Signaling Pathway In Breast Cancer Research Articles

Thidiazuron decreases epithelial-mesenchymal transition activity through the NF-kB and PI3K/AKT signalling pathways in breast cancer.

Breast cancer is the major type among the women population globally. The treatment of cancer metastasis has made modest progress due to multiple factors. Thidiazuron (TDZ) is a novel plant growth regulator that has been shown to have anticancer effects. Therefore, we explored the anti‐metastatic potentials of TDZ in cell lines by assessing its potential to suppress the epithelial‐mesenchymal transition (EMT). We pretreated the BEAS‐2B and breast cancer (MDA‐MB‐231) cells with TDZ and deliberated alteration in a cell viability, mammosphere, migration, NF‐кB signalling, PI3K/AKT signalling and matrix metalloproteinase (MMP) expression and analysed the EMT induction by TGF‐β/TNF‐α‐stimulated BEAS‐2B cells. Treatment with TDZ (5‐50 μmol) diminished the migration and invasion of the extremely metastatic MDA‐MB‐231 cells. Additionally, TDZ treatment led to down‐regulation of uPAR, uPA, VEGF and MMP‐2/‐9 expression and up‐regulation of TIMP‐1/2 expression in these cells. Furthermore, TDZ treatment blocked invasion and EMT in non‐tumorigenic BEAS‐2B epithelial cells stimulated with TGF‐β/TNF‐α.TDZ prevents EMT and may thus block metastasis of breast cancer cells.

An in-silico method leads to recognition of hub genes and crucial pathways in survival of patients with breast cancer

Breast cancer is a highly heterogeneous disorder characterized by dysregulation of expression of numerous genes and cascades. In the current study, we aim to use a system biology strategy to identify key genes and signaling pathways in breast cancer. We have retrieved data of two microarray datasets (GSE65194 and GSE45827) from the NCBI Gene Expression Omnibus database. R package was used for identification of differentially expressed genes (DEGs), assessment of gene ontology and pathway enrichment evaluation. The DEGs were integrated to construct a protein–protein interaction network. Next, hub genes were recognized using the Cytoscape software and lncRNA–mRNA co-expression analysis was performed to evaluate the potential roles of lncRNAs. Finally, the clinical importance of the obtained genes was assessed using Kaplan–Meier survival analysis. In the present study, 887 DEGs including 730 upregulated and 157 downregulated DEGs were detected between breast cancer and normal samples. By combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 2 hub genes including MAD2L1 and CCNB1 were selected. We also identified 12 lncRNAs with significant correlation with MAD2L1 and CCNB1 genes. According to The Kaplan–Meier plotter database MAD2L1, CCNA2, RAD51-AS1 and LINC01089 have the most prediction potential among all candidate hub genes. Our study offers a framework for recognition of mRNA–lncRNA network in breast cancer and detection of important pathways that could be used as therapeutic targets in this kind of cancer.

WAVE3 phosphorylation regulates the interplay between PI3K, TGF-\u03b2, and EGF signaling pathways in breast cancer

Both TGF-β and the PI3K-AKT signaling pathways are known activators of various intracellular pathways that regulate critical cellular functions, including cancer cell survival and proliferation. The interplay between these two oncogenic pathways plays a major role in promoting the initiation, growth, and progression of tumors, including breast cancers. The molecular underpinning of the inter-relationship between these pathways is, however, not fully understood, as is the role of WAVE3 phosphorylation in the regulation of tumor growth and progression. WAVE3 has been established as a major driver of the invasion–metastasis cascade in breast cancer and other tumors of epithelial origin. WAVE3 phosphorylation downstream of PI3K was also shown to regulate cell migration. Here we show that, in addition to PI3K, WAVE3 tyrosine phosphorylation can also be achieved downstream of TGF-β and EGF and that WAVE3 tyrosine phosphorylation is required for its oncogenic activity. Our in vitro analyses found loss of WAVE3 phosphorylation to significantly inhibit cell migration, as well as tumorsphere growth and invasion. In mouse models for breast cancer, loss of WAVE3 phosphorylation inhibited tumor growth of two aggressive breast cancer cell lines of triple-negative subtype. More importantly, we found that WAVE3 phosphorylation is also required for the activation of PI3K, TGF-β, and EGF signaling and their respective downstream effectors. Therefore, our study identified a novel function for WAVE3 in the regulation of breast cancer development and progression through the modulation of a positive feedback loop between WAVE3 and PI3K-TGF-β-EGF signaling pathways.

Comprehensive analysis of the NME gene family functions in breast cancer.

BackgroundAn increasing amount of research over recent years on the anti-metastasis function of the non-metastatic (NME) gene family has been challenged, with some studies identifying its involvement in the promotion of oncogenesis. Therefore, the specific functions of the NME gene family require redefining through a comprehensive analysis of tumor heterogeneity and survival benefit. However, the functions of NME genes have not been comprehensively investigated in breast cancer (BC).MethodsIn this study, ONCOMINE, GEPIA, Kaplan-Meier plotter, cBioPortal, String, and metascape databases were utilized for comparison of the mRNA expression, patient survival and network analysis of NME-associated signaling pathways in BC patients.ResultsThe mRNA expression of NME1 and NME2 was significantly increased in BC. Additionally, high NME 1 and NME2 levels were related to poor overall survival (OS), while the upregulated expression of NME3, NME5, and NME7 indicated prolonged survival. Moreover, increased mRNA level, amplification, or deep deletions in the NME gene family were identified in approximately 41% (450/1098) of all included BC specimens. NME1 and NME2 genes displayed the highest correlation with genetic correlations of the human NME genes in BC. The following pathways were regulated by NME gene upregulation: R-HAS-380270: Recruitment of mitotic centrosome and complexes; GO:0006228: UTP biosynthetic process; R-HAS-380259: Loss of NlP from mitotic centrosomes; hsa03410: Base excision repair; and CORUM:3714: Pericenrin-GCP complex, which was significantly modulated by changes influencing the NME genes.ConclusionsCollectively, our findings revealed that the elevated expression of NME1 and NME2 could act as a biomarker and predictive tool for BC patients with poor prognosis. Furthermore, our findings indicated that NME3, NME5, and NME7 might play the roles of tumor suppressor genes, which require validation through further experiments.

Introduction of hsa-miR-512-3p as a new regulator of HER2 signaling pathway in breast cancer.

Dysregulation of HER2 signaling pathway in breast cancer is well documented. Our bioinformatics analysis predicted hsa-miR-512-3p (miR-512-3p) as a bona fide regulator of HER2 as well as HER3, PIK3R2, and AKT1 genes. Then, we intended to examine the effect of miR-512-3p on the predicted target genes that are involved in HER2 signaling pathway. RT-qPCR results indicated lower expression of miR-512-3p in breast cancer specimens, compared to their normal pairs. Overexpression of miR-512-3p resulted in HER2, HER3, PIK3R2, and AKT1 gene downregulation, detected by RT-qPCR and the result was confirmed by western analysis and ELIZA test against p-AKT, BAX, FADD, and HER2 proteins in SKBR3 cells, respectively. Then, dual-luciferase assay supported the direct interaction of miR-512-3p with 3'UTR sequences of HER2, HER3, PIK3R2, and AKT1 target genes. When miR-512-3p was overexpressed, BAX/BCL2 expression ratio and proportion of sub-G1 cell population were increased in transfected SKBR3 cells, detected by RT-qPCR and flow cytometry, respectively. These results were consistent with the decreased viability of transfected cells, documented by MTT assay. In addition, results were consistent with the upregulation of BAX, BAK, BOK, PTEN, P53, and P21 genes and downregulation of CCND1 gene in SKBR3 cells. Although the overexpression of miR-512 resulted in cell cycle arrest at Sub-G1 stage in MDA-MB-231 cells, this effect seemed independent of targeting HER2, HER3, PIK3R2, and AKT1 target genes. Overall, results indicated that miR-512-3p acts as a cell-type-specific tumor suppressor, through targeting HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-512-3p as a potential candidate marker for breast cancer diagnosis.

Fibrinogen-Like Protein 2 (FGL2) is a Novel Biomarker for Clinical Prediction of Human Breast Cancer

BackgroundFibrinogen-like protein 2 (FGL2) is a member of the fibrinogen-like protein family and possesses important regulatory functions in both innate and adaptive immune responses. FGL2 is overexpressed in glioma, and its expression level is negatively associated with the prognosis of glioma patients. However, the diagnostic value of FGL2 is unknown in breast carcinoma.Material/MethodsWe comprehensively analyzed the expression pattern of FGL2 in breast cancer. Several online databases – TCGA, Oncomine, GEPIA, Kaplan-Meier plotter, and PrognoScan – were used in this study.ResultsBased on the TCGA dataset and Oncomine database, we found that the expression level of FGL2 was remarkably lower in breast cancer compared with adjacent normal tissues. Clinical data showed that the expression level of FGL2 was significantly associated with radiation therapy, PR status, and tumor stage. Bioinformatics analysis of the GEPIA, Kaplan-Meier plotter, and PrognoScan databases showed that lower FGL2 expression levels were associated with a worse prognosis in breast cancer patients. Furthermore, the expression level of FGL2 was positively correlated with the immune cell infiltrations in breast cancer, especially those cells with high antitumor activities. GO, KEGG, and GSEA analyses also validated that FGL2 was closely related to genes involved in the immune response, signal transduction, and T cell receptor signaling pathway in breast cancer.ConclusionsThe results demonstrated that high expression of FGL2 is a useful marker for breast cancer treatment and appears to be correlated with enhanced antitumor activities in breast cancer patients.

PdpaMn inhibits fatty acid synthase-mediated glycolysis by down-regulating PI3K/Akt signaling pathway in breast cancer.

Novel manganese complex, PdpaMn ([(Pdpa)MnCl2]), was developed to induce apoptosis in breast cancer cells. The impact of phosphoinositide-(3)-kinase pathway onto fatty acid synthase (FASN) has an effect on cellular metabolism in breast cancer. However, reverse actions from FASN towards PI3K/Akt are still indefinable. Perhaps, loss of FASN could regulate glycolysis. Previously we established that PdpaMn inhibits FASN and involve in mitochondrial function. This study investigated the activity of PdpaMn on glycolysis and its mechanism. PdpaMn was used to suppress FASN expression in tumor. Expression of ATP and lactic acid level was measured to investigate the glycolysis variance in cells and animals. MCF-7 and 4T1 cells were treated with G28UCM, an inhibitor of FASN and PdpaMn, western blotting to detect PI3K/Akt signaling pathway. The capacity of proliferation was investigated by western blotting and immunohistochemistry. PdpaMn selectively inhibits cancer cells and tumor growth but also block FASN expression and suppresses the content of free fatty acid. Lactate dehydrogenase (LDHA) protein level was down-regulated as G28UCM and PdpaMn inhibited FASN, glucose transporter (Glut1), and pyruvate kinase (PKM2) proteins level were not affected. PI3K, p-Akt in the experimental group evidently declined compared to the control group. Proliferation was suppressed in FASN-arbitrated glycolysis. Our study supports the hypothesis that loss of FASN by PdpaMn suppressed glycolysis via down-regulating PI3K/Akt signaling pathway revealing the direct link between FASN and glycolysis. The results have paved the way to unravel the mechanisms of FASN and mitochondrial will be useful for designing novel co-targeting strategies for breast cancer.

A:Role of Notch Signaling Pathway in Breast Cancer and Chemo-resistance

Despite the advanced treatment strategies, breast cancer remains the second cancer causing mortality across women worldwide. Notch signaling pathway has been found to be emerged in several cancers as well as in acquired and innate drug resistance. Therefore, understanding the role of Notch molecular mechanism could harbor beneficial impact to overcome chemoresistance and developing novel treatment strategies for cancer. Cisplatin (CIS) is an effective chemotherapeutic agent that is widely used against cancer but resistance is frequently occurs with limited therapeutic efficacy. One approach to overcome such unfavorable resistance is using complementary therapies with CIS. Thymoquinone (TQ) is a main compound in the essential oil of Nigella sativa. It has potent oncostatic activity by modulation of multiple regulatory pathways. Pentoxifylline (PTX) is a methylated xanthine derivative with remarkable anti-inflammatory and immunomodulatory actions. Combining TQ and PTX with CIS could be a promising strategy to suppress Notch signaling and overcome CIS resistance.

Notch Signaling Pathway and Endocrine Resistance in Breast Cancer.

Nearly 70% of breast cancers express the estrogen receptor (ER) and are hormone-dependent for cell proliferation and survival. Anti-estrogen therapies with aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs) or selective estrogen receptor down regulators (SERDs) are the standard endocrine therapy approach for ER positive breast cancer patients. However, about 30% of patients receiving endocrine therapy will progress during the therapy or become endocrine resistance eventually. The intrinsic or acquired endocrine resistance has become a major obstacle for endocrine therapy. The mechanism of endocrine resistance is very complicated and recently emerging evidence indicates dysregulation of Notch signaling pathway contributes to endocrine resistance in breast cancer patients. The potential mechanisms include regulation of ER, promotion of cancer stem cell (CSC) phenotype and mesenchymal cell ratio, alteration of the local tumor microenvironment and cell cycle. This review will summarize the latest progress on the investigation of Notch signaling pathway in breast cancer endocrine resistance.

Anticancer bioactive peptide combined with docetaxel and its mechanism in the treatment of breast cancer.

Breast cancer remains a worldwide public-health issue. Novel drugs that increase the sensitivity and reduce the toxic side effects of chemotherapeutic agents are urgently required. The present study investigated the effect and mechanism of the short-term intermittent administration of an anticancer bioactive peptide (ACBP), docetaxel (DTX), ACBP combined with DTX (MIX) and ACBP combined with low dose DTX (L-MIX) to nude mice bearing human breast cancer tumors. The body weight, tumor length, tumor diameter, diet and water consumption of the tumor-bearing nude mice were calculated. The protein and mRNA expression levels of p53, p21 and Ki67 were detected via immunohistochemistry and reverse transcription-quantitative PCR, respectively. The results revealed that the activity level of each group of mice was consistent. However, the food and water consumption of the ACBP group was significantly increased compared with the NS group. Compared with the normal saline group, the tumor weights and volumes of the treatment groups were significantly decreased, indicating an inhibitory effect of the treatment. However, the MIX group exhibited lower tumor weights and volumes compared with the ACBP and DTX groups. Furthermore, no significant cell necrosis, edema or inflammatory cell infiltration was observed upon hematoxylin & eosin staining of the liver and spleen in all groups. The results also revealed that the p21, p53 and Ki67 protein and mRNA levels were decreased in the ACBP, DTX and MIX groups compared with the control group. Additionally, when compared with those in the MIX and L-MIX groups, the p21 and Ki67 protein, and p53 and Ki67 mRNA levels in the ACBP and DTX groups were significantly increased. The results suggested that the short-term intermittent use of ACBP alone had an inhibitory effect on tumor growth and improved the food and water consumption of tumor-bearing nude mice. Furthermore, the combination of ACBP and DTX reduced toxic side effects and the dosage requirement of drugs to achieve therapeutic effects on the tumor-bearing nude mice. Therefore, the antitumor effect of ACBP may be associated with the improvement of immune function in tumor-bearing nude mice and ACBP may serve an antitumor role via the p53-p21 signaling pathway in breast cancer.

Breast Cancer Prevention-Is there a Future for Sulforaphane and Its Analogs?

Breast cancer is the most prevalent type of cancer among women worldwide. There are several recommended methods of breast cancer prevention, including chemoprevention. There are several approved drugs used to prevent breast cancer occurrence or recurrence and metastasizing. There are also a number of new substances undergoing clinical trials and at the stage of initial study. Studies suggest that dietary factors play a crucial role in breast cancer etiology. Epidemiological studies indicate that in particular vegetables from the Brassicaceae family are a rich source of chemopreventive substances, with sulforaphane (SFN) being one of the most widely studied and characterized. This review discusses potential applicability of SFN in breast cancer chemoprevention. A comprehensive review of the literature on the impact of SFN on molecular signalling pathways in breast cancer and breast untransformed cells is presented. The presented results of in vitro and in vivo studies show that this molecule has a potential to act as a preventive molecule either to prevent disease development or recurrence and metastasizing, and as a compound protecting normal cells against the toxic effects of cytostatics. Finally, the still scanty attempts to develop an improved analog are also presented and discussed.

Logic-based analysis of gene expression data predicts association between TNF, TGFB1 and EGF pathways in basal-like breast cancer.

For breast cancer, clinically important subtypes are well characterized at the molecular level in terms of gene expression profiles. In addition, signaling pathways in breast cancer have been extensively studied as therapeutic targets due to their roles in tumor growth and metastasis. However, it is challenging to put signaling pathways and gene expression profiles together to characterize biological mechanisms of breast cancer subtypes since many signaling events result from post-translational modifications, rather than gene expression differences. We designed a logic-based computational framework to explain the differences in gene expression profiles among breast cancer subtypes using Pathway Logic and transcriptional network information. Pathway Logic is a rewriting-logic-based formal system for modeling biological pathways including post-translational modifications. Our method demonstrated its utility by constructing subtype-specific path from key receptors (TNFR, TGFBR1 and EGFR) to key transcription factor (TF) regulators (RELA, ATF2, SMAD3 and ELK1) and identifying potential association between pathways via TFs in basal-specific paths, which could provide a novel insight on aggressive breast cancer subtypes. Codes and results are available at http://epigenomics.snu.ac.kr/PL/.

Dysregulation of TLR2 Serves as a Prognostic Biomarker in Breast Cancer and Predicts Resistance to Endocrine Therapy in the Luminal B Subtype.

Background: Breast cancer (BCa) is a serious global health burden among females, and the development of resistance represents an important challenge to BCa treatment. Here, we examined the expression of toll-like receptor 2 (TLR2) in BCa patients and the prognostic value of TLR2 for predicting endocrine resistance.Methods: The study included 150 BCa patients, of which 82 underwent endocrine therapy. TLR2 mRNA expression was measured by quantitative Real-Time PCR, and its prognostic value was determined by Kaplan-Meier survival analysis. Changes in the expression of TLR2 in BCa patients with endocrine resistance were assessed, and the value of TLR2 for predicting endocrine resistance was evaluated using the receiver operating characteristic curve analysis.Results: TLR2 expression was higher in BCa tissue than in normal tissue and associated with tumor size, HER2 status, tumor subtype, and TNM stage. TLR2 upregulation was associated with poor prognosis in patients with BCa, as well as endocrine resistance, and TLR2 upregulation was more prevalent among HER2-positive BCa cases. The predictive performance of TLR2 for endocrine resistance was higher in HER2-positive BCa than in other hormone receptor-positive BCa cases.Conclusion: TLR2 upregulation is a promising biomarker for prognosis and predicting resistance to endocrine therapy. The relationship between TLR2 and HER2 indicates that TLR2 may be involved in endocrine resistance through the HER2 signaling pathway in BCa.

ADGRF1 signaling pathways in Breast Cancer

BackgroundAdhesion G‐protein‐coupled receptors (GPCRs) form the second largest GPCR subfamily. They control many cellular processes involved in cancer, such as cell adhesion, invasion/migration, and stem cell function. Most of adhesion GPCRs are still orphans without any known endogenous ligands. In breast cancer, ADGRF1, previously called GPR110, is overexpressed in 2 aggressive subtypes of breast cancers, HER2+ and triple‐negative. We have found that HER2+ cells with ADGRF1 overexpression in doxycycline‐inducible manner formed more secondary mammospheres and more colonies in the soft agar assay and had higher % of Aldefluor+ cell population. However, ADGRF1‐overexpressing BT474 cells formed fewer tumors in mice and had reduced tumor growth compared to control.AimTo understand the downstream signaling of ADGRF1 that may contribute to the opposing findings in vitro and in vivo.MethodsThe experiments were conducted using two HER2+ breast cancer cell lines: BT474 and SKBR3 stable cells overexpressing ADGRF1 in doxycycline‐inducible manner and MDA‐MB‐231, a triple‐negative breast cancer cell line with endogenous ADGRF1 overexpression. The coupling of ADGRF1 to Gαs and Gαq pathway was assessed by measuring cAMP (AlphaScreen assay) and IP1 (Homogeneous Time‐Resolved Fluorescence assay), respectively and by co‐immunoprecipitation and immunoblotting. The effect of synaptamide, an ADGRF1 agonist, was also assessed on cAMP and IP1. The effects of Gαs or Gαq pathway activators were assessed on the potential of mammosphere formation (using Mammosphere assay).ResultsThe basal levels of cAMP and IP1 was significantly higher in doxycycline‐treated (+Dox) BT474 cells overexpressing ADGRF1 compared to cells without doxycycline (−Dox). Synaptamide (1–100 nM) did not further increase cAMP or IP1 in these cells. On the other hand, synaptamide increased cAMP but not IP1 in a concentration‐dependent manner in MDA‐MB‐231 cells. Immunoprecipitation with both anti‐Gαs and anti‐Gαq antibodies pulled down ADGRF1 in +Dox but not −Dox treated BT474 and SKBR3 cells. Forskolin, an activator of adenylyl cyclase that increases cAMP, reduced mammosphere formation in MDA‐MB‐231 cells. The effect of Gαs or Gαq pathway activators on mammosphere formation in BT474 and SKBR3 clones is being assessed in ongoing experiments.ConclusionOur results show that ADGRF1 couples to both Gαs and Gαq pathways. However, our data do not indicate that differential coupling leads to opposing in vitro versus in vivo effects. Additional studies with pathway activators as well as acute versus chronic activation of ADGRF1 may elucidate ADGRF1 pathways relevant in breast cancer biology.Support or Funding InformationDepartment of Defense Grants W81XWH‐14‐1‐0340 and W81XWH‐14‐1‐0341 to Drs. Trivedi and Schiff, respectively.

Tumour angiogenesis and c-Met pathway activation - implications in breast cancer.

Breast cancer is a heterogeneous disease with wide range of clinical behaviour. Tumour angiogenesis and metastasis have been considered as prognostic markers of the breast carcinoma, and c-Met, a transmembrane receptor tyrosine kinase has been implicated in both these processes of tumour progression. Thisstudy was conductedto elucidate c-Met and downstream signalling pathways in breast cancer and correlate with angiogenesis as assessed by microvessel density (MVD) and other prognostic parameters including lymph node metastases. Microvessel density (MVD) was assessed by endothelial cell (CD34) marker in breast cancers. c-Met was evaluated by immunohistochemistry for protein expression and by copy number assay for amplification at gene level. PCR array for gene expression related to c-Met, RAS-MAPK, PI3K-AKT and angiogenesis pathway was performed by real-time PCR. c-Met protein, copy number and mRNA expression did not differ significantly with the lymph node status or MVD. However, Her-2 overexpressing group showed c-Met protein overexpression and amplification. c-Met protein overexpression was also noted in the Luminal B subtype though no amplification was noted. Thus, the c-Met immunohistochemistry score and the c-MET copy numbers did not correlate with each other. c-Met downstream pathway genes (RAS-MAPK, PI3K-AKT and angiogenesis pathway) showed significant upregulation in Luminal B molecular subtype, lymph node-positive cases and cases with high MVD. The downstream signalling pathways (angiogenesis, RAS-MAPK and PI3K-AKT) were associated high MVD, lymph node metastases, and Her-2 and Luminal B subtype. Since inhibitors of these pathways are commercially available, these can be of therapeutic significance.

Abstract P3-11-20: Treatment sequencing of HR+/HER2- metastatic breast cancer (mBC) patients based on PIK3CA alteration status - A retrospective analysis of a US clinicogenomics database

Abstract Background: The PIK3CA gene encodes phosphatidylinositol-3-kinase (PI3K-α), which plays a central role in the growth, division, and many cellular processes critical for tumorigenesis. PIK3CA alteration is present in up to 40% of patients with HR+/HER2- mBC. Recent advances in therapies targeting the PI3K signaling pathway in breast cancer demonstrate improved outcomes; however, additional understanding on the implication of PIK3CA mutations in treatment sequencing decisions for patients with HR+/HER2- mBC is required. Methods: A retrospective analysis of the Flatiron Health/Foundation Medicine Clinico-Genomics Database, a nationwide database comprising de-identified patient-level structured and unstructured clinical data curated via technology-enabled abstraction and linked to genomic information, was conducted to characterize treatment sequencing among HR+/HER2- mBC patients, stratified by PIK3CA alteration status (PIK3CA+, PIK3CA-), as well as timing of NGS testing. The cohort included patients with follow-up through December 31, 2018, with no patients having received a PI3K inhibitor for mBC during this time. Patients in the cohort had ≥2 clinic visits since Jan 1 2011, chart confirmed diagnosis of HR+/HER2- mBC, and ≥1 Foundation Medicine next-generation sequencing (NGS) test conducted no earlier than 30 days before the mBC diagnosis date (index date). Treatment sequencing analysis included a sub-analysis by de-novo or relapsed mBC and based on sites of metastases and menopausal status. Results are presented as PIK3CA+ vs PIK3CA-, respectively (where applicable). Results: A total of 3,804 BC patients were in the database at the time of the analysis. After applying inclusion criteria, 1,293 patients were included for analysis (521 (40.3%) PIK3CA+, 772 (59.7%) PIK3CA-), median age 59yrs (Q1-Q3: 49-66), 98.8% female. PIK3CA+ patients were slightly older (median 60 vs. 56yrs). The majority of patients received their NGS test during or after 2L of mBC therapy (58% vs 54.9%), while only 5.6% of PIK3CA+ and 9.3% of PIK3CA- patients received NGS before commencing 1L therapy. The most common 1L mBC regimens were chemotherapy (CT) (36%), endocrine therapy (ET) (35%) and CDK4/6-based regimen (CDK4/6) (18%). PIK3CA status by NGS did not have a significant influence on treatment sequencing, however PIK3CA- patients had numerically higher CT than ET as 1L regimen. Common treatments were similar in 2L mBC, with less use of ET and increased use of CDK4/6 across groups. When assessing by menopausal status, the top three 1L treatments were unchanged, however there was a slight increased use of ET (43% vs 35%) and CDK4/6 (21% vs 20%) in post-menopausal patients. The most common sequence of treatments from 1L to 2L (post mBC diagnosis) was two lines of ET for the PIK3CA+ patients (14%) and two lines of CT for the PIK3CA- patients (19%). Two lines of CT was also the most common sequence for 2L to 3L for both groups (12% vs 18%). Remaining treatment sequences demonstrated significant heterogeneity and similar patterns in subgroups assessed. Conclusions: This analysis demonstrated significant heterogeneity of treatment patterns and sequences for both the PIK3CA+ and PIK3CA- groups, which may be expected given the limited available targeted therapies for patients with PIK3CA mutations during the study period. Mutation status is known late in the course of metastatic disease and does not appear to be associated with choice of treatment at this time. Citation Format: Stuart J Turner, Iris Wang, Jinhee Park, Hemanth Kanakamedala, Ines Lorenzo. Treatment sequencing of HR+/HER2- metastatic breast cancer (mBC) patients based on PIK3CA alteration status - A retrospective analysis of a US clinicogenomics database [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-20.

TRIP6 enhances stemness property of breast cancer cells through activation of Wnt/\u03b2-catenin

BackgroundThe urgent problem in the treatment of breast cancer is the recurrence induced by breast cancer stem cells (CSCs). Understanding the role and molecular mechanism of specific molecules in breast cancer stem cells can provide a theoretical basis for better treatment. TRIP6 is an adapter protein which belongs to the zyxin family of LIM proteins and is important in regulating the functions of CSCs. The present study aims to investigate the effects and mechanism of TRIP6 in breast cancer.MethodsTRIP6 expression in breast cancer cells and tissues were detected by Real-Time PCR, western blot and immunohistochemistry (IHC). MTT assays, colony formation assays, Xenografted tumor model and mammosphere formation assays were performed to investigate the oncogenic functions of TRIP6 in the tumorigenic capability and the tumor-initiating cell-like phenotype of breast cancer cells in vitro and in vivo. Luciferase reporter, subcellular fractionation and immunofluorescence staining assays were performed to determine the underlying mechanism of TRIP6-mediated stemness of breast cancer cells.ResultsTRIP6 expression was significantly upregulated in breast cancer, and was closely related to the clinicopathologic characteristics, poor overall survival (OS), relapse-free survival (RFS) and poor prognosis of breast cancer patients. Functional studies revealed that overexpression of TRIP6 significantly enhanced proliferative, tumorigenicity capability and the cancer stem cell-like properties of breast cancer in vitro and in vivo. On the contrary, silencing TRIP6 achieved the opposite results. Notably, we found that TRIP6 promoted Wnt/β-catenin signaling pathway in breast cancer to strengthen the tumor-initiating cell-like phenotype of breast cancer cells.ConclusionsThis study indicates that TRIP6 plays an important role in maintaining the stem cell-like characteristics of breast cancer cells, supporting the significance of TRIP6 as a novel potential prognostic biomarker and therapeutic target for diagnosis and treatment of breast cancer.

ROR2 promotes the epithelial-mesenchymal transition by regulating MAPK/p38 signaling pathway in breast cancer.

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a tyrosine-protein kinase receptor highly implicated in the growth plate and cartilage development, which may be involved in epithelial-mesenchymal transition (EMT) in breast cancer (BC) cells. Although ROR2 is known to promote the migration of BC cells, the detailed mechanism of this event is still not clear. Here, we found that ROR2 expression was significantly increased in BC lymphatic metastatic tissue as well as BC samples compared to normal adjacent breast tissues. A higher expression of ROR2 in MDA-MB-231 and a lower expression of ROR2 in MCF-7 cells were observed. MDA-MB-231-siROR2 cells with ROR2 knockdown inhibited MDA-MB-231 cell invasion, migration, and clonal formation, while MCF-7-OvROR2 cells with overexpression showed the opposite results. The underlying mechanisms involved in ROR2-induced EMT in MDA-MB-231 and MCF-7 cells were further investigated. ROR2 may activate EMT progression in BC cells by altering MAPK kinase 3/6 (MKK3/6) expression. The expressions of transforming growth factor-β, matrix metalloproteinase-2 (MMP-2), and MMP-9, which were related to tumor cell invasion activities, were notably increased in MCF-7-OvROR2 cells. The EMT markers, including snail, N-cadherin, tissue inhibitor of metalloproteinases-1, and vimentin, were significantly upregulated in MCF-7-OvROR2 cells. On the contrary, E-cadherin was obviously reduced expressed in MCF-7-OvROR2 cells. ROR2 may regulate the malignant phenotype of BC cells possibly via activation of mitogen-activated protein kinase (MAPK)/p38 signaling pathway. Collectively, ROR2 promotes BC carcinogenesis by mediating the MAPK/p38 pathway, which is independent of Wnt5α.

LncRNA NONHSAT141924 promotes paclitaxel chemotherapy resistance through p-CREB/Bcl-2 apoptosis signaling pathway in breast cancer.

Breast cancer is the most prevalent malignant neoplasm among women worldwide. Despite continuous improvement of breast cancer individualized comprehensive therapy, local recurrence and distant metastasis still remain the challenges due to the development of acquired drug-resistance. Long non-coding RNAs (LncRNAs) is known to participated in the development of breast cancer. However, the mechanisms of LncRNAs involving in drug-resistance of breast cancer during chemotherapy remain to be further elucidated. Aiming to screen for candidate LncRNAs responsible for breast cancer mechanism, we first investigated the expression patterns of LncRNAs and mRNAs in paired breast cancer tissues and normal tissues using Agilent Human lncRNA array. The microarray results clearly demonstrated multiple differentially expressed mRNAs and LncRNAs including LncRNA NONHSAT141924. The remarkable up-regulation of LncRNA NONHSAT141924 in breast cancer MCF-7 was further confirmed by quantitative real-time PCR. GO and KEGG pathway analysis demonstrated that LncRNA NONHSAT141924 was most closely associated with paclitaxel (PTX)-resistant phenotype. To further explore the mechanism by which LncRNA NONHSAT141924 contributes to PTX-resistant characteristics, LncRNA NONHSAT141924 was transfected into MCF-7 breast cancer cell line. Overexpression of LncRNA NONHSAT141924 significantly reduced MCF-7 cell survivability through modulation of p-CREB/Bcl-2 apoptosis signaling pathway, one of the major pathways participated in LncRNAs-mediated chemotherapy resistance. Taken together, our study provides a new LncRNA-mediated regulatory mechanism for PTX-resistance of breast cancer and suggests that therapeutic inhibition of LncRNA NONHSAT141924 might be an efficient strategy for PTX-resistant breast cancer treatment.

CCL18 promotes the invasion and metastasis of breast cancer through Annexin A2.

Chemokine (C‑C motif) ligand 18 (CCL18) is derived from breast tumor‑associated macrophages (TAMs), which are primarily a macrophage subpopulation with an M2 phenotype. CCL18 binds to its receptor, PYK2 N‑terminal domain interacting receptor 1 (Nir1), and promotes tumor progression and metastasis by inducing epithelial‑mesenchymal transition (EMT) via the PI3K/Akt/GSK3β/Snail signaling pathway in breast cancer cells. Recent research shows that Annexin A2 (AnxA2) plays a significant role in the invasion, metastasis, angiogenesis, proliferation, F‑actin polymerization and multidrug resistance to chemotherapy of breast cancer. The present study aimed to elucidate the molecular mechanisms by which CCL18 promotes breast cancer progression through AnxA2 which are not fully understood. Western blot analysis showed that the expression of AnxA2 was upregulated in highly invasive breast cancer cell lines and invasive ductal carcinoma. Furthermore, through chemotaxis, scratch, Matrigel invasion, and spontaneous metastasis assays, it was demonstrated that AnxA2 enhanced the invasion of breast cancer cells and the metastasis of human breast cancer cells to lungs of SCID mice with CCL18 stimulation. Cellular F‑actin measurement assay showed that reduction of AnxA2 suppressed CCL18‑induced F‑actin polymerization though phosphorylation of integrin β1 in breast cancer cells. Immunofluorescence and western blot analysis revealed that AnxA2 promoted CCL18‑induced EMT via the PI3K/Akt/GSK3β/Snail signaling pathway, and LY294002 inhibited the phosphorylation of AnxA2 invitro. In brief, AnxA2, as a downstream molecule of Nir 1 binding to CCL18, promotes invasion and metastasis by EMT through the PI3K/Akt/GSK3β/Snail signaling pathway in breast cancer. This study suggests that AnxA2 is a potential anti‑invasion/metastasis target for therapeutic intervention in breast cancer.