Sensitive MCF-7 Cells Research Articles

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together.

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug–drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity.

Exosomes from tamoxifen-resistant breast cancer cells transmit drug resistance partly by delivering miR-9-5p

BackgroundResistance to drug therapy is a major impediment for successful treatment of patients suffering from breast cancer (BC). Tamoxifen (TAM) is an extensively used therapeutic agent, which substantially reduces the risk of recurrence and associated mortality in BC. This study demonstrated that exosomal transfer of microRNA-9-5p (miR-9-5p) enhanced the resistance of MCF-7 cells to TAM.MethodsInitially, BC-related differentially expressed genes (DEGs) and their upstream regulatory miRNAs were identified. The TAM-resistant MCF-7 (MCF-7/TAM) cell line and the non-medicated sensitive MCF-7 cell line were formulated, followed by isolation of the exosomes. Next, the apoptosis rate of exosome-treated MCF-7 cells was determined after co-culture with TAM. The interaction between miR-9-5p and ADIPOQ was identified by a combination of bioinformatic analysis and luciferase activity assay. In order to validate the effect of miR-9-5p and ADIPOQ on TAM resistance in the MCF-7 cells in vitro and in vivo, miR-9-5p was delivered into the exosomes. ADIPOQ and miR-9-5p were identified as the BC-related DEG and upstream regulatory miRNA.ResultsExosomes derived from the MCF-7/TAM cells could increase the resistance of MCF-7 cells to TAM. Notably, miR-9-5p altered the sensitivity of BC cells to TAM. In addition, ADIPOQ was negatively regulated by miR-9-5p. Furthermore, MCF-7/TAM cell-derived miR-9-5p inhibited the apoptosis of MCF-7 cells, and promoted the cell resistance to TAM. In vivo experiments in nude mice ascertained that the tumor injected with exosomal miR-9-5p showed improved resistance to TAM.ConclusionsExosomal transfer of miR-9-5p augmented the drug resistance of BC cells to TAM by down-regulating ADIPOQ, suggesting its functionality as a candidate molecular target for the management of BC.

Enhancement of cisplatin sensitivity in human breast cancer MCF-7 cell line through BiP and 14-3-3ζ co-knockdown.

Cisplatin treatment confers the relative resistance to MCF-7 cells as compared to other breast cancer cell lines. One principal reason is that chemotherapeutic agents induce autophagy in these cells to inhibit apoptosis. Binding immunoglobulin protein (BiP), a master regulator of unfolded protein response (UPR) and 14-3-3ζ are two critical proteins upregulated in breast cancer rendering resistance to anticancer drugs. They also play pivotal roles in autophagy with crosstalk with the apoptotic pathways of UPR through certain regulators. Thus, BiP and 14-3-3ζ were selected as the candidate targets to enhance cell death and apoptosis. First, cisplatin resistance was induced and determined by MTT assay and qPCR in MCF-7 cells. Then, the apoptosis axis of UPR was activated by knocking down either BiP or 14-3-3ζ and overactivated by co-knockdown of BiP and 14-3-3ζ. Apoptosis assays were performed using flow cytometry, TUNEL assays utilized confocal microscopy followed by western blot analysis and caspase-3 and JNK activities were investigated to assess the outcomes. Finally, an autophagy assay followed by western blotting was performed to study the effects of co-knockdown genes on cell autophagy in the presence and absence of cisplatin. The present data indicated the enhancement of cisplatin sensitivity in MCF-7 cells co-knocked down in BiP and 14-3-3ζ compared with either gene knockdown. Upregulation of JNK and cleaved-PARP1 protein levels as well as caspase-3 and JNK overactivation confirmed the results. A marked attenuation of autophagy and Beclin1 as well as ATG5 downregulation were detected in co-knockdown cells compared to knockdown with either BiP or 14-3-3ζ. Cisplatin sensitization of MCF-7 cells through double-knockdown of BiP and 14-3-3ζ highlights the potential of targeting UPR and autophagy factors to increase the effect of chemotherapy.

Activating transcription factor-2 (ATF2) is a key determinant of resistance to endocrine treatment in an in vitro model of breast cancer

BackgroundActivating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment.Materials and methodsWe knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR.ResultsBy silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells.ConclusionThese data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

14P Modulation of MCF-7 cell sensitivity to anticancer agents by targeting glycolysis

Energy imbalance is one of the tumor cells' key properties in some cases supporting rapid progression and resistance to therapies. Simultaneous effects on glycolysis and other signaling pathways seem to be a promising strategy for anticancer therapy. Objectives: evaluation of the effects of glucose deprivation and glycolysis inhibitor (2-Deoxy-D-Glucose, 2DOG) on the antiproliferative activities of various anticancer agents, searching for promising combinations.

Aconitase 2 sensitizes MCF-7 cells to cisplatin eliciting p53-mediated apoptosis in a ROS-dependent manner

Aconitase 2 (ACO2) belongs to the tricarboxylic acid (TCA) cycle, which represents a key metabolic hub for cellular metabolism that is frequently altered in cancer for satisfying bioenergetic and biosynthetic requirements of proliferating cells. The promotion of ACO2 activity in breast cancer cell lines was shown to slow down proliferation imposing a switch from aerobic glycolysis to oxidative metabolism. The alteration of metabolic pathways in cancer also impinges on the sensitivity to chemotherapeutic interventions. In this work, we evidence that the presence of ACO2 sensitizes cells to the treatment with the genotoxic agents cisplatin (CDDP) and doxorubicin activating the apoptotic cell death mechanism. This response was driven by the accumulation of reactive oxygen species (ROS) following both ACO2 overexpression and CDDP exposure that permit the stabilization/activation of p53 in nuclear and mitochondrial compartments. Collectively, our results highlight that in ACO2 overexpressing cells the promotion of mitochondrial metabolism accounts for increased ROS production that was buffered by p53 mitochondrial recruitment and autophagy induction. However, these systems are not able to counteract the CDDP-mediated oxidative stress that becomes the Achilles heel for increasing susceptibility to apoptotic cell death.

Sensitivity of MCF-7 cells with differential expression of S6K1 isoforms to the regulatory impact of fibroblasts

Sensitivity of MCF-7 cells with differential expression of S6K1 isoforms to the regulatory impact of fibroblasts

Overexpression of ABCC2 and NF-Κβ/p65 with Reduction in Cisplatin and 4OH-Tamoxifen Sensitivity in MCF-7 Breast Cancer Cells: The Influence of TNF-α

Background: TNF-α, as a pro-inflammatory cytokine in the tumor microenvironment is able to regulate the expression and function of various ATP binding cassette (ABC) transporters involved in clinical drug resistance and among them, ABCC2 transporter is represented to contribute to cancer multidrug resistance (MDR) by drug efflux. Methods: In this study, we aimed to evaluate the effects of TNF-α and/or E2 (17β-estradiol) on the mRNA and protein expression levels of ABCC2 and NF-κB (p65) transcription factor in estrogen receptor positive (ER+) MCF-7 cells by QRT-PCR and Western blot analysis. Also, we used MTT assay to study the cell sensitivity against the active form of tamoxifen (4OH-TAM), a hypothetical substrate and Cisplatin (Cis), a well-known substrate for ABCC2 used in endocrine and chemo-therapy of breast cancers, respectively. Data were analyzed by one-way ANOVA and Tukey tests. Significance was considered in P-values < 0.05. Results: The expression levels of ABCC2 and the active form of NF-κB (p65) were significantly increased following 20-day concomitant treatment with TNF-α and E2, compared to untreated cells as control. Also, the viability assay showed that 20-day TNF-α+E2 treatment led to more sensitivity reduction of MCF-7 cells to Cis and 4OH-TAM compared to E2-treated and untreated cells. Conclusion: Based on our findings, there is a positive correlation between ABCC2 overexpression, over-activity of NF-ҡB/p65 and decreasing the sensitivity of MCF-7 cells to Cis and 4OH-TAM following TNF-α treatment in MCF-7 cells. Further experiments are needed to elucidate possible mechanistic relationship of these findings and their clinical significance in order to circumvent the drug-resistance in breast tumors.

MiR-520h Stimulates Drug Resistance to Paclitaxel by Targeting the OTUD3-PTEN Axis in Breast Cancer.

MicroRNAs (miRNAs) have been identified as negative posttranscriptional regulators of target genes and are involved directly in the pathological processes of tumors, including drug resistance. However, the exact function of miR-520h in breast cancer remains poorly understood. The aim of this study was to investigate the molecular mechanisms of miR-520h in paclitaxel resistance in the MCF-7 breast cancer cell line. Ectopic expression of miR-520h could promote the proliferation of breast cancer cells and inhibit paclitaxel-induced cell apoptosis. Inhibiting the expression of miR-520h could enhance the sensitivity to paclitaxel in paclitaxel-resistant MCF-7/Taxol cells. Furthermore, luciferase reporter assays showed that OTUD3 was a direct target of miR-520h. OTUD3 plays a necessary role in the paclitaxel resistance effect of miR-520h, and cotreatment with a miR-520h inhibitor and OTUD3 overexpression significantly enhanced MCF-7 cell sensitivity to paclitaxel. Moreover, miR-520h substantially inhibited the protein expression of PTEN via OTUD3 and subsequently affected downstream p-AKT pathway activity. In a clinical study, we also found that high miR-520h expression was associated with more aggressive pathological characteristic and poor prognosis. Therefore, our findings showed that miR-520h targeted the OTUD3-PTEN axis to drive paclitaxel resistance, and this miR might be an important potential target for breast cancer treatment.

Intracellular delivery of drugs by chitosan-based multi-liposomal complexes

Positively charged linear chitosan molecules were cross-linked with sulfate-anions to form chitosan nanoparticles which were used as a scaffold of negatively charged cardiolipin/egg lecithin liposomes loaded with doxorubicin (DOX). Thus formed multi-liposomal complexes (MLCs) containing 55 liposomes/chitosan and bearing a slight positive net charge effectively transmitted DOX into the cytoplasm of cells in culture. The efficiency of DOX delivery increased 4–5-fold upon drug incorporation in MLCs. Inside the cells, the penetrated MLCs released DOX thus enabling its accumulation in the nuclei and interaction with its intracellular target DNA leading to a decrease in cell survival. The effects were demonstrated on 3T3 line of mouse fibroblasts, drug sensitive tumor MCF-7 cells and drug resistant human ovarian carcinoma OVCAR-8 cells (formerly called MCF-7/ADR). Thus, MLC particles can be used for effective delivery of payloads in cells of different origin.

Dual in vitro invasion/migration suppressing and tamoxifen response modulating effects of a recombinant anti-ALCAM scFv on breast cancer cells.

It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.

Abstract P6-04-16: Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1

Abstract Despite the availability of effective drugs that target ERα-positive breast cancer, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is a potent and clinically relevant metabolite of tamoxifen. Therefore, characterization of endoxifen resistance mechanisms may be crucial to understanding tamoxifen resistance. We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen over a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. We subjected endoxifen resistant cells to a genome-wide, CRISPR-mediated knockout screen to identify genes involved in mediating endoxifen resistance. Subsequent analyses revealed that disruption of genes regulated by the glucocorticoid receptor (GR) enhanced cells’ ability to survive and proliferate in the presence of endoxifen, suggesting that GR signaling blocks growth of resistant cells. We provide evidence that GR activation via dexamethasone significantly inhibits the proliferation rates of endoxifen resistant cells by 50-60%, with little to no inhibitory effects in endoxifen sensitive models. Follow-up studies suggested that alpha zinc-glycoprotein 1 (AZGP1), a secreted lipolytic enzyme induced by GR, plays a role in mediating this phenotype. AZGP1 RNA and protein are highly expressed in normal breast tissue, but corresponding levels are lower in ERα-positive breast malignancies. In these tumors, low AZGP1 expression correlates with worse prognosis. Additionally, high expression of AZGP1 is associated with improved outcomes in patients with prostate, pancreatic, gastric and breast cancer. AZGP1 is expressed in endocrine sensitive MCF7 and T47D cells, but is silenced in endoxifen resistant models at the mRNA and protein level. Further, treatment of resistant cells with conditioned medium containing AZGP1 resulted in significant inhibition of cell proliferation and migration, and antibody-mediated depletion of AZGP1 from conditioned medium completely reversed these effects. While there are no reports regarding the molecular functions of AZGP1 in cancer we provide evidence that AZGP1 directly inhibits oncogenic signaling in endocrine resistant breast cancer through interaction with, and inhibition of, β6 integrin. Through the analysis of endoxifen resistant breast cancer cells, we have identified novel roles for GR, AZGP1 and β6 integrin in the development and progression of endocrine resistant breast cancer. We demonstrate that activation of GR may elicit therapeutic benefit in endocrine resistant breast cancer, specifically in tumors expressing AZGP1 and β6 integrin. Further, AZGP1 and β6 integrin may represent novel prognostic and/or predictive biomarkers in endocrine resistant disease. Citation Format: Calley J. Jones, Santiago Acero Bedoya, Anna V. Mykytyn, Matthew P. Goetz, John R. Hawse. Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-16.

Abstract P5-02-14: The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance

Abstract Background: Based on preclinical research, the MAPK growth factor pathway is thought to be an oncogenic pathway driving breast cancer, potentially in association with the ER pathway. In patients it is unclear how functional activity of the MAPK pathway activity relates to activity of the ER pathway to drive cancer growth. We have reported before on the development of assays, e.g. for the ER pathway, that enable quantitative measurement of activity of signal transduction pathways based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945). We now present (1) development of the MAPK-AP1 pathway model, (2) MAPK-AP1 pathway activity and its relation to ER pathway activity in breast cancer; (3) emergence of MAPK pathway activity associated with hormonal therapy resistance. Methods: The MAPK-AP1 pathway model was developed and biologically validated on Affymetrix mRNA data from samples (different cell types) with a known MAPK pathway activity. MAPK-AP1 and ER pathway activity scores were measured on Affymetrix expression data of primary untreated breast cancer samples (GSE45827, n=141) and of fulvestrant sensitive and resistant MCF7 cell line clones (GSE74391). Results: In clinical breast cancer samples, average MAPK pathway activity was increased in all subtypes compared to normal breast tissue, with a large dynamic range indicating that in each subtype there are samples with high and with low pathway activity; ER pathway activity in Luminal A and B subtypes ranged from high to low, and was low in all other subtypes. MAPK-AP1 pathway activity did not correlate with ER pathway activity. Fulvestrant-sensitive MCF7 cells had high ER and low MAPK pathway activity scores, while resistant MCF7 clones exhibited increased MAPK pathway activity and complete loss of ER pathway activity, explaining therapy resistance. Discussion: The MAPK-AP1 pathway is frequently activated in all breast cancer subtypes, independent of ER pathway activity. We hypothesize that in case of resistance to hormonal therapy measurement of both pathway activities may be informative as to the underlying resistance mechanism, i.e. loss of ER pathway activity and/or gain of MAPK pathway activity. Future clinical studies are needed to confirm the role of these signaling pathways in hormonal resistance. Citation Format: Anja van de Stolpe, Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh. The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-14.

Differential expression, function and prognostic value of miR-17–92 cluster in ER-positive and triple-negative breast cancer

Recent evidence has shown that the miR-17–92 cluster can function either as oncogene or tumor suppressor in human cancers. The function of miR-17–92 in subtypes of breast cancer remains largely unknown. The expression of miR-17–92 is elevated in triple negative breast cancer (TNBC) but reduced in estrogen receptor (ER)-positive breast cancer (ERPBC). We show that increased expression of miRNAs belonging to the miR-17–92 cluster is associated with poor outcome in TNBC, whereas the expression of miR-17–92 miRNAs is with good outcome in ERPBC. We show that ectopic expression of miR-17–92 inhibited cell growth and invasion of ER-positive and HER2-enriched cells. On the contrary, miR-17–92 expression enhanced cell growth and invasion of TNBC cells. Further, we found that miR-17–92 expression sensitized MCF7 cells to chemotherapeutic compounds, whereas it rendered SKBR3 cells resistant to them. We found that expression of ADORA1 was reduced by miR-17–92-expressing breast cancer cells, specifically in ERPBC. We observed an inverse correlation between the expression of ADORA1 and miR-17–92 in human breast cancer. Treatment with DPCPX, a selective ADORA1 antagonist, abolished the difference in the growth of control and miR-17–92 overexpressing MCF7 cells and identified ADORA1 as a key functional target of miR-17–92 in ERPBC. Furthermore, increased expression of ADORA1 in ERPBC is associated with a poor outcome. Our observations underscore the context-dependent role of miR-17–92 in breast cancer subtypes and suggest that miR-17–92 could serve as novel prognostic markers in breast cancer.

Napabucasin Attenuates Resistance of Breast Cancer Cells to Tamoxifen by Reducing Stem Cell-Like Properties.

BackgroundTamoxifen (TAM) is the first-line drug for estrogen receptor-positive (ER+) breast cancer (BC) treatment. However, its resistance is a main obstacle in clinical practice. Thus, new therapeutic agents are urgently needed to fight TAM resistance.Material/MethodsHere, we constructed TAM-resistant ER+BC cells with TAM resistance, named MCF-7-R. Western blot, quantitative real-time PCR (qRT-PCR), ALDH1 activity analysis, and spheroid-forming detection were used to detect the stemness of cells and the effects of napabucasin (NP) on BC cell stemness. Cell counting kit-8 (CCK8) assay was used to evaluate the effects of NP on cell viability.ResultsMCF-7-R cells exhibited higher stemness compared with the parental MCF-7 cells, which was evident by the increased spheroid formation ability at diluted concentration, aldehyde dehydrogenase (ALDH) activity, and expression of stemness critical biomarkers (Oct4, Nanog, and Sox2). Additionally, it was found that napabucasin (NP) specifically killed MCF-7-T cells, characterized by remarkably decreased IC50 value. Notably, NP reduced MCF-7-R cell stemness, which was evident as the decreased stemness marker expression, spheroid-forming capacity, and ALDH1 activity. Importantly, NP attenuated TAM resistance of MCF-7-R cells and enhanced sensitivity of MCF-7 cells to TAM. Mechanistic study showed that NP inhibited STAT3 activation, and overexpression of STAT3 rescued NP-mediated inhibition of the stemness-like characteristics of MCF-7-R cells.ConclusionsNP might be used as an adjuvant therapy for ER+ BC patients with TAM resistance.

Induced Tamoxifen Resistance is Mediated by Increased Methylation of E-Cadherin in Estrogen Receptor-Expressing Breast Cancer Cells

Estrogen receptor-positive breast cancers are treated with tamoxifen, a drug that competitively inhibits the binding of estrogen to its receptor. Resistance to tamoxifen is a major hurdle in effective management of target breast cancer patient population. A number of dynamic changes within the tumor microenvironment, including the phenomenon of epithelial to mesenchymal transition (EMT), determine the response to endocrine therapy. EMT is marked by silencing or suppression of epithelial marker, E-Cadherin and we found significantly down-regulated E-Cadherin, among other epithelial markers, and a significantly up-regulated mesenchymal marker, Twist, among other mesenchymal markers, in a model system that comprised of tamoxifen sensitive MCF-7 cells and their tamoxifen-resistant counterparts, MCF-7-TAM, developed by chronic and escalating exposure of parental cells to tamoxifen. Further, E-cadherin, but not Twist, was differentially expressed in MCF-7-TAM cells because of differential methylation. Treatment with demethylating agent 5-azacytidine increased the expression of E-cadherin thus verifying a role of methylation in its silencing and, moreover, 5-azacytidine treatment also re-sensitized MCF-7-TAM cells to tamoxifen, as evaluated by assays for viability, apoptosis and migration potential. The 5-azacytidine effects were similar to effects of E-cadherin overexpression in MCF-7-TAM cells. This work describes novel mechanism of E-cadherin downregulation in tamoxifen resistant breast cancer cells. Further studies are needed to exploit this information for betterment of breast cancer therapy.

Inhibition of PI3K pathway using BKM120 intensified the chemo-sensitivity of breast cancer cells to arsenic trioxide (ATO)

Although conventional therapeutic approaches have brought remarkable advantages for the treatment of breast cancer (BC), drug-resistance still remains as a leading cause of tumor recurrence in this malignancy. In the present study, we designed experiments to evaluate the therapeutic value of PI3K inhibition in combination with Arsenic trioxide (ATO) in MCF-7 cells. The results of our study manifested that BKM120 sensitized MCF-7 cells to the lower concentrations of ATO. The significant anti-cancer effect of PI3K inhibition became even more evident when we found that BKM120, either as a single agent or in combination with ATO, reduced clonogenic ability of anoikis-resistant stem-like MCF-7 cells. Our findings also showed that BKM120 augmented ATO-induced anti-proliferative effects through inducing G1 arrest and reducing the incorporation of BrdU into the synthesized DNA of drugs-treated cells, which was coupled with c-Myc-mediated suppression of hTERT expression. Moreover, we found that in the presence of PI3K inhibitor, ATO is able to more profoundly induce apoptosis in MCF-7 cells, as revealed by the increment in the percentage of haplodiploid sub-G1 cells and the externalization of phosphatidylserine. Real-time PCR analysis also revealed that probably down-regulation of survivin coupled with up-regulation of forkhead family transcription factors is responsible for the enhancive effect of drugs in this cell line. Conclusively, this study shed lights on the effect of PI3K inhibition in chemo-sensitivity of MCF-7 cells, disclosing that combination of BKM120 and ATO could be a promising therapeutic approach in BC.

Role of Cytochrome P450 Enzymes in the Maintenance of Breast Cancer Metastasized to the Bone [30D

INTRODUCTION: Metastasis of cancer cells causes most cancer-related deaths, prompting interest in its molecular mechanisms. Cancer homeostasis depends on the tumor microenvironment. Interactions between malignant cells and surrounding stroma contribute to cancer processes, including carcinogenesis and resistance to therapy. For example, multiple myeloma and leukemia cells can be protected from systemic chemotherapy by stromal cytochrome P450 enzymes (CYPs) that are found in the bone marrow (BM). We hypothesized that stromal CYPs protect solid tumors metastases to the BM. METHODS: We used a conditioned media model to mimic tumor microenvironment, asking whether a human BM mesenchymal stroma cell line can affect growth of a breast adenocarcinoma metastatic cell line (MCF7). RESULTS: We first established that the IC50 of Doxorubicin of MCF7 cells is 50nM, a concentration which also preserved growth of stromal cells. We found that concomitant treatment of Doxorubicin and Clarithromycin (a CYP3A4 inhibitor) sensitized MCF7 cells in stromal conditioned media more than treatment with only Doxorubicin, while it had no such effect in stroma-free conditions. Preliminary results also show that the concomitant treatment of Doxorubicin and Clarithromycin rescued the sensitivity of MCF7 cell line to Doxorubicin in stromal conditioned media, while it had no rescue effect in stroma-free cultures. CONCLUSION: Our results suggest that breast carcinoma cells are relatively resistant to Doxorubicin in the presence of BM stroma and that this resistance can be overcome by Clarithromycin. Our in vitro data suggest that like its role in hematological malignancies, stroma in the BM microenvironment creates biochemical niches that protect metastases from systemic chemotherapy.

Computational analysis of data from a genome-wide screening identifies new PARP1 functional interactors as potential therapeutic targets.

Knowledge of interaction network between different proteins can be a useful tool in cancer therapy. To develop new therapeutic treatments, understanding how these proteins contribute to dysregulated cellular pathways is an important task. PARP1 inhibitors are drugs used in cancer therapy, in particular where DNA repair is defective. It is crucial to find new candidate interactors of PARP1 as new therapeutic targets in order to increase efficacy of PARP1 inhibitors and expand their clinical utility. By a yeast-based genome wide screening, we previously discovered 90 candidate deletion genes that suppress growth-inhibition phenotype conferred by PARP1 in yeast. Here, we performed an integrated and computational analysis to deeply study these genes. First, we identified which pathways these genes are involved in and putative relations with PARP1 through g:Profiler. Then, we studied mutation pattern and their relation to cancer by interrogating COSMIC and DisGeNET database; finally, we evaluated expression and alteration in several cancers with cBioPortal, and the interaction network with GeneMANIA. We identified 12 genes belonging to PARP1-related pathways. We decided to further validate RIT1, INCENP and PSTA1 in MCF7 breast cancer cells. We found that RIT1 and INCENP affected PARylation and PARP1 protein level more significantly in PARP1 inhibited cells. Furthermore, downregulation of RIT1, INCENP and PSAT1 affected olaparib sensitivity of MCF7 cells.Our study identified candidate genes that could have an effect on PARP inhibition therapy. Moreover, we also confirm that yeast-based screenings could be very helpful to identify novel potential therapy factors.

Cancer Radiotherapy: Enhanced Radiosensitization by Gold Nanoparticles with Acid‐Triggered Aggregation in Cancer Radiotherapy (Adv. Sci. 8/2019)

In article number 1801806, Xing-Jie Liang, Jianfeng Liu, and co-workers develop a gold nanoparticles (GNPs) system with acid-triggered aggregation as a radiosensitizer. With a sensitizer enhancement ratio (SER) value of 1.730 (vs. single GNPs of 1.16), this GNPs system improves sensitivity of MCF-7 cells and MCF-7 tumor xenograft to ionizing radiation. This work provides a strategy for fabricating radiosensitizer by in situ acid-triggered nano-aggregates formation.