Breast Epithelial Cell Line MCF-10A Research Articles

Abstract 197: The GPR52-MCAM axis as a novel regulatory mechanism of breast cancer cell clustering and metastatic potential

Abstract Background: Breast cancer is the most diagnosed cancer worldwide and is the second leading cause of cancer death among women in the United States. Despite breakthroughs in targeted therapies for breast cancer, the 5-year survival rate for women with metastatic breast cancer is 30%. There is a strong rationale to explore the therapeutic potential of G-protein coupled receptors (GPCRs), which comprise the largest class of proteins successfully targeted by FDA-approved drugs. Importantly, at least one-third of GPCRs are “orphans” and remain largely uncharacterized. One of these orphan GPCRs is GPR52. To date, there is no literature on the role of GPR52 in any cancer type, but we find from publicly available clinical data that low mRNA expression of GPR52 in breast tumors correlates with reduced overall survival (hazard ratio=0.67 [0.53-0.83], logrank P= 0.00035) and that GPR52 mRNA levels tend to be lower in tumor metastases compared to primary tumor (n>80, p=4.45e-17). Therefore, we hypothesize that loss of GPR52 may support breast cancer metastasis. Results: We used CRISPR-Cas9 to knockout (KO) GPR52 in triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line MCF10A, and confirmed the generation of frameshift mutations by Sanger sequencing. Interestingly, we observed that GPR52 KO cells clustered together in vitro in 2D and hypothesized that this characteristic may allow GPR52 KO breast cancer cells to increase their metastatic potential. We conducted invasion assays though Matrigel and found that GPR52 KO cells were more likely to invade as sheets or clusters. To determine the in vivo impact of GPR52 loss, we injected MDA-MB-468 GPR52 KO cells in zebrafish (Danio rerio) two days post-fertilization and observed that GPR52 loss led to increased metastatic foci and total cancer surface area thirty hours post-injection (n≥12, p<0.05). We determined from RNA-sequencing (n=3, p<.0001) and confirmed with western blotting that the melanoma cell adhesion molecule (MCAM) is upregulated in the MCF10A and MDA-MB-468 GPR52 KO cells. Increased mRNA expression of MCAM has been associated with reduced overall survival in breast cancer patients (hazard ratio=1.33 [1.05-1.62], logrank P= 0.02) Conclusion: Knockout of GPR52 from breast cancer cells promotes increased cell clustering in vitro and metastasis in zebrafish. These data support the investigation of GPR52 as a biomarker of cancer aggression and a potential therapeutic target for breast cancer patients whose tumors express GPR52. Citation Format: Sarah Z. Hanif, CheukMan C. Au, Ingrid Torregroza, Bhavneet Bhinder, Lukas Dow, Olivier Elemento, Todd Evans, Kristy A. Brown. The GPR52-MCAM axis as a novel regulatory mechanism of breast cancer cell clustering and metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 197.

Orai1α and Orai1β support calcium entry and mammosphere formation in breast cancer stem cells

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells.

Dysregulated Expression of LncRNA-SChLAP1 in Breast Cancer

This study aimed to investigate the expression and clinical significance of the long chain non-coding RNA SCHLAP1 in breast cancer tissues. The research included 60 breast cancer patients treated between June 2017 and September 2019. Cancer and adjacent tissues were collected for analysis. Furthermore, breast cancer cell lines MCF-7 and HCC1937, along with normal breast epithelial cell line MCF10A, were used to study the impact of LncRNA SCHLAP1 on breast cancer cell phenotypes. qRT-PCR was employed to measure LncRNA SCHLAP1 expression levels in cells and tissues. The results demonstrated that LncRNA SCHLAP1 was significantly up-regulated in breast cancer cells and patient tissues (P <0.01). Moreover, differences in LncRNA SCHLAP1 expression were observed in patients with varying age, lymph node invasion, TNM staging, HER-2, and Ki-67 expression levels (P <0.01). Patients with high LncRNA SCHLAP1 expression had a significantly lower two-year survival rate (P <0.01). In vitro experiments revealed that down-regulated LncRNA SCHLAP1 inhibited the proliferation, migration, and invasion of MCF-7 cells, while promoting apoptosis (P <0.01). This study suggests that LncRNA SCHLAP1 is associated with breast cancer progression and patient survival, serving as an independent predictor for breast cancer progression.

A TGF-β-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration.

The non-receptor tyrosine kinase, SRC, is overexpressed and/or hyperactivated in various human cancers and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrated that transforming growth factor-β (TGF-β) induces SRC expression at the transcriptional level by activating an intragenic the SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-β1 stimulation upregulated the SRC1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that SMAD complex is recruited to three enhancer regions ∼15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-β. JUN, a member of the activator protein (AP)-1 family, localises to the enhancer and regulates TGF-β-induced SRC expression. Furthermore, TGF-β-induced SRC upregulation plays a crucial role in epithelial-mesenchymal transition (EMT)-associated cell migration by activating the SRC/focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-β-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.

Effect of microRNA-21 blockade in human triple negative breast cancer allografts on progression and immune checkpoint proteins.

e13097 Background: Triple negative breast cancer (TNBC) is an orphan disease that attacks > 46,000 US women every year. TNBC recurs after standard-of-care surgery, chemotherapy and radiation, killing half its victims within 4 years. Poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates extend the survival of a fraction of TNBC patients for a few months. Thus, TNBC shows a critical need for molecularly-targeted therapy. Most TNBC cells show high microRNA 21 (miR-21), identical in humans and mice, which decreases tumor suppressor proteins that keep cell growth in check. Hypothesis: Short anti-miR-21 RNA analogs conjugated to a receptor ligand will direct TNBC cell uptake and slow the growth of TNBC orthotopic allografts with immune activation and minimal toxicity. Methods: We designed an anti-miR-21 RNA analog using aminomethyl bridged nucleic acid (BNA), exhibiting low toxicity, and serum stability. We lipofected a concentration ramp of the anti-miR-21 RNA analog into human TNBC cell lines (MDA-MB-231, MDA-MB-157, MDA-MB-436, MDA-MB-468, BT-20, BT-549, HCC1806, HCC1937, HCC1806) representing a spectrum of TNBC subtypes, then measured proliferation and biochemical markers. We also conjugated a peptide analog for endocytosis by the insulin-like growth factor 1 receptor (IGF1R), overexpressed in most TNBC lesions. The peptide ligand for IGF1R provides a unique strategy for delivering the anti-miR-21 RNA analog preferentially into TNBC cells. We administered 5 mg/kg of the anti-miR-21 RNA-peptide analog in sterile saline intraperitoneally twice a week for 14 days into immunocompetent female Balb/c mice bearing syngeneic EMT6 TNBC tumors in their mammary fat pads. Results: The anti-miR-21 RNA analog slowed proliferation, increased apoptosis, elevated tumor suppressor proteins, and suppressed immune checkpoint gene expression in multiple human TNBC lines. IC50 for inhibiting cell proliferation correlated with miR-21 copies/cell in 7 TNBC cell lines, but not in the non-tumorigenic breast epithelial cell line MCF-10A. The anti-miR-21 RNA-peptide analog stopped orthotopic TNBC allograft growth in syngeneic immunocompetent female Balb/c mice during 14 days of therapy, with apparent safety. Conclusions: This strategy optimizes cancer cell-specific delivery to block proliferation and immune checkpoints. The results support further preclinical proof-of-concept studies to enable an eIND submission for the anti-miR-21 RNA-peptide analog.

AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

New bis(thieno[2,3-b]pyridine) hybrids linked to arene units as potential bacterial biofilm and MRSA inhibitors

Using sonication and mediated by piperazine, new arene-linked bis(thieno[2,3- b]pyridine) hybrids were efficiently prepared in this study. The target hybrids were prepared by reacting bis(α-haloketone) with two equivalents of the appropriate pyridine-2(1H)-thiones in the presence of 1.4 equivalents of piperazine. The reaction mixture was subjected to sonication at 60 °C for 30–40 min to produce the desired products in 88%–95% yields. When tested against six different ATCC bacterial strains, the new products demonstrated a wide range of antibacterial activity. The 4-(4-methoxyphenyl)-linked hybrids 1i and 1j, attached to 6-(4-methoxyphenyl) and 6-( p-tolyl) units, respectively, had the best efficacy against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa strains. Both hybrids showed more effective potency than the reference ciprofloxacin with MIC and MBC values in the ranges from 2.0 to 2.1, and 4.1 to 4.2 µM, respectively. Additionally, hybrids 1i and 1j demonstrated stronger efficacy than linezolid with MIC values ranging from 2.0 to 4.2 µM, and MBC values ranging from 8.2 to 8.5 µM, respectively, against the MRSA ATCC:33 591 and ATCC:43 300 strains. Furthermore, hybrids 1i and 1j showed inhibitory antibacterial biofilm activity comparable to the standard ciprofloxacin. They had IC50 values ranging from 3.8 to 4.6 µM against S. aureus, E. faecalis, E. coli, and P. aeruginosa strains. The cytotoxicity of hybrids 1i and 1j against the human breast epithelial cell line MCF-10A lends credence to both hybrids’ potential as safe antibacterial agents.

Abstract 4784: Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer

Abstract Early breast carcinogenesis is characterized by an intermittently hypoxic, acidic and nutrient-deprived microenvironment, eventually selecting for the invasive, aerobically glycolytic phenotype known as Warburg Effect (WE), a universal cancer hallmark. We have reproduced the emergence of WE in vitro, by subjecting the nontumorigenic breast epithelial cell line MCF10A to multiple intermittent cycles of media starvation followed by recovery in growth media. Clonal expansion of surviving cells demonstrated increased lactate production in aerobic conditions (WE). A comprehensive molecular characterization, through WES and single cell multiomics, was performed to study the relationship between mutations, CNA, cytogenetic abnormalities, transcriptional dysregulation, and epigenetic modifications. Mutational analysis of 6 WE clones identified 249 shared mutations, including HRAS(G12V), BRAF(P523L) and ALK(Q180X). COSMIC mutational signature 18 (ROS-induced) was identified as the main DNA lesion across all WE clones, suggesting oxidative stress as a main source of mutagenesis associated with WE development. Non-synonymous mutations in FUS, HDAC3, EZH2, MAZ, KMTD2, MET, and other oncogenes were found in the clones, consisting in secondary events in the WE transformation. Geneset enrichment analysis identified over-expression of MYC/E2F targets, MTORC1 signaling, glycolysis and cell cycle pathways, as well as under-expression of EMT/cell adhesion pathways, in WE clones. CNA, based on a consensus from WES, scRNAseq (inferCNV) and scATACseq (epiAneufinder), confirmed amplification of MYC, E2F1 and MYBL2, as well as Gain(19q) and Gain(20) in all WE clones, as well as loss of TP63 in 5 out of 6 WE clones. Integrative single cell transcriptome/chromatin accessibility analysis (ArchR) identified decreased accessibility and expression of EMT-related genes, as a consequence of differential expression of pioneer transcription factors (i.e. reduced expression of TP63, due to deletion, led to silencing of TP53/63/73 targeted genes), as well as well-established EMT regulators (ZEB1, SNAI1/2, NFKB1), among others. We propose that the WE phenotype emerges after intermittent nutrient-derived oxidative stress induces increased expression of HRAS-regulated genes, acutely increasing mutation rate and transcriptionally inducing an EMT-like state. Once HRAS(G12V), MYC/E2F1 amplification and TP53 deletions are fixed in the population, the clones will maintain the ROS production and EMT-like state, even in optimum growth media conditions, completing the transition from an acute response to a chronic-inheritable phenotype known as WE. These findings reinforce the importance of microenvironmental stress driving early carcinogenesis, bridges the gap between environment and genomic-driven causes of malignant transformation and provides testable hypotheses of causal events in connection with WE. Citation Format: Rafael Renatino Canevarolo, Praneeth Reddy Sudalagunta, Joon-Hyun Song, Erez Persi, Mehdi Damaghi, Ariosto Siqueira Silva. Multiomic landscape of evolution of Warburg effect identifies drivers of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4784.

Abstract P5-11-03: Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT

Abstract Objectives: The incidence of breast cancer has jumped to first place in the global malignant tumor. As an essential component of the TMEM family, TMEM45A is abnormally highly expressed in some malignant tumors, and plays a role in promoting and regulating the occurrence and development of tumors. This study analyzed the expression of TMEM45A in breast cancer tissues and breast cancer cell lines, and explored the relationship with clinical subtypes. To examine the effects of TMEM45A on TNBC cell proliferation, migration, invasion and other biological functions, and to preliminarily clarify the molecular mechanism of TMEM45A regulating the epithelial-mesenchymal transition of TNBC cell lines. Methods: 1. Download and analyze the TCGA database, analyze the difference in the expression of TMEM45A in normal breast tissue and different subtypes of breast cancer tissue, and detect the protein expression level of TMEM45A in 8 pairs of cancer tissues and adjacent tissues by western blot experiment. 2. Detection of TMEM45A protein expression in normal breast epithelial cell line MCF10A and five breast cancer cell lines (MCF-7, MDA-MB-231, BT-549, MDA-MB-468, SK-BR-3) by western blot experiment. 3. Using small interfering RNA technology to down-regulate the protein expression levels of MDA-MB-231 and BT-549 TNBC cell lines with relatively high TMEM45A expression. The changes in cell proliferation ability were detected by CCK8 assay and clone formation assay, and the changes in cell migration ability and invasion ability were detected by wound healing assay and transwell assay, respectively. 4. After down-regulating TMEM45A in two TNBC cell lines, the expression changes of epithelial cell phenotype marker E-cadherin, mesenchymal phenotype marker N-cadherin, Vimentin, and critical molecules in TGF-β/Smad signal transduction pathway (TGFβ1, Smad2, Smad3, p-Smad2, p-Smad3) were detected by western blot experiment. Results: 1. Based on the TCGA database, the results showed that the expression level of TMEM45A mRNA in breast cancer was significantly higher than in normal breast tissue. The expression level of TMEM45A mRNA in any breast cancer subtype was higher than that in normal breast tissue, and it was the highest in the TNBC subtype. In the results of western blot experiments of clinical breast cancer specimens, the expression of TMEM45A in breast cancer tissues was significantly higher than that in the corresponding adjacent tissues, which further confirmed the reliability of the results of the TCGA database. 2. In normal breast epithelial cell line MCF10A and five breast cancer cell lines, MCF10A has the lowest expression level of TMEM45A protein. Among the five breast cancer cell lines, MDA-MB-231 had the highest expression of TMEM45A, followed by BT-549. 3. CCK8 assay, clone formation assay, wound healing assay and transwell assay showed that down-regulating TMEM45A protein levels in the two TNBC cell lines could significantly reduce cell proliferation, migration and invasion abilities. 4. After down-regulating TMEM45A protein levels in two TNBC cell lines, EMT-related indicators E-cadherin protein expression levels increased, N-cadherin and Vimentin protein expressions decreased, and the expression levels of essential protein molecules Smad2 and Smad3 in the TGF-β/Smad signal transduction pathway did not change significantly, while p-Smad2, p-Smad3 and TGF-β1 protein expressions decreased. Conclusions: TMEM45A is relatively highly expressed in breast cancer tissues and cell lines, especially in the subtype of TNBC. Down-regulation of TMEM45A expression can inhibit the proliferation, migration and invasion of TNBC cell lines. Mechanistically, TMEM45A may reverse EMT by inhibiting the activity of the TGF-β/Smad pathway. Citation Format: Yingkun Xu, Shengchun Liu. Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-11-03.

Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression.3vPKP8VT_3CNKdhee9few9Video

Wnt-pathway inhibitors with selective activity against triple-negative breast cancer: From thienopyrimidine to quinazoline inhibitors.

The Wnt-pathway has a critical role in development and tissue homeostasis and has attracted increased attention to develop anticancer drugs due to its aberrant activation in many cancers. In this study, we identified a novel small molecule series with a thienopyrimidine scaffold acting as a downstream inhibitor of the β-catenin-dependent Wnt-pathway. This novel chemotype was investigated using Wnt-dependent triple-negative breast cancer (TNBC) cell lines. Structure activity relationship (SAR) exploration led to identification of low micromolar compounds such as 5a, 5d, 5e and a novel series with quinazoline scaffold such as 9d. Further investigation showed translation of activity to inhibit cancer survival of HCC1395 and MDA-MB-468 TNBC cell lines without affecting a non-cancerous breast epithelial cell line MCF10a. This anti-proliferative effect was synergistic to docetaxel treatment. Collectively, we identified novel chemotypes acting as a downstream inhibitor of β-catenin-dependent Wnt-pathway that could expand therapeutic options to manage TNBC.

Polyamine synthesis enzyme AMD1 is closely related to the tumorigenesis and prognosis of human breast cancer

Adenosylmethionine decarboxylase 1 (AMD1) has been implicated in carcinogenesis and tumor progression. However, the potential biomechanism and biological implications of AMD1 in breast cancer (BC) remain unclear. The purpose of this study was to investigate the effect of abnormal expression of AMD1 in BC. The expression of AMD1 in different human BC cell lines was studied by using western blotting and qRT–PCR. In vitro cell proliferation, clone formation, cell cycle and apoptosis assays were performed to explore the effect of AMD1 on cellular proliferation. Xenograft mouse models were established to elucidate the role of AMD1 in BC growth. The expression profiles of AMD1 in 28 pairs of BC tissues and adjacent noncancerous tissues (ANTs) were investigated by using western blotting and immunohistochemistry. The clinical implication and prognostic evaluation of AMD1 in BC were examined by excavating the online database. We found that the expression levels of AMD1 in BC cell lines were significantly higher than those in the normal human breast epithelial cell line MCF-10A. In addition, AMD1 potentiated proliferation, induced cell cycle progression and inhibited apoptosis in BC cells. Subcutaneous tumor xenografts also supported the promotive role of AMD1 in BC growth. We discovered that the level of AMD1 in BC tissues was significantly higher than that in ANTs. Using the online database, increased AMD1 was found to be associated with clinical indicators and predicted a poor prognosis in patients with BC. Our findings indicate that AMD1 elicits potent oncogenic effects on the malignant progression of BC. AMD1 might serve as a promising diagnostic biomarker and therapeutic target for patients with BC.

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

Reduction of Metastasis via Epigenetic Modulation in a Murine Model of Metastatic Triple Negative Breast Cancer (TNBC).

Simple SummaryWe investigate the use of the small molecule epigenetic modulator 4SC-202 as a potential cancer therapeutic or adjuvant for Triple Negative Breast Cancer (TNBC). Epigenetic modulation involves alteration of the cellular phenotype without altering the genotype. Here, we investigate how 4SC-202 affects tumor growth, the ability of tumor cells to migrate, and the growth of tumors in vivo. This study demonstrates that 4SC-202 kills tumor cells, reduces their ability to migrate, and decreases metastasis of the tumor cells and tumor burden in a highly metastatic murine model of TNBC. These preclinical studies provide critical information on the potential efficacy of 4SC-202 as a potential therapeutic or adjuvant for TNBC.This study investigates the effects of a dual selective Class I histone deacetylase (HDAC)/lysine-specific histone demethylase 1A (LSD1) inhibitor known as 4SC-202 (Domatinostat) on tumor growth and metastasis in a highly metastatic murine model of Triple Negative Breast Cancer (TNBC). 4SC-202 is cytotoxic and cytostatic to the TNBC murine cell line 4T1 and the human TNBC cell line MDA-MB-231; the drug does not kill the normal breast epithelial cell line MCF10A. Furthermore, 4SC-202 reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model, which closely mimics human TNBC in terms of sites of metastasis, reveal reduced tumor burden and lung metastasis. The mechanism of action of 4SC-202 may involve effects on cancer stem cells (CSC) which can self-renew and form metastatic lesions. Approximately 5% of the total 4T1 cell population grown in three-dimensional scaffolds had a distinct CD44high/CD24low CSC profile which decreased after treatment. Bulk transcriptome (RNA) sequencing analyses of 4T1 tumors reveal changes in metastasis-related pathways in 4SC-202-treated tumors, including changes to expression levels of genes implicated in cell migration and cell motility. In summary, 4SC-202 treatment of tumors from a highly metastatic murine model of TNBC reduces metastasis and warrants further preclinical studies.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling

Simple SummaryThe recent discovery of the genetic identity of the tubulin carboxypeptidase (TCP) provides a unique opportunity to study the role of the detyrosination of α-tubulin (deTyr-Tub), as performed by the TCP, in breast epithelial cells and breast cancer cells. Previous research has shown that elevated deTyr-Tub conveys a poor prognosis in breast cancer and is upregulated in a coordinated manner at the invasive margin of patient tumor samples. Using TCP expression constructs, we have shown that increased deTyr-Tub promotes apoptosis in normal breast epithelial cells, that does not occur in the same cells with an oncogenic KRas mutation or Bcl-2/Bcl-xL overexpression. Furthermore, the addition of the TCP to the breast cancer cell lines MDA-MB-231 and Hs578t, also harboring Ras mutations, leads to increased focal gelatin degradation.Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Structure-activity relationship studies on O-alkylamino-tethered salicylamide derivatives with various amino acid linkers as potent anticancer agents

In our continued SAR study efforts, a series of O-alkylamino-tethered salicylamide derivatives with various amino acid linkers has been designed, synthesized, and biologically evaluated as potent anticancer agents. Five selected compounds with different representative chemical structures were found to show broad anti-proliferative activities, effective against all tested ER-positive breast cancer (BC) and triple-negative breast cancer (TNBC) cell lines with low micromolar IC50 values. Among these compounds, compound 9a (JMX0293) maintained good potency against MDA-MB-231 cell line (IC50 = 3.38 ± 0.37 μM) while exhibiting very low toxicity against human non-tumorigenic breast epithelial cell line MCF-10A (IC50 > 60 μM). Further mechanistic studies showed that compound 9a could inhibit STAT3 phosphorylation and contribute to apoptosis in TNBC MDA-MB-231 cells. More importantly, compound 9a significantly suppressed MDA-MB-231 xenograft tumor growth in vivo without significant toxicity, indicating its great potential as a promising anticancer drug candidate for further clinical development.

Arene-ruthenium(II) complexes with tetracyclic oxime derivatives: synthesis, structure and antiproliferative activity against human breast cancer cells

Six new arene-ruthenium(II) complexes containing two different η6-arene ligands – benzene and hexamethylbenzene, with indenoquinoxalinone oxime analogues (11H-indeno[1,2-b]quinoxalin-11-one oxime, 6H-indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one oxime and 6–(hydroxyimino)indolo[2,1-b]quinazolin-12(6H)-one oxime (tryptanthrin-6-oxime)) as N,N’- chelating ligands are reported. The complexes were characterized by elemental analysis, IR, UV–VIS, 1H and 13C NMR spectroscopy and by single-crystal X-ray structure analysis. Complexes adopt a half-sandwich «piano-stool» geometry with oxime ligands in anionic form, the ruthenium(II) center coordinates one arene, one N,N-bidentate oximate and one chloride ligand. The computational analysis of non-covalent intermolecular interactions revealed weak attraction between the oxime oxygen atoms and the nearest hydrogen atoms of the oxime and the arene ligands. The cytotoxic activity of the complexes was evaluated against human breast cancer cell line MCF-7 and cisplatin-resistant human breast cancer cell line MCF-7CR as well as non-cancerous human breast epithelial cell line MCF10A. The cytotoxicity tests show micromolar IC50 values and tryptanthrin-6-oxime hexamethylbenzene-ruthenium(II) complex was found to exhibit the best antiproliferative activity among the studied compounds against the MCF-7 and MCF-7CR cell lines (IC50 9.0 ± 4.4 and 8.9 ± 1.5 µM, respectively).

Ajuba Overexpression Promotes Breast Cancer Chemoresistance and Glucose Uptake through TAZ-GLUT3/Survivin Pathway.

The LIM protein Ajuba has been implicated in the development of human cancers. To date, its expression pattern and biological significance in breast cancers (BC) have not been fully investigated. In the current study, we examined Ajuba protein levels in 93 invasive ductal carcinoma specimens by immunohistochemistry. The Ajuba expression level was elevated in breast cancer tissue compared with normal tissue. Ajuba overexpression is correlated with advanced tumor-node-metastasis (TNM) stage, positive node status, and adverse patient outcomes. The Ajuba protein level was also higher in BC cell lines compared to normal breast epithelial cell line MCF-10A. Ectopically expressed Ajuba in MCF-7 cells stimulated in vitro and in vivo cell growth, invasion, cell cycle progression, and decreased paclitaxel-induced apoptosis. RNA-sequencing (RNA-seq) followed by gene set enrichment analysis (GSEA) analysis showed that Ajuba overexpression regulated the Hippo signaling pathway. Ajuba overexpression also increased glucose uptake and increased expression of TAZ, GLUT3, and Survivin. TAZ knockdown abolished the role of Ajuba on GLUT3 and Survivin induction. The ChIP assay showed that TEAD4, a major TAZ binding transcription factor, could bind to the GLUT3 and Survivin promoter regions. In conclusion, our data demonstrated that elevated Ajuba expression is correlated with poor BC prognosis and regulated malignant behavior through TAZ-GLUT3/Survivin signaling in BC cells.

Breast adipose regulation of premenopausal breast epithelial phenotype involves interleukin 10.

Epidemiological studies inversely associate BMI with breast cancer risk in premenopausal women, but the pathophysiological linkage remains ill-defined. Despite the documented relevance of the 'local' environment to breast cancer progression and the well-accepted differences in transcriptome and metabolic properties of anatomically distinct fat depots, specific breast adipose contributions to the proliferative potential of non-diseased breast glandular compartment are not fully understood. To address early breast cancer causation in the context of obesity status, we compared the cellular and molecular phenotypes of breast adipose and matched breast glandular tissue from premenopausal non-obese (mean BMI = 27 kg/m2) and obese (mean BMI = 44 kg/m2) women. Breast adipose from obese women showed higher expression levels of adipogenic, pro-inflammatory, and estrogen synthetic genes than from non-obese women. Obese breast glandular tissue displayed lower proliferation and inflammatory status and higher expression of anti-proliferative/pro-senescence biomarkers TP53 and p21 than from non-obese women. Transcript levels for T-cell receptor and co-receptors CD3 and CD4 were higher in breast adipose of obese cohorts, coincident with elevated adipose interleukin 10 (IL10) and FOXP3 gene expression. In human breast epithelial cell lines MCF10A and HMEC, recombinant human IL10 reduced cell viability and CCND1 transcript levels, increased those of TP53 and p21, and promoted (MCF10A) apoptosis. Our findings suggest that breast adipose-associated IL10 may mediate paracrine interactions between non-diseased breast adipose and breast glandular compartments and highlight how breast adipose may program the local inflammatory milieu, partly by recruiting FOXP3+ T regulatory cells, to influence premenopausal breast cancer risk.