Decoding LINC00052 role in breast cancer by bioinformatic and experimental analyses.

LncRNA is a group of transcripts with a length exceeding 200 nucleotides that contribute to tumour development. Our research group found that LINC00052 expression was repressed during the formation of breast cancer (BC) multicellular spheroids. Intriguingly, LINC00052 precise role in BC remains uncertain. We explored LINC00052 expression in BC patients` RNA samples (TCGA) in silico, as well as in an in-house patient cohort, and inferred its cellular and molecular mechanisms. In vitro studies evaluated LINC00052 relevance in BC cells viability, cell cycle and DNA damage. Results. Bioinformatic RNAseq analysis of BC patients showed that LINC00052 is overexpressed in samples from all BC molecular subtypes. A similar LINC00052 expression pattern was observed in an in-house patient cohort. In addition, higher LINC00052 levels are related to better BC patient´s overall survival. Remarkably, MCF-7 and ZR-75-1 cells treated with estradiol showed increased LINC00052 expression compared to control, while these changes were not observed in MDA-MB-231 cells. In parallel, bioinformatic analyses indicated that LINC00052 influences DNA damage and cell cycle. MCF-7 cells with low LINC00052 levels exhibited increased cellular protection against DNA damage and diminished growth capacity. Furthermore, in cisplatin-resistant MCF-7 cells, LINC00052 expression was downregulated. Conclusion. This work shows that LINC00052 expression is associated with better BC patient survival. Remarkably, LINC00052 expression can be regulated by Estradiol. Additionally, assays suggest that LINC00052 could modulate MCF-7 cells growth and DNA damage repair. Overall, this study highlights the need for further research to unravel LINC00052 molecular mechanisms and potential clinical applications in BC.

Transcriptomic Changes in Cisplatin-Resistant MCF-7 Cells.

Breast cancer is a leading cause of cancer-related deaths among women. Cisplatin is used for treatment, but the development of resistance in cancer cells is a significant concern. This study aimed to investigate changes in the transcriptomes of cisplatin-resistant MCF7 cells. We conducted RNA sequencing of cisplatin-resistant MCF7 cells, followed by differential expression analysis and bioinformatic investigations to identify changes in gene expression and modified signal transduction pathways. We examined the size and quantity of extracellular vesicles. A total of 724 genes exhibited differential expression, predominantly consisting of protein-coding RNAs. Notably, two long non-coding RNAs (lncRNAs), NEAT1 and MALAT, were found to be dysregulated. Bioinformatic analysis unveiled dysregulation in processes related to DNA synthesis and repair, cell cycle regulation, immune response, and cellular communication. Additionally, modifications were observed in events associated with extracellular vesicles. Conditioned media from resistant cells conferred resistance to wild-type cells in vitro. Furthermore, there was an increase in the number of vesicles in cisplatin-resistant cells. Cisplatin-resistant MCF7 cells displayed differential RNA expression, including the dysregulation of NEAT1 and MALAT long non-coding RNAs. Key processes related to DNA and extracellular vesicles were found to be altered. The increased number of extracellular vesicles in resistant cells may contribute to acquired resistance in wild-type cells.

Coexpression of Smac/DIABLO and Estrogen Receptor in breast cancer.

BACKGROUND: Smac/DIABLO is a proapoptotic protein deregulated in breast cancer, with a controversial role as a tumor marker, possibly due to a lack of correlative mRNA and protein analyses. OBJECTIVE: To investigate the association of Smac/DIABLO gene and protein levels with clinical variables in breast cancer patients. METHODS: Smac/DIABLO mRNA expression was analyzed by qPCR in 57 frozen tissues, whereas protein levels were assessed by immunohistochemistry in 82 paraffin-embedded tissues. Survivin mRNA levels were also measured. In vitro assays were performed to investigate possible regulators of Smac/DIABLO. RESULTS: Higher levels of Smac/DIABLO mRNA and protein were found in estrogen receptor (ER)-positive samples (p= 0.0054 and p= 0.0043, respectively) in comparison to ER-negative tumors. A negligible positive association was found between Smac/DIABLO and survivin expression. In vitro assays showed that Smac/DIABLO is not regulated by ER and, conversely, it does not participate in ER expression modulation. CONCLUSIONS: mRNA and protein levels of Smac/DIABLO were increased in ER-positive breast tumors in comparison with ER-negative samples, although the mechanism of this regulation is still unknown. Public databases showed a possible clinical relevance for this association.

Basal-Type Breast Cancer Stem Cells Over-Express Chromosomal Passenger Complex Proteins.

(1) Aim: In the present paper we analyzed the transcriptome of CSCs (Cancer Stem Cells), in order to find defining molecular processes of breast cancer. (2) Methods: We performed RNA-Seq from CSCs isolated from the basal cell line MDA-MB-468. Enriched processes and networks were studied using the IPA (Ingenuity Pathway Analysis) tool. Validation was performed with qRT-PCR and the analysis of relevant genes was evaluated by overexpression, flow cytometry and in vivo zebrafish studies. Finally, the clinical relevance of these results was assessed using reported cohorts. (3) Results: We found that CSCs presented marked differences from the non-CSCs, including enrichment in transduction cascades related to stemness, cellular growth, proliferation and apoptosis. Interestingly, CSCs overexpressed a module of co-regulated Chromosomal Passenger Proteins including BIRC5 (survivin), INCENP and AURKB. Overexpression of BIRC5 increased the number of CSCs, as assessed by in vitro and in vivo zebrafish xenotransplant analyses. Analysis of previously published cohorts showed that this co-regulated module was not only overexpressed in basal breast tumors but also associated with relapse-free and overall survival in these patients. (4) Conclusions: These results underline the importance of Cancer Stem Cells in breast cancer progression and point toward the possible use of chromosomal passenger proteins as prognostic factors.

MT4-MMP Modulates the Expression of miRNAs in Breast Cancer Cells.

BACKGROUND: MT4-MMP is a member of the metalloproteinases family, although with a controversial role in the extracellular matrix remodelation. Overexpression of this metalloproteinase has been observed in breast cancer and it has been suggested that it can regulate tumor growth and cancer progression. The mechanisms by which MT4-MMP participates in breast cancer includes tumor blood vessels desestabilization, the activation of an angiogenic switch, and increase of EGFR signaling. However, all the mechanisms by which MT4-MMP participates in breast cancer are still unknowns. AIM OF THE STUDY: To study if MT4-MMP could modulate the expression of microRNAs (miRNAs) related to biological processes associated with tumor formation and progression. METHODS: MT4-MMP was ectopically overexpressed in MDA-MB-231 cells and the miRNAs expression profile modulated by the metalloproteinase was studied by using miRNAs microarrays. Microarray data were analyzed with different tools to find the molecular and cellular functions related to the differentially expressed miRNAs. The clinical relevance of some miRNAs was analyzed using a public database. RESULTS: MT4-MMP overexpression in breast cancer cells induced the modulation of 65 miRNAs, which were related to the alteration of pathways dependent of p53, TGF-ß, MAPK, ErbB, and Wnt, as well as processes such as cell cycle, adherens junctions, apoptosis, and focal adhesion. Several of the upregulated miRNAs were associated to a worse prognosis in breast cancer patients. CONCLUSIONS: In breast cancer cells, the overexpression of MT4-MMP modulates the expression of miRNAs involved in several biological processes associated with tumor formation and progression and with clinical relevance.

NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells.

BACKGROUND: NF-κB is a transcription factor involved in cancer stem cells maintenance of many tumors. Little is known about the specific stem-associated upstream regulators of this pathway in ovarian cancer. The Aim of the study was to analyze the role of the canonical and non-canonical NF-κB pathways in stem cells of ovarian cancer cell lines. METHODS: Stem cells were isolated using sorting cytometry. Western blot and RT-PCR were used to quantify protein and messenger RNA levels. Loss and gain of function assays were performed using siRNAs and dominant-negative proteins, respectively. NF-κB binding activity was measured with a reporter gene assay. The stem phenotype was estimated with clonogenic assays using soft agar, colony formation, ovospheres formation and in vivo tumorigenicity assays. RESULTS: The CD44+ subpopulation of SKOV3 ovarian cancer cell line presented higher mRNA levels of key stemness genes, an increased tumorigenic capacity and higher expression of the RelA, RelB and IKKα. When the canonical pathway was inhibited by means of a dominant-negative version of IkBα, the stem cell population was reduced, as shown by a reduced CD44+ subpopulation, a decrease in the expression of the stemness genes and a reduction of the stem phenotype. In addition, IKKα, the main upstream non-canonical kinase, was highly expressed in the CSC population. Accordingly, when IKKα was inhibited using shRNAs, the expression of the stemness genes was reduced. CONCLUSIONS: This report is the first to show the importance of several elements of both NF-κB pathway in maintaining the ovarian cancer stem cell population.

Protein Sam68 regulates the alternative splicing of survivin DEx3.

Messenger RNA alternative splicing (AS) regulates the expression of a variety of genes involved in both physiological and pathological processes. AS of the anti-apoptotic and proliferation-associated survivin (BIRC5) gene generates six isoforms, which regulate key aspects of cancer initiation and progression. One of the isoforms is survivin DEx3, in which the exclusion of exon 3 generates a unique carboxyl terminus with specific anti-apoptotic functions. This isoform is highly expressed in advanced stages of breast and cervical tumors. Therefore, understanding the mechanisms that regulate survivin DEx3 mRNA AS is clearly important. To this end, we designed a minigene (M), and in combination with a series of deletions and site-directed mutations, we determined that the first 22 bp of exon 3 contain cis-acting elements that enhance the exclusion of exon 3 to generate the survivin DEx3 mRNA isoform. Furthermore, using pulldown assays, we discovered that Sam68 is a possible trans-acting factor that binds to this region and regulates exon 3 splicing. This result was corroborated using a cell line in which the Sam68 binding site in the survivin gene was mutated with the CRISPR/Cas system. This work provides the first clues regarding the regulation of survivin DEx3 mRNA splicing.

miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation.

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT-PCR analysis. One of the most altered miRNAs was miR-10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR-10b in MCF-7 cells (miR-10b-OE cells) promoted higher self-renewal and expression of stemness and epithelial-mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR-10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self-renewal. Bioinformatics analyses identified several potential miR-10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self-renewal. The targeting of PTEN by miR-10b was confirmed using a luciferase reporter, qRT-PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR-10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self-renewal ability of CSCs and breast cancer cell lines overexpressing miR-10b. In conclusion, miR-10b regulates the self-renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.

Tissue inhibitor of metalloproteinases-4 (TIMP-4) regulates stemness in cervical cancer cells.

Tissue inhibitor of metalloproteinase-4 (TIMP-4) belongs to a family of extracellular matrix (ECM) metalloproteinases inhibitors that are overexpressed in several cancers. However, the role of TIMP-4 during carcinogenesis is poorly understood. To evaluate TIMP-4 functions in carcinogenesis, stably transfected cells overexpressing this tissue inhibitor were used. Xenograft tumor growth, stem cell enrichment, colony formation, and gene regulation were investigated. Microarrays and in silico analysis were carried out to elucidate TIMP-4 molecular mechanisms. In the present report, we show that in nude mice, cervical cancer cells that overexpress TIMP-4 formed tumors faster than control cell-derived tumors. Furthermore, in vivo limiting dilution assays showed that fewer TIMP-4 overexpressing cells are needed for tumor formation. In vitro analyses demonstrated that TIMP-4 overexpression or exposure to human recombinant TIMP-4 (hrTIMP4) caused an enrichment of the tumor progenitor cell (TPC) population. Accordingly, genome-wide expression and signaling pathway analyses showed that hrTIMP-4 modulated cell survival, cell proliferation, inflammation, and epithelial-mesenchymal transition (EMT) signaling networks. Notably, NFκB signaling pathway appeared to be globally activated upon hrTIMP-4 treatment. Overall, this report provides the first example that TIMP-4 regulates carcinogenesis through enriching the TPC population in cervical cancer cells. Understanding TIMP-4 effects on tumorigenesis may provide clues for future therapies design. © 2015 Wiley Periodicals, Inc.

Tissue Inhibitor of Metalloproteinase-4 Triggers Apoptosis in Cervical Cancer Cells.

Tissue inhibitor of metalloproteinase-4 (TIMP-4) is a member of extracellular matrix (ECM) metalloproteinases inhibitors that has pleiotropic functions. However, TIMP-4 roles in carcinogenesis are not well understood. Cell viability and flow cytometer assays were employed to evaluate cell death differences between H-Vector and H-TIMP-4 cell lines. Immunobloting and semi-quantitative RT-PCR were used to evaluate the expression of apoptosis regulators. We showed that TIMP-4 has apoptosis-sensitizing effects towards several death stimuli. Consistent with these findings, regulators of apoptosis from Inhibitors of Apoptosis Proteins (IAP), FLICE-like inhibitor proteins (FLIP) and Bcl-2 family members were modulated by TIMP-4. In addition, TIMP-4 knockdown resulted in cell survival increase after serum deprivation, as assessed by clonogenic cell analyses. This report shows that TIMP-4 regulates carcinogenesis through apoptosis activation in cervical cancer cells. Understanding TIMP-4 effects in tumorigenesis may provide clues for future therapies.

Tissue inhibitor of metalloproteases-4 (TIMP-4) modulates adipocyte differentiation in vitro.

Tissue inhibitors of metalloproteases (TIMPs) are multifunctional proteins that inhibit matrix metalloproteases (MMPs). The latest described member of the family, TIMP-4, is expressed mainly in adipose tissue, with detectable levels in the brain and heart. Besides its high expression in fat, the role of this inhibitor in adipose tissue is unknown. In order to study the role of TIMP-4 during adipogenesis in vitro, 3T3-L1 cells were stably transfected with a TIMP-4 specific shRNA or a control shRNA. Unexpectedly, upon TIMP-4 knockdown, 3T3-L1 cells differentiated faster into mature adipocytes. To get better insight of TIMP-4's role in adipogenesis, microarray expression analyses were performed. Network enrichment analyses uncovered 25 significant upstream signaling pathways, among which the NFκB cascade was found. Previous works have shown that NFκB is a key regulator of adipogenesis. In accordance, we found that TIMP-4 knockdown decreased NFκB activity during adipogenesis. The present work suggests that TIMP-4 might act as a negative regulator of adipogenesis through NFκB cascade modulation.

Ectopic expression of new alternative splice variant of Smac/DIABLO increases mammospheres formation.

Smac-α is a mitochondrial protein that, during apoptosis, is translocated to the cytoplasm, where it negatively regulates members of the inhibitor of apoptosis (IAP) family via the IAP-binding motif (IBM) contained within its amino-terminus. Here, we describe a new alternative splice variant from Smac gene, which we have named Smac-ε. Smac-ε lacks both an IBM and a mitochondrial-targeting signal (MTS) element. Smac-ε mRNA exhibits a tissue-specific expression pattern in healthy human tissues as well as in several cancer cell lines. The steady-state levels of endogenous Smac-ε protein is regulated by the proteasomal pathway. When ectopically expressed, this isoform presents a cytosolic localization and is unable to associate with or to regulate the expression of X-linked Inhibitor of apoptosis protein, the best-studied member of IAP family. Nevertheless, over-expression of Smac-ε increases mammosphere formation. Whole genome expression analyses from these mammospheres show activation of several pro-survival and growth pathways, including Estrogen-Receptor signaling. In conclusion, our results support the functionality of this new Smac isoform.

Survivin isoform Delta Ex3 regulates tumor spheroid formation.

Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.

Molecular modeling used to evaluate CYP2C9-dependent metabolism: homology modeling, molecular dynamics and docking simulations.

Cytochrome P450 (CYP) 2C9 is the principal isoform of the CYP2C subfamily in the human liver and is involved in the oxidation of several endogenous and xenobiotic compounds, including many therapeutic drugs. The metabolism of drugs by CYP2C9 can yield either safe or toxic products, which may be related to the recognition and binding modes of the substrates to this isoform. These interactions can be studied using in silico methods such as quantum chemistry, molecular dynamics and docking simulations, which can also be useful for predicting the structure of metabolites. In these types of studies, the ligand and the protein must be tridimensional models; thus, the protein can be built by homology modeling or retrieved from the Protein Data Bank. Therefore, the current review emphasizes the importance of using in silico methods to predict the metabolism of CYP2C9 because these computational tools have allowed the description of the principal characteristics of the active site of this isoform at the molecular level and the chemical properties of its ligands.

Depletion of the oncoprotein Bcl-3 induces centrosome amplification and aneuploidy in cancer cells.

Bcl-3 is an atypical member of the inhibitor of NF-kappa B family of proteins since it can function as a coactivator of transcription. Although this oncogene was described in leukemia, it is overexpressed in a number of solid tumors as well. The oncogenic potential of Bcl-3 has been associated with its capacity to increase proliferation by means of activating the cyclin D1 promoter and to its antiapoptotic role mediated by the inhibiton of p53 activity. In the course of dissecting these properties, we found that depleting Bcl-3 protein using shRNAs induce a decrease of proliferation and clonogenic survival associated with the induction of multinucleation and increased ploidy. These effects were associated with a DNA damage response, a delay in G2/M checkpoint and the induction of centrosome amplification.

Role of Smac/DIABLO in cancer progression.

Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is a proapoptogenic mitochondrial protein that is released to the cytosol in response to diverse apoptotic stimuli, including commonly used chemotherapeutic drugs. In the cytosol, Smac/DIABLO interacts and antagonizes inhibitors of apoptosis proteins (IAPs), thus allowing the activation of caspases and apoptosis. This activity has prompted the synthesis of peptidomimetics that could potentially be used in cancer therapy. For these reasons, several authors have analyzed the expression levels of Smac/DIABLO in samples of patients from different tumors. Although dissimilar results have been found, a tissue-specific role of this protein emerges from the data. The objective of this review is to present the current knowledge of the Smac/DIABLO role in cancer and its possible use as a marker or therapeutic target for drug design.