Somatic Copy Number Alterations in Colorectal Cancer Lead to a Differentially Expressed ceRNA Network (ceRNet).

Colorectal cancer (CRC) represents the second deadliest malignancy worldwide. Around 75% of CRC patients exhibit high levels of chromosome instability that result in the accumulation of somatic copy number alterations. These alterations are associated with the amplification of oncogenes and deletion of tumor-ppressor genes and contribute to the tumoral phenotype in different malignancies. Even though this relationship is well known, much remains to be investigated regarding the effect of said alterations in long non-coding RNAs (lncRNAs) and, in turn, the impact these alterations have on the tumor phenotype. The present study aimed to evaluate the role of differentially expressed lncRNAs coded in regions with copy number alterations in colorectal cancer patient samples. We downloaded RNA-seq files of the Colorectal Adenocarcinoma Project from the The Cancer Genome Atlas (TCGA) repository (285 sequenced tumor tissues and 41 non-tumor tissues), evaluated differential expression, and mapped them over genome sequencing data with regions presenting copy number alterations. We obtained 78 differentially expressed (LFC > 1|< -1, padj < 0.05) lncRNAs, 410 miRNAs, and 5028 mRNAs and constructed a competing endogenous RNA (ceRNA) network, predicting significant lncRNA-miRNA-mRNA interactions. Said network consisted of 30 lncRNAs, 19 miRNAs, and 77 mRNAs. To understand the role that our ceRNA network played, we performed KEGG and GO analysis and found several oncogenic and anti-oncogenic processes enriched by the molecular players in our network. Finally, to evaluate the clinical relevance of the lncRNA expression, we performed survival analysis and found that C5orf64, HOTAIR, and RRN3P3 correlated with overall patient survival. Our results showed that lncRNAs coded in regions affected by SCNAs form a complex gene regulatory network in CCR.

Pathogenic variant profile in DNA damage response genes correlates with metastatic breast cancer progression-free survival in a Mexican-mestizo population.

Introduction: Metastatic breast cancer causes the most breast cancer-related deaths around the world, especially in countries where breast cancer is detected late into its development. Genetic testing for cancer susceptibility started with the BRCA 1 and 2 genes. Still, recent research has shown that variations in other members of the DNA damage response (DDR) are also associated with elevated cancer risk, opening new opportunities for enhanced genetic testing strategies. Methods: We sequenced BRCA1/2 and twelve other DDR genes from a Mexican-mestizo population of 40 metastatic breast cancer patients through semiconductor sequencing. Results: Overall, we found 22 variants -9 of them reported for the first time- and a strikingly high proportion of variations in ARID1A. The presence of at least one variant in the ARID1A, BRCA1, BRCA2, or FANCA genes was associated with worse progression-free survival and overall survival in our patient cohort. Discussion: Our results reflected the unique characteristics of the Mexican-mestizo population as the proportion of variants we found differed from that of other global populations. Based on these findings, we suggest routine screening for variants in ARID1A along with BRCA1/2 in breast cancer patients from the Mexican-mestizo population.

Non-Coding RNAs Associated With Radioresistance in Triple-Negative Breast Cancer.

The resistance that Triple-Negative Breast Cancer (TNBC), the most aggressive breast cancer subtype, develops against radiotherapy is a complex phenomenon involving several regulators of cell metabolism and gene expression; understanding it is the only way to overcome it. We focused this review on the contribution of the two leading classes of regulatory non-coding RNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), against ionizing radiation-based therapies. We found that these regulatory RNAs are mainly associated with DNA damage response, cell death, and cell cycle regulation, although they regulate other processes like cell signaling and metabolism. Several regulatory RNAs regulate multiple pathways simultaneously, such as miR-139-5p, the miR-15 family, and the lncRNA HOTAIR. On the other hand, proteins such as CHK1 and WEE1 are targeted by several regulatory RNAs simultaneously. Interestingly, the study of miRNA/lncRNA/mRNA regulation axes increases, opening new avenues for understanding radioresistance. Many of the miRNAs and lncRNAs that we reviewed here can be used as molecular markers or targeted by upcoming therapeutic options, undoubtedly contributing to a better prognosis for TNBC patients.

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.

Interplay Between Autophagy and Wnt/ß-Catenin Signaling in Cancer: Therapeutic Potential Through Drug Repositioning.

The widespread dysregulation that characterizes cancer cells has been dissected and many regulation pathways common to multiple cancer types have been described in depth. Wnt/ß-catenin signaling and autophagy are among these principal pathways, which contribute to tumor growth and resistance to anticancer therapies. Currently, several therapeutic strategies that target either Wnt/ß-catenin signaling or autophagy are in various stages of development. Targeted therapies that block specific elements that participate in both pathways; are subject to in vitro studies as well as pre-clinical and early clinical trials. Strikingly, drugs designed for other diseases also impact these pathways, which is relevant since they are already FDA-approved and sometimes even routinely used in the clinic. The main focus of this mini-review is to highlight the importance of drug repositioning to inhibit the Wnt/ß-catenin and autophagy pathways, with an emphasis on the interplay between them. The data we found strongly suggested that this field is worth further examination.

A Multi-Center Study of BRCA1 and BRCA2 Germline Mutations in Mexican-Mestizo Breast Cancer Families Reveals Mutations Unreported in Latin American Population.

The presence of germline and somatic deleterious mutations in the BRCA1 and BRCA2 genes has important clinical consequences for breast cancer (BC) patients. Analysis of the mutational status in BRCA genes is not yet common in public Latin American institutions; thus, our objective was to implement high-performance technology with highly reliable results with the possibility of analyzing several patients simultaneously, therefore reducing cost and work time. A prospective cohort of 252 unrelated sporadic breast cancer patients from the Mexican-mestizo population were analyzed using next generation sequencing (NGS) based on ion semiconductor sequencing. We found 28 pathogenic mutations (25 in BRCA1 and 13 in BRCA2), 11 of which had not been reported previously in Hispanic or Latin American populations. A total of 38 patients were positive for a pathogenic mutation representing 15% of our Mexican women cohort with breast cancer; 25 for BRCA1; and 13 for BRCA2. Our results revealed that there are mutations not analyzed by mutations panels, and our findings support the suitability of massive sequencing approaches in the public institutions of developing countries. Hence, BRCA screening should be offered to patients with breast cancer regardless of their family history of cancer in order to identify unaffected family carriers.

Crosstalk Between Long Non-coding RNAs, Micro-RNAs and mRNAs: Deciphering Molecular Mechanisms of Master Regulators in Cancer.

Cancer is a complex disease, and its study requires deep understanding of several biological processes and their regulation. It is an accepted fact that non-coding RNAs are vital components of the regulation and cross-talk among cancer-related signaling pathways that favor tumor aggressiveness and metastasis, such as neovascularization, angiogenesis, and vasculogenic mimicry. Both long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs) have been described as master regulators of cancer on their own; yet there is accumulating evidence that, besides regulating mRNA expression through independent mechanisms, these classes of non-coding RNAs interact with each other directly, fine-tuning the effects of their regulation. While still relatively scant, research on the lncRNA-miRNA-mRNA axis regulation is growing at a fast rate, it is only in the last 5 years, that lncRNA-miRNA interactions have been identified in tumor-related vascular processes. In this review, we summarize the current progress of research on the cross-talk between lncRNAs and miRNAs in the regulation of neovascularization, angiogenesis and vasculogenic mimicry.

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.

BRCA mutations: is everything said?

BACKGROUND: Mutations in the BRCA1 and BRCA2 genes constitute a risk factor for breast cancer development. BRCA mutation research has been an active field since the discovery of the genes, and new mutations in both genes are constantly described and classified according to several systems. AIM: We intend to provide an overview of the current state of BRCA1 and BRCA2 mutation description and classification. We wanted to know whether there was a trend towards a more frequently described mutation type and what the proportion of pathogenic mutations was. RESULTS: We found that, although new mutations are described each year as reflected in current database records, very few of them are reported in papers. Classification systems are highly heterogeneous and a consensus among them is still under development. Regarding their function, a large number of mutations are yet to be analyzed, a very complex task, due to the great number of possible variations and their diverse effect in the BRCA gene functions. After individual analysis, many variants of unknown significance turn out to be pathogenic, and many can disrupt interactions with other proteins involved in mechanisms such as DNA damage repair pathways. Recent data suggest that looking for mutation patterns or combinations would shed a wider light on BRCA-derived cancer susceptibility in the upcoming years.

Long Non-Coding RNAs as New Master Regulators of Resistance to Systemic Treatments in Breast Cancer.

Predicting response to systemic treatments in breast cancer (BC) patients is an urgent, yet still unattained health aim. Easily detectable molecules such as long non-coding RNAs (lncRNAs) are the ideal biomarkers when they act as master regulators of many resistance mechanisms, or of mechanisms that are common to more than one treatment. These kinds of markers are pivotal in quasi-personalized treatment selection, and consequently, in improvement of outcome prediction. In order to provide a better approach to understanding development of disease and resistance to treatments, we reviewed current literature searching for lncRNA-associated systemic BC treatments including endocrine therapies, aromatase inhibitors, selective estrogen receptor modulators (SERMs), trastuzumab, paclitaxel, docetaxel, 5-fluorouracil (5-FU), anthracyclines, and cisplatin. We found that the engagement of lncRNAs in resistance is well described, and that lncRNAs such as urotelial carcinoma-associated 1 (UCA1) and regulator of reprogramming (ROR) are indeed involved in multiple resistance mechanisms, which offers tantalizing perspectives for wide usage of lncRNAs as treatment resistance biomarkers. Thus, we propose this work as the foundation for a wide landscape of functions and mechanisms that link more lncRNAs to resistance to current and new treatments in years of research to come.

Alternative splicing regulation in tumor necrosis factor-mediated inflammation.

It is generally accepted that alternative splicing has an effect on disease when it leads to conspicuous changes in relevant proteins, but that the combinatorial effect of several small modifications can have marked outcomes as well. Inflammation is a complex process involving numerous signaling pathways, among which the tumor necrosis factor (TNF) pathway is one of the most studied. Signaling pathways are commonly represented as intricate cascades of molecular interactions that eventually lead to the activation of one or several genes. Alternative splicing is a common means of controlling protein expression in time and space; therefore, it can modulate the outcome of signaling pathways through small changes in their elements. Notably, the overall process is tightly regulated, which is easily overlooked when analyzing the pathway as a whole. The present review summarizes recent studies of the alternative splicing of key players of the TNF pathway leading to inflammation, and hypothesizes on the cumulative results of those modifications and the impact on cancer development.

Micro-RNAs as Potential Predictors of Response to Breast Cancer Systemic Therapy: Future Clinical Implications.

Despite advances in diagnosis and new treatments such as targeted therapies, breast cancer (BC) is still the most prevalent tumor in women worldwide and the leading cause of death. The principal obstacle for successful BC treatment is the acquired or de novo resistance of the tumors to the systemic therapy (chemotherapy, endocrine, and targeted therapies) that patients receive. In the era of personalized treatment, several studies have focused on the search for biomarkers capable of predicting the response to this therapy; microRNAs (miRNAs) stand out among these markers due to their broad spectrum or potential clinical applications. miRNAs are conserved small non-coding RNAs that act as negative regulators of gene expression playing an important role in several cellular processes, such as cell proliferation, autophagy, genomic stability, and apoptosis. We reviewed recent data that describe the role of miRNAs as potential predictors of response to systemic treatments in BC. Furthermore, upon analyzing the collected published information, we noticed that the overexpression of miR-155, miR-222, miR-125b, and miR-21 predicts the resistance to the most common systemic treatments; nonetheless, the function of these particular miRNAs must be carefully studied and further analyses are still necessary to increase knowledge about their role and future potential clinical uses in BC.

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.

Targeting Metabolic Remodeling in Triple Negative Breast Cancer in a Murine Model.

Background: Chemotherapy is the backbone of systemic treatment for triple negative breast cancer (TNBC), which is one of the most relevant breast cancers molecular types due to the ability of tumor cells to develop drug resistance, highlighting the urgent need to design newer and safer drug combinations for treatment. In this context, to overcome tumor cell drug resistance, we employed a novel combinatorial treatment including Doxorubicin, Metformin, and Sodium Oxamate (DoxMetOx). Such pharmacological combination targets indispensable hallmarks of cancer-related to aerobic glycolysis and DNA synthesis. Materials and Methods: Thirty-five female nude mice were transplanted subcutaneously with MDA-MB-231 triple negative human cancer cell line. Once tumors were visible, mice were treated with doxorubicin, metformin, oxamate or all possible pharmacologic combinations. Treatments were administered daily for 15 days and tumors were measured by calipers every day. MicroPET images were taken in three different occasions, basal state, in the middle of the treatment, and at the end of treatment. Western blot analyses, qRT-PCR, flow cytometry, and cytotoxicity assays were performed to elucidate the mechanism of cell death promoted by the drugs in vitro. Results: In this work we assessed the proof of concept of metabolic correction in solid tumors as an effective drug treatment; hence, mice bearing tumors treated with the DoxMetOx therapy showed a complete inhibition of the tumor mass growing in 15 days of treatment depicted by the micro PET images. In vitro studies displayed that the three drugs together act by inhibiting both, mTOR-phosphorylation and expression of LDH-A gene, promoting apoptosis via dependent on the caspase-3 pathway, accompanied by cleavage of PARP. Moreover, induction of autophagy process was observed by the accumulation of LC3-II, a primordial protein implicated in the conformation and elongation of the autophagolysosome. Conclusions: The lack of effective drugs to inhibit TNBC growth is the main cause of therapy failure and tumor relapse. We have showed that targeting crucial molecular pathways in cancer by the combination of Doxorubicin, Metformin, and Oxamate resulted as an efficient and rapid tumor growth inhibitor in a triple negative xenograft model. Our findings are promising for patients diagnosed with TNBC tumors, for which unfortunately there are no reliable drug therapies.

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.

PAX8 is transcribed aberrantly in cervical tumors and derived cell lines due to complex gene rearrangements.

The transcription factor PAX8, a member of the paired box-containing gene family with an important role in embryogenesis of the kidney, thyroid gland and nervous system, has been described as a biomarker in tumors of the thyroid, parathyroid, kidney and thymus. The PAX8 gene gives rise to four isoforms, through alternative mRNA splicing, but the splicing pattern in tumors is not yet established. Cervical cancer has a positive expression of PAX8; however, there is no available data determining which PAX8 isoform or isoforms are present in cervical cancer tissues as well as in cervical carcinoma-derived cell lines. Instead of a differential pattern of splicing isoforms, we found numerous previously unreported PAX8 aberrant transcripts ranging from 378 to 542 bases and present in both cervical carcinoma-derived cell lines and tumor samples. This is the first report of PAX8 aberrant transcript production in cervical cancer. Reported PAX8 isoforms possess differential transactivation properties; therefore, besides being a helpful marker for detection of cancer, PAX8 isoforms can plausibly exert differential regulation properties during carcinogenesis.

MicroRNAs are involved in cervical cancer development, progression, clinical outcome and improvement treatment response (Review).

Cervical cancer (CC) is the third most diagnosed cancer among females worldwide and the fourth cause of cancer-related mortality. Prophylactic HPV vaccines and traditional pap-smear screening are undoubtedly capable of decreasing the incidence and mortality of CC. However, a large number of females succumb to the disease each year due to late diagnosis and resistance to conventional treatments. Thus, it is necessary to identify new molecular markers to predict the clinical outcome and to design powerful treatments. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are involved in the modulation of several cell pathways associated with progression from pre-malignant to invasive and metastatic disease, increasing tumor malignancy. The aim of this review was to summarize the recent data that describe the important role of miRNAS involved in CC in order to determine their potential as prognostic biomarkers and as therapy targets. Studies of >40 miRNAs with roles in cancer regulation were identified. We also identified 17 miRNAs associated with progression, 12 involved with clinical outcome and 7 that improved CC treatment response. The present review is expected to broaden understanding of the functional role and potential clinical uses of miRNAs in CC.

Transcript profiling distinguishes complete treatment responders with locally advanced cervical cancer.

Cervical cancer (CC) mortality is a major public health concern since it is the second cause of cancer-related deaths among women. Patients diagnosed with locally advanced CC (LACC) have an important rate of recurrence and treatment failure. Conventional treatment for LACC is based on chemotherapy and radiotherapy; however, up to 40% of patients will not respond to conventional treatment; hence, we searched for a prognostic gene signature able to discriminate patients who do not respond to the conventional treatment employed to treat LACC. Tumor biopsies were profiled with genome-wide high-density expression microarrays. Class prediction was performed in tumor tissues and the resultant gene signature was validated by quantitative reverse transcription-polymerase chain reaction. A 27-predictive gene profile was identified through its association with pathologic response. The 27-gene profile was validated in an independent set of patients and was able to distinguish between patients diagnosed as no response versus complete response. Gene expression analysis revealed two distinct groups of tumors diagnosed as LACC. Our findings could provide a strategy to select patients who would benefit from neoadjuvant radiochemotherapy-based treatment.