Integrated Quantitative Analysis of the Phosphoproteome and Transcriptome in Tamoxifen-resistant Breast Cancer*

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Breast Cancer ◽  
High Rate ◽  
System Level ◽  
Clinical Samples ◽  
Transcriptome Data ◽  
Data Set ◽  
Reporter Assays ◽  
Treated Breast ◽  
Mcf 7 ◽  
Resistant Cells

Quantitative phosphoproteome and transcriptome analysisof ligand-stimulated MCF-7 human breast cancer cells wasperformed to understand the mechanisms of tamoxifen resistanceat a system level. Phosphoproteome data revealed thatWT cells were more enriched with phospho-proteins thantamoxifen-resistant cells after stimulation with ligands.Surprisingly, decreased phosphorylation after ligand perturbationwas more common than increased phosphorylation.In particular, 17?-estradiol induced down-regulation inWT cells at a very high rate. 17?-Estradiol and the ErbBligand heregulin induced almost equal numbers of up-regulatedphospho-proteins in WT cells. Pathway and motifactivity analyses using transcriptome data additionallysuggested that deregulated activation of GSK3? (glycogensynthasekinase 3?) and MAPK1/3 signaling might be associatedwith altered activation of cAMP-responsive elementbindingprotein and AP-1 transcription factors intamoxifen-resistant cells, and this hypothesis was validatedby reporter assays. An examination of clinical samples revealedthat inhibitory phosphorylation of GSK3? at serine 9was significantly lower in tamoxifen-treated breast cancerpatients that eventually had relapses, implying that activationof GSK3? may be associated with the tamoxifen-resistantphenotype. Thus, the combined phosphoproteomeand transcriptome data set analyses revealed distinct signal

scholarly journals Lidocaine and Bupivacaine Downregulate MYB and DANCR lncRNA by Upregulating miR-187-5p in MCF-7 Cells

2022 ◽  
Vol 8 ◽  
Author(s):  
Chiao-Yi Lin ◽  
Wen-Ting Tseng ◽  
Yao-Yin Chang ◽  
Mong-Hsun Tsai ◽  
Eric Y. Chuang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Expression Profiles ◽  
Luciferase Reporter ◽  
Functional Assay ◽  
Next Generation ◽  
Potential Biomarker ◽  
Reporter Assays ◽  
Generation Sequencing ◽  
Mcf 7

Background: Breast cancer is the most common malignancy and a leading cause of death among women. The majority of patients require surgery, and retrospective studies have revealed an association between anaesthetic techniques during surgery and clinical outcomes. Local anaesthetics (LAs) influence carcinogenesis by interacting with non-coding RNAs (ncRNAs). However, the detailed mechanisms underlying the association between LAs and ncRNAs remain unclear.Methods: In this study, the effects of two commonly used LAs, lidocaine and bupivacaine, on the malignancy of MCF-7 breast cancer cells were investigated. The expression profiles of the microRNAs (miRNAs) that responded to treatment with LAs were determined through next-generation sequencing.Results: Data from the functional assay revealed that the LAs suppressed the proliferation of MCF-7 cells. The result of next-generation sequencing revealed that 131 miRNAs were upregulated, following treatment with the LAs. Validation using polymerase chain reaction (PCR) identified miR-187-5p as a potential biomarker, and it was selected for further analyses. Prediction with bioinformatics tools and luciferase reporter assays revealed that MYB is a direct target gene of miR-187-5p. Based on the hypothesis that lncRNAs acts as miRNA sponges, the target lncRNA, DANCR, of miR-187-5p was predicted using DIANA-LncBase v2 and validated using luciferase reporter assays. In addition, the reciprocal suppressive effect between DANCR and miR-187-5p was determined.Conclusions: This study suggests that one of the anti-tumour mechanisms of lidocaine and bupivacaine is mediated through the DANCR-miR-187-5p-MYB axis. This may provide a novel molecular mechanism of tumour suppression in breast cancer.

scholarly journals Role of Differentially Expressed Proteins in Acquired Resistance to Cdk4/6 Inhibitor in Breast Cancer

2021 ◽  
Author(s):  
Binayak Kumar ◽  
Peeyush Prasad ◽  
Ragini Singh ◽  
Ram Krishana Sahu ◽  
Ashutosh Singh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Drug Targets ◽  
Acquired Resistance ◽  
Therapy Resistance ◽  
Resistant Cell ◽  
Molecular Signature ◽  
Mcf 7 ◽  
Resistant Cells

Abstract CDK4/6 inhibitors (Abemaciclib, Ab and Palbociclib, Pb) stop the G1-phase in cell-cycle being used to cure advanced stage of breast cancer (BC). Acquired resistance is a major challenge in BC therapy. The molecular signature of the therapy resistance for Ab and Pb drugs in BC should be explored. Here, we developed Ab/Pb-resistant cell-models and explored the molecular changes. Drug’s resistance cells were developed in MCF-7 cells by continuous drug treatment and it was confirmed by MTT-assay, PI-staining-microscopy, and real-time-qPCR. Global proteome profiling done by Labelled-free-Proteome-Orbitrap-Fusion-MS-MS technique. Bioinformatics tools used to analyse the proteome data. Ab-resistant and Pb-resistant MCF-7 cells showed increased tolerance for the respective drug. The BCL-2 and MCL-1 survival genes were up-regulated, while the apoptosis genes BAD, BAX, CASP-3 and PARP-1were down-regulated in the resistant cells. Expression of the MDR-1, ABCG2, ESR-1, CDK4, CDK6, and Cyclin-D1 genes were increased in both resistance cells. For proteomics, 237 and 239 proteins were expressed differently in the resistant Ab and Pb cells, respectively. The NUDT5, PEPD, ABAT, ATP1B1, GGCT, and SELENBP1 proteins were down-regulated and the SBSN, HSD17B10, CD9, PDIA3, PSMB4, SLC2A1, and VTN proteins were up-regulated in Ab-resistant cells. The NUDT5, PEPD, and GGCT proteins were down-regulated, while CD47, HIST1H2BN, LMNA, VTN, PSMB5, HBB, PSMA7, FLNB, PRDX4, VDAC1, GOT2, HSPA5, SERPINH1, EIF4A2, FTH, and VIM proteins were up-regulated in Pb-resistant cells. These proteins are a new set of prognostic markers and drug targets for overcoming the respective drug resistance. However, it is necessary to perform an in vivo or clinical assessment.

The enrichment of CD44 in exosomes from breast cancer cells treated with doxorubicin promotes chemoresistance

2020 ◽  
Author(s):  
xiaohong wang ◽  
kai cheng ◽  
guoqiang zhang ◽  
yue yu ◽  
song liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Chemotherapy Resistance ◽  
Clinical Samples ◽  
Cd44 Expression ◽  
Candidate Target ◽  
And Function ◽  
Cancer Chemoresistance ◽  
Mcf 7

Abstract Background: Exosomes have been shown to be associated with chemotherapy resistance transmission between cancer cells. However, the cargo and function of exosomes changed in response to doxorubicin remains unclear. Methods: We compared proteome profiles of exosomes extracted from the supernatant of MCF-7(S/Exo) and MCF-7/ADR(A/Exo) cells. We confirmed the differential expression of the candidate target-exosomic-CD44 by immune gold staining and western blot. We further studied the changes of chemosensitivity and CD44 expression in MCF-7 cells co-incubated with A/Exo. We analyzed the levels of exosomal CD44 from patient plasma, and compared the sensitivity and specificity of exosomic CD44 and plasma CD44 on diagnosis of chemoresistance. We modified the MCF-7-derived exosomes loaded with siRNA against CD44 to observe the effects of targeting reduced CD44 expression in lumimal A breast cancer cells. Results: DOX increased the exosomes release from MCF-7/ADR cells and the exosomes mediated proteins intercellular transfer in breast cancer chemoresistance regulation. The candidate target of CD44 in A/Exo was much higher than in S/Exo and the increase levels of exosomic CD44 (21.65-fold) was much higher than cellular CD44 (6.55-fold). The same results were obtained in clinical samples. Exosome-siRNA targeted CD44 (Exos-siCD44) could efficiently targeted to silence its expression. When co-cultured on Exos-siCD44, breast cancer cells exhibited reduced cell proliferation and enhanced susceptibility to DOX and the same phenomenon was observed in mice. Conclusion：Drug-resistant breast cancer cells spread resistance capacity to sensitive ones by releasing exosomes to transfer proteins in intercellular.

Oestrogen treatment enhances the sensitivity of hormone-resistant breast cancer cells to doxorubicin

2010 ◽  
Vol 31 (2) ◽  
pp. 137-143 ◽  
Author(s):  
Alexander M. Scherbakov ◽  
Yulia S. Lobanova ◽  
Olga E. Andreeva ◽  
Valentina A. Shatskaya ◽  
Mikhail A. Krasil'nikov
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Nuclear Factor Κb ◽  
Apoptotic Response ◽  
Oestrogen Treatment ◽  
Interfering Rna ◽  
Mcf 7 ◽  
Resistant Cells

Recently, it was shown that the resistance of breast cancer cells to growth-stimulating oestrogen action may be accompanied with the paradoxical tumour sensitization to oestrogen apoptotic action. In the present paper, we studied the influence of oestrogens on the sensitivity of resistant breast tumours to cytostatic drugs, and to evaluate the role of NF-κB (nuclear factor κB) signalling in the regulation of the apoptotic response of the resistant cells. The experiments were carried out on the oestrogen-dependent MCF-7 breast cancer cells and resistant MCF-7/LS subline generated through long-term cultivation of the parental cells in the absence of oestrogen. The cell treatment with the combination of oestradiol and Dox (doxorubicin) was found to enhance the apoptotic action of Dox in MCF-7/LS cells but not in the parent cells. MCF-7/LS cells were characterized by the increased level of ROS (reactive oxygen species) and decreased NF-κB activity. Oestradiol in combination with Dox leads to significant NF-κB stimulation and its accumulation in the nucleus of MCF-7/LS cells. The knockdown of NF-κB with siRNA (small interfering RNA) increased the apoptotic response of the MCF-7/LS cells to both Dox and oestradiol demonstrating the important role of NF-κB in the protection of the MCF-7/LS cells against apoptosis. In general, the results obtained show that: (i) oestradiol enhances the apoptotic action of Dox in the resistant breast cancer cells; and (ii) suppression of NF-κB signalling amplifies the apoptotic response of the resistant cells to both oestrogen and Dox, demonstrating that NF-κB may serve as a potential target in the therapy of the resistant breast cancer.

scholarly journals The Impact of Extracellular Ca2+ and Nanosecond Electric Pulses on Sensitive and Drug-Resistant Human Breast and Colon Cancer Cells

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3216
Author(s):  
Julita Kulbacka ◽  
Nina Rembiałkowska ◽  
Anna Szewczyk ◽  
Helena Moreira ◽  
Anna Szyjka ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Colon Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Calcium Ions ◽  
Colon Cancer Cells ◽  
Human Breast ◽  
The Impact ◽  
Mcf 7 ◽  
Resistant Cells

(1) Background: Calcium electroporation (CaEP) is based on the application of electrical pulses to permeabilize cells (electroporation) and allow cytotoxic doses of calcium to enter the cell. (2) Methods: In this work, we have used doxorubicin-resistant (DX) and non-resistant models of human breast cancer (MCF-7/DX, MCF-7/WT) and colon cancer cells (LoVo, LoVo/DX), and investigated the susceptibility of the cells to extracellular Ca2+ and electric fields in the 20 ns–900 ns pulse duration range. (3) Results: We have observed that colon cancer cells were less susceptible to PEF than breast cancer cells. An extracellular Ca2+ (2 mM) with PEF was more disruptive for DX-resistant cells. The expression of glycoprotein P (MDR1, P-gp) as a drug resistance marker was detected by the immunofluorescent (CLSM) method and rhodamine-123 efflux as an MDR1 activity. MDR1 expression was not significantly modified by nanosecond electroporation in multidrug-resistant cells, but a combination with calcium ions significantly inhibited MDR1 activity and cell viability. (4) Conclusions: We believe that PEF with calcium ions can reduce drug resistance by inhibiting drug efflux activity. This phenomenon of MDR mechanism disruption seems promising in anticancer protocols.

scholarly journals TRIM47 activates NF-κB signaling via PKC-ε/PKD3 stabilization and contributes to endocrine therapy resistance in breast cancer

2021 ◽  
Vol 118 (35) ◽  
pp. e2100784118
Author(s):  
Kotaro Azuma ◽  
Kazuhiro Ikeda ◽  
Takashi Suzuki ◽  
Kenjiro Aogi ◽  
Kuniko Horie-Inoue ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Kinase ◽  
Cancer Patients ◽  
Endocrine Therapy ◽  
Cancer Biology ◽  
Therapy Resistance ◽  
Clinical Samples ◽  
Breast Cancer Patients ◽  
Endocrine Therapy Resistance ◽  
Mcf 7

Increasing attention has been paid to roles of tripartite motif–containing (TRIM) family proteins in cancer biology, often functioning as E3 ubiquitin ligases. In the present study, we focus on a contribution of TRIM47 to breast cancer biology, particularly to endocrine therapy resistance, which is a major clinical problem in breast cancer treatment. We performed immunohistochemical analysis of TRIM47 protein expression in 116 clinical samples of breast cancer patients with postoperative endocrine therapy using tamoxifen. Our clinicopathological study showed that higher immunoreactivity scores of TRIM47 were significantly associated with higher relapse rate of breast cancer patients (P = 0.012). As functional analyses, we manipulated TRIM47 expression in estrogen receptor–positive breast cancer cells MCF-7 and its 4-hydroxytamoxifen (OHT)-resistant derivative OHTR, which was established in a long-term culture with OHT. TRIM47 promoted both MCF-7 and OHTR cell proliferation. MCF-7 cells acquired tamoxifen resistance by overexpressing exogenous TRIM47. We found that TRIM47 enhances nuclear factor kappa-B (NF-κB) signaling, which further up-regulates TRIM47. We showed that protein kinase C epsilon (PKC-ε) and protein kinase D3 (PKD3), known as NF-κB–activating protein kinases, are directly associated with TRIM47 and stabilized in the presence of TRIM47. As an underlying mechanism, we showed TRIM47-dependent lysine 27–linked polyubiquitination of PKC-ε. These results indicate that TRIM47 facilitates breast cancer proliferation and endocrine therapy resistance by forming a ternary complex with PKC-ε and PKD3. TRIM47 and its associated kinases can be a potential diagnostic and therapeutic target for breast cancer refractory to endocrine therapy.

scholarly journals miR-186 Regulates the Initiation and Development of Breast Cancer via SHP-1 Methylation

2020 ◽  
Author(s):  
Kun Wang ◽  
Chunxia Zhou ◽  
Di Wang ◽  
Bin Zhang ◽  
Yongjian Zheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Promoter Region ◽  
Breast Cancer Cells ◽  
Methylation Status ◽  
Luciferase Reporter ◽  
Molecular Diagnostic ◽  
Double Labeling ◽  
Reporter Assays ◽  
Mcf 7

Abstract Background Change in the methylation status of genomic DNA, especially in CpG islands in the promoter region, is considered to be an early event in tumor initiation, leading to silencing of gene expression, subsequent abnormalities in gene structure and function, and malignant transformation of the cell. Due to the abnormal expression of miR-186 and SHP-1 in breast cancer tissues and cells, we propose that miR-186 is closely related to the methylation of SHP-1Method Using 5-azacytidine as a de-methylation agent and Validating with Setylation-specific polymerase chain reaction (MSP) after treatment. Measurement of the viability of breast cancer cells using the CCK-8 method Measurement of the apoptotic rate of breast cancer cells using annexin V-FITC/PI double labeling. Cell metastasis were measured by wound healing assay. Luciferase reporter assays was used to confirm the target of MiR-186. SHP-1 and miR-186 expression was measured by RT-PCR and western blot.Results In the present study, we found that SHP-1 expression was reduced to various degrees in all 5 cell lines (UACC-812, MDA-MB-213, MDA-MB-468, SK-RB-3 and MCF-7). 5-azacytidine can remove the methylation from the SHP-1 promoter region. Apoptosis was observed in MCF-7 cells after demethylation of the SHP-1 gene promoter region by 5-azacytidine, and the effect was time- and concentration-dependent. Luciferase reporter assays showed that miR-186 promotes methylation through binding with the 3’ UTR of the SHP-1 promoter region.Western blot showed miR-186 regulates the initiation and development of tumor cells through the SHP-1-JAK-STAT axis. In animal models, low expression of miR-186 can cause significantly limited tumor growth.Conclusion The low SHP-1 expression may be an important factor in the initiation of breast cancer, and that miR-186 could serve as an excellent molecular diagnostic marker and a possible therapeutic target.

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