Sequential Salinomycin Treatment Results in Resistance Formation through Clonal Selection of Epithelial-Like Tumor Cells

Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC)–specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.     

miR-200c-3p靶向FOSL1增加乳腺癌细胞化疗敏感性

化疗耐受是乳腺癌复发转移率居高不下、综合治疗效果难以提高的主要瓶颈。前期研究证实,miR-200c-3p在乳腺癌敏感细胞MCF-7中的表达量显著高于耐药细胞MCF-7/5Fu,提示miR-200c-3p可能参与乳腺癌化疗增敏,但是具体机制不详。生物信息学预测联合双荧光素酶报告基因实验证实,miR-200c-3p靶向调控FOSL1,且在多种肿瘤中miR-200c-3p与FOSL1表达负相关。实时荧光定量PCR技术和Western印迹技术证实,FOSL1在耐药细胞MCF-7/5Fu中的表达量显著高于亲本细胞MCF-7。在MCF-7细胞中,过表达FOSL1能够显著提高该细胞对5-Fu的化疗耐受;在MCF-7/5Fu中,使用siRNA技术沉默FOSL1,将提高该细胞对5-Fu的化疗敏感性。此外,MTT实验还发现,miR-200c-3p抑制剂能够显著上调MCF-7细胞对5-Fu的耐受,但是在此细胞中干扰FOSL1的表达,又可以增加其对5-Fu的化疗敏感性;miR-200c-3p mimics显著增加MCF-7/5Fu细胞的化疗敏感性,上调FOSL1表达后又可逆转miR-200c-3p mimics的化疗增敏作用。总之,miR-200-3p能够通过靶向FOSL1增加乳腺癌细胞对5-fluorouridine化疗敏感性。     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.     

Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen.     

Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. Here we reinforced other''s report that human breast cancer cell line MCF-7/S could acquire increased survival potential from its resistant variants MCF-7/Adr and MCF-7/Doc. Additionally, exosomes of the latter, A/exo and D/exo, significantly modulated the cell cycle distribution and drug-induced apoptosis with respect to S/exo. Exosomes pre-treated with RNase were unable to regulate cell cycle and apoptosis resistance, suggesting an RNA-dependent manner. Microarray and polymerase chain reaction for the miRNA expression profiles of A/exo, D/exo, and S/exo demonstrated that they loaded selective miRNA patterns. Following A/exo and D/exo transfer to recipient MCF-7/S, the same miRNAs were significantly increased in acquired cells. Target gene prediction and pathway analysis showed the involvement of miR-100, miR-222, and miR-30a in pathways implicated in cancer pathogenesis, membrane vesiculation and therapy failure. Furthermore, D/exo co-culture assays and miRNA mimics transfection experiments indicated that miR-222-rich D/exo could alter target gene expression in MCF-7/S. Our results suggest that drug-resistant breast cancer cells may spread resistance capacity to sensitive ones by releasing exosomes and that such effects could be partly attributed to the intercellular transfer of specific miRNAs.     

MicroRNA-155 Regulates Cell Survival,Growth, and Chemosensitivity by Targeting FOXO3a in Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Despite improvement in treatment over the past few decades, there is an urgent need for development of targeted therapies. miR-155 (microRNA-155) is frequently up-regulated in breast cancer. In this study, we demonstrate the critical role of miR-155 in regulation of cell survival and chemosensitivity through down-regulation of FOXO3a in breast cancer. Ectopic expression of miR-155 induces cell survival and chemoresistance to multiple agents, whereas knockdown of miR-155 renders cells to apoptosis and enhances chemosensitivity. Further, we identified FOXO3a as a direct target of miR-155. Sustained overexpression of miR-155 resulted in repression of FOXO3a protein without changing mRNA levels, and knockdown of miR-155 increases FOXO3a. Introduction of FOXO3a cDNA lacking the 3′-untranslated region abrogates miR-155-induced cell survival and chemoresistance. Finally, inverse correlation between miR-155 and FOXO3a levels were observed in a panel of breast cancer cell lines and tumors. In conclusion, our study reveals a molecular link between miR-155 and FOXO3a and presents evidence that miR-155 is a critical therapeutic target in breast cancer.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

circ-ZEB1 regulates epithelial-mesenchymal transition and chemotherapy resistance of colorectal cancer through acting on miR-200c-5p

Circular RNAs (circRNAs) have been demonstrated to be important regulators in human malignant tumors, including colorectal cancer (CRC). While the role circ-ZEB1 played in CRC remains unclear. In this study, we aim to explore the biological function and the underlying mechanism of circ-ZEB1 in CRC. RNAscope was used to analyze the expression and localization of circ-ZEB1 in CRC tissues. Loss of function experiments were conducted, including CCK-8, transwell assays, flow cytometry analysis, and murine xenograft models, so as to detect the effect of circ-ZEB1 on CRC cells. IC50 assay was used to evaluate the influence of circ-ZEB1 on the chemoresistance of CRC cells. Epithelial-mesenchymal transition (EMT) related markers were detected. The relationship between circ-ZEB1 and miR-200c-5p was investigated by FISH, dual-luciferase reporter assay, and RIP assay. We found in our study that circ-ZEB1 was significantly upregulated in CRC tissues. Downregulation of circ-ZEB1 inhibited cell proliferation, colony formation, as well as cell migration and invasion abilities of CRC cell lines. In vivo experiments indicated that knockdown of circ-ZEB1 suppressed tumorigenesis and distant metastasis of CRC cells in nude mice. What's more, EMT and chemoresistance of CRC cells were also attenuated following circ-ZEB1 knockdown. Mechanistically, we proved that circ-ZEB1 could directly bind with miR-200c and functioned as miR-200c sponge to exert its biological functions in CRC cells. In conclusion, circ-ZEB1 could promote CRC cells progression, EMT, and chemoresistance via acting on miR-200c, elucidating a potential therapeutic target to inhibit CRC progression.     

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling

We have previously demonstrated that extracellular adenosine 5''-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.Subject terms: Breast cancer, Cell signalling     

Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

The presence of cancer stem-like cells (CSCs) is one of the mechanisms responsible for chemoresistance that has been a major hindrance towards lung adenocarcinoma (LAD) treatment. Recently, we have identified microRNA (miR)-200b as a key regulator of chemoresistance in human docetaxel-resistant LAD cells. However, whether miR-200b has effects on regulating CSCs remains largely unclear and needs to be further elucidated. Here, we showed that miR-200b was significantly downregulated in CD133⁺/CD326⁺ cells that exhibited properties of CSCs derived from docetaxel-resistant LAD cells. Also, restoration of miR-200b could inhibit maintenance and reverse chemoresistance of CSCs. Furthermore, suppressor of zeste-12 (Suz-12) was identified as a direct and functional target of miR-200b, and silencing of Suz-12 phenocopied the effects of miR-200b on CSCs. Additionally, overexpression of histone deacetylase (HDAC) 1 was identified as a pivotal mechanism responsible for miR-200b repression in CSCs through a specificity protein (Sp) 1-dependent mechanism, and restoration of miR-200b by HDAC1 repression significantly suppressed CSCs formation and reversed chemoresistance of CSCs by regulating Suz-12-E-cadherin signaling. Also, downregulation of HDAC1 or upregulation of miR-200b reduced the in vivo tumorigenicity of CSCs. Finally, Suz-12 was inversely correlated with miR-200b, positively correlated with HDAC1 and up-regulated in docetaxel-resistant LAD tissues compared with docetaxel-sensitive tissues. Taken together, the HDAC1/miR-200b/Suz-12-E-cadherin signaling might account for maintenance of CSCs and formation of chemoresistant phenotype in docetaxel-resistant LAD cells.     

MiR-124 Radiosensitizes Human Colorectal Cancer Cells by Targeting PRRX1

One of the challenges in the treatment of colorectal cancer patients is that these tumors show resistance to radiation. MicroRNAs (miRNAs) are involved in essential biological activities, including chemoresistance and radioresistance. Several research studies have indicated that miRNA played an important role in sensitizing cellular response to ionizing radiation (IR). In this study, we found that miR-124 was significantly down-regulated both in CRC-derived cell lines and clinical CRC samples compared with adjacent non-tumor colorectal tissues, MiR-124 could sensitize human colorectal cancer cells to IR in vitro and in vivo. We identified PRRX1, a new EMT inducer and stemness regulator as a novel direct target of miR-124 by using target prediction algorithms and luciferase assay. PRRX1 knockdown could sensitize CRC cells to IR similar to the effects caused by miR-124. Overexpression of PRRX1 in stably overexpressed-miR-124 cell lines could rescue the effects of radiosensitivity enhancement brought by miR-124. Taking these observations into consideration, we illustrated that miR-124 could increase the radiosensitivity of CRC cells by blocking the expression of PRRX1, which indicated miR-124 could act as a great therapeutic target for CRC patients.     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.     

Circulating MiR-125b as a marker predicting chemoresistance in breast cancer

Background

Chemotherapy is an important component in the treatment paradigm for breast cancers. However, the resistance of cancer cells to chemotherapeutic agents frequently results in the subsequent recurrence and metastasis. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of circulating microRNAs (miRNAs) can predict clinical outcome in breast cancer patients treated with adjuvant chemotherapy.

Methodology/Principal Findings

Circulating miRNAs in blood serum prior to treatment were determined by quantitative Real-Time PCR in 56 breast cancer patients with invasive ductal carcinoma and pre-operative neoadjuvant chemotherapy. Proliferating cell nuclear antigen (PCNA) immunostaining and TUNEL were performed in surgical samples to determine the effects of chemotherapy on cancer cell proliferation and apoptosis, respectively. Among the miRNAs tested, only miR-125b was significantly associated with therapeutic response, exhibiting higher expression level in non-responsive patients (n = 26, 46%; p = 0.008). In addition, breast cancers with high miR-125b expression had higher percentage of proliferating cells and lower percentage of apoptotic cells in the corresponding surgical specimens obtained after neoadjuvant chemotherapy. Increased resistance to anticancer drug was observed in vitro in breast cancer cells with ectopic miR-125b expression; conversely, reducing miR-125b level sensitized breast cancer cells to chemotherapy. Moreover, we demonstrated that the E2F3 was a direct target of miR-125b in breast cancer cells.

Conclusions/Significance

These data suggest that circulating miR-125b expression is associated with chemotherapeutic resistance of breast cancer. This finding has important implications in the development of targeted therapeutics for overcoming chemotherapeutic resistance in novel anti-cancer strategies.     

Identification of miR-200a as a novel suppressor of connexin 43?in breast cancer cells

Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3′-untranslated region (3′-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.