m6A-TCPred: a web server to predict tissue-conserved human m6A sites using machine learning approach.

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification in eukaryotic cells that plays a crucial role in regulating various biological processes, and dysregulation of m6A status is involved in multiple human diseases including cancer contexts. A number of prediction frameworks have been proposed for high-accuracy identification of putative m6A sites, however, none have targeted for direct prediction of tissue-conserved m6A modified residues from non-conserved ones at base-resolution level. RESULTS: We report here m6A-TCPred, a computational tool for predicting tissue-conserved m6A residues using m6A profiling data from 23 human tissues. By taking advantage of the traditional sequence-based characteristics and additional genome-derived information, m6A-TCPred successfully captured distinct patterns between potentially tissue-conserved m6A modifications and non-conserved ones, with an average AUROC of 0.871 and 0.879 tested on cross-validation and independent datasets, respectively. CONCLUSION: Our results have been integrated into an online platform: a database holding 268,115 high confidence m6A sites with their conserved information across 23 human tissues; and a web server to predict the conserved status of user-provided m6A collections. The web interface of m6A-TCPred is freely accessible at: www.rnamd.org/m6ATCPred .

Surgical margins and prognosis of borderline and malignant phyllodes tumors.

PURPOSE: To investigate the optimal surgical margin and prognostic risk factors for borderline and malignant phyllodes tumors (PTs). METHODS: A retrospective analysis was conducted on patients with borderline and malignant PTs at our hospital from 2011 to 2022. Univariate and multivariate Cox proportional hazard models were employed to analyze the effects of various variables on local recurrence-free survival (LRFS) and disease-free survival (DFS). RESULTS: This study comprised 150 patients, 85 classified as borderline and 65 as malignant. During a median follow-up of 66 months (range: 3-146 months), 34 cases (22.7%) experienced local recurrence, 9 cases (6.0%) exhibited distant metastasis, and 7 cases (4.7%) resulted in death. Irrespective of the histological subtypes, patients with surgical margins ≥ 1 cm exhibit significantly higher 5-year LRFS and 5-year DFS rates compared to those with margins < 1 cm. Among patients with initial margins < 1 cm, LRFS (P = 0.004) and DFS (P = 0.003) were improved in patients reoperated to achieve margins ≥ 1 cm. Surgical margin < 1 cm (HR = 2.567, 95%CI 1.137-5.793, P = 0.023) and age < 45 years (HR = 2.079, 95%CI 1.033-4.184, P = 0.040) were identified as independent risk factors for LRFS. Additionally, surgical margin < 1 cm (HR = 3.074, 95%CI 1.622-5.826, P = 0.001) and tumor size > 5 cm (HR = 2.719, 95%CI 1.307-5.656, P = 0.007) were determined to be independent risk factors for DFS. CONCLUSIONS: A negative surgical margin of at least 1 cm (with secondary resection if necessary) should be achieved for borderline and malignant PTs. Tumor size > 5 cm and age < 45 years were predictive of recurrence, suggesting multiple therapy modalities may be considered for these high-risk patients.

Long noncoding RNA KB-1980E6.3 promotes breast cancer progression through the PI3K/AKT signalling pathway.

This research aims to investigate the effect of lncRNA KB-1980E6.3 on the biological behaviour of breast cancer cells under normoxic conditions and the underlying molecular mechanism. The expression of KB-1980E6.3 in breast cancer tissues and cells was detected by RT-qPCR. The proliferation, migration and invasion of cells were evaluated by CCK-8, colony formation, scratch and Transwell assays; KB-1980E6.3-related xenograft models were established for in vivo studies. The protein expression of PI3K, p-PI3K, AKT and p-AKT was validated by western blotting analysis. The levels of KB-1980E6.3 are significantly upregulated in breast cancer tissues and cells and are related to the poor prognosis. Functional research both in vivo and in vitro revealed that the downregulation of KB-1980E6.3 expression significantly decreased cell proliferation, invasion and migration, while ectopic KB-1980E6.3 expression obviously promoted these biological phenotypes. In terms of the mechanism, KB-1980E6.3 is involved in the activation of the PI3K/AKT signalling pathway. Knockdown of KB-1980E6.3 reduced the expression of the p-PI3K and p-AKT proteins, whereas KB-1980E6.3 overexpression showed the opposite result. The agonist 740Y-P and inhibitor LY294002 reversed the effect of KB-1980E6.3 knockdown and overexpression on the PI3K/AKT pathway in BC cells. KB-1980E6.3 promotes the proliferation, invasion and migration of breast cancer cells by activating PI3K/AKT signalling, which can be used as a potential target for breast cancer therapy.

Pyroptosis-Related lncRNAs for Predicting the Prognosis and Identifying Immune Microenvironment Infiltration in Breast Cancer Lung Metastasis.

Breast cancer (BC) is the second leading cause of death among women and is highly heterogeneous. Three pyroptosis (PR) subtypes were identified in patients with BC from the Cancer Genome Atlas Database (TCGA) cohorts using 20 PR-related regulators, which illustrate a strong association between BC prognosis and PR. Lung metastasis commonly occurs in the advanced stages of BC, resulting in a poor quality of life. Eight differentially expressed (DE) lncRNAs were identified using LASSO-Cox analysis between PR-related and BC lung metastasis. Moreover, a BRCA risk-score (RS) model was established using multivariate Cox regression, which correlated with prognosis in TCGA-BRCA. Clinical characteristics, tumor mutation burden, and tumor immune cell infiltration were extensively investigated between high- and low-RS groups. Similarly, a lower RS implied longer overall survival, greater inflammatory cell infiltration, and better immunotherapeutic response to PD-1 blockers. Our findings provide a foundation for future studies targeting PR and confirme that RS could predict the prognosis of patients with BC.

Exploring the influencing factors of the pathologic complete response in estrogen receptor-positive, HER2-negative breast cancer after neoadjuvant chemotherapy: a retrospective study.

BACKGROUND: Pathological complete response (pCR) is the goal of neoadjuvant chemotherapy (NACT). We aimed to develop a nomogram to predict the probability of achieving pCR in estrogen receptor-positive (ER+), HER2-negative (HER2-) breast cancer patients. METHODS: A total of 273 ER+, HER2- breast cancer patients who received 4 cycles of thrice-weekly standard NACT in the First Affiliated Hospital of Chongqing Medical University were retrospectively enrolled. Univariate and multivariate logistic regression analyses were used to screen the predictive factors to develop the nomograms. The discrimination and calibration abilities were assessed by the C-index, receiver operating characteristic curve (AUC), and calibration plot. RESULTS: There were 28 patients (10.3%) with overall pCR, 38 patients (13.9%) with breast pCR after NACT. ER expression, PgR expression, the neutrophil-to-lymphocyte ratio (NLR) and the Ki-67 index were independent predictive factors for achieving overall pCR. These indicators had good discrimination and calibration ability (AUC 0.843). The nomogram for breast pCR was established based on ER expression, PgR expression, the NLR, and the Ki-67 index and showed great discriminatory ability, with an AUC of 0.810. The calibration curve showed that the predictive ability of the nomogram was a good fit to actual observations. CONCLUSION: The nomograms exhibited a sufficient discriminatory ability for predicting pCR after NACT in ER+, HER2- breast cancer patients. Utilizing these nomograms will enable us to identify patients at high probability for pCR after NACT and provide a reference for preoperative adjuvant therapy.

Genetic Variants Associated with Thyroid Cancer Risk: Comprehensive Research Synopsis, Meta-Analysis, and Cumulative Epidemiological Evidence.

PURPOSE: With the increasing incidence of thyroid cancer (TC), associations between genetic polymorphisms and TC risk have attracted a lot of attention. Considering that the results of associations of genetic variants with TC were usually inconsistent based on publications until now, we attempted to comprehensively evaluate the real evidence of associations between single nucleotide polymorphisms (SNPs) and TC risk. METHOD: We performed meta-analyses on 36 SNPs in 23 genes associated with TC susceptibility based on the data from 99 articles and comprehensively valued the epidemiological evidence of significant associations through the Venice criteria and false-positive report probability (FPRP) test. OR and P value were also calculated for 19 SNPs in 13 genes based on the insufficient data from 22 articles. RESULTS: 19 SNPs were found significantly associated with TC susceptibility. Of these, strong epidemiological evidence of associations was identified for the following seven SNPs: POU5F1B rs6983267, FOXE1 rs966423, TERT rs2736100, NKX2-1 rs944289, FOXE1 rs1867277, FOXE1 rs2439302, and RET rs1799939, in which moderate associations were found in four SNPs and weak associations were found in eight SNPs. In addition, probable significant associations with TC were found in nine SNPs. CONCLUSION: Our study systematically evaluated associations between SNPs and TC risk and offered reference information for further understanding of polymorphisms and TC susceptibility.

A pseudo-targeted metabolomics study based on serum bile acids profiling for the differential diagnosis of benign and malignant breast lesions.

INTRODUCTION: Breast cancer (BC) has become the most commonly diagnosed cancer worldwide. It is very critical for the differential diagnosis between BC and benign breast diseases (BBD). The characteristics of serum bile acids (BAs) profiling in patients with BBD and BC was elucidated so that potential biomarkers could be find out for the differential diagnosis of BC and BBD. METHODS: A pseudo-targeted approach was used to perform BAs metabolomics analysis in serum of 29 patients with BBD and 47 patients with BC by ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Partial least squares-discriminant analysis (PLS-DA) was used to establish a differential diagnostic model for BC, and the receiver operating characteristic (ROC) curve and logistic regression analysis were used to screen out bile acids as biomarkers for the differential diagnosis of BC and BBD. RESULTS: The serum BAs profile in BC group was quite different from that in BBD group. Compared with the BBD group, BC group had higher level of chenodeoxycholic acid (CDCA), while they had lower levels of dihydroxy tauro-conjugated BA (Tdi-1) and sulfated dihydroxy glyco-conjugated BA (Gdi-S-1). The sensitivity and specificity of PLS-DA model for patients classification were 100% and 92.3%, respectively. The combined biomarker, CDCA and Tdi-1, had high efficacy for the differential diagnosis (area under the curve was 0.954, 95% CI: 0.880-1.000) of BC. Besides, the performance was superior to traditional biomarkers in the differential diagnosis of BC with or without comorbidities. CONCLUSION: The profile of serum BAs in women with BC was quite different from that in patients with BBD. Serum BAs profiling analysis could be used as an effective tool for the differential diagnosis of BC and BBD.

GPR30-mediated HMGB1 upregulation in CAFs induces autophagy and tamoxifen resistance in ERα-positive breast cancer cells.

Tamoxifen (TAM) resistance constitutes a challenge in managing estrogen receptor (ER)α+ breast cancer patients. G-protein-coupled estrogen receptor (GPR30/GPER), which reportedly initiates TAM resistance in ERα+/ GPR30+ breast cancers, is detected in the breast cancer microenvironment, especially cancer associated fibroblasts (CAFs). Herein, considering that GPR30 mediates transcriptional regulation in different cell backgrounds, a microarray strategy was applied in immortalized CAFs derived from primary breast cancer samples, resulting in the identification of 165 GPR30 target genes, among which HMGB1 was confirmed to be upregulated by 17-ß estradiol(E2)- and TAM-triggered GPR30 activation in CAFs. Activated GPR30 increased extracellular HMGB1 secretion by CAFs, which was reduced by blocking PI3K/AKT signaling using G15 or LY294002. GPR30-induced HMGB1 upregulation triggered MEK/ERK signaling, leading to increased autophagic behavior to protect cancer cells from TAM-induced apoptosis, mimicking the recombinant HMGB1-mediated increase in cancer cell resistance potential to TAM. MEK/ERK signaling blockage by U0126 decreased the autophagic behavior and resistance ability of cancer cells to TAM. CAF-expressed GPR30 induced TAM resistance via HMGB1 in vivo. Overall, TAM upregulated HMGB1 expression and secretion in CAFs via GPR30/PI3K/AKT signaling, and the secreted HMGB1 induced autophagy to enhance TAM resistance in MCF-7 cells in an ERK-dependent manner. Thus, targeting GPR30 and downstream cascades may be an effective strategy to attenuate the resistance of ERα-positive breast tumors to endocrine therapy.

A novel Lnc408 maintains breast cancer stem cell stemness by recruiting SP3 to suppress CBY1 transcription and increasing nuclear ß-catenin levels.

Tumor initiation, development, and relapse may be closely associated with cancer stem cells (CSCs). The complicated mechanisms underlying the maintenance of CSCs are keeping in illustration. Long noncoding RNAs (lncRNAs), due to their multifunction in various biological processes, have been indicated to play a crucial role in CSC renewal and stemness maintenance. Using lncRNA array, we identified a novel lncRNA (named lnc408) in epithelial-mesenchymal transition-related breast CSCs (BCSCs). The lnc408 is high expressed in BCSCs in vitro and in vivo. The enhanced lnc408 is critical to BCSC characteristics and tumorigenesis. Lnc408 can recruit transcript factor SP3 to CBY1 promoter to serve as an inhibitor in CBY1 transcription in BCSCs. The high expressed CBY1 in non-BCSC interacts with 14-3-3 and ß-catenin to form a ternary complex, which leads a translocation of the ternary complex into cytoplasm from nucleus and degradation of ß-catenin in phosphorylation-dependent pattern. The lnc408-mediated decrease of CBY1 in BCSCs impairs the formation of 14-3-3/ß-catenin/CBY1 complex, and keeps ß-catenin in nucleus to promote CSC-associated CD44, SOX2, Nanog, Klf4, and c-Myc expressions and contributes to mammosphere formation; however, restoration of CBY1 expression in tumor cells reduces BCSC and its enrichment, thus lnc408 plays an essential role in maintenance of BCSC stemness. In shortly, these findings highlight that the novel lnc408 functions as an oncogenic factor by recruiting SP3 to inhibit CBY1 expression and ß-catenin accumulation in nucleus to maintain stemness properties of BCSCs. Lnc408-CBY1-ß-catenin signaling axis might serve as a new diagnostic and therapeutic target for breast cancer.

SLC6A8-mediated intracellular creatine accumulation enhances hypoxic breast cancer cell survival via ameliorating oxidative stress.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia. METHODS: RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo. RESULTS: SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3. CONCLUSIONS: Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.

GPER-Induced ERK Signaling Decreases Cell Viability of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with a poor prognosis. Effective biomarkers and specific therapeutic targets for HCC are therefore urgently needed. G protein-coupled estrogen receptor (GPER) plays a crucial role in numerous cancer types; however, its functions in HCC require further exploration. In the present study, we found a remarkable difference in GPER staining between tumor tissue (100/141, 70.9%) and matched non-tumor tissue (27/30, 90.0%). Compared with the GPER-negative patients, the GPER-positive patients with HCC were closely associated with female sex, negative hepatitis B surface antigen, small tumor size, low serum alpha fetoprotein level, and longer overall survival. Treatment with GPER-specific agonist G1 led to the sustained and transient activation of the EGFR/ERK and EGFR/AKT signaling pathways, respectively, in the HCC cell lines HCCLM3 and SMMC-7721, which express high levels of GPER. Interestingly, G1-induced EGFR/ERK signaling, rather than EGFR/AKT signaling mediated by GPER, was involved in decreasing cell viability by blocking cell cycle progression, thereby promoting apoptosis and inhibiting cell growth. Clinical analysis indicated that simultaneous high expression of GPER and phosphorylated-ERK (p-ERK) predicted improved prognosis for HCC. Finally, the activation of GPER/ERK signaling remarkably suppressed tumor growth in an HCC xenograft model, and this result was consistent with the in vitro data. Our findings suggest that specific activation of the GPER/ERK axis may serve as a novel tumor-suppressive mechanism and that this axis could be a therapeutic target for HCC.

A novel hypoxic long noncoding RNA KB-1980E6.3 maintains breast cancer stem cell stemness via interacting with IGF2BP1 to facilitate c-Myc mRNA stability.

The hostile hypoxic microenvironment takes primary responsibility for the rapid expansion of breast cancer tumors. However, the underlying mechanism is not fully understood. Here, using RNA sequencing (RNA-seq) analysis, we identified a hypoxia-induced long noncoding RNA (lncRNA) KB-1980E6.3, which is aberrantly upregulated in clinical breast cancer tissues and closely correlated with poor prognosis of breast cancer patients. The enhanced lncRNA KB-1980E6.3 facilitates breast cancer stem cells (BCSCs) self-renewal and tumorigenesis under hypoxic microenvironment both in vitro and in vivo. Mechanistically, lncRNA KB-1980E6.3 recruited insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to form a lncRNA KB-1980E6.3/IGF2BP1/c-Myc signaling axis that retained the stability of c-Myc mRNA through increasing binding of IGF2BP1 with m6A-modified c-Myc coding region instability determinant (CRD) mRNA. In conclusion, we confirm that lncRNA KB-1980E6.3 maintains the stemness of BCSCs through lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis and suggest that disrupting this axis might provide a new therapeutic target for refractory hypoxic tumors.

Although c­MYC contributes to tamoxifen resistance, it improves cisplatin sensitivity in ER­positive breast cancer.

Tamoxifen (TAM) resistance is a major challenge in the treatment of estrogen receptor­positive (ER+) breast cancer. To date, to the best of our knowledge, there are only a few studies available examining the response of patients with TAM­resistant breast cancer to chemotherapy, and the guidelines do not specify recommended drugs for these patients. In the present study, TAM­resistant cells were shown to exhibit increased proliferation and invasion compared with the parent cells, and the increased expression of c­MYC was demonstrated to play an important role in TAM resistance. Furthermore, the TAM­resistant cells were significantly more sensitive to cisplatin compared with the parent cells, and the silencing of c­MYC expression desensitized the cells to cisplatin through the inhibition of the cell cycle. An increased c­MYC expression was observed in 28 pairs of primary and metastatic tumors from patients treated with TAM, and the clinical remission rate of cisplatin­based chemotherapy was significantly higher compared with other chemotherapy­based regimens in 122 patients with TAM resistant breast cancer. Taken together, the data of the present study demonstrated that although c­MYC was involved in TAM resistance, it increased the sensitivity of ER+ breast cancer to cisplatin. Thus, cisplatin may be a preferred chemotherapeutic agent for the treatment of patients with TAM­resistant breast cancer, particularly in patients where the rapid control of disease progression is required.

Does the prohibition on open burning of straw mitigate air pollution? An empirical study in Jilin Province of China in the post-harvest season.

Straw open burning is common practice in agricultural production, and has been identified as highly relevant to regional haze. A variety of control measures for straw open burning have been adopted in agricultural areas to mitigate air pollution, but their contribution to air quality improvement is difficult to estimate, and has been addressed by few studies. Using Jilin Province during the post-harvest season (PHS) as a study area, we empirically evaluated the contribution of the Straw Open Burning Prohibition Plan (SOBP) to air quality improvement by response surface methodology (RMS) modeling combined with scenario analysis, and constructed and introduced a stagnant index to the RMS model. The results indicated that the adoption of the SOBP by Jilin Province in the 2018 PHS mitigated air pollution effectively. The contribution of the plan to air quality improvement was also evaluated under two scenarios: if the SOBP had been implemented during the 2015 PHS, 2016 PHS, and 2017 PHS, the cumulative PM2.5 concentration would have declined in these years by 23%, 15%, and 22%, respectively, compared with their actual values; if the SOBP had not been implemented in the 2018 PHS, the cumulative PM2.5 concentration would have increased by 10%-14% compared with the actual value. The results can provide decision makers with a reference to assess the environmental performance of the SOBP, and guidance for establishing scientific environmental regulations for cleaner agricultural production and sustainable development.

Identification of candidate RNA signatures in triple-negative breast cancer by the construction of a competing endogenous RNA network with integrative analyses of Gene Expression Omnibus and The Cancer Genome Atlas data.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is characterized by aggressive and metastatic clinical characteristics and generally leads to earlier distant recurrence and poorer prognosis than other molecular subtypes. Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) serve a crucial role in a wide variety of biological processes by interacting with microRNAs (miRNAs) as competing endogenous RNAs (ceRNAs) and, thus, affect the expression of target genes in multiple types of cancer. Seven datasets from the Gene Expression Omnibus (GEO) database, including 444 tumor and 88 healthy tissue samples, were utilized to investigate the underlying mechanisms of TNBC and identify prognostic biomarkers. Differentially expressed genes (DEGs) were further validated in The Cancer Genome Atlas database and the associations between their expression levels and clinical information were analyzed to identify prognostic values. A potential lncRNA-miRNA-mRNA ceRNA network was also constructed. Finally, 69 mRNAs from the integrated Gene Expression Omnibus datasets were identified as DEGs using the robust rank aggregation method with |log2FC|>1 and adjusted P<0.01 set as the significance cut-off levels. In addition, 29 lncRNAs, 21 miRNAs and 27 mRNAs were included in the construction of the ceRNA network. The present study elucidated the mechanisms underlying the progression of TNBC and identified novel prognostic biomarkers for TNBC.

GPER-regulated lncRNA-Glu promotes glutamate secretion to enhance cellular invasion and metastasis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a group of breast cancer with heterogeneity and poor prognosis and effective therapeutic targets are not available currently. TNBC has been recognized as estrogen-independent breast cancer, while the novel estrogen receptor, namely G protein-coupled estrogen receptor (GPER), was claimed to mediate estrogenic actions in TNBC tissues and cell lines. Through mRNA microarrays, lncRNA microarrays, and bioinformatics analysis, we found that GPER is activated by 17ß-estradiol (E2) and GPER-specific agonist G1, which downregulates a novel lncRNA (termed as lncRNA-Glu). LncRNA-Glu can inhibit glutamate transport activity and transcriptional activity of its target gene VGLUT2 via specific binding. GPER-mediated reduction of lncRNA-Glu promotes glutamate transport activity and transcriptional activity of VGLUT2. Furthermore, GPER-mediated activation of cAMP-PKA signaling contributes to glutamate secretion. LncRNA-Glu-VGLUT2 signaling synergizes with cAMP-PKA signaling to increase autologous glutamate secretion in TNBC cells, which activates glutamate N-methyl-D-aspartate receptor (NMDAR) and its downstream CaMK and MEK-MAPK pathways, thus enhancing cellular invasion and metastasis in vitro and in vivo. Our data provide new insights into GPER-mediated glutamate secretion and its downstream signaling NMDAR-CaMK/MEK-MAPK during TNBC invasion. The mechanisms we discovered may provide new targets for clinical therapy of TNBC.

Autocrine TGF-ß1/miR-200s/miR-221/DNMT3B regulatory loop maintains CAF status to fuel breast cancer cell proliferation.

Cancer-associated fibroblasts (CAFs) remain active even in the absence of cancer cells. However, the molecular mechanism underlying the sustained active status of CAFs is largely unrevealed. We found that in CAFs, DNMT3B was not only a target of miR-200b, miR-200c and miR-221, but was able to induce DNA methylation of miR-200s promoters. DNMT3B eventually reached a stably high level by the counteracting effect of decreasing miR-200b/c and increasing miR-221 in normal fibroblasts (NFs) with long-term exogenous TGF-ß1 treatment, and DNMT3B further led to a low level of miR-200s which established CAF activation. Meanwhile, miR-200s/miR-221/DNMT3B signaling sustained autocrine TGF-ß1 maintaining active CAF status. Destruction of the autocrine TGF-ß1/miR-200s/miR-221/DNMT3B signaling led to demethylation of miR-200s promoters and further restored the NF phenotypes. Moreover, we confirmed that TCF12, the target of miR-141, stimulated c-Myc/Cyclin D1 axis in breast cancer cells to promote cancer growth by enhancing CXCL12 of CAFs. The current study reveals that the TGF-ß1/miR-200s/miR-221/DNMT3B regulatory loop is responsible for the maintenance of CAFs status and is also necessary for CAF function in promoting malignance of breast cancer, which provides a potential target for CAF-driven therapeutic strategy.

ATM-Mediated Phosphorylation of Cortactin Involved in Actin Polymerization Promotes Breast Cancer Cells Migration and Invasion.

BACKGROUND/AIMS: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear. METHODS: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay. RESULTS: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion. CONCLUSION: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.

MiR-205/YAP1 in Activated Fibroblasts of Breast Tumor Promotes VEGF-independent Angiogenesis through STAT3 Signaling.

Tumor microenvironment contributes to tumor angiogenesis. However, the role of the activated cancer associated-fibroblasts (CAFs) in angiogenesis is still unclear. Here we report that miR-205/YAP1 signaling in the activated stromal fibroblasts plays a critical role in VEGF-independent angiogenesis in breast tumor. Methods: miR-205 expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR); YAP1 expression by qRT-PCR, western blotting and immunohistochemistry; IL11 and IL15 expression by qRT-PCR, western blotting and ELISA. Tube formation and three-dimensioned sprouting assays in vitro, and orthotopic Xenografts in vivo were conducted as angiogenesis experiments. The mechanism of miR-205/YAP1-mediated tumor angiogenesis was analyzed via overexpression and shRNA, siRNA, or antibody neutralization experiments in combination with anti-VEGF antibody or Axitinib. Results: miR-205/YAP1 signaling axis activates breast normal fibroblasts (NFs) into CAFs, promotes tubule formation and sprouting of Human Umbilical Vein Endothelial Cells (HUVECs). Rescue of miR-205 in CAFs blunts angiogenesis processes. YAP1, a target of miR-205, does not regulate VEGF expression but specifically enhances IL11 and IL15 expressions, maintaining tumor angiogenesis even in the presence of Axitinib or after exhaustion of VEGF by neutralizing VEGF antibody. IL11 and IL15 released from CAFs activate STAT3 signaling in HUVECs. Blockage of IL11 and IL15 expression in CAFs results in the inactivation of STAT3-signaling in HUVECs and repression of the CAF-induced angiogenesis. The blunt angiogenesis halts the invasion and metastasis of breast cancer cells in vivo. Conclusions: These results provide a novel insight into breast CAF-induced tumor angiogenesis in a VEGF-independent manner.