The Use of Zebrafish Xenotransplant Assays to Analyze the Role of lncRNAs in Breast Cancer.

Breast cancer represents a great challenge since it is the first cause of death by cancer in women worldwide. LncRNAs are a newly described class of non-coding RNAs that participate in cancer progression. Their use as cancer markers and possible therapeutic targets has recently gained strength. Animal xenotransplants allows for in vivo monitoring of disease development, molecular elucidation of pathogenesis and the design of new therapeutic strategies. Nevertheless, the cost and complexities of mice husbandry makes medium to high throughput assays difficult. Zebrafishes (Danio rerio) represent a novel model for these assays, given the ease with which xenotransplantation trials can be performed and the economic and experimental advantages it offers. In this review we propose the use of xenotransplants in zebrafish to study the role of breast cancer lncRNAs using low to medium high throughput assays.

Integrative analysis of transcriptional profile reveals LINC00052 as a suppressor of breast cancer cell migration.

BACKGROUND: Long-non-coding RNAs, a class of transcripts with lengths > 200 nt, play key roles in tumour progression. Previous reports revealed that LINC00052 (long intergenic non-coding RNA 00052) was strongly downregulated during breast cancer multicellular spheroids formation and suggested a role in cell migration and oxidative metabolism. OBJECTIVE: To examine the function of LINC00052 in MCF-7 breast cancer cells. METHODS: Loss-of-function studies were performed to evaluate LINC00052 role on MCF-7 breast cancer cells. Microarray expression assays were performed to determine genes and cellular functions modified after LINC00052 knockdown. Next, the impact of LINC00052 depletion on MCF-7 cell respiration and migration was evaluated. RESULTS: 1,081 genes were differentially expressed upon LINC00052 inhibition. Gene set enrichment analysis, Gene Ontology and Key Pathway Advisor analysis showed that signalling networks related to cell migration and oxidative phosphorylation were enriched. However, whereas LINC00052 knockdown in MCF-7 cells revealed marginal difference in oxygen consumption rates when compared with control cells, LINC00052 inhibition enhanced cell migration in vitro and in vivo, as observed using a Zebrafish embryo xenotransplant model. CONCLUSION: Our data show that LINC00052 modulates MCF-7 cell migration. Genome-wide microarray experiments suggest that cancer cell migration is affected by LINC00052 through cytoskeleton modulation and Notch/ß-catenin/NF-κB signalling pathways.

Adipose-derived mesenchymal stem cells promote the malignant phenotype of cervical cancer.

Epidemiological studies indicate that obesity negatively affects the progression and treatment of cervical-uterine cancer. Recent evidence shows that a subpopulation of adipose-derived stem cells can alter cancer properties. In the present project, we described for the first time the impact of adipose-derived stem cells over the malignant behavior of cervical cancer cells. The transcriptome of cancer cells cultured in the presence of stem cells was analyzed using RNA-seq. Changes in gene expression were validated using digital-PCR. Bioinformatics tools were used to identify the main transduction pathways disrupted in cancer cells due to the presence of stem cells. In vitro and in vivo assays were conducted to validate cellular and molecular processes altered in cervical cancer cells owing to stem cells. Our results show that the expression of 95 RNAs was altered in cancer cells as a result of adipose-derived stem cells. Experimental assays indicate that stem cells provoke an increment in migration, invasion, angiogenesis, and tumorigenesis of cancer cells; however, no alterations were found in proliferation. Bioinformatics and experimental analyses demonstrated that the NF-kappa B signaling pathway is enriched in cancer cells due to the influence of adipose-derived stem cells. Interestingly, the tumor cells shift their epithelial to a mesenchymal morphology, which was reflected by the increased expression of specific mesenchymal markers. In addition, stem cells also promote a stemness phenotype in the cervical cancer cells. In conclusion, our results suggest that adipose-derived stem cells induce cervical cancer cells to acquire malignant features where NF-kappa B plays a key role.

lncMat2B regulated by severe hypoxia induces cisplatin resistance by increasing DNA damage repair and tumor-initiating population in breast cancer cells.

Multicellular tumor spheroids (MCTSs) constitute a three-dimensional culture system that recapitulates the in vivo tumor microenvironment. Tumor cells cultured as MCTSs present antineoplastic resistance due to the effect of microenvironmental signals acting upon them. In this work, we evaluated the biological function of a new microenvironment-regulated long non-coding RNA, lncMat2B, in breast cancer. In MCTSs, the expression of lncMat2B presented an increase and a zonal heterogeneity, as it was expressed principally in quiescent cells of hypoxic regions of the MCTSs. As expected, functional assays supported the role of severe hypoxia in the regulation of lncMat2B. Moreover, gain- and loss-of-function assays using a transcriptional silencing CRISPR/Cas9 system and gBlock revealed that lncMAT2B regulates the tumor-initiating phenotype. Interestingly, lncMat2B is overexpressed in a cisplatin-resistant MCF-7 cell line, and its ectopic expression in wild type MCF-7 cells increased survival to cisplatin exposure by reducing DNA damage and reactive oxygen species accumulation. lncMAT2B is a possible link between severe hypoxia, tumor-initiating phenotype and drug resistance in breast cancer cells.

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.

Microenvironment-regulated lncRNA-HAL is able to promote stemness in breast cancer cells.

Multicellular Tumor Spheroids culture (MCTS) is an in vitro model mimicking the characteristics of the tumor microenvironment, such as hypoxia and acidosis, resulting in the presence of both proliferating and quiescent cell populations. lncRNA's is a novel group of regulatory molecules that participates in the acquisition of tumorigenic phenotypes. In the present work we evaluated the oncogenic association of an uncharacterized lncRNA (lncRNA-HAL) in the tumorigenic phenotype induced by the MCTS microenvironment. We measured lncRNA-HAL expression level in MCF-7-MCTS populations and under different hypoxic conditions by RT-qPCR. Afterwards, we silenced lncRNA-HAL expression by shRNAs and evaluated its effect in MCF-7 transcriptome (by RNAseq) and validated the modified cellular processes by proliferation, migration, and stem cells assays. Finally, we analyzed which proteins interacts with lncRNA-HAL by ChIRP assay, to propose a possible molecular mechanism for this lncRNA. We found that lncRNA-HAL is overexpressed in the internal quiescent populations (p27 positive populations) of MCF-7-MCTS, mainly in the quiescent stem cell population, being hypoxia one of the microenvironmental cues responsible of its overexpression. Transcriptome analysis of lncRNA-HAL knockdown MCF7 cells revealed that lncRNA-HAL effect is associated with proliferation, migration and cell survival mechanisms; moreover, lncRNA-HAL silencing increased cell proliferation and impaired cancer stem cell proportion and function, resulting in decreased tumor grafting in vivo. In addition, we found that this lncRNA was overexpressed in triple-negative breast cancer patients. Analysis by ChIRP assay showed that this nuclear lncRNA binds to histones and hnRNPs suggesting a participation at the chromatin level and transcriptional regulation. The results obtained in the present work suggest that the function of lncRNA-HAL is associated with quiescent stem cell populations, which in turn is relevant due to its implications in cancer cell survival and resistance against treatment in vivo. Altogether, our data highlights a new lncRNA whose expression is regulated by the tumor microenvironment and associated to stemness in breast cancer.

IKKε regulates the breast cancer stem cell phenotype.

The Inhibitor of Nuclear Factor Kappa B Kinase Subunit Epsilon (IKKε) is an oncogenic protein that is up-regulated in various types of human cancers, including breast tumors. This kinase regulates diverse processes associated with malignant progression including proliferation, invasion, and metastasis. To delve into the molecular mechanisms regulated by this kinase we performed RNA-seq and network analysis of breast cancer cells overexpressing IKKε. We found that the TNF/NF-κB cascade was clearly enriched, and in accordance, NF-κB pathway inhibition in these cells resulted in a decreased expression of IKKε target genes. Interestingly, we also found an enrichment of a mammary stemness functional pathway. Upregulation of IKKε led to an increase of a stem CD44+/CD24-/low population accompanied by a high expression of stem markers such as ALDH1A3, NANOG, and KLF4 and with an increased clonogenic ability and mammosphere formation capacity. These results were corroborated with in vivo dilution assays in zebrafish embryos which showed a significant increase in the number of Cancer Stem Cells (CSCs). Finally, we found that Triple-Negative breast tumors, which are enriched in CSCs, display higher levels of IKKε than other breast tumors, supporting the association of this kinase with the stem phenotype. In conclusion, our results highlight the role of IKKε kinase in the regulation of the stem cell phenotype in breast cancer cells, as assessed by expression, functional and in vivo assays. These results add to the potential use of this kinase as a therapeutic target in this neoplasia.

CTCF knockout reveals an essential role for this protein during the zebrafish development.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

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.

Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience.

Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development.

BACKGROUND: Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. RESULTS: There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord. CONCLUSIONS: This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development.