Phenotype Of Breast Cancer Cells Research Articles

Abstract LB208: An intricate connection between canonical Wnt and MAPK/ERK signaling drives the partial hybrid EMT state involving FOXC2/p63 activation leading to cancer stemness and metastasis

Abstract The epithelial to mesenchymal transition (EMT) is a dynamic process underlying metastasis. Here we show that TCF1, a TCF7 gene product, promotes a hybrid epithelial/mesenchymal (E/M) phenotype in basal breast cancer cells that was enriched in stemness and metastasis as compared to cells residing in the M-like state. E/M cells (CD104highCD44high) were enriched in canonical Wnt signaling, thereby upregulating FOXC2 which was turn able of converting M into E/M cells. Consistent with the E/M state, FOXC2 upregulated TA and ΔNp63 isoforms which control epithelial differentiation and Wnt signaling via β4 integrin/CD104 and Frizzled 7 upregulation respectively. Importantly, Wnt3a was able of stimulating ERK phosphorylation in E/M cells leading to FOXC2 and p63 upregulation thereby preventing E/M to M differentiation. These findings were further corroborated by ATAC-sequencing pointing predominantly to FOXC2 as a master regulator of the E/M state that was suppressed by ERK inhibition. Together, these results demonstrate that TCF7 stimulates Wnt3a/ERK activation which in turn activates the FOXC2/p63 axis, thereby promoting the hybrid EMT state underlying stemness and metastasis. Citation Format: Huizhi Liang, Outhiriaradjou Benard, Zuen Ren, Kimita Suyama, Jingli Wang, Anthony Griffen, Hao Wang, Lindsay LeFave, Larry Norton, Rachel B. Hazan. An intricate connection between canonical Wnt and MAPK/ERK signaling drives the partial hybrid EMT state involving FOXC2/p63 activation leading to cancer stemness and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB208.

Ropivacaine as a novel AKT1 specific inhibitor regulates the stemness of breast cancer

BackgroundRopivacaine, a local anesthetic, exhibits anti-tumor effects in various cancer types. However, its specific functions and the molecular mechanisms involved in breast cancer cell stemness remain elusive.MethodsThe effects of ropivacaine on breast cancer stemness were investigated by in vitro and in vivo assays (i.e., FACs, MTT assay, mammosphere formation assay, transwell assays, western blot, and xenograft model). RNA-seq, bioinformatics analysis, Western blot, Luciferase reporter assay, and CHIP assay were used to explore the mechanistic roles of ropivacaine subsequently.ResultsOur study showed that ropivacaine remarkably suppressed stem cells-like properties of breast cancer cells both in vitro and in vivo. RNA-seq analysis identified GGT1 as the downstream target gene responding to ropivacaine. High GGT1 levels are positively associated with a poor prognosis in breast cancer. Ropivacaine inhibited GGT1 expression by interacting with the catalytic domain of AKT1 directly to impair its kinase activity with resultant inactivation of NF-κB. Interestingly, NF-κB can bind to the promoter region of GGT1. KEGG and GSEA analysis indicated silence of GGT1 inhibited activation of NF-κB signaling pathway. Depletion of GGT1 diminished stem phenotypes of breast cancer cells, indicating the formation of NF-κB /AKT1/GGT1/NF-κB positive feedback loop in the regulation of ropivacaine-repressed stemness in breast cancer cells.ConclusionOur finding revealed that local anesthetic ropivacaine attenuated breast cancer stemness through AKT1/GGT1/NF-κB signaling pathway, suggesting the potential clinical value of ropivacaine in breast cancer treatment.

Abstract 6937: High throughput single cell simultaneous DNA and RNA sequencing identified cell-of-origin of breast cancer

Abstract Understanding the relationship between genotype and phenotype in breast cancer cells has been challenging at single cell resolution, mainly because existing high-throughput methods are limited to measuring a single modality and data must be integrated indirectly via computational methods. To address this challenge, we developed wellDR-seq, a high-throughput single cell method that can simultaneously measure the single cell whole genome and transcriptome directly from thousands of single cells in parallel. Using this method, we profiled 17,427 single cells in 6 different ER+ breast cancer patients with either premalignant disease (Ductal-carcinoma-in-situ) or invasive ductal carcinoma (IDC). From these data we identified the epithelial cell-of-origin in 3 cases, showing that ER+ breast cancer cells originated from Luminal Hormone Responsive (LumHR) in the normal breast tissues. We also found that autosomal somatic copy number aberrations were exclusively present in LumSec (< 2%) and LumHR epithelial cells, while chrX gain or loss also occurred in stromal cells (eg fibroblast and endothelial) but a low frequency (<2%) in our datasets. Additionally, our data show the impact of subclonal copy number profiles on gene expression programs, which reflects both gene dosage effects in CNA regions and more complex deregulation in copy-neutral regions. wellDR-seq offers a powerful tool for large-scale single cell genome and transcriptome simultaneous sequencing across different clinical samples, opens new avenue to investigate genotype and phenotypes interactions, identify tumor cells origins, reveal somatic copy number and mutation events, quantify the gene dosage effect and discerning differential genes expression between tumor subclones. Citation Format: Kaile Wang, Rui Ye, Shanshan Bai, Zhenna Xiao, Lei Yano, Jianzhuo Li, Yiyun Lin, Emi Sei, Steven Lin, Alastair Thompson, Savitri Krishnamurthy, Nicholas Navin. High throughput single cell simultaneous DNA and RNA sequencing identified cell-of-origin of breast cancer [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 6937.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer

PurposeDe novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear.MethodsA compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA.ResultsData from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ −, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression.ConclusionHMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

Retracted] Long non‑coding RNA MALAT‑1 contributes to maintenance of stem cell‑like phenotypes in breast cancer cells.

[This retracts the article DOI: 10.3892/ol.2017.7557.].

The impact of tumor microenvironment: unraveling the role of physical cues in breast cancer progression.

Metastasis accounts for the vast majority of breast cancer-related fatalities. Although the contribution of genetic and epigenetic modifications to breast cancer progression has been widely acknowledged, emerging evidence underscores the pivotal role of physical stimuli in driving breast cancer metastasis. In this review, we summarize the changes in the mechanics of the breast cancer microenvironment and describe the various forces that impact migrating and circulating tumor cells throughout the metastatic process. We also discuss the mechanosensing and mechanotransducing molecules responsible for promoting the malignant phenotype in breast cancer cells. Gaining a comprehensive understanding of the mechanobiology of breast cancer carries substantial potential to propel progress in prognosis, diagnosis, and patient treatment.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

The effect of DNA methyltransferase 3A suppression in progression of the resistance phenotype in breast cancer cells

Introduction. Rearrangement of molecular pathways and activation of bypass signaling determine the progression of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential for breast and other cancer beneficial treatments.Materials and methods. The present work was performed on estrogen receptor α ERα-positive (ERα – estrogen receptor α) McF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERα-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing.Results. We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression of cell resistance.Conclusion. Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.

Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-β pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.

Analysis of Interleukin-6 Gene Variants (rs1800795, rs1800796, rs1554606, rs1800797, rs2069840, rs12700386, and rs2069861) as Prognostic Markers in Breast Cancer: A Systematic Review, Meta-Analysis, and Network Analysis.

Interleukin-6 (IL-6) has obviously tumor-promoting and tumor-inhibitory effects and can induce an epithelial-mesenchymal transition phenotype in human breast cancer (BC) cells and implicate its potential to promote BC metastasis. Herein, we aimed to evaluate the association of IL-6 variants (rs1800795, rs1800796, rs1554606, rs1800797, rs2069840, rs12700386, and rs2069861) with the susceptibility to BC. The databases of PubMed/Medline, Web of Science, Scopus, and Cochrane Library were searched until December 19, 2022, without any restrictions. The quality assessment of each study was performed based on the Newcastle-Ottawa Scale tool. The Review Manager 5.3 software presented the effect sizes including odds ratio (OR) along with a 95% confidence interval (CI). Both publication bias and sensitivity analyses were carried out by the Comprehensive Meta-Analysis version 2.0 software. A total of 2,508 records were identified among databases and at last, 27 articles were entered into the meta-analysis. Seven polymorphisms of IL-6 were entered into the analyses. Just rs1800797 polymorphism in the dominant model (OR = 1.51; 95% CI = 1.15-2.00; P = 0.003) and rs2069840 polymorphism in heterozygous (OR = 0.89; 95% CI = 0.81-0.97; P = 0.008) and dominant (OR = 0.91; 95% CI = 0.84-0.99; P = 0.02) models had a significant association with the BC risk. In conclusion, among 7 polymorphisms and despite a few included cases, the present meta-analysis recommended that the AA+GA genotype of rs1800797 polymorphism had a significantly elevated risk and the GC and the CC+GC genotypes of rs2069840 polymorphism had a protective role in the BC patients.

Therapeutic influence of simvastatin on MCF-7 and MDA-MB-231 breast cancer cells via mitochondrial depletion and improvement in chemosensitivity of cytotoxic drugs

BackgroundBreast cancer is the most commonly diagnosed cancer worldwide with 2.26 million cases in 2020. Cancer heterogeneity is the major challenge before existing therapeutic modalities due to metabolic variability of the cells as Warburg and anti-Warburg both type of metabolic phenotypes has been reported as a major contributing factors for cancer progression, invasion, metastasis and relapse. Also, this metabolic variability is associated with chemo and radio-resistance and poor therapeutic outcomes. Therefore, in present study we put an attempt to understand how simvastatin exert its effects on two metabolically different cell types and second how this drug can affect mitochondrial biomass, mt-DNA and glycolysis in both the cell types. MethodsWe have observed effects of simvastatin on MCF-7 (dependent more on OXPHOS) and MDA-MB-231 (TNBC; more glycolytic with defected mitochondria) cells alone and after simvastatin pre-treatment followed by cytotoxic drugs including cisplatin, doxorubicin, gemcitabine, vincristine. We have conducted MTT assay for viability, cell death detection assay, apoptotic morphology study, scratch assay, transwell migration assay, lactate estimation in media (glycolysis parameter), mt-DNA to n-DNA ratio, mitotracker red (for mitochondrial membrane potential) and mitotracker green staining (for mitochondrial biomass) and qPCR to study expression of mitochondrial transcription factors and apoptotic genes including PGC-1α, NRF-1, NRF-2, TFAM, Bcl-2 and Bax. ResultsWe observed that 20 μM simvastatin (SIM) was most efficient dose for MCF-7, whereas 12.5 μM for MDA-MB-231 cells. Simvastatin itself caused a significant decrease in viability, increased cell death, and diminished wound closure in scratch assay as well as inhibited transwell migration. Also, the cells pre-treated with simvastatin for 72 h followed by treatment with cytotoxic drugs for 48 h increased chemo-sensitivity of cisplatin (CIS), doxorubicin (DOX), gemcitabine (GEM) and vincristine (VIN). SIM alone and in pre-treatment followed by cytotoxic drug treatment studies, there was a significant decrease in mitochondrial biomass and mitochondrial membrane potential (MMP), but also decreased glycolysis as evidenced by decrease in lactate levels in culture media. For inhibition of migratory potential, it was in the following order: CIS ˃ VIN ˃DOX˃ GEM, which was in the same order to diminish mitochondrial functionality (mt-DNA/n-DNA ratio, mitotracker green staining and a significant decrease in the expression of transcriptional factors of mitochondrial biogenesis). Contrastingly a decrease in the same order was observed in lactate concentration independent to the mitochondrial loss, but probably via inherent ability of the drugs to reduce lactate and glycolysis. However, for cell death, apoptotic phenotype, diminished expression of Bcl-2 along with increase in Bax and loss of viability, the efficiency of simvastatin alone and in pre-treatment studies was in the following order: VIN ˃ DOX˃GEM˃CIS, which was supported by loss of fluorescence of mitotracker red, suggested decrease in MMP; marker of cell death. ConclusionWe conclude that by using different doses simvastatin can target different metabolic phenotypes of breast cancer cells and can also increase the chemo-sensitivity of cytotoxic drugs, so that they can work efficiently at lower doses which will ultimately diminish the cost and toxicity issues.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

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.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

The focus of the study is to examine the function of TYMSOS in immune escape of breast cancer, which is the most frequently diagnosed malignancy among women globally. Our study demonstrated that upregulated TYMSOS was associated with unfavorable prognosis and immune escape in breast cancer. TYMSOS promoted the malignant phenotypes of breast cancer cells, and reduced the cytotoxicity of NK92 cells on these cells. CBX3 was a downstream effector in TYMSOS-induced malignant phenotypes in breast cancer cells. Mechanistic studies showed that TYMSOS facilitated CBX3-mediated transcriptional repression of ULBP3, and it also promoted SYVN1-mediated ubiquitin-proteasomal degradation of ULBP3. TYMSOS promoted cell growth, metastasis, and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis. The invivo studies further showed that silencing of TYMSOS repressed tumor growth and boosted NK cell cytotoxicity. In sum, TYMSOS boosted breast cancer metastasis and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis.

Differential miRNA expression of hypoxic MCF7 and PANC-1 cells.

Hypoxia plays a critical role in the tumor microenvironment by affecting cellular proliferation, metabolism, apoptosis, DNA repair, and chemoresistance. Since hypoxia provokes a distinct shift of microRNA, it is important to illustrate the relative contribution of each hypoxamiR to cancer progression. The present study aims to shed light on the hypoxamiRs that are involved in pancreatic and breast cancer progression to highlight novel targets for the development of new therapies. For 20 cycles, MCF7 breast cancer cells and PANC-1 pancreatic cancer cells were subjected to chronic cyclic hypoxia, which consisted of 72 hours of hypoxia followed by 24 hours of reoxygenation. After 10 and 20 cycles of hypoxia, miRNA expression alterations were profiled using RT-PCR array and further analyzed using a visual analytics platform. The MTT cell proliferation assay was used to determine hypoxic cells' chemoresistance to doxorubicin. Under chronic cyclic hypoxia, hypoxic PANC-1 cells have a comparable doubling time with their normoxic counterparts, whereas hypoxic MCF7 cells show a massive increase in doubling time when compared to their normoxic counterparts. Both hypoxic cell lines developed EMT-like phenotypes as well as doxorubicin resistance. According to the findings of miRNet, 6 and 10 miRNAs were shown to play an important role in enriching six hallmarks of pancreatic cancer in the 10th and 20th cycles of hypoxia, respectively, while 7 and 11 miRNAs were shown to play an important role in enriching the four hallmarks of breast cancer in the 10th and 20th cycles of hypoxia, respectively. miR-221, miR-21, miR-155, and miR-34 were found to be involved in the potentiation of hypoxic PANC-1 hallmarks at both the 10th and 20th cycles, while miR-93, miR-20a, miR-15, and miR-17 were found to be involved in the potentiation of hypoxic MCF7 hallmarks at both the 10th and 20th cycles. This variation in miRNA expression was also connected to the emergence of an EMT-like phenotype, alterations in proliferation rates, and doxorubicin resistance. The chemosensitivity results revealed that chronic cyclic hypoxia is critical in the formation of chemoresistant phenotypes in pancreatic and breast cancer cells. miR-181a and let-7e expression disparities in PANC1, as well as miR-93, miR-34, and miR-27 expression disparities in MCF7, may be associated with the formation of chemoresistant MCF7 and PANC-1 cells following 20 cycles of chronic cyclic hypoxia. Indeed, further research is needed since the particular mechanisms that govern these processes are unknown.

Targeting SHP2 with an Active Site Inhibitor Blocks Signaling and Breast Cancer Cell Phenotypes.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an oncogenic protein for which targeted therapies are being sought. In line with this idea, we have previously reported the development of a specific active site inhibitor named CNBDA that showed effectivity in suppressing the transformation phenotypes of breast cancer cells. To improve efficacy, we introduced limited modifications to the parent compound and tested potency in vitro and under cell culture conditions. Of these modifications, removal of one of the butyric acid groups led to the production of a compound named CNBCA, which showed a 5.7-fold better potency against the SHP2 enzyme activity in vitro. In addition, CNBCA showed better selectivity to SHP2 than the control PTPs (SHP1 and PTP1B) as determined by the phosphatase assay. Furthermore, CNBCA binds and inhibits enzyme activity of full-length SHP2 in cellular contexts, downregulates SHP2 mediated signaling, and suppresses breast cancer cell phenotypes, including cell proliferation, colony formation, and mammosphere growth. These findings show that targeting SHP2 with CNBCA is effective against the cancerous properties of breast cancer cells.

Overexpression of Long Non-coding RNA uc.246 Facilitates Angiogenesis, Migration, and EMT Phenotype of Human Breast Cancer Cells.

Breast cancer is the most malignant subtype of gynecological tumors and with aggressive biological behavior and poor outcome. Ultra-conserved non-coding RNA (ucRNA) is a newly discovered class of long non-coding RNAs (lncRNAs) which involved in the regulation of interaction network of genes. However, the exact function and mechanism by which ucRNA modulates breast cancer aggressive has not yet to be completely elucidated. In the present study, we demonstrated that the expression of uc.246 was significantly upregulated in metastatic breast cancer patients and TNBC cell lines, compared with those in controls. Furthermore, overexpression of uc.246 in MCF-7 cell lines enhanced the capacity of breast cancer cells to induce tube formation and migration of HUVECs, and, finally, enhanced breast cancer cells metastasis. Meanwhile, uc.246 overexpressing enhances the EMT phenotype of TNBC cells. Mechanistically, we found that uc.246 promoted malignant progression of breast cancer via upregulating the levels of VEGF-C and increased the levels of mesenchymal marker protein. Our results demonstrated that uc.246 induced angiogenesis, migration, and EMT phenotype and may represent a novel prognostic biomarker and therapeutic target for patients with breast cancer.

The Synergistic Effect of Proanthocyanidin and HDAC Inhibitor Inhibit Breast Cancer Cell Growth and Promote Apoptosis.

Histone deacetylase inhibitor (HDACi) is a drug mainly used to treat hematological tumors and breast cancer, but its inhibitory effect on breast cancer falls short of expectations. Grape seed proanthocyanidin extract (GSPE) with abundant proanthocyanidins (PAs) has been explored for its inhibition of HDAC activity in vitro and in vivo. To enhance HDACi's effectiveness, we investigated the potential of PA to synergistically enhance HDACi chidamide (Chi), and determined the underlying mechanism. We evaluated the half-inhibitory concentration (IC50) of PA and Chi using the cell counting kit 8 (CCK8), and analyzed drugs' synergistic effect with fixed-ratio combination using the software Compusyn. Breast cancer cell's phenotypes, including short-term and long-term proliferation, migration, invasion, apoptosis, and reactive oxygen species (ROS) levels, were assessed via CCK8, clone-formation assay, wound-healing test, Transwell Matrigel invasion assay, and flow-cytometry. Protein-protein interaction analysis (PPI) and KEGG pathway analysis were used to determine the underlying mechanism of synergy. PA + Chi synergistically inhibited cell growth in T47D and MDA-MB-231 breast cancer cell lines. Short-term and long-term proliferation were significantly inhibited, while cell apoptosis was promoted. Ten signaling pathways were identified to account for the synergistic effect after RNA sequencing. Their synergism may be closely related to the steroid biosynthesis and extracellular matrix (ECM) receptor interaction pathways. PA + Chi can synergistically inhibit breast cancer cell growth and proliferation, and promote apoptosis. These effects may be related to steroid biosynthesis or the ECM receptor pathway.