of thyroid cancer (TC) with recurrence or distant metastasis (DMs). However, some studies emerged with conflicting results. Therefore, we provide a meta-analysis to evaluate the diagnostic performance of CTC for detection of recurrence in patients of TC. METHODS: We searched PubMed, Web of Science, Cochrane library with the keywords "thyroid cancer" and "circulating tumor cells". Data extraction and risk of bias assessment were performed independently by two reviewers. The summary receiver operating characteristic curve (SROC) and other parameters were adopted to summarize the overall test performance. The sensitivity of CTCs in the detection of recurrent TC was reviewed. All analyses were performed by STATA 12.0 and Meta-disc software. RESULTS: For CTCs expressing epithelial cell adhesion molecule (EpCAM), seven studies were included in our meta-analysis. Pooled sensitivity, specificity, and diagnostic odds ratio were 0.71 (95% CI: 0.63-0.78), 0.89 (95% CI: 0.84-0.94), and 26.75 (95% CI: 9.11-78.53); 0.78 (95% CI: 0.65-0.89), 0.88 (95% CI: 0.76-0.96), and 40.01 (95% CI: 10.49-152.63) for CTCs expressing thyroid stimulating hormone receptor (TSHR). The area under the SROC for EpCAM and TSHR were both 0.91. CONCLUSION: CTC was a reliable marker for the diagnosis of TC patients with recurrence and DMs, and the sensitivity of CTCs expressing TSHR was higher than that of EpCAM. Additional research is warranted in order to establish uniformity in international guidelines, make up the drawbacks of conventional diagnostic methods and to prevent futile surgery.

The pathological and clinical landscape of refractory metastatic triple negative breast cancer: a narrative review.

Background and Objective: Triple negative breast cancer (TNBC) refers to a special subtype of breast cancer that is negative for the estrogen receptor, the progesterone receptors, and human epidermal growth factor receptor 2. As a group of diseases, it has strong heterogeneity. Refractory metastatic triple negative breast cancer (mTNBC) has even greater heterogeneity, more susceptibility to drug resistance, and faster progression, which makes it more difficult to treat effectively and significantly reduces a patient's overall survival. Therefore, in order to overcome this difficulty in clinical practice, we need to deeply understand the special subgroup by analyzing definition and prognostic factors of refractory mTNBC and describing the therapeutic status and future treatment directions. Methods: Recent domestic and foreign guidelines, as well as clinical studies related to refractory mTNBC on PubMed and the China National Knowledge Infrastructure (CNKI) databases were retrospectively analyzed. The six keywords we selected were used for literature search. Two authors performed database searches independently, and disagreements over the results were mediated by a third reviewer. Key Content and Findings: According to the guidelines, refractory mTNBC has not been clearly defined. Related studies indicated that tumor heterogeneity may be one of the main mechanisms of early relapse or drug resistance in refractory mTNBC. The clinical treatment options for refractory mTNBC are very limited. Although chemotherapy is the standard treatment, it is limited by poor efficacy and intolerance in the clinical stage. Therefore, in recent years, many studies have explored novel treatment options. Both immunotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors have been selected as first-line treatment in clinical studies, but gained limited benefits. Indeed, clinical studies have shown good efficacy with novel ADCs, which may be promising in the clinical treatment of refractory mTNBC. Conclusions: Currently, improving the survival time and quality of life of refractory mTNBC are major challenges for clinicians. Novel therapies including immunosuppressive agents, PARP inhibitors, and ADCs rather than chemotherapy alone have achieved good results in the exploration of first-line treatment for refractory TNBC patients, but this warrants further research and investigation.

Diagnostic accuracy of de-escalated surgical procedure in axilla for node-positive breast cancer patients treated with neoadjuvant systemic therapy: A systematic review and meta-analysis.

BACKGROUND: More initial clinical node-positive breast cancer patients achieve axillary pathological complete response (ax-pCR) after neoadjuvant systemic therapy (NST). Restaging axillary status and performing de-escalated surgical procedures to replace routine axillary lymph nodes dissection (ALND) is urgently needed. Targeted axillary lymph node biopsy (TLNB) is a novel de-escalated surgical strategy marking metastatic axillary nodes before NST and targeted dissection and biopsy intraoperatively to tailor individual axillary management. METHODS: This study provided a systematic review and meta-analysis to evaluate the feasibility and diagnosis accuracy of TLNB. Prospective and retrospective clinical trials on TLNB were searched from Pubmed, Embase, and Cochrane. Identification rate (IFR), false-negative rate (FNR), negative predictive value (NPV), and rate of ax-pCR were the outcomes of this meta-analysis. RESULTS: One thousand nine hundred and twenty patients attempted TLNB, with an overall IFR of 93.5% (95% confidence interval [CI] 90.1%-96.2%). IFR of three nodal marking methods, namely iodine seeds, clips, and carbon dye, was 95.6% (95% CI 91.2%-98.7%), 91.7% (95% CI 87.3%-95.4%), and 97.1% (95% CI 89.1%-100.0%), respectively. Of them, 847 patients received ALND, with an overall FNR of 5.5% (95% CI 3.3%-8.0%), and NPV ranged from 90.1% to 96.1%. Regression analysis showed that the overlap of targeted and sentinel biopsied nodes might associate with IFRs and FNRs. CONCLUSION: TLNB is a novel, less invasive surgical approach to distinguish initial node-positive breast cancer that achieves negative axillary conversion after NST. It yields an excellent IFR with a low FNR and a high NPV. A combination of preoperative imaging, intraoperative TLNB with SLNB, and postoperative nodal radiotherapy might affect the future treatment paradigm of primary breast cancer with nodal metastases.

Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

PURPOSE: To investigate the mechanism through which hyperthermia promotes exosome secretion and drug sensitivity in adriamycin-resistant breast cancer. MATERIALS AND METHODS: We first evaluated the effect of hyperthermia on adriamycin-resistant breast cancer viability and used transmission electron microscopy, nanoparticle tracking analysis, and a bicinchoninic acid kit to validate the effect of hyperthermia on exosome secretion. The effective targeting molecules and pathways changed by hyperthermia were explored by RNA microarray and verified in vitro. The adriamycin-resistant MCF-7/ADR cells co-incubated with the exosomes produced by MCF-7/ADR cells after hyperthermia were assessed. The uptake of exosomes by MCF-7/ADR cells after hyperthermia treatment was evaluated by confocal microscopy. Finally, the mechanism through which hyperthermia promotes exosome secretion by hyperthermia was determined. RESULTS: Hyperthermia significantly suppressed the growth of adriamycin-resistant breast cancer cells and increased drug sensitivity by upregulating FOS and CREB5, genes related to longer overall survival in breast cancer patients. Moreover, hyperthermia promoted exosome secretion through Rab7b, a small GTPase that controls endosome transport. The upregulated FOS and CREB5 antioncogenes can be transferred to MCF-7/ADR cells by hyperthermia-treated MCF-7/ADR cell-secreted exosomes. CONCLUSIONS: Our results demonstrated a novel function of hyperthermia in promoting exosome secretion in adriamycin-resistant breast cancer cells and revealed the effects of hyperthermia on tumor cell biology. These hyperthermia-triggered exosomes can carry antitumor genes to the residual tumor and tumor microenvironment, which may be more beneficial to the effects of hyperthermia. These results represent an exploration of the relationship between therapeutic strategies and exosome biology.

Circulating metabolites serve as diagnostic biomarkers for HER2-positive breast cancer and have predictive value for trastuzumab therapy outcomes.

Human epidermal growth factor receptor 2 (HER2)-positive is a particularly aggressive type of the breast cancer. Trastuzumab-based therapy is a standard treatment for HER2-positive breast cancer, but some patients are resistance to the therapy. There are no known diagnostic biomarkers to improve the early diagnosis of HER2-positive breast cancer and the clinical utility of trastuzumab therapy. Using ultrahigh-performance liquid time of flight mass spectrometry (UPLC-TOF-MS)-based serum metabolomics and multivariate statistical analysis, we investigated and identified the circulating metabolites L-arginine and arachidonic acid were elevated in trastuzumab-responsive and trastuzumab-resistant HER2-positive breast cancer patients, and increased until reaching their peaks in trastuzumab-resistant HER2-positive breast cancer patients. Moreover, an equation for assessing the risk scores based on linear logistic regression models involving L-arginine and arachidonic acid was created, which was beneficial for revealing metabolic changes in HER2-positive breast cancer and enhancing current trastuzumab-based therapy. In summary, we develop serum-based metabolic biomarkers for diagnosis of HER2-positive breast cancers and predicts the therapeutic effects of trastuzumab therapy.

Screening of the novel immune-suppressive biomarkers of TMED family and whether knockdown of TMED2/3/4/9 inhibits cell migration and invasion in breast cancer.

Background: Transmembrane p24 trafficking protein (TMED) family members are implicated in several solid tumors, but their clinical relevance for breast cancer (BC) remains unclear. This study aimed to probe their prognostic values and relations with tumor immunity in BC. Methods: TMED family mRNA expression was assessed in five microarray datasets (GSE65212, GSE42568, GSE5364, GSE22820 and GSE45827) from Gene Expression Omnibus (GEO) database and invasive breast cancer (BRCA) cohort from The Cancer Genome Atlas (TCGA). Receiver operating characteristic (ROC) curve was performed to determine the predictive values of filtered members of the TMED family. The protein expressions of screen genes were validated by Clinical Proteomic Tumor Analysis Consortium (CPTAC) data from University of ALabama at Birmingham CANcer data analysis portal (UALCAN) and detected in the clinical specimens by western blot assay. Clinicopathologic variables were analyzed with bc-GenExMiner, and patient prognostic data were obtained with Kaplan-Meier Plotter. In vitro wound healing and invasion assays were performed on siRNA-transfected BC cell lines. TIMER 2.0, SangerBox, and ImmPort were used to evaluate tumor immune infiltration, immune checkpoints, and other immune-related genes. CbioPortal, Metascape, Expression2kinases, and LinkedOmics were used to explore gene regulatory network. Results: BC tissues expressed TMED2/3/4/9 at a higher level than normal tissues, providing diagnostic potential. All the areas under the ROC curve for TMED2/3/4/9 were more than 0.7. TMED2/3/4/9 correlated with numerous clinical variables, including lymph node status, Scarff-Bloom-Richardson score (SBR), Nottingham Prognostic Index (NPI), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and triple-negative breast cancer (TNBC) status, and their high expression predicted the poor prognosis of BC patients. TMED2/3/4/9 knockdown drastically inhibited the migratory and invasive capacities of MDA-MB-231 and HCC1937 cells. TMED2/3/4/9 expressions correlated negatively with the infiltration of tumor-suppressive immune cells such as CD8+ T cells, dendritic cells, and natural killer cells, and was inversely related to a variety of immune checkpoint genes, including programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4). A set of kinases, transcription factors, and microRNAs (miRNAs) may regulate TMED2/3/4/9 abnormalities at the genome level. Conclusions: TMED2/3/4/9 may serve as diagnostic, prognostic, and immune-suppressive biomarkers in BC.

Exosomal circRNAs: a new communication method in cancer.

Exosomes are extracellular vesicles with unique membrane markers and components that participate in cellular communication. The contents of exosomes, including growth factors, microRNAs, long noncoding RNAs, and circular RNAs (circRNAs), have been recognized as prognostic biomarkers and promote cancer progression through cancer cell growth, metastasis, angiogenesis, and cancer development. One of the components of exosomes, circRNAs, are covalently closed and prevented from degrading, which results in their continually accumulating in exosomes. Evidence suggests that exosomal circRNAs are abundant and stable in body fluids and have been implicated in many diseases. In this article we summarize the biogenesis and function of circRNAs and explore the expressions of exosomal circRNAs in cancer, emphasizing the fact that exosomal circRNAs are a novel diagnostic biomarker in the early stages of cancer and/or a therapeutic target in further cancer treatment.