Cell migration and proliferation are regulated by miR-26a in colorectal cancer via the PTEN-AKT axis.

BACKGROUND: Invasion and metastasis are determinant events in the prognosis of Colorectal cancer (CRC), a common neoplasm worldwide. An important factor for metastasis is the acquired capacity of the cell to proliferate and invade adjacent tissues. In this paper, we explored the role of micro-RNA-26a in the regulation of proliferation and migration in CRC-derived cells through the negative regulation of PTEN, a key negative regulator of the AKT pathway. METHODS: Expression levels of PTEN and mir-26a were surveyed in normal and CRC-derived cell lines; paraffin embedded human tissues, TCGA CRC expression data and a Balb/c mice orthotopic induced CRC model. CRC was induced by an initial intraperitoneal dose of the colonic carcinogen Azoxymethane followed by inflammatory promoter Dextran Sulfate Sodium Salt. Luciferase assays provide information about miR-26a-PTEN 3'UTR interaction. Proliferation and migration by real time cell analysis and wound-healing functional analyses were performed to assess the participation of mir-26a on important hallmarks of CRC and its regulation on the PTEN gene. RESULTS: We observed a negative correlation between PTEN and mir-26a expression in cell lines, human tissues, TCGA data, and tissues derived from the CRC mouse model. Moreover, we showed that negative regulation of PTEN exerted by miR-26a affected AKT phosphorylation levels directly. Functional assays showed that mir-26a directly down-regulates PTEN, and that mir-26a over-expressing cells had higher proliferation and migration rates. CONCLUSIONS: All this data proposes an important role of mir-26a as an oncomir in the progression and invasion of CRC. Our data suggested that mir-26a could be used as a biomarker of tumor development in CRC patients, however more studies must be conducted to establish its clinical role.

MiR-26a downregulates retinoblastoma in colorectal cancer.

MicroRNAs are non-coding short RNAs that target the 3' untranslated region of messenger RNAs (mRNAs) and lead to their degradation or to translational repression. Several microRNAs have been designated as oncomirs, owing to their regulating tumor suppressor genes. Interestingly, a few of them have been found to target multiple genes whose simultaneous suppression contributes to the development of a tumoral phenotype. Here, we have showed that miR-26a is overexpressed in colorectal cancer data obtained from TCGA Research Network and in human colon cancer pathological specimens; moreover, an orthotopic in vivo model of colon cancer showed overexpression of miR-26a, while Rb1 expression inversely correlated to miR-26a in TCGA Research Network data, pathological samples, and the in vivo model. Then, by means of luciferase assay, we demonstrated that miR-26a targets the 3' untranslated region of Rb1 mRNA directly. This is, to our knowledge, the first report of miR-26a targeting Rb1 in colon cancer. The results of this study suggested that miR-26a could serve as a progression biomarker in colorectal cancer. Further validation studies are still needed to confirm our findings.

Laherradurin Inhibits Tumor Growth in an Azoxymethane/Dextran Sulfate Sodium Colorectal Cancer Model In Vivo.

Colorectal cancer (CRC) is the third most common neoplasia in the world. Its mortality rate is high due to the lack of specific and effective treatments, metastasis, and resistance to chemotherapy, among other factors. The natural products in cancer are a primary source of bioactive molecules. In this research, we evaluated the antitumor activity of an acetogenin (ACG), laherradurin (LH), isolated from the Mexican medicinal plant Annona macroprophyllata Donn.Sm. in a CRC murine model. The CRC was induced by azoxymethane-dextran sulfate sodium (AOM/DSS) in Balb/c mice and treated for 21 days with LH or cisplatin. This study shows for the first time the antitumor activity of LH in an AOM/DSS CRC model. The acetogenin diminished the number and size of tumors compared with cisplatin; the histologic studies revealed a recovery of the colon tissue, and the blood toxicity data pointed to less damage in animals treated with LH. The TUNEL assay indicated cell death by apoptosis, and the in vitro studies exhibited that LH inhibited cell migration in HCT116 cells. Our study provides strong evidence of a possible anticancer agent for CRC.

HypoxaMIRs: Key Regulators of Hallmarks of Colorectal Cancer.

Hypoxia in cancer is a thoroughly studied phenomenon, and the logical cause of the reduction in oxygen tension is tumor growth itself. While sustained hypoxia leads to death by necrosis in cells, there is an exquisitely regulated mechanism that rescues hypoxic cells from their fatal fate. The accumulation in the cytoplasm of the transcription factor HIF-1α, which, under normoxic conditions, is marked for degradation by a group of oxygen-sensing proteins known as prolyl hydroxylases (PHDs) in association with the von Hippel-Lindau anti-oncogene (VHL) is critical for the cell, as it regulates different mechanisms through the genes it induces. A group of microRNAs whose expression is regulated by HIF, collectively called hypoxaMIRs, have been recognized. In this review, we deal with the hypoxaMIRs that have been shown to be expressed in colorectal cancer. Subsequently, using data mining, we analyze a panel of hypoxaMIRs expressed in both normal and tumor tissues obtained from TCGA. Finally, we assess the impact of these hypoxaMIRs on cancer hallmarks through their target genes.

Aberrant Metabolism as Inductor of Epigenetic Changes in Breast Cancer: Therapeutic Opportunities.

Aberrant metabolism is arising interest in the scientific community not only because of the role it plays in the development and establishment of the tumor mass but also the possibility of drug poisoning of key enzymes overexpressed in tumor cells. Moreover, tumor metabolism provides key molecules to maintain the epigenetic changes that are also an undisputed characteristic of each tumor type. This metabolic change includes the Warburg effect and alterations in key pathways involved in glutaminolysis, pentose phosphate, and unsaturated fatty acid biosynthesis. Modifications in all these pathways have consequences that impact genetics and epigenetics processes such as DNA methylation patterns, histone post-translational modifications, triggering oncogenes activation, and loss in tumor suppressor gene expression to lead the tumor establishment. In this review, we describe the metabolic rearrangement and its association with epigenetic regulation in breast cancer, as well as its implication in biological processes involved in cancer progression. A better understanding of these processes could help to find new targets for the diagnosis, prognosis, and treatment of this human health problem.

Inhibition of Wnt-ß-Catenin Signaling by ICRT14 Drug Depends of Post-Transcriptional Regulation by HOTAIR in Human Cervical Cancer HeLa Cells.

BACKGROUND: In Cervical cancer (CC), in addition to HPV infection, the most relevant alteration during CC initiation and progression is the aberrant activation of Wnt/ß-catenin pathway. Several inhibitory drugs of this pathway are undergoing preclinical and clinical studies. Long non-coding RNAs (lncRNAs) are associated with resistance to treatments. In this regard, understanding the efficiency of drugs that block the Wnt/ß-catenin pathway in CC is of relevance to eventually propose successful target therapies in patients with this disease. METHODS: We analyzed the levels of expression of 249 components of the Wnt/ß-catenin pathway in a group of 109 CC patients. Three drugs that blocking specific elements of Wnt/ß-catenin pathway (C59, NSC668036 and ICRT14) by TOP FLASH assays and qRT-PCR were tested in vitro in CC cells. RESULTS: 137 genes of the Wnt/ß-catenin pathway were up-regulated and 112 down-regulated in CC patient's samples, demonstrating that this pathway is dysregulated. C59 was an efficient drug to inhibit Wnt/ß-catenin pathway in CC cells. NSC668036, was not able to inhibit the transcriptional activity of the Wnt/ß-catenin pathway. Strikingly, ICRT14 was neither able to inhibit this pathway in HeLa cells, due to HOTAIR interaction with ß-catenin, maintaining the Wnt/ß-catenin pathway activated. CONCLUSIONS: These results demonstrate a mechanism by which HOTAIR evades the effect of ICRT14, a Wnt/ß-catenin pathway inhibitory drug, in HeLa cell line. The emergence of these mechanisms reveals new scenarios in the design of target therapies used in cancer.

Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide in both sexes. Current therapies include surgery, chemotherapy, and targeted therapy; however, prolonged exposure to chemical agents induces toxicity in patients and drug resistance. So, we implemented a therapeutic strategy based on the combination of doxorubicin, metformin, and sodium oxamate called triple therapy (Tt). We found that Tt significantly reduced proliferation by inhibiting the mTOR/AKT pathway and promoted apoptosis and autophagy in CRC derived cells compared with doxorubicin. Several autophagy genes were assessed by western blot; ULK1, ATG4, and LC3 II were overexpressed by Tt. Interestingly, ULK1 was the only one autophagy-related protein gradually overexpressed during Tt administration. Thus, we assumed that there was a post-transcriptional mechanism mediating by microRNAs that regulate UKL1 expression during autophagy activation. Through bioinformatics approaches, we ascertained that ULK1 could be targeted by mir-26a, which is overexpressed in advanced stages of CRC. In vitro experiments revealed that overexpression of mir-26a decreased significantly ULK1, mRNA, and protein expression. Contrariwise, the Tt recovered ULK1 expression by mir-26a decrease. Due to triple therapy repressed mir-26a expression, we hypothesized this drug combination could be involved in mir-26a transcription regulation. Consequently, we analyzed the mir-26a promoter sequence and found two HIF-1α transcription factor recognition sites. We developed two different HIF-1α stabilization models. Both showed mir-26a overexpression and ULK1 reduction in hypoxic conditions. Immunoprecipitation experiments were performed and HIF-1α enrichment was observed in mir-26a promoter. Surprisingly, Tt diminished HIF-1α detection and restored ULK1 mRNA expression. These results reveal an important regulation mechanism controlled by the signaling that activates HIF-1α and that in turn regulates mir-26a transcription.

Negative Regulation of ULK1 by microRNA-106a in Autophagy Induced by a Triple Drug Combination in Colorectal Cancer Cells In Vitro.

Colorectal cancer (CRC) is among the top three most deadly cancers worldwide. The survival rate for this disease has not been reduced despite the treatments, the reason why the search for therapeutic alternatives continues to be a priority issue in oncology. In this research work, we tested our successful pharmacological combination of three drugs, metformin, doxorubicin, and sodium oxamate (triple therapy, or TT), as an autophagy inducer. Firstly, we employed western blot (WB) assays, where we observed that after 8 h of stimulation with TT, the proteins Unc-51 like autophagy activating kinase 1(ULK1), becline-1, autophagy related 1 protein (Atg4), and LC3 increased in the CRC cell lines HCT116 and SW480 in contrast to monotherapy with doxorubicin. The overexpression of these proteins indicated the beginning of autophagy flow through the activation of ULK1 and the hyperlipidation of LC3 at the beginning of this process. Moreover, we confirm that ULK1 is a bona fide target of hsa-miR-106a-5p (referred to from here on as miR-106a) in HCT116. We also observed through the GFP-LC3 fusion protein that in the presence of miR-106a, the accumulation of autophagy vesicles in cells stimulated with TT is inhibited. These results show that the TT triggered autophagy to modulate miR-106a/ULK1 expression, probably affecting different cellular pathways involved in cellular proliferation, survivance, metabolic maintenance, and cell death. Therefore, considering the importance of autophagy in cancer biology, the study of miRNAs that regulate autophagy in cancer will allow a better understanding of malignant tumors and lead to the development of new disease markers and therapeutic strategies.

Identification of miRNA Master Regulators in Breast Cancer.

Breast cancer is the neoplasm with the highest number of deaths in women. Although the molecular mechanisms associated with the development of this tumor have been widely described, metastatic disease has a high mortality rate. In recent years, several studies show that microRNAs or miRNAs regulate complex processes in different biological systems including cancer. In the present work, we describe a group of 61 miRNAs consistently over-expressed in breast cancer (BC) samples that regulate the breast cancer transcriptome. By means of data mining from TCGA, miRNA and mRNA sequencing data corresponding to 1091 BC patients and 110 normal adjacent tissues were downloaded and a miRNA-mRNA network was inferred. Calculations of their oncogenic activity demonstrated that they were involved in the regulation of classical cancer pathways such as cell cycle, PI3K-AKT, DNA repair, and k-Ras signaling. Using univariate and multivariate analysis, we found that five of these miRNAs could be used as biomarkers for the prognosis of overall survival. Furthermore, we confirmed the over-expression of two of them in 56 locally advanced BC samples obtained from the histopathological archive of the National Cancer Institute of Mexico, showing concordance with our previous bioinformatic analysis.

Negative Regulation of Serine Threonine Kinase 11 (STK11) through miR-100 in Head and Neck Cancer.

BACKGROUND: Serine Threonine Kinase 11 (STK11), also known as LKB1, is a tumor suppressor gene that regulates several biological processes such as apoptosis, energetic metabolism, proliferation, invasion, and migration. During malignant progression, different types of cancer inhibit STK11 function by mutation or epigenetic inactivation. In Head and Neck Cancer, it is unclear what mechanism is involved in decreasing STK11 levels. Thus, the present work aims to determine whether STK11 expression might be regulated through epigenetic or post-translational mechanisms. METHODS: Expression levels and methylation status for STK11 were analyzed in 59 cases of head and neck cancer and 10 healthy tissue counterparts. Afterward, we sought to identify candidate miRNAs exerting post-transcriptional regulation of STK11. Then, we assessed a luciferase gene reporter assay to know if miRNAs directly target STK11 mRNA. The expression levels of the clinical significance of mir-100-3p, -5p, and STK11 in 495 HNC specimens obtained from the TCGA database were further analyzed. Finally, the Kaplan-Meier method was used to estimate the prognostic significance of the miRNAs for Overall Survival, and survival curves were compared through the log-rank test. RESULTS: STK11 was under-expressed, and its promoter region was demethylated or partially methylated. miR-17-5p, miR-106a-5p, miR-100-3p, and miR-100-5p could be negative regulators of STK11. Our experimental data suggested evidence that miR-100-3p and -5p were over-expressed in analyzed tumor patient samples. Luciferase gene reporter assay experiments showed that miR-100-3p targets and down-regulates STK11 mRNA directly. With respect to overall survival, STK11 expression level was significant for predicting clinical outcomes. CONCLUSION: This is, to our knowledge, the first report of miR-100-3p targeting STK11 in HNC. Together, these findings may support the importance of regulation of STK11 through post-transcriptional regulation in HNC and the possible contribution to the carcinogenesis process in this neoplasia.

Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer.

Colorectal cancer (CRC) is an important health issue worldwide, accounting for the third place of cancer incidence. Chronic inflammation, as seen in Crohn's disease and ulcerative colitis, is the most important risk factor for developing CRC, as it favours neoplastic transformation by enhancing epithelial cell turnover in the colonic mucosa. Treatments for CRC need to be improved; currently they are not specific and have several secondary effects in patients. The main objective of this work was to evaluate a new therapeutic strategy against a colitis-related colorectal cancer in vivo and in vitro by targeting mTOR-signaling and lactate dehydrogenase A. Together, these mechanisms directly affect tumor energetics. In this study we evaluated a better and more efficient triple therapy against a chronic inflammation-associated CRC in vivo and in vitro. After the development of tumors, mice were treated intraperitoneally during a forty-day period with single drugs or different combinations of Metformin, Sodium Oxamate and Doxorubicin. Targeted inhibition of the mTOR pathway, lactate dehydrogenase A and the concurrent use of Doxorubicin (called in this work as triple therapy), leaded to a notable reduction in the number and size of tumors in mice, and, a significant pro-inflammatory cytokines reduction Besides, we showed that treated cells were induced to early autophagy, and apoptosis cell death. Our results represent a novel and robust therapeutic strategy for overcoming CRC by means of targeting central molecular pathways in cancer by the combination of Metformin, Oxamate, and Doxorubicin leading to a rapid tumor growth inhibition and a dramatic colorectal crypt restoration. Besides, drug combination resulted in a notable reduction of anti-inflammatory cytokines.