Hongyu Shen

Exosomes in development, metastasis and drug resistance of breast cancer

Transport through the cell membrane can be divided into active, passive and vesicular types (exosomes). Exosomes are nano‐sized vesicles released by a variety of cells. Emerging evidence shows that exosomes play a critical role in cancers. Exosomes mediate communication between stroma and cancer cells through the transfer of nucleic acid and proteins. It is demonstrated that the contents and the quantity of exosomes will change after occurrence of cancers. Over the last decade, growing attention has been paid to the role of exosomes in the development of breast cancer, the most life‐threatening cancer in women. Breast cancer could induce salivary glands to secret specific exosomes, which could be used as biomarkers in the diagnosis of early breast cancer. Exosome‐delivered nucleic acid and proteins partly facilitate the tumorigenesis, metastasis and resistance of breast cancer. Exosomes could also transmit anti‐cancer drugs outside breast cancer cells, therefore leading to drug resistance. However, exosomes are effective tools for transportation of anti‐cancer drugs with lower immunogenicity and toxicity. This is a promising way to establish a drug delivery system.

MiR-222 promotes drug-resistance of breast cancer cells to adriamycin via modulation of PTEN/Akt/FOXO1 pathway.

BACKGROUND AND PURPOSE Acquisition of resistance to adriamycin (ADR) is one of the most important clinical obstacles in the treatment of breast cancer, but the molecular mechanisms underlying sensitivity to ADR remain elusive. In our previous study, through miRNA microarray and experiments, we have emphasized that miR-222 could promote the ADR-resistance in breast cancer cells. The aim of this study was to explore the possible mechanism by which miR-222 affects sensitivity to ADR. METHODS Through pathway enrichment analyses for miR-222, we found that PTEN/Akt/FOXO1 signaling pathway may be of importance. RT-qPCR analyses and western blot assays confirmed the relationship between miR-222 expression and target genes. Immunofluorescence further visually displayed the location of FOXO1. When blocking PTEN/Akt/FOXO1 signaling pathway, we demonstrated the effects of miR-222-mediated ADR resistance by MTT and apoptosis assays. RESULTS RT-qPCR and Western blot results showed that miR-222 expression was negatively correlated with FOXO1 expression. In addition, the subcellular translocation of FOXO1 due to the altered expression of miR-222 was observed from immunofluorescence. Moreover, upregulation of miR-222 expression in MCF-7/S cells is associated with decreased PTEN expression levels and increased phospho-Akt (p-Akt) expression. Conversely in MCF-7/ADR cells, inhibition of miR-222 resulted in increased PTEN expression and decreased p-Akt expression. For further validation, results of the present study also demonstrated that PTEN/Akt/FOXO1 signaling was responsible for the ADR-resistance of breast cancer cells since LY294002, an inhibitor of Akt signaling, partially increased the sensitivity of MCF-7/S cells to ADR. More importantly, we postulated that high expression of miR-222 is closely related to poor overall survival by TCGA database validation. CONCLUSIONS Taken together, these data elucidated that miR-222 mediated ADR-resistance of breast cancer cells partly through regulation of PTEN/Akt/FOXO1 signaling pathway and inhibition of miR-222 may improve the prognosis of breast cancer patients.

Exosomes from adriamycin-resistant breast cancer cells transmit drug resistance partly by delivering miR-222

Breast cancer (BCa) is one of the major deadly cancers in women. However, treatment of BCa is still hindered by the acquired-drug resistance. It is increasingly reported that exosomes take part in the development, metastasis, and drug resistance of BCa. However, the specific role of exosomes in drug resistance of BCa is poorly understood. In this study, we investigate whether exosomes transmit drug resistance through delivering miR-222. We established an adriamycin-resistant variant of Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line (MCF-7/Adr) from a drug-sensitive variant (MCF-7/S). Exosomes were isolated from cell supernatant by ultracentrifugation. Cell viability was assessed by MTT assay and apoptosis assay. Individual miR-222 molecules in BCa cells were detected by fluorescence in situ hybridization (FISH). Then, FISH was combined with locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH). Individual miR-222 could be detected as bright photostable fluorescent spots and then the quantity of miR-222 per cell could be counted. Stained exosomes were taken in by the receipt cells. MCF-7/S acquired drug resistance after co-culture with exosomes from MCF-7/Adr (A/exo) but did not after co-culture with exosomes from MCF-7/S (S/exo). The quantity of miR-222 in A/exo-treated MCF-7/S was significantly greater than in S/exo-treated MCF-7/S. MCF-7/S transfected with miR-222 mimics acquired adriamycin resistance while MCF-7/S transfected with miR-222 inhibitors lost resistance. In conclusion, exosomes are effective in transmitting drug resistance and the delivery of miR-222 via exosomes may be a mechanism.

MicroRNA expression profiles of drug-resistance breast cancer cells and their exosomes

Exosomes have been shown to transmit drug resistance through delivering miRNAs. We aimed to explore their roles in breast cancer. Three resistant sublines were established by exposing parental MDA-MB-231 cell line to docetaxel, epirubicin and vinorelbine, respectively. Preneoadjuvant chemotherapy biopsies and paired surgically-resected specimens embedded in paraffin from 23 breast cancer patients were collected. MiRNA expression profiles of the cell lines and their exosomes were evaluated using microarray. The result showed that most miRNAs in exosomes had a lower expression level than that in cells, however, some miRNAs expressed higher in exosomes than in cells, suggesting a number of miRNAs is concentrated in exosomes. Among the dysregulated miRNAs, 22 miRNAs were consistently up-regulated in exosomes and their cells of origin. We further found that 12 of the 22 miRNAs were significantly up-regulated after preneoadjuvant chemotherapy. Further study of the role of these 12 miRNAs in acquisition of drug resistance is needed to clarify their contribution to chemoresistance.

MicroRNA-29a contributes to drug-resistance of breast cancer cells to adriamycin through PTEN/AKT/GSK3β signaling pathway.

PURPOSE Acquisition of resistance to adriamycin (ADR) during the treatment of breast cancer is still a major clinical obstacle. MicroRNAs (miRNAs) are a class of short noncoding RNAs which associate with cancer chemoresistance through regulating gene expression by targeting mRNAs. Our previous microarray found that miR-29a may strongly confer the ADR resistance of breast cancer cells. Here, we aim to explore the possible mechanism by which miR-29a affects sensitivity to ADR. METHODS ADR-resistant MCF-7 breast cancer cell subline (MCF-7/ADR) was successfully established in vitro through a stepwise increase of ADR concentrations in the culture based on parental MCF-7 cell lines (MCF-7/S). We used TargetScan (a wide use of target prediction algorithms) in conjunction with pathway enrichment analyses to predict the mRNAs that were most likely to involve in miR-29a-mediated drug resistance in cancers. We confirmed the effects of miR-29a-mediated ADR resistance through MTT and apoptosis assays, and further investigated the activities of two target genes, PTEN and GSK3β, by RT-qPCR analyses and western blot assays. RESULTS The expression level of miR-29a in MCF-7/ADR cells was remarkablely higher than in MCF-7/S cells. Further MTT and apoptosis assays revealed that transfection of miR-29a inhibitors into MCF-7/ADR cells resulted in prominent reduction of the drug resistance, in contrast, transfection of miR-29a mimics into MCF-7/S cells obviously increased their drug resistance. Through pathway enrichment analyses for miR-29a, we found that PTEN/AKT/GSK3β signaling pathway may be of importance. RT-qPCR and Western blot results showed that downregulation of miR-29a expression in MCF-7/ADR cells increased PTEN expression levels, resulting in decreased phospho-Akt (p-Akt) and phospho-GSK3β (p-GSK3β) expression. Conversely, upregulation of miR-29a expression in MCF-7/S cells is associated with decreasing PTEN expression and increasing p-Akt and p-GSK3β expression. CONCLUSIONS PTEN and GSK3β are targeted by miR-29a, and miR-29a may contribute to ADR resistance through inhibition of the PTEN/AKT/GSK3β pathway in breast cancer cells. Thus, miR-29a may be a potential target for the patients who acquired ADR-resistance during the treatment of breast cancer.

The miR-30 family: Versatile players in breast cancer:

The microRNA family, miR-30, plays diverse roles in regulating key aspects of neoplastic transformation, metastasis, and clinical outcomes in different types of tumors. Accumulating evidence proves that miR-30 family is pivotal in the breast cancer development by controlling critical signaling pathways and relevant oncogenes. Here, we review the roles of miR-30 family members in the tumorigenesis, metastasis, and drug resistance of breast cancer, and their application to predict the prognosis of breast cancer patients. We think miR-30 family members would be promising biomarkers for breast cancer and may bring a novel insight in molecular targeted therapy of breast cancer.

Curcumin inhibits cancer progression through regulating expression of microRNAs

Curcumin, a major yellow pigment and spice in turmeric and curry, is a powerful anti-cancer agent. The anti-tumor activities of curcumin include inhibition of tumor proliferation, angiogenesis, invasion and metastasis, induction of tumor apoptosis, increase of chemotherapy sensitivity, and regulation of cell cycle and cancer stem cell, indicating that curcumin maybe a strong therapeutic potential through modulating various cancer progression. It has been reported that microRNAs as small noncoding RNA molecules are related to cancer progression, which can be regulated by curcumin. Dysregulated microRNAs play vital roles in tumor biology via regulating expressions of target genes and then influencing multiple cancer-related signaling pathways. In this review, we focused on the inhibition effect of curcumin on various cancer progression by regulating expression of multiple microRNAs. Curcumin-induced dysregulation of microRNAs may activate or inactivate a set of signaling pathways, such as Akt, Bcl-2, PTEN, p53, Notch, and Erbb signaling pathways. A better understanding of the relation between curcumin and microRNAs may provide a potential therapeutic target for various cancers.

Predictive role of GSTP1-containing exosomes in chemotherapy-resistant breast cancer

Anthracycline/taxane-based chemotherapy regimens are usually used as neoadjuvant chemotherapies to decrease tumour size and prevent metastasis of advanced breast cancer. However, patients have a high risk of developing chemo-resistance during treatment through still unknown mechanisms. Glutathione S-transferase P1 (GSTP1), which belongs to the family of phase II metabolic enzymes, has been reported to function in detoxifying several anti-cancer drugs by conjugating them with glutathione. Previous studies have identified GSTP1 as a predictor of prognosis and chemo-resistance in breast cancer patients, but the mechanisms governing GSTP1-dependent drug resistance are still unclear. We have found that GSTP1 expression is much higher in adriamycin-resistant cells and their corresponding exosomes. The role of GSTP1-containing exosomes in conferring drug resistance was analysed through cell apoptosis and immunofluorescence staining assays. Furthermore, we analysed 42 cases of paired breast cancer tissues collected before and after anthracycline/taxane-based neoadjuvant chemotherapy by immunohistochemistry. Higher GSTP1 expression was shown in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in the samples collected before and after the chemotherapy treatment. Interestingly, GSTP1 partly re-localized from the cell nucleus to the cytoplasm upon treatment, and similar results were obtained for the exosomal marker Tumour susceptibility gene 101 protein (TSG101), which also increased in the cytoplasm after chemotherapy. After analysing the serum exosomes of 30 patients treated with anthracycline/taxane-based neoadjuvant chemotherapy, we discovered that the levels of GSTP1 in exosomes from patients in the PD/SD group were significantly higher than those in the PR/CR group. Here, for the first time, we investigated a novel role for GSTP1-containing exosomes and their capability to transfer drug resistance and evaluated their clinical use in predicting chemo-resistance.

MiRNAs-mediated cisplatin resistance in breast cancer

Cisplatin is a widely used chemotherapeutic agent in breast cancer treatments with inevitable rapidly acquired resistance or intrinsically resistance. Enormous evidence points to the bioprocesses of resistant formation consisting of diverse miRNAs direct and indirect actions on relevant encoding genes. In this report, we overview detailed information on the miRNAs effect on cisplatin-induced resistance, including alterations in cell survival, modification of DNA damage response, changes in cellular uptake or efflux of the drug, altered DNA methylation, and perturbations in the miRNA biogenesis pathway. This will provide potential miRNA-targeted strategies for the treatment of breast cancer therapy and requires further clinical application.

miR-222 confers the resistance of breast cancer cells to Adriamycin through suppression of p27kip1 expression

Adriamycin (Adr) is a potent chemotherapeutic agent for chemotherapy of breast cancer patients. Despite impressive initial clinical responses, some developed drug resistance to Adr-based therapy and the mechanisms underlying breast cancer cells resistance to Adr are not well known. In our previous study, in vitro, we verified that miR-222 was upregulated in Adr-resistant breast cancer cells (MCF-7/Adr) compared with the sensitive parental cells (MCF-7/S). Here, miR-222 inhibitors or mimics were transfected into MCF-7 cell lines. RT-qPCR and western blot were used to detect the expression of p27(kip1). Immunofluorescence showed that miR-222 altered the subcellular location of p27(kip1) in nucleus. MTT was employed to verify the sensitivity of breast cancer cell lines to Adr. Flow cytometry showed the apoptosis and cell cycles of the cells after adding Adr. The results showed that downregulation of miR-222 in MCF-7/Adr increased sensitivity to Adr and Adr-induced apoptosis, and arrested the cells in G1 phase, accompanied by more expressions of p27(kip1), especially in nucleus. Furthermore, overexpressed miR-222 in MCF-7/S had the inverse results. Taken together, the results found that miR-222 induced Adr-resistance at least in part via suppressing p27(kip1) expression and altering its subcellular localization, and miR-222 inhibitors could reverse Adr-resistance of breast cancer cells. These results disclosed that the future holds much promise for the targeted therapeutic in the treatment of Adr-resistant breast cancer.