Biomarkers of breast cancer apoptosis induced by chemotherapy and TRAIL

Treatment of breast cancer is complex and challenging due to the heterogeneity of the disease. To avoid significant toxicity and adverse side-effects of chemotherapy in patients who respond poorly, biomarkers predicting therapeutic response are essential. This study has utilized a proteomic approach integrating 2D-DIGE, LC-MS/MS, and bioinformatics to analyze the proteome of breast cancer (ZR-75-1 and MDA-MB-231) and breast epithelial (MCF-10A) cell lines induced to undergo apoptosis using a combination of doxorubicin and TRAIL administered in sequence (Dox-TRAIL). Apoptosis induction was confirmed using a caspase-3 activity assay. Comparative proteomic analysis between whole cell lysates of Dox-TRAIL and control samples revealed 56 differentially expressed spots (≥2-fold change and p < 0.05) common to at least two cell lines. Of these, 19 proteins were identified yielding 11 unique protein identities: CFL1, EIF5A, HNRNPK, KRT8, KRT18, LMNA, MYH9, NACA, RPLP0, RPLP2, and RAD23B. A subset of the identified proteins was validated by selected reaction monitoring (SRM) and Western blotting. Pathway analysis revealed that the differentially abundant proteins were associated with cell death, cellular organization, integrin-linked kinase signaling, and actin cytoskeleton signaling pathways. The 2D-DIGE analysis has yielded candidate biomarkers of response to treatment in breast cancer cell models. Their clinical utility will depend on validation using patient breast biopsies pre- and post-treatment with anticancer drugs.     

Fluvastatin Mediated Breast Cancer Cell Death: A Proteomic Approach to Identify Differentially Regulated Proteins in MDA-MB-231 Cells

Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.     

DC Electric Fields Direct Breast Cancer Cell Migration, Induce EGFR Polarization, and Increase the Intracellular Level of Calcium Ions

Migration of cancer cells leads to invasion of primary tumors to distant organs (i.e., metastasis). Growing number of studies have demonstrated the migration of various cancer cell types directed by applied direct current electric fields (dcEF), i.e., electrotaxis, and suggested its potential implications in metastasis. MDA-MB-231 cell, a human metastatic breast cancer cell line, has been shown to migrate toward the anode of dcEF. Further characterizations of MDA-MB-231 cell electrotaxis and investigation of its underlying signaling mechanisms will lead to a better understanding of electrically guided cancer cell migration and metastasis. Therefore, we quantitatively characterized MDA-MB-231 cell electrotaxis and a few associated signaling events. Using a microfluidic device that can create well-controlled dcEF, we showed the anode-directing migration of MDA-MB-231 cells. In addition, surface staining of epidermal growth factor receptor (EGFR) and confocal microscopy showed the dcEF-induced anodal EGFR polarization in MDA-MB-231 cells. Furthermore, we showed an increase of intracellular calcium ions in MDA-MB-231 cells upon dcEF stimulation. Altogether, our study provided quantitative measurements of electrotactic migration of MDA-MB-231 cells, and demonstrated the electric field-mediated EGFR and calcium signaling events, suggesting their involvement in breast cancer cell electrotaxis.     

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor,CTGF) expression

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

Benzyl isothiocyanate inhibits basal and hepatocyte growth factor-stimulated migration of breast cancer cells

Benzyl isothiocyanate (BITC), which is found in cruciferous vegetables, has been shown to have anti-carcinogenic properties. Hepatocyte growth factor (HGF) has the ability to stimulate dissociation, migration, and invasion in various tumor cells, and abnormally increased expressions of HGF and its transmembrane tyrosine kinase receptor, c-Met, have previously been detected in human breast cancer, and are associated with high tumor grade and poor prognosis. In this study, in order to assess the mechanisms relevant to the BITC-induced regulation of breast cancer cell migration and invasion, MDA-MB-231 human breast cancer cells and 4T1 murine mammary carcinoma cells were cultured in the presence of 0-4?μmol/l BITC with or without 10?μg/l of HGF. BITC inhibited both the basal and HGF-induced migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner. In MDA-MB-231 cells, BITC reduced both basal and HGF-induced secretion and activity of urokinase-type plasminogen activator (uPA). In addition, BITC increased the protein levels of plasminogen activator inhibitor-1. HGF stimulated c-Met and Akt phosphorylation, but did not affect the phosphorylation of extracellular signal-regulated kinase-1/2 or stress-activated protein/c-jun N-terminal kinase. BITC suppressed NF-κB activity and reduced the HGF-induced phosphorylation of c-Met and Akt in a dose-dependent manner. LY294002, a specific Akt inhibitor, reduced both basal and HGF-induced uPA secretion and migration of MDA-MB-231 cells. In this study, we demonstrated that BITC profoundly inhibits the migration and invasion of MDA-MB-231 cells, which is associated with reduced uPA activity, and also that these phenomena are accompanied by the suppression of Akt signaling.     

整合素αvβ3对MDA-MB-231BO乳腺癌细胞AKT信号通路的调控

旨在探究整合素αvβ3的单克隆抗体LM609在BSP不同表达水平的乳腺癌细胞中对AKT(蛋白激酶B)信号通路的影响。利用免疫细胞化学法检测BSP不同表达水平的乳腺癌细胞中整合素αvβ3的表达量。BSP基因沉默乳腺癌MDA-MB-231BO细胞,Western blotting在蛋白水平检测磷酸化AKT的表达,MTT试验和细胞划痕试验分别检测细胞增殖、迁移能力的变化。结果显示,与231BO-Scrambled细胞相比,231BO-BSP27细胞中BSP蛋白水平明显降低,抑制率达到(59.43±1.71)%;LM609分别处理两株细胞后,与对照组231BO-Scrambled细胞相比,BSP基因沉默组21BO-BSP27细胞中AKT磷酸化水平下调明显,为(33.78±1.51)%(P<0.01);231BO-BSP27细胞和对照组231BO-Scrambled中细胞的增殖和迁移能力均有不同程度的下降(P<0.05)。LM609能够抑制胞内整合素αvβ3功能的表达,进而对AKT信号通路进行调控,并影响细胞增殖和迁移的发生。     

Adenosine arrests breast cancer cell motility by A3 receptor stimulation

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.     

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K_d = 48 nM and the related sFRP-2 with a K_d = 95 nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3β1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.     

Mitotic apparatus and nucleoli compartmentalization of 50,000-dalton type II regulatory subunit of cAMP-dependent protein kinase in estrogen receptor negative MDA-MB-231 human breast cancer cells

Affinity purified RI and RII antibodies of regulatory subunits (R) of type I (RI) and type II (RII) cAMP-dependent protein kinase were utilized to determine the immunological characterization and specific compartmentalization of R in estrogen receptor negative MDA-MB-231 human breast cancer cells. The 8-azido-(32P)-cAMP binding analysis of MDA-MB-231 cell extracts exhibited 47,000- and 50,000-dalton cAMP receptor proteins. RI and RII antibodies, by immunoprecipitation, detected the 47,000- and 50,000-dalton proteins, respectively. The 47,000-dalton protein was identified as RI as it showed a similar molecular weight as of bovine RI on SDS-polyacrylamide gel electrophoresis. Although 50,000-dalton protein did not co-migrate with bovine heart 54,000-dalton RII, it was identified as RII of MDA-MB-231 cells since it was specifically precipitated with RII antibody but not with RI antibody. An indirect immunofluorescence revealed that during different phases of growth of MDA-MB-231 cells, 50,000-dalton RII was specifically compartmentalized in the mitotic spindle and nucleoli of the cells whereas RI did not exhibit a specific compartmentalization in the cells, but was distributed throughout the cell components. These results suggest specific role(s) of 50,000-dalton RII at the nuclei of MDA-MB-231 cells.     

Artemisia absinthium (AA): a novel potential complementary and alternative medicine for breast cancer

Natural products have become increasingly important in pharmaceutical discoveries, and traditional herbalism has been a pioneering specialty in biomedical science. The search for effective plant-derived anticancer agents has continued to gain momentum in recent years. The present study aimed to investigate the role of crude extracts of the aerial parts of Artemisia absinthium (AA) extract in modulating intracellular signaling mechanisms, in particular its ability to inhibit cell proliferation and promote apoptosis in a human breast carcinoma estrogenic-unresponsive cell line, MDA-MB-231, and an estrogenic-responsive cell line, MCF-7. Cells were incubated with various concentrations of AA, and anti-proliferative activity was assessed by MTT assays, fluorescence microscopy after propidium iodide staining, western blotting and cell cycle analysis. Cell survival assays indicated that AA was cytotoxic to both MDA-MB-231 and MCF-7 cells. The morphological features typical of nucleic staining and the accumulation of sub-G1 peak revealed that the extract triggered apoptosis. Treatment with 25 μg/mL AA resulted in activation of caspase-7 and upregulation of Bad in MCF-7 cells, while exposure to 20 μg/mL AA induced upregulation of Bcl-2 protein in a time-dependent response in MDA-MB-231 cells. Both MEK1/2 and ERK1/2 was inactivated in both cell lines after AA treatment in a time-dependent manner. These results suggest that AA-induced anti-proliferative effects on human breast cancer cells could possibly trigger apoptosis in both cell lines through the modulation of Bcl-2 family proteins and the MEK/ERK pathway. This might lead to its possible development as a therapeutic agent for breast cancer following further investigations.     

Toll-like receptor 2 mediates invasion via activating NF-κB in MDA-MB-231 breast cancer cells

MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB-231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-κB and induced phosphorylation of TAK1 and IκBα in the TLR2/NF-κB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-β, VEGF and MMP9 secretion, which are associated with TLR2-NF-κB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-κB signaling activity and highly invasive capacity of MDA-MB-231 cells.     

Dysregulation of lncRNA-CCRR contributes to brain metastasis of breast cancer by intercellular coupling via regulating connexin 43 expression

Cardiac conduction regulatory RNA (CCRR) is down-regulated in the pathogenesis of heart failure (HF), which accordingly suppresses cardiac conduction while promoting arrhythmogenicity. Meanwhile, CX43 was reported to play a role in the pathogenesis of metastatic breast cancer and melanoma brain colonization. In this study, we studied the role of long non-coding RNA CCRR and its interaction with CX43 in brain metastasis of breast cancer. Breast cancer patients were grouped according to the metastasis status. Real-time PCR and IHC assay were used to measure the expression of lncRNA-CCRR and CX43 in patients. Western blot was conducted to observe the effect of lncRNA-CCRR on the expression of CX43 in MDA-MB-231BR and BT-474BR cells. Compared with the non-metastasis group, the mRNA expression of tissue lncRNA-CCRR, cerebrospinal fluid (CSF) lncRNA-CCRR, tissue CX43 and tissue protein expression of CX43 were both evidently up-regulated in metastasis patients, especially in patients with brain metastasis. The expression of lncRNA-CCRR was positively correlated with the up-regulated expression of CX43. Moreover, CX43 expression was significantly lower in MDA-MB-231WT cells compared with that in MDA-MB-231BR cells. Also, the overexpression of lncRNA-CCRR evidently increased dye transfer rate from astrocytes to MDA-MB-231BR/BT-474BR cells but reduced lncRNA-CCRR expression and suppressed the transmigration of MDA-MB-231BR/BT-474BR cells in a blood-brain barrier (BBB) model. In this study, we demonstrated that the presence of lncRNA-CCRR could up-regulate the expression of CX43, which promoted gap junction formation in brain metastasis of breast cancer. Accordingly, the communication between breast cancer cells and astrocytes was also promoted.     

Lung Epithelial Cells Induce Both Phenotype Alteration and Senescence in Breast Cancer Cells

Purpose

The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.

Methods

Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.

Results

Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.

Conclusions

Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.