Changes in the molecular nodes of the Notch and NRF2 pathways in cervical cancer tissues from the precursor stages to invasive carcinoma.

Cancer is a multifactorial disease characterized by the loss of control in the expression of genes known as cancer driver genes. Cancer driver genes trigger uncontrolled cell replication, which leads to the development of malignant tumors. A cluster of signal transduction pathways that contain cancer driver genes involved in cellular processes, such as cell proliferation, differentiation, apoptosis and dysregulated organ growth, are associated with cancer initiation and progression. In the present study, three signal transduction pathways involved in cervical cancer (CC) development were analyzed: The Hippo pathway (FAT atypical cadherin, yes-associated protein 1, SMAD4 and TEA domain family member 2), the Notch pathway [cellular-MYC, cAMP response element-binding binding protein (CREBBP), E1A-associated cellular p300 transcriptional co-activator protein and F-Box and WD repeat domain containing 7] and the nuclear factor erythroid 2-related factor 2 (NRF2) pathway [NRF2, kelch-like ECH-associated protein 1 (KEAP1), AKT and PIK3-catalytic subunit α]. Tumor samples from patients diagnosed with various stages of CC, including cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3, in situ CC and invasive CC, were analyzed. The mRNA expression levels were analyzed using reverse transcription-quantitative PCR assays, whereas protein expression levels were assessed through immunohistochemical tissue microarrays. High mRNA expression levels of c-MYC and AKT and low expression levels of NRF2 and KEAP1 were associated with a decreased survival time of patients with CC. Additionally, increased expression levels of c-MYC were detected in the invasive CC stage. At the protein level, increased NRF2 expression levels were observed in all five stages of CC samples compared with those in the cancer-free control samples. AKT1 was found to be dysregulated in the CIN 1 and CIN 2 stages, PI3K in the in situ and invasive stages, and CREBBP in the CIN 3 and in situ stages. In summary, the present study demonstrated significant changes in proteins of the Notch and NRF2 pathways in CC. NRF2 was overexpressed in all cervical cancer stages (cervical intraepithelial neoplasia, in situ CC and invasive CC). The present study makes an important contribution to the possible biomarker proteins to be analyzed for the presence of premalignant and malignant lesions in the cervix.

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.

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.

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.

Changes in the transcriptome profile of breast cancer cells grown as spheroids.

BACKGROUND: Multicellular tumor spheroids mimic the functional organization of tumors in vivo, providing biological readouts that predict the behavior of cancer cells more accurately. The current study aimed to evaluate the transcriptome (mRNAs and long non-coding RNAs) of multicellular tumor spheroids from breast cancer cells. METHODS: MCF-7 cell spheroids were used; the transcriptome was analyzed using RNAseq and RNA microarrays; the secretion of macrophage migration inhibitor (MIF), a cytokine exported by the cholesterol efflux regulatory protein, was measured by ELISA. Linc00052 was inhibited using short-hairpin RNAs (shRNAs). RESULTS: We found several differentially regulated mRNAs and lncRNAs in MCF-7 cell spheroids. We also found significant enrichment of the Wnt/B-catenin death receptor and the cholesterol metabolic processes. Interestingly, we also found an increased concentration of MIF. Further, at 12 and 20 days of 3D culture we found 221 and 1146 dysregulated lncRNAs, respectively; including linc00052 (long intergenic non-protein coding RNA 52), which has been involved in breast cancer. Linc00052 knock-down experiments suggest that it could be a key regulator of cholesterol pathways in breast cancer. CONCLUSIONS: Our data shows that tumor spheroids can induce changes in the transcriptome of the cultured cells, including both mRNAs and ncRNA. One of the major changes included the deregulation of cholesterol pathways, of which linc00052 is apparently a key regulator.

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.

Bcl-3 regulates UVB-induced apoptosis.

B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.

Tissue inhibitor of metalloproteinases-2 growth-stimulatory activity is mediated by nuclear factor-kappa B in A549 lung epithelial cells.

Tissue inhibitors of metalloproteinases (TIMPs) are pleiotropic factors that function as key regulators of extracellular matrix remodeling. They exhibit multifunctional roles including cell growth-stimulating activities and protection from apoptosis. In the present study, we showed that human recombinant TIMP-2 (hrTIMP-2) promotes growth of A549 lung cells. This effect was accompanied by increase in nuclear factor-kappa B (NF-kappaB) activity 24h after exposure as determined by electrophoretic mobility shift assay (EMSA) analysis. This effect was correlated with downregulation of IkappaBalpha and beta proteins and later increases in Bcl-3, IkappaB, and cyclin D1 proteins. Blocking induction of NF-kappaB activity using a dominant-negative mutated version of IkappaBalpha abrogated NF-kappaB activation and cell proliferation.