Sensitivity Of Breast Cancer Cells Research Articles

Upregulation of microRNA-181a-5p increases the sensitivity of HS578T breast cancer cells to cisplatin by inducing vitamin D receptor-mediated cell autophagy.

Breast cancer (BC) is the leading cause of death in females worldwide. Although cisplatin is a strong-effect and broad-spectrum chemotherapy drug, resistance to cisplatin remains a significant factor effecting clinical efficacy. The underlying mechanism of cancer cell resistance to cisplatin is not fully understood. MicroRNAs (miRs/miRNAs), as a regulator, are involved in regulating chemosensitivity to numerous chemotherapeutic drugs. The present study aimed to investigate the function of miR-181a-5p as a potential tumor suppressor in improving the efficiency of cisplatin in BC. The IC50 of cisplatin and miR-181a-5p expression were determined in five BC cell lines, and HS578T was selected as an appropriate cell line for subsequent experiments. The sensitivity of HS578T cells to cisplatin was assessed using cell proliferation, migration and apoptosis assays. Western blotting was performed to detect the expression of vitamin D receptor (VDR) and autophagy in HS578T cells. It was found that the increase in autophagy resulted in increased apoptosis and sensitivity to cisplatin in HS578T cells. miR-181a-5p transfection also inhibited the proliferation and migration ability of HS578T cells and induced apoptosis. Meanwhile, HS578T cells have increased sensitivity to cisplatin. VDR, as a target gene and autophagy regulator of miR-181a-5p, was negatively regulated by miR-181a-5p. Upon the decrease in VDR expression, the autophagy in HS578T cells was increased. These results indicate that the increase in autophagy enhanced the chemosensitivity of cisplatin by inducing apoptosis of HS578T cells and by inhibiting proliferation and migration. The present study showed that miR-181a-5p increased the chemical sensitivity of HS578T cells to cisplatin by inhibiting VDR to promote autophagy. The use of miR-181a-5p/autophagy/VDR-based treatment strategies may be a potential method to overcome cisplatin resistance in BC.

Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1

SUMMARYAberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC. XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor.

Investigating differences between tamoxifen resistant and sensitive breast cancer cells with flow cytometry.

The active metabolite of tamoxifen, 4‐hydroxytamoxifen, functions as an anti‐estrogen in breast cancer cells and thus inhibits proliferation. While tamoxifen continues to be successfully used to treat estrogen‐dependent breast cancer, most patients receiving treatment will develop chemoresistance over time. Two commonly reported biomarkers of tamoxifen resistance are decreased expression of insulin‐like growth factor 1 receptor (IGF‐1R) and increased expression of epidermal growth factor receptor (EGFR). In prior work we have shown that these receptors facilitate chemoresistance and have unique regulatory functions measurable in resistant cell lines compared with nonresistant. Thus, we hypothesized that these receptors and a newly identified biomarker, integrin β1, may be used to search for the presence of resistant breast cancer cells within a population of cells that are sensitive to tamoxifen therapy. We tested this by designing a straightforward cell‐labeling approach to measure differences in the receptor expression of resistant vs. sensitive cells cytometrically. Our results show that separation is possible when observing the expression of IGF‐1R as well as integrin β1. Interestingly, we found no detectable difference in EGFR expression between tamoxifen resistant and ‐sensitive cells when measured with cytometry despite the fact that EGFR is upregulated in resistant cells. Our long‐term goal is to utilize sorting to isolate tamoxifen resistant subpopulations of cells by receptor expression level. Isolating rare resistant cells that reside within a population of drug‐sensitive cells will offer new insights into why chemoresistance occurs.

LncRNA CBR3-AS1 regulates of breast cancer drug sensitivity as a competing endogenous RNA through the JNK1/MEK4-mediated MAPK signal pathway

BackgroundAdriamycin (ADR) resistance is one of the main obstacles to improving the clinical prognosis of breast cancer patients. Long noncoding RNAs (lncRNAs) can regulate cell behavior, but the role of these RNAs in the anti-ADR activity of breast cancer remains unclear. Here, we aim to investigate the imbalance of a particular long noncoding RNA, lncRNA CBR3 antisense RNA 1 (CBR3-AS1), and its role in ADR resistance.MethodsMicroarray analysis of ADR-resistant breast cancer cells was performed to identify CBR3-AS1. CCK-8 and colony formation assays were used to detect the sensitivity of breast cancer cells to ADR. Dual-luciferase reporter, RNA pulldown, IHC and western blot analyses were used to verify the relationship between the expression of CBR3-AS1, miRNA and target genes. For in vivo experiments, the effect of CBR3-AS1 on breast cancer resistance was observed in a xenograft tumor model. The role of CBR3-AS1 in influencing ADR sensitivity was verified by clinical breast cancer specimens from the TCGA, CCLE, and GDSC databases.ResultsWe found that CBR3-AS1 expression was significantly increased in breast cancer tissues and was closely correlated with poor prognosis. CBR3-AS1 overexpression promoted ADR resistance in breast cancer cells in vitro and in vivo. Mechanistically, we identified that CBR3-AS1 functioned as a competitive endogenous RNA by sponging miR-25-3p. MEK4 and JNK1 of the MAPK pathway were determined to be direct downstream proteins of the CBR3-AS1/miR-25-3p axis in breast cancer cells.ConclusionsIn summary, our findings demonstrate that CBR3-AS1 plays a critical role in the chemotherapy resistance of breast cancer by mediating the miR-25-3p and MEK4/JNK1 regulatory axes. The potential of CBR3-AS1 as a targetable oncogene and therapeutic biomarker of breast cancer was identified.

CircGFRA1 affects the sensitivity of triple-negative breast cancer cells to paclitaxel via the miR-361-5p/TLR4 pathway.

In recent years, the role of circular RNAs (circRNAs) in tumours has attracted widespread attention. Some circRNAs have been reported to play a role in triple-negative breast cancer (TNBC). However, circRNAs have rarely been reported in terms of TNBC resistance. This study aimed to clarify that circGFRA1 affects the sensitivity of TNBC cells to paclitaxel (PTX) by the miR-361-5p/TLR4 pathway. Compared with the non-PTX-resistant TNBC cell line MDA-MB-231, the expression of circGFRA1 in the PTX-resistant TNBC cell line MDA-MB-231.PR was significantly increased. The small hairpin RNA-mediated circGFRA1 knockdown inhibited the resistance of TNBC cells to PTX. RNA pull-down assay and luciferase reporter gene assay confirmed the binding between circGFRA1 and miR-361-5p and between miR-361-5p and TLR4. It has been proven that circGFRA1 knockdown can inhibit the resistance of TNBC cells to PTX by promoting the expression of miR-361-5p, and subsequently reduce the expression of TLR4.

Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells

Cisplatin (CDDP) is currently one of the most effective FDA-approved treatments for breast cancer. Previous studies have shown that CDDP-induced cell death in human breast cancer (MCF-7) cells is associated with disruption of calcium homeostasis. However, whether the sensitivity of breast cancer cells to cisplatin is associated with dysregulation of the expression of calcium-binding proteins (CaBPs) remains unknown. In this study, we evaluated the effect of the intracellular calcium chelator (BAPTA-AM) on viability of MCF-7 cells in the presence of toxic and sub-toxic doses of cisplatin. Furthermore, this study assessed the expression of CaBPs, calmodulin, S100A8, and S100A14 in MCF-7 cells treated with cisplatin. Cell viability was determined using MTT-based in vitro toxicity assay. Intracellular calcium imaging was done using Fluo-4 AM, a cell-permeant fluorescent calcium indicator. Expression of CaBPs was tested using real-time quantitative PCR. Exposure of cells to increasing amounts of CDDP correlated with increasing fluorescence of the intracellular calcium indicator, Fluo-4 AM. Conversely, treating cells with cisplatin significantly decreased mRNA levels of calmodulin, S100A8, and S100A14. Treatment of the cells with calcium chelator, BAPTA-AM, significantly enhanced the cytotoxic effects of sub-toxic dose of cisplatin. Our results indicated a statistically significant negative correlation between calmodulin, S100A8, and S100A14 expression and sensitivity of breast cancer cells to a sub-toxic dose of cisplatin. We propose that modulating the activity of calcium-binding proteins, calmodulin, S100A8, and S100A14, could be used to increase cisplatin efficacy, lowering its treatment dosage while maintaining its chemotherapeutic value.

Resuming Sensitivity of Tamoxifen-Resistant Breast Cancer Cells to Tamoxifen by Tetrandrine.

Background:Tamoxifen is one of the medicines for adjuvant endocrine therapy of hormone-dependent breast cancer. However, development of resistance to tamoxifen occurs inevitably during treatment. This study aimed to determine whether sensitivity of tamoxifen-resistant breast cancer cells (TAM-R) could be reinstated by tetrandrine (Tet).Methods:All experiments were conducted in TAM-R cells derived from the MCF-7 breast cancer cell line by long-term tamoxifen exposure. Cell growth, apoptosis, and autophagy were end-points that evaluated the effect of Tet (0.9 μg/ml, 1.8 μg/ml, and 3.75 μg/ml) alone or in combination with TAM (1 μM). Cell apoptosis was determined by an ELISA assay and autophagy was determined by fluorescent staining using the Enzo autophagy detection kit. Immunoblotting was used to evaluate markers for apoptosis, autophagy, and related signal pathway molecules.Results:Growth of TAM-R cells was significantly inhibited by Tet. Combination of Tet with tamoxifen induced a greater inhibition on cell growth than tamoxifen alone, which was predominantly due to enhancement of pro-apoptotic effect of TAM by Tet. Autophagy was significantly inhibited in TAM-R cells treated with Tet plus TAM as shown by increased autophagosomes and the levels of LC3-II and p62. At 0.9 μg/ml, Tet increased the levels of both apoptosis and autophagy markers. Among them increase in p53 levels was more dramatic.Conclusions:Tet as a monotherapy inhibits TAM-R cells. Tet potentiates the pro-apoptotic effect of TAM via inhibition of autophagy.

Aspirin Suppresses Breast Cancer Metastasis by Targeting Anoikis Resistance

Background: Aspirin intake lowers the risk of breast cancer metastasis, but mechanisms remain unclear. Anoikis resistance has been recently implicated in metastasis. Here we investigated whether aspirin might prevent breast cancer metastasis by targeting anoikis resistance. MethodsA:noikis was induced by culturing breast cancer cells on 1% agar treated plate for 24 h, and apoptotic cell death was determined by Annexin V and PI staining. Chemoprevention of breast cancer metastasis was evaluated by using a 4T1 mouse mammary tumor model. Findings: Aspirin sensitized breast cancer cells to anoikis in vitro, and lowered the circulating tumor cells as well as distant metastasis in vivo. Thromboxane A2 (TXA2) pathway was identified as the relevant molecular target for aspirin in anoikis sensitization. Mechanistically, thromboxane A2 receptor (TP) was up-regulated in detached breast cancer cells, conferred anoikis resistance through Akt activation, thereby facilitated breast cancer metastasis to lung. Interpretation: TXA2 pathway plays a critical role in anoikis resistance, and might serve as potential target for chemoprevention of breast cancer metastasis. Funding Information: his work was supported by the National Natural Science Foundation of China (81773064, 31972973 and 32021005). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: All animal study was conducted under protocols approved by the Institutional Animal Ethics Committee of Jiangnan University. (Issue No. 20200430c0501101[020], 20190330c0700615[58]).

Glucose Stress Induces Early Onset of Metastasis in Hormone-Sensitive Breast Cancer Cells

Cell adhesion is the backbone of many events in the cancer cell life cycle, including proliferation, metastasis, migration, invasion and even cell survival. In a tumor, usually the cells in the core have high migratory potential though they constantly suffer from glucose starvation. Our study was aimed at understanding events such as attachment to the surfaces at one site and then mobility to the secondary sites during progression of cancer in the hormone sensitive breast cancer cells MCF7, following their exposure to different concentrations of glucose in the environment. We have shown that low glucose availability is detected within 3 h of shortage which is then translated into variable expression of genes for cell-to-cell adhesion such as cadherins and Ig-like cell adhesion molecules, and matrix-associated genes such as integrins and metalloproteases. We also found that low glucose concentrations induced cell adhesion, whereas higher concentrations stimulated cell migration. In addition, several regulatory molecules involved in mitochondrial metabolism, proliferation, and glucose uptake as demonstrated respectively by MTT assay, BrdU uptake, glucose uptake and pyruvate kinase activity showed varied expression during epithelial to mesenchymal transition. Cytoskeleton staining demonstrated development of lamellipodia in glucose starved medium indicating cascade of physiological and molecular events in the cells to find a more nutrient-rich environment for the development of secondary tumor. Further studies on protein markers with a 3D spheroid culturing approach are likely to expand our understanding of onset of metastasis in tumor tissues.

Role of GPX4-Mediated Ferroptosis in the Sensitivity of Triple Negative Breast Cancer Cells to Gefitinib.

Gefitinib resistance in triple negative breast cancer (TNBC) is a growing important concern. Glutathione peroxidase 4 (GPX4) is a main regulator of ferroptosis, which is pivotal for TNBC cell growth. We investigated GPX4-mediated ferroptosis in gefitinib sensitivity in TNBC. Gefitinib resistant TNBC cells MDA-MB-231/Gef and HS578T/Gef were constructed and treated with lentivirus sh-GPX4 and ferroptosis inhibitor ferrostatin-1. GPX4 expression, cell viability and apoptosis were detected. Malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS) levels were evaluated. The levels of ferroptosis-related proteins were detected. Subcutaneous tumor model was established in nude mice, and gefitinib was intraperitoneally injected to evaluate tumor growth, apoptosis, and Ki-67 expression. GPX4 was increased in gefitinib-resistant cells. After silencing GPX4, the inhibition rate of cell viability was increased, the limitation of colony formation ability was reduced, apoptosis rate was increased, and the sensitivity of cells to gefitinib was improved. After silencing GPX4, MDA and ROS production were increased, while GSH was decreased. Silencing GPX4 promoted ferroptosis. Inhibition of GPX4 promoted gefitinib sensitivity by promoting cell ferroptosis. In vivo experiments also revealed that inhibition of GPX4 enhanced the anticancer effect of gefitinib through promoting ferroptosis. Overall, inhibition of GPX4 stimulated ferroptosis and enhanced TNBC cell sensitivity to gefitinib.

MiR-146a Enhances the Sensitivity of Breast Cancer Cells to Paclitaxel by Downregulating IRAK1.

Objective: To investigate the effect of miR-146a on the sensitivity of breast cancer cells to paclitaxel (PTX). Materials and Methods: The mRNA expressions of miR-146a in normal breast cancer cells, MCF-7, and PTX-resistant breast cancer cells, MCF-7/PTX, were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). MTS was used to analyze the cytotoxicity treated with different concentrations of PTX. Overexpressed and silenced cell lines of miR-146a and interleukin-1 receptor-associated kinase 1 (IRAK1) were constructed, respectively. Cells were treated with PTX and observed the changes of cell morphology. Proliferation was detected by clone formation assay. Invasion and migration were measured by transwell. RT-PCR was applied to detect the expression of IRAK1 gene. Dual luciferase report was performed to validate the target relationship between miR-146a and IRAK1. Salvage experiments were used to further verify the relationship between miR-146a and IRAK1. Results: PTX reduces the viability of MCF-7 and MCF-7/PTX cells in a dose-dependent manner. The IC50 of PTX in MCF-7 cells was significantly lower compared with MCF-7/PTX cells (p < 0.05). Compared with MCF-7/PTX cells, the expression of miR-146a gene in MCF-7 cells was significantly increased, while the expression of IRAK1 gene was significantly reduced (p < 0.05). Cell proliferation, invasion, and migration were decreased after miR-146a overexpression or IRAK1 silencing. Whereas, miR-146a silencing and IRAK1 overexpression can increase cell proliferation, invasion, and migration ability. Salvage experiments further verify that IRAK1 can weaken the role of miR-146a. Conclusion: miR-146a can enhance the sensitivity of breast cancer cells to PTX; the mechanism may be related to the downregulation of IRAK1.

Tamoxifen attenuates reactive astrocyte-induced brain metastasis and drug resistance through the IL-6/STAT3 signaling pathway.

Brain metastasis affects approximately 20%-30% of patients with triple-negative breast cancers (TNBCs). Even small metastatic lesions in the brain can trigger severe neurological impairments and result in extremely short survival time. Recently, active astrocytes were reported to be associated with brain metastases. However, how activated astrocytes regulate the behaviors of disseminated breast cancer cells in the brain remains unknown. In this study, human primary astrocytes were stimulated with IL-1β to form active astrocytes to study the cross-talk between stromal cells (astrocytes) and TNBC cells in brain metastases. Our results showed that active astrocytes significantly increase the malignancy of TNBC cells and prevent them from undergoing apoptosis caused by doxorubicin. We also found that the high level of IL-6 secreted by activated astrocytes was responsible for the drug resistance of breast cancer, which could be abolished by treatment of astrocytes with tamoxifen (TAM). The blockage of active astrocyte-derived IL-6 by a neutralizing antibody resulted in the attenuation of drug resistance, consequently enhancing the sensitivity of breast cancer cells to doxorubicin. Furthermore, the possible involved TAM-modulated drug resistance mechanism may be associated with a decrease in IL-6 expression in astrocytes and the downregulation of MAPK and JAK2/STAT3 signaling in cancer cells. Our data suggested that TAMs might reduce drug resistance through the IL-6/JAK2/STAT3 signaling pathway, providing a possible therapy to treat brain metastasis in TNBCs, as estrogen receptor inhibitors (TAMs, etc.) can cross the blood-brain barrier.

2-methoxyestradiol sensitizes breast cancer cells to taxanes by targeting centrosomes.

Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC50 values in all cells tested ranging from 28–44% (P < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; P < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; P < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.

New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis, and therapy

KLF4 plays a critical role in determining cell fate responding to various stresses or oncogenic signaling. Here, we demonstrated that KLF4 is tightly regulated by poly(ADP‐ribosyl)ation (PARylation). We revealed the subcellular compartmentation for KLF4 is orchestrated by PARP1‐mediated PARylation. We identified that PARylation of KLF4 is critical to govern KLF4 transcriptional activity through recruiting KLF4 from soluble nucleus to the chromatin. We mapped molecular motifs on KLF4 and PARP1 that facilitate their interaction and unveiled the pivotal role of the PBZ domain YYR motif (Y430, Y451 and R452) on KLF4 in enabling PARP1‐mediated PARylation of KLF4. Disruption of KLF4 PARylation results in failure in DNA damage response. Depletion of KLF4 by RNA interference or interference with PARP1 function by KLF4YYR/AAA (a PARylation‐deficient mutant) significantly sensitizes breast cancer cells to PARP inhibitors. We further demonstrated the role of KLF4 in modulating homologous recombination through regulating BRCA1 transcription. Our work points to the synergism between KLF4 and PARP1 in tumorigenesis and cancer therapy, which provides a potential new therapeutic strategy for killing BRCA1‐proficient triple‐negative breast cancer cells.

SIPA1 enhances SMAD2/3 expression to maintain stem cell features in breast cancer cells.

SIPA1, a GTPase activating protein that negatively regulates Ras-related protein (Rap), is a potential modulator of tumor metastasis and recurrence. In this study, we first showed that SIPA1 facilitated the stemness features of breast cancer cells, such as of tumorsphere formation capability and the expression of stemness marker CD44. In addition, SIPA1 promoted the expression of four stemness-associated transcription factors through increasing the expression of SMAD2 and SMAD3 in vitro and in vivo. The stemness features were abolished by blocking the phosphorylation of SMAD3 with its specific inhibitor SIS3. Furthermore, SIPA1 decreased the breast cancer cell sensitivity to chemotherapy drugs. This effect was, however, competitively reversed by blocking the SMAD3 phosphorylation by SIS3 treatment in breast cancer cells. Taken together, SIPA1 promotes and sustains the stemness of breast cancer cells and their resistance to chemotherapy by increasing the expression of SMAD2 and SMAD3, and blocking SMAD3 phosphorylation could suppress the cancer cell stemness and increase the sensitivity to chemotherapy in breast cancer cells expressing a high level of SIPA1.

Hypoxia Alters the Response to Anti-EGFR Therapy by Regulating EGFR Expression and Downstream Signaling in a DNA Methylation–Specific and HIF-Dependent Manner

Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. SIGNIFICANCE: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4998/F1.large.jpg.

APOBEC3 enzymes mediate efficacy of cisplatin and are epistatic with base excision repair and mismatch repair in platinum response.

Identifying the mechanisms mediating cisplatin response is essential for improving patient response. Previous research has identified base excision repair (BER) and mismatch repair (MMR) activity in sensitizing cells to cisplatin. Cisplatin forms DNA adducts including interstrand cross-links (ICLs) that distort the DNA helix, forcing adjacent cytosines to become extrahelical. These extrahelical cytosines provide a substrate for cytosine deaminases. Herein, we show that APOBEC3 (A3) enzymes are capable of deaminating the extrahelical cytosines to uracils and sensitizing breast cancer cells to cisplatin. Knockdown of A3s results in resistance to cisplatin and induction of A3 expression in cells with low A3 expression increases sensitivity to cisplatin. We show that the actions of A3s are epistatic with BER and MMR. We propose that A3-induced cytosine deamination to uracil at cisplatin ICLs results in repair of uracils by BER, which blocks ICL DNA repair and enhances cisplatin efficacy and improves breast cancer outcomes.

LncRNA MALAT1 promotes breast cancer progression and doxorubicin resistance via regulating miR-570–3p

BackgroundMetastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that regulates disease progression in various types of cancers. The aim of this study was to explore the role of MALAT1 in breast cancer (BC) progression and doxorubicin resistance. MethodsReal-time polymerase chain reaction (RT-PCR) was used to determine the expression of MALAT1 in BC tissues and cells; MTT and Transwell assay were used to detect the proliferation, migration and invasion of BC cells, respectively; drug resistance test was performed to assess the sensitivity of BC cells to doxorubicin; dual-luciferase reporter gene assay was conducted to verify the interaction between MALAT1 and miR-570–3p. ResultsMALAT1 was highly expressed in BC tissues compared with normal tissues adjacent to cancer as well as in BC cells. In addition, inhibition the expression of MALAT1 could significantly suppress the proliferation, migration and invasion of BC cells. Meanwhile, down-regulation of MALAT1 sensitized BC cells to doxorubicin. Moreover, bioinformatics analysis suggested that miR-570–3p was the potential downstream target of MALAT1. Dual-luciferase reporter gene assay confirmed that MALAT1 could directly target miR-570–3p. Additionally, miR-570–3p was lowly expressed in BC tissues and cells. Up-regulation of miR-570–3p not only significantly inhibited the proliferation, metastasis, and invasion of BC cells, but also increased the sensitivity of BC cells to doxorubicin. ConclusionMALAT1 functions as a novel oncogenic lncRNA in regulating the progression and doxorubicin resistance of BC by targeting miR-570–3p.

Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy

Oncogenic activation of the mTOR signaling pathway occurs frequently in tumor cells and contributes to the devastating features of cancer, including breast cancer. mTOR inhibitors rapalogs are promising anticancer agents in clinical trials; however, rapalogs resistance remains an unresolved clinical challenge. Therefore, understanding the mechanisms by which cells become resistant to rapalogs may guide the development of successful mTOR-targeted cancer therapy. In this study, we found that eEF-2K, which is overexpressed in cancer cells and is required for survival of stressed cells, was involved in the negative-feedback activation of Akt and cytoprotective autophagy induction in breast cancer cells in response to mTOR inhibitors. Therefore, disruption of eEF-2K simultaneously abrogates the two critical resistance signaling pathways, sensitizing breast cancer cells to rapalogs. Importantly, we identified mitoxantrone, an admitted anticancer drug for a wide range of tumors, as a potential inhibitor of eEF-2K via a structure-based virtual screening strategy. We further demonstrated that mitoxantrone binds to eEF-2K and inhibits its activity, and the combination treatment of mitoxantrone and mTOR inhibitor resulted in significant synergistic cytotoxicity in breast cancer. In conclusion, we report that eEF-2K contributes to the activation of resistance signaling pathways of mTOR inhibitor, suggesting a novel strategy to enhance mTOR-targeted cancer therapy through combining mitoxantrone, an eEF-2K inhibitor.

Retinoic Acid Sensitivity of Triple-Negative Breast Cancer Cells Characterized by Constitutive Activation of the notch1 Pathway: The Role of Rarβ.

Simple SummaryWe provide experimental evidence that the rare subgroup of triple-negative breast cancer characterized by constitutive activation of the NOTCH1 signaling pathway is sensitive to the anti-tumor action of all-trans retinoic acid, the active metabolite of vitamin A. In this tumor context, all-trans retinoic acid exerts not only an effective action on its own, but it also stimulates the inhibitory activity of γ-secretase inhibitors, a series of therapeutic agents targeting NOTCH1, on cancer cell growth. From a basic and mechanistic standpoint, an important result of the study regards the specific involvement of the retinoid receptor RARβ in the anti-tumor action exerted by all-trans retinoic acid in sensitive triple-negative breast cancer cells. From an applicative point of view the study represents the basis for the design of clinical trials on the efficacy of combinations between all-trans retinoic acid and γ-secretase inhibitors in the treatment of patients affected by a specific subtype of triple-negative breast cancer.Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks effective therapeutic options. In this study, we profile eighteen TNBC cell lines for their sensitivity to the anti-proliferative action of all-trans retinoic acid (ATRA). The only three cell lines (HCC-1599, MB-157 and MDA-MB-157) endowed with ATRA-sensitivity are characterized by genetic aberrations of the NOTCH1-gene, causing constitutive activation of the NOTCH1 γ-secretase product, N1ICD. N1ICD renders HCC-1599, MB-157 and MDA-MB-157 cells sensitive not only to ATRA, but also to γ-secretase inhibitors (DAPT; PF-03084014). Combinations of ATRA and γ-secretase inhibitors produce additive/synergistic effects in vitro and in vivo. RNA-sequencing studies of HCC-1599 and MB-157 cells exposed to ATRA and DAPT and ATRA+DAPT demonstrate that the two compounds act on common gene sets, some of which belong to the NOTCH1 pathway. ATRA inhibits the growth of HCC-1599, MB-157 and MDA-MB-157 cells via RARα, which up-regulates several retinoid target-genes, including RARβ. RARβ is a key determinant of ATRA anti-proliferative activity, as its silencing suppresses the effects exerted by the retinoid. In conclusion, we demonstrate that ATRA exerts a significant anti-tumor action only in TNBC cells showing constitutive NOTCH1 activation. Our results support the design of clinical trials involving combinations between ATRA and γ-secretase inhibitors for the treatment of this TNBC subtype.