Xiao-Ting Gu

Essential role for TrpC5-containing extracellular vesicles in breast cancer with chemotherapeutic resistance

Significance A critical challenge for chemotherapy is development of chemoresistance, but underlying molecular mechanisms remain unclear. In this study, we found that drug-resistant adriamycin-resistant human breast cancer cells possessed numerous transient receptor potential channel 5 (TrpC5) -containing extracellular vesicles (EVs) on the cell surface. Suppressing TrpC5 expression diminished the formation of EVs. Incubation of drug-sensitive recipient cells with EVs endowed recipients with drug-resistant properties. In both human samples and a mouse model of breast cancer, the expression of TrpC5 proteins was high in the tumor, and the levels of TrpC5-positive EVs were high in the circulation. These data suggest a critical role of TrpC5-containing EVs in the transfer of drug resistance. In the future, monitoring TrpC5-containing EVs in the circulation could potentially be used to predict the clinical outcome of chemotherapy. A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. However, the underlying mechanisms and validated predictors remain unclear. Extracellular vesicles (EVs) have gained attention as potential means for cancer cells to share intracellular contents. In adriamycin-resistant human breast cancer cells (MCF-7/ADM), we analyzed the role of transient receptor potential channel 5 (TrpC5) in EV formation and transfer as well as the diagnostic implications. Up-regulated TrpC5, accumulated in EVs, is responsible for EV formation and trapping of adriamycin (ADM) in EVs. EV-mediated intercellular transfer of TrpC5 allowed recipient cells to acquire TrpC5, consequently stimulating multidrug efflux transporter P-glycoprotein production through a Ca2+- and activated T-cells isoform c3-mediated mechanism and thus, conferring chemoresistance on nonresistant cells. TrpC5-containing circulating EVs were detected in nude mice bearing MCF-7/ADM tumor xenografts, and the level was lower after TrpC5–siRNA treatment. In breast cancer patients who underwent chemotherapy, TrpC5 expression in the tumor was significantly higher in patients with progressive or stable disease than in patients with a partial or complete response. TrpC5-containing circulating EVs were found in peripheral blood from patients who underwent chemotherapy but not patients without chemotherapy. Taken together, we found that TrpC5-containing circulating EVs may transfer chemoresistance property to nonchemoresistant recipient cells. It may be worthwhile to further explore the potential of using TrpC5-containing EVs as a diagnostic biomarker for chemoresistant breast cancer.

A Methylation-Based Regulatory Network for MicroRNA 320a in Chemoresistant Breast Cancer

We previously demonstrated that the overexpression of transient receptor potential channel C5 (TRPC5) and nuclear factor of activated T-cells isoform c3 (NFATC3) are essential for cancer chemoresistance, but how TRPC5 and NFATC3 are regulated was still unclear. In this study, microRNA 320a (miR-320a) was found to be down-regulated in chemoresistant cancer cells. MiR-320a directly targeted TRPC5 and NFATC3, and down-regulation of miR-320a triggered TRPC5 and NFATC3 overexpression. In chemoresistant cells, down-regulation of miR-320a was associated with regulation by methylation, which implicated promoter methylation of the miR-320a coding sequence. Furthermore, the transcription factor v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS-1), which inhibited miR-320a expression, was activated in chemoresistant cancer cells; such activation was associated with hypomethylation of the ETS-1 promoter. Lastly, the down-regulation of miR-320a and high expression of TRPC5, NFATC3, and ETS-1 were verified in clinically chemoresistant samples. Low expression of MiR-320a was also found to be a significant unfavorable predictor for clinic outcome. In conclusion, miR-320a is a mediator of chemoresistance by targeting TRPC5 and NFATC3. Expression of miR-320a is regulated by methylation of its promoter and that of ETS-1.

Methylation-regulated miR-149 modulates chemoresistance by targeting GlcNAc N-deacetylase/N-sulfotransferase-1 in human breast cancer

Dysregulation of microRNA is strongly implicated in the chemoresistance of cancer. In this study, we found that miR‐149 was downregulated and involved in chemoresistance in adriamycin (ADM)‐resistant human breast cancer cells (MCF‐7/ADM). Downregulation of miR‐149 was related to hypermethylation of its 5′‐UTR; this methylation also affected the expression of the glypican 1 gene, which is both the host and the target gene of miR‐149. Furthermore, we found that miR‐149 modulated chemoresistance through targeting the expression of GlcNAc N‐deacetylase/N‐sulfotransferase‐1 (NDST1). With downregulated miR‐149, NDST1 expression was increased in chemoresistant MCF‐7/ADM cells versus control MCF‐7 wild‐type cells. The increased NDST1 then activated a heparan sulfate‐related pathway involving activation of heparanase. Finally, expression of miR‐149 and NDST1 was confirmed in clinical chemoresistant samples of breast cancers receiving anthracycline/taxane‐based chemotherapies. The high expression of NDST1 was also an unfavorable predictor for distant relapse‐free survival in Her2 and basal breast cancers. Taken together, our findings demonstrate that miR‐149 is regulated by methylation, and is a modulator of cancer chemoresistance by targeting NDST1.

A new agent developed by biotransformation of polyphyllin VII inhibits chemoresistance in breast cancer

Biotransformation by the endophytes of certain plants changes various compounds, and this ‘green’ chemistry becomes increasingly important for finding new products with pharmacological activity. In this study, polyphyllin VII (PPL7) was biotransformed by endophytes from the medicinal plant Paris polyphylla Smith, var. yunnanensis. This produced a new compound, ZH-2, with pharmacological activity in vitro and in vivo. ZH-2 was more potent than PPL7 in selectively killing more chemoresistant than chemosensitive breast cancer cells. ZH-2 also re-sensitized chemoresistant breast cancer cells, as evidenced by the improved anti-cancer activity of commonly-used chemotherapeutic agent in vitro, in vivo, and in clinical samples. This anti-chemoresistance effect of ZH-2 was associated with inhibiting the epithelial-mesenchymal transition (EMT) pathway. Taken together, our findings are the first one to link biotransformation with a biomedicine. The results provide insights into developing new pharmacologically-active agents via biotransformation by endophytes.

Genome-wide profiling of long non-coding RNA expression patterns in anthracycline-resistant breast cancer cells.

Long non-coding RNAs (lncRNAs) are involved in cancer progression. In the present study, we analyzed the lncRNA profiles in adriamycin-resistant and -sensitive breast cancer cells and found a group of dysregulated lncRNAs in the adriamycin-resistant cells. Expression of the dysregulated lncRNAs was correlated with dysregulated mRNAs, and these were enriched in GO and KEGG pathways associated with cancer progression and chemoresistance development. Among these lncRNA-mRNA interactions, some lncRNAs may cis‑regulate neighboring protein-coding genes and be involved in chemoresistance. We then validated that the lncRNA NONHSAT028712 regulated nearby CDK2 and interfered with the cell cycle and chemoresistance. Furthermore, we identified another group of lncRNAs that trans-regulated genes by interacting with different transcription factors. For example, NONHSAT057282 and NONHSAG023333 modulated chemoresistance and most likely interacted with the transcription factors ELF1 and E2F1, respectively. In conclusion, in the present study, we report for the first time the lncRNA expression patterns in adriamycin-resistant breast cancer cells, and provide a group of novel lncRNA targets that mediate chemoresistance development in both cis- and trans-action modes.

Methylation of Notch3 modulates chemoresistance via P-glycoprotein.

The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells (MCF-7/PTX) and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells.

A transcription/translation-based gene signature predicts resistance to chemotherapy in breast cancer.

Although chemotherapy is widely used to treat human cancers, most chemotherapeutic agents only benefit a small fraction of patients because of the heterogeneity of cancers. Therefore, identifying of the sensitivity of cancers toward various chemotherapies would be important for choosing of chemotherapeutic regime. In this study, a 23-gene chemoresistance signature was developed from chemoresistant breast cancers. Functions of the genes in the signature were related with transcription and translation. The signature was indicative of chemoresistance and associated with poor prognosis in multiple chemotherapeutic agents and cancer types. Furthermore, by applying computational approaches, we identified several compounds that might specifically affect the chemoresistant signature. Decitabine (DAC) was the compound most likely to target the signature. In vitro and clinical analysis confirmed effect of DAC toward both breast cancer cell lines and ovarian cancers respectively. In conclusion, our study identified a chemoresistant signature that is both predictive and prognostic, and the signature-related chemoresistance could be suppressed by DAC treatment.

Notch3 negatively regulates chemoresistance in breast cancers

To define the role of the NOTCH signaling pathway in the development of chemoresistance and the associated epithelial–mesenchymal transition (EMT), we investigated the effect of Notch3 on adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM cells). We found that Notch3 was downregulated and involved in the chemoresistance of MCF-7/ADM cells, while forced expression of Notch3 reversed the chemoresistance. Furthermore, fos-related antigen 1 (Fra1) was negatively regulated by Notch3 and was highly expressed in MCF-7/ADM cells. Increased Fra1 activated the EMT process. Finally, Notch3 expression was confirmed in clinically chemoresistant samples of breast cancers from patients receiving anthracycline-based chemotherapy. Low expression of Notch3 was an unfavorable predictor of distant relapse-free survival in ER positive breast cancers. Taken together, our findings demonstrate that the Notch3-Fra1 signaling pathway mediates chemoresistance via the EMT.

Genome-wide analysis of the three-way interplay among gene expression, estrogen receptor expression and chemotherapeutic sensitivity in breast cancer

The expression of estrogen receptor α (ER) in breast cancers may be indicative of a favorable prognosis and most of these cancers respond to anti-estrogens or aromatase inhibitors. However, ER-positive (ER+) breast cancers receiving anti-hormone and/or chemotherapy sometimes lose their ER expression, which leads to the evolution of the disease to higher aggressiveness and drug resistance. In the present study, an ER-modified signature (EMS) was developed from the expression profile of a chemoresistant MCF-7 breast cancer cell line that lost ER expression during long-term treatment with a chemotherapeutic agent. The EMS could discriminate the ER-negative (ER−) breast cancer cells from the ER+ ones, which included seven pathways essential for the ER− cell development. Furthermore, the EMS indicated a more malignant subgroup of the ER− cells by discriminating the chemoresistant ER− cells from the chemosensitive ones. In addition, the classified chemoresistant ER− patients demonstrated worse prognosis. In conclusion, we developed a new method to discriminate subgroups of ER− breast cancer cells.