MicroRNA-204/CREB5 axis regulates vasculogenic mimicry in breast cancer cells.

BACKGROUND: Vasculogenic mimicry (VM) is characterized by formation of three-dimensional (3D) channels-like structures by tumor cells, supplying the nutrients needed for tumor growth. VM is stimulated by hypoxic tumor microenvironment, and it has been associated with increased metastasis and clinical poor outcome in cancer patients. cAMP responsive element (CRE)-binding protein 5 (CREB5) is a hypoxia-activated transcription factor involved in tumorigenesis. However, CREB5 functions in VM and if its regulated by microRNAs remains unknown in breast cancer. OBJECTIVE: We aim to study the functional relationships between VM, CREB5 and microRNA-204-5p (miR-204) in breast cancer cells. METHODS: CREB5 expression was evaluated by mining the public databases, and using RT-qPCR and Western blot assays. CREB5 expression was silenced using short-hairpin RNAs in MDA-MB-231 and MCF-7 breast cancer cells. VM formation was analyzed using matrigel-based cultures in hypoxic conditions. MiR-204 expression was restored in cancer cells by transfection of RNA mimics. Luciferase reporter assays were performed to evaluate the binding of miR-204 to 3'UTR of CREB5. RESULTS: Our data showed that CREB5 mRNA expression was upregulated in a set of breast cancer cell lines and clinical tumors, and it was positively associated with poor prognosis in lymph nodes positive and grade 3 basal breast cancer patients. Silencing of CREB5 impaired the hypoxia-induced formation of 3D channels-like structures representative of the early stages of VM in MDA-MB-231 cells. In contrast, VM formation was not observed in MCF-7 cells. Interestingly, we found that CREB5 expression was negatively regulated by miR-204 mimics in breast cancer cells. Functional analysis confirmed that miR-204 binds to CREB5 3'-UTR indicating that it's an ulterior effector. CONCLUSIONS: Our findings suggested that CREB5 could be a potential biomarker of disease progression in basal subtype of breast cancer, and that perturbations of the miR-204/CREB5 axis plays an important role in VM development in breast cancer cells.

Angiogenesis Analysis by In Vitro Coculture Assays in Transwell Chambers in Ovarian Cancer.

Angiogenesis is an important biological process in tumor growth and metastasis of tumor cells, and it has been associated with poor clinical outcomes in ovarian cancer. In vitro assays are useful tools for understanding the complex mechanisms of angiogenesis under a variety of conditions. Capillary-like formation and transwell migration assays are two of the most common techniques used in angiogenesis research. Here, we show an easy coculture model to study the role of microRNAs on angiogenesis that combines tube formation and cell migration assays. Recently, we reported that miR-204 is repressed in breast cancer and restoration in cancer cell lines results in angiogenesis inhibition. Here, we restored the expression of miR-204 by transfection of precursor molecule in the tumorigenic SKOV3 ovarian cancer cell line, and analyzed the effects in cell migration, invasion, and tube formation of endothelial cells using matrigel-coated transwell chambers.

Tumor suppressor miR-29c regulates radioresistance in lung cancer cells.

Radiotherapy is an important treatment option for non-small cell lung carcinoma patients. Despite the appropriate use of radiotherapy, radioresistance is a biological behavior of cancer cells that limits the efficacy of this treatment. Deregulation of microRNAs contributes to the molecular mechanism underlying resistance to radiotherapy in cancer cells. Although the functional roles of microRNAs have been well described in lung cancer, their functional roles in radioresistance are largely unclear. In this study, we established a non-small cell lung carcinoma Calu-1 radioresistant cell line by continuous exposure to therapeutic doses of ionizing radiation as a model to investigate radioresistance-associated microRNAs. Our data show that 50 microRNAs were differentially expressed in Calu-1 radioresistant cells (16 upregulated and 34 downregulated); furthermore, well-known and novel microRNAs associated with resistance to radiotherapy were identified. Gene ontology and enrichment analysis indicated that modulated microRNAs might regulate signal transduction, cell survival, and apoptosis. Accordingly, Calu-1 radioresistant cells were refractory to radiation by increasing cell survival and reducing the apoptotic response. Among deregulated microRNAs, miR-29c was significantly suppressed. Reestablishment of miR-29c expression in Calu-1 radioresistant cells overcomes the radioresistance through the activation of apoptosis and downregulation of Bcl-2 and Mcl-1 target genes. Analysis of The Cancer Genome Atlas revealed that miR-29c is also suppressed in tumor samples of non-small cell lung carcinoma patients. Notably, we found that low miR-29c levels correlated with shorter relapse-free survival of non-small cell lung carcinoma patients treated with radiotherapy. Together, these results indicate a new role of miR-29c in radioresistance, highlighting their potential as a novel biomarker for outcomes of radiotherapy in lung cancer.

Differential proteomic analysis reveals that EGCG inhibits HDGF and activates apoptosis to increase the sensitivity of non-small cells lung cancer to chemotherapy.

PURPOSE: To search for regulated proteins in response to green tea (-)-epigallocatechin-3-gallate (EGCG) in A549 lung cancer cells. EXPERIMENTAL DESIGN: 2DE and ESI/multistage MS (ESI-MS/MS) were performed to identify modulated proteins in A549 cells treated with EGCG. Cell migration was evaluated by transwell assays. RNA interference was used to silence the hepatoma-derived growth factor (HDGF). Caspase-3, caspase-9, and HDGF were immunodetected by Western blot assays. Flow cytometry was used for detection of mitochondrial membrane potential and apoptosis. RESULTS: We found that HDGF expression was threefold suppressed by EGCG treatment. Downregulation of HDGF by EGCG was confirmed using anti-HDGF antibodies in three lung cancer cell lines. EGCG treatment and HDGF abrogation by RNA interference resulted in a decreased migration of A549 cells. In addition, EGCG induced a marked synergistic effect with cisplatin in cell death. Consistently, an enhanced cytotoxicity in HDGF-silenced cells was also found. Cell death was associated to increased apoptosis, disruption of the mitochondrial membrane potential, and activation of caspase-3 and caspase-9. CONCLUSION AND CLINICAL RELEVANCE: Our data suggest for the first time that abrogation of HDGF by EGCG enhances cisplatin-induced apoptosis and sensitize A549 cells to chemotherapy. Therefore, we propose that decreasing the HDGF levels by using EGCG may represent a novel strategy in lung cancer therapy.

Comparative proteomic profiling of triple-negative breast cancer reveals that up-regulation of RhoGDI-2 is associated to the inhibition of caspase 3 and caspase 9.

There are no targeted therapeutic modalities for triple-negative breast cancer (TNBC), thus it is associated with poor prognosis and worst clinical outcome. Here, our aim was to identify deregulated proteins in TNBC with potential therapeutic applications. Proteomics profiling of TNBC and normal breast tissues through two-dimensional electrophoresis and ESI-MS/MS mass spectrometry revealed the existence of 16 proteins (RhoGDI-2, HSP27, SOD1, DJ1, UBE2N, PSME1, FTL, SH3BGRL, and eIF5A-1) with increased abundance in carcinomas. We also evidenced for the first time the deregulation of COX5, MTPN and DB1 proteins in TNBC that may represent novel tumor markers. Particularly, we confirmed the overexpression of the Rho-GDP dissociation inhibitor 2 (RhoGDI-2) in distinct breast cancer subtypes, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Remarkably, targeted disruption of RhoGDI-2 by RNA interference induced mitochondrial dysfunction, and facilitated caspase-3 and -9 activation in two breast cancer cell lines. Moreover, suppression of RhoGDI-2 resulted in a robust sensitization of breast cancer cells to cisplatin therapy. In conclusion, we identified novel proteins deregulated in TNBC, and confirmed the overexpression of RhoGDI-2. We propose that RhoGDI-2 inhibition may be exploited as a potential therapeutic strategy along cisplatin-based chemotherapy in breast cancer. BIOLOGICAL SIGNIFICANCE: There are no useful biomarkers neither targeted therapeutic modalities for triple-negative breast cancer, which highly contributes to the poor prognosis of this breast cancer subtype. In this work, we used two-dimensional electrophoresis and ESI-MS/MS spectrometry to identify novel deregulated proteins in breast cancer tissues. Particularly, our results showed that RhoGDI-2, a protein that has been associated to metastasis and poor survival in human cancers, is overexpressed in different subtypes of breast tumors, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Our data also provided novel insights about the role of RhoGDI-2 in apoptosis through intrinsic pathway inhibition. Importantly, they suggested that targeted modulation of RhoGDI-2 levels might be a useful strategy for breast cancer therapy.

RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy.

In tumor cells the effectiveness of anti-neoplastic agents that cause cell death by induction of DNA damage is influenced by DNA repair activity. RAD50 protein plays key roles in DNA double strand breaks repair (DSBs), which is crucial to safeguard genome integrity and sustain tumor suppression. However, its role as a potential therapeutic target has not been addressed in breast cancer. Our aim in the present study was to analyze the expression of RAD50 protein in breast tumors, and evaluate the effects of RAD50-targeted inhibition on the cytotoxicity exerted by cisplatin and anthracycline and taxane-based therapies in breast cancer cells. Immunohistochemistry assays on tissue microarrays indicate that the strong staining intensity of RAD50 was reduced in 14% of breast carcinomas in comparison with normal tissues. Remarkably, RAD50 silencing by RNA interference significantly enhanced the cytotoxicity of cisplatin. Combinations of cisplatin with doxorubicin and paclitaxel drugs induced synergistic effects in early cell death of RAD50-deficient MCF-7, SKBR3, and T47D breast cancer cells. Furthermore, we found an increase in the number of DSBs, and delayed phosphorylation of histone H2AX after cisplatin treatment in RAD50-silenced cells. These cellular events were associated to a dramatical increase in the frequency of chromosomal aberrations and a decrease of cell number in metaphase. In conclusion, our data showed that RAD50 abrogation impairs DNA damage response and sensitizes breast cancer cells to cisplatin-combined therapies. We propose that the development and use of inhibitors to manipulate RAD50 levels might represent a promising strategy to sensitize breast cancer cells to DNA damaging agents.

microRNA-18b is upregulated in breast cancer and modulates genes involved in cell migration.

microRNAs are small non-coding RNAs of ~22 nucleotides that function at post-transcriptional level as negative regulators of gene expression. Aberrant expression of microRNAs could promote uncontrolled proliferation, migration and invasion of human cancer cells. In this study, we analyzed the expression of microRNA-18b (miR-18b) in breast cancer cell lines and in a set of clinical specimens. Our results showed that miR-18b was upregulated in four out of five breast cancer cell lines and also in breast tumors. In order to identify potential gene targets, we carried out transcriptional profiling of MDA-MB-231 breast cancer cells that ectopically expressed miR-18b. Our results showed that 263 genes were significantly modulated in miR-18b-deficient cells (fold change >1.5; P≤0.05). We found that knock-down of miR-18b induced the upregulation of 55 olfactory receptor (OR) genes and nine genes (NLRP7, KLK3, OLFM3, POSTN, MAGED4B, KIR3DL3, CRX, SEMG1 and CEACAM5) with key roles in cell migration and metastasis. Consistently, we found that ectopic inhibition of miR-18b suppressed the migration of two breast cancer cell models in vitro. In conclusion, we have uncovered genes directly or indirectly modulated by miR-18b which may represent potential therapeutic targets in breast cancer. Our data also pointed out a role of miR-18b in migration of breast cancer cells.

Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy.

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural alternative agents, as resveratrol, may be an effective adjuvant in breast cancer therapy.