Zinc associated nanomaterials and their intervention in emerging respiratory viruses: Journey to the field of biomedicine and biomaterials.

Respiratory viruses represent a severe public health risk worldwide, and the research contribution to tackle the current pandemic caused by the SARS-CoV-2 is one of the main targets among the scientific community. In this regard, experts from different fields have gathered to confront this catastrophic pandemic. This review illustrates how nanotechnology intervention could be valuable in solving this difficult situation, and the state of the art of Zn-based nanostructures are discussed in detail. For virus detection, learning from the experience of other respiratory viruses such as influenza, the potential use of Zn nanomaterials as suitable sensing platforms to recognize the S1 spike protein in SARS-CoV-2 are shown. Furthermore, a discussion about the antiviral mechanisms reported for ZnO nanostructures is included, which can help develop surface disinfectants and protective coatings. At the same time, the properties of Zn-based materials as supplements for reducing viral activity and the recovery of infected patients are illustrated. Within the scope of noble adjuvants to improve the immune response, the ZnO NPs properties as immunomodulators are explained, and potential prototypes of nanoengineered particles with metallic cations (like Zn2+) are suggested. Therefore, using Zn-associated nanomaterials from detection to disinfection, supplementation, and immunomodulation opens a wide area of opportunities to combat these emerging respiratory viruses. Finally, the attractive properties of these nanomaterials can be extrapolated to new clinical challenges.

Development of an alternative saliva test for diagnosis of SARS-CoV-2 using TRIzol: Adapting to countries with lower incomes looking for a large-scale detection program.

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities. We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 µL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol. We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.

Corrigendum: HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells.

[This corrects the article DOI: 10.3389/fonc.2019.00381.].

HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells.

HOX transcript antisense RNA (HOTAIR) is an oncogenic long non-coding RNA frequently overexpressed in cancer. HOTAIR can enhance the malignant behavior of tumors by sponging microRNAs with tumor suppressor functions. Vasculogenic mimicry is a hypoxia-activated process in which tumor cells form three-dimensional (3D) channel-like networks, resembling endothelial blood vessels, to obtain nutrients. However, the role of HOTAIR in vasculogenic mimicry and the underlying mechanisms are unknown in human cancers. In the current study, we investigated the relevance of HOTAIR in hypoxia-induced vasculogenic mimicry in metastatic MDA-MB-231 and invasive Hs-578t triple negative breast cancer cells. Analysis of The Cancer Genome Atlas (TCGA) database using cBioPortal confirmed that HOTAIR was upregulated in clinical breast tumors relative to normal mammary tissues. Our quantitative RT-PCR assays showed a significant increase in HOTAIR levels after 48 h hypoxia relative to normoxia in breast cancer cell lines. Remarkably, knockdown of HOTAIR significantly abolished the hypoxia-induced vasculogenic mimicry which was accompanied by a reduction in the number of 3D channel-like networks and branch points. Likewise, HOTAIR silencing leads to reduced cell migration abilities of cancer cells. Bioinformatic analysis predicted that HOTAIR has a potential binding site for tumor suppressor miR-204. Luciferase reporter assays confirmed that HOTAIR is a competitive endogenous sponge of miR-204. Congruently, forced inhibition of HOTAIR in cells resulted in augmented miR-204 levels in breast cancer cells. Further bioinformatic analysis suggested that miR-204 can bind to the 3' untranslated region of focal adhesion kinase 1 (FAK) transcript involved in cell migration. Western blot and luciferase reporter assays confirmed that FAK is a novel target of miR-204. Finally, silencing of HOTAIR resulted in low levels of cytoplasmic FAK protein and alterations in the organization of cellular cytoskeleton and focal adhesions. In summary, our results showed, for the first time, that HOTAIR mitigates cell migration and vasculogenic mimicry by targeting the miR-204/FAK axis in triple negative breast cancer cells.

National Clinical Practice Guidelines for the management of non-small cell lung cancer in early, locally advanced and metastatic stages. Extended version. / Guía de Práctica Clínica Nacional para el manejo del cáncer de pulmón de células no pequeñas en estadios tempranos, localmente avanzados y metastásicos.

OBJECTIVE: Lung cancer is one the leading causes of mortality worldwide. Symptomatic manifestations of the disease generally occur in the advanced-stage setting, and therefore an important number of patients have advanced or metastatic disease by the time they are diagnosed. This situation contributes to a poor prognosis in the treatment of lung cancer. Evidencebased clinical recommendations are of great value to support decision-making for daily practice, and thus improving health care quality and patient outcomes. MATERIALS AND METHODS: This document was an initiative of the Mexican Society of Oncology (SMEO) in collaboration with Mexican Center of Clinical Excellence (Cenetec) according to Interna- tional Standards. Such standards included those described by the IOM, NICE, SIGN and GI-N. An interdisciplinary Guideline Development Group (GDG) was put together which included medical oncologists, surgical oncologistsc, radiation therapists, and methodologists with expertise in critical appraisal, sys- tematic reviews and clinical practice guidelines development. RESULTS: 62 clinical questions were agreed among members of the GDG. With the evidence identified from systematic reviews, the GDG developed clinical recommendations using a Modified Delphi Panel technique. Patients' representatives validated them. CONCLUSIONS: These Clinical Practice Guideline aims to support the shared decision-making process for patients with different stages of non-small cell lung cancer. Our goal is to improve health-care quality on these patients.

OBJETIVO: El cáncer de pulmón es una de las principales causas de mortalidad alrededor del mundo. Su historia natural, con la manifestación de síntomas en etapas avanzadas y el retraso en su diagnóstico hacen que una gran proporción de pacientes se diagnostiquen en estadios tardíos de la enfermedad, lo que hace muy complicado el tratamiento exitoso de la misma. De esto deriva la importancia de dar origen a recomendaciones basadas en evidencia para soportar la toma de decisiones clínicas por parte de los grupos interdisicplinarios que se encargan del manejo de este padecimiento. MATERIAL Y MÉTODOS: Este documento se desarrolló por parte de la Sociedad Mexicana de Oncología en colaboración con el Centro Nacional de Excelencia Tec- nológica de México (Cenetec) a través de la dirección de integración de Guías de Práctica Clínica en cumplimiento a estándares internacionales como los descritos por el Ins- tituto de Medicina de EUA (IOM, por sus siglas en inglés), el Instituto de Excelencia Clínica de Gran Bretaña (NICE, por sus siglas en inglés), la Red Colegiada para el Desarrollo de Guías de Escocia (SIGN, por sus siglas en inglés), la Red Internacional de Guías (G-I-N, por sus siglas en inglés); entre otros. Se integró en representación de la Sociedad Mexicana de Oncología un Grupo de Desarrollo de la Guía (GDG) de manera interdisciplinaria, considerando oncólogos médicos, cirujanos oncólogos, cirujanos de tórax, radio-oncólogos, y metodólogos con experiencia en revisiones sistemáticas de la literatura y guías de práctica clínica. RESULTADOS: Se consensuaron 62 preguntas cllínicas que abarcaron lo establecido previamente por el GDG en el documento de alcances de la Guía. Se identificó la evidencia científica que responde a cada una de estas preguntas clínicas y se evaluó críticamente la misma, antes de ser incorporada en el cuerpo de evidencia de la Guía. El GDG acordó mediante la técnica de consenso formal de expertos Panel Delphi la redacción final de las recomendaciones clínicas. C. CONCLUSIONES: Esta Guía de Práctica Clínica pretende proveer recomendaciones clínicas para el manejo de los distintos estadios de la enfermedad y que asistan en el proceso de toma de decisiones compartida. El GDG espera que esta guía contribuya a mejorar la calidad de la atención clínica en las pacientes con cáncer de pulmón de células no pequeñas.

HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells

Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3'UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells.

A Novel OsteomiRs Expression Signature for Osteoblast Differentiation of Human Amniotic Membrane-Derived Mesenchymal Stem Cells.

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are a potential source of cells for therapeutic applications in bone regeneration. Recent evidence reveals a role for microRNAs (miRNAs) in the fine-tuning regulation of osteogenesis (osteomiRs) suggesting that they can be potential targets for skeleton diseases treatment. However, the functions of osteomiRs during differentiation of hAM-MSCs to osteogenic lineage are poorly understood. In this investigation, we discovered a novel miRNAs expression signature corresponding to the matrix maturation (preosteoblast) and mineralization (mature osteoblast) stages of dexamethasone-induced osteoblastic differentiation of hAM-MSCs. Comprehensive miRNAs profiling using TaqMan Low Density Arrays showed that 18 miRNAs were significantly downregulated, whereas 3 were upregulated in the matrix maturation stage (7 days after osteogenic induction) in comparison to undifferentiated cells used as control. Likewise, 47 miRNAs were suppressed and 25 were overexpressed at mineralization stage (14 days after osteogenic induction) in comparison to osteoprogenitors cells. Five out 93 miRNAs (miR-19b-3p, miR-335-3p, miR-197-3p, miR-34b-39, and miR-576-3p) were regulated at both 7 and 14 days suggesting a role in coordinated guidance of osteoblastic differentiation. Exhaustive bioinformatic predictions showed that the set of modulated miRNAs may target multiple genes involved in regulatory networks driving osteogenesis including key members of BMP, TGF-ß, and WNT/ß-catenin signaling pathways. Of these miRNAs, we selected miR-204, a noncoding small RNA that was expressed at matrix maturation phase and downregulated at maturation stage, for further functional studies. Interestingly, gain-of-function analysis showed that restoration of miR-204 using RNA mimics at the onset of mineralization stage dramatically inhibited deposition of calcium and osteogenic maturation of hAM-MSCs. Moreover in silico analysis detected a conserved miR-204 binding site at the 3'UTR of TGF-ßR2 receptor gene. Using luciferase assays we confirmed that TGF-ßR2 is a downstream effector of miR-204. In conclusion, we have identified a miRNAs signature for osteoblast differentiation of hAM-MSCs. The results from this study suggested that these miRNAs may act as potential inhibitors or activators of osteogenesis. Our findings also points towards the idea that miR-204/TGF-ßR2 axis has a regulatory role in differentiation of hAM-MSCs committed to osteoblastic lineage.

Dietary Compounds as Epigenetic Modulating Agents in Cancer.

Epigenetic mechanisms control gene expression during normal development and their aberrant regulation may lead to human diseases including cancer. Natural phytochemicals can largely modulate mammalian epigenome through regulation of mechanisms and proteins responsible for chromatin remodeling. Phytochemicals are mainly contained in fruits, seeds, and vegetables as well as in foods supplements. These compounds act as powerful cellular antioxidants and anti-carcinogens agents. Several dietary compounds such as catechins, curcumin, genistein, quercetin and resveratrol, among others, exhibit potent anti-tumor activities through the reversion of epigenetic alterations associated to oncogenes activation and inactivation of tumor suppressor genes. In this review, we summarized the actual knowledge about the role of dietary phytochemicals in the restoration of aberrant epigenetic alterations found in cancer cells with a particular focus on DNA methylation and histone modifications. Furthermore, we discussed the mechanisms by which these natural compounds modulate gene expression at epigenetic level and described their molecular targets in diverse types of cancer. Modulation of epigenetic activities by phytochemicals will allow the discovery of novel biomarkers for cancer prevention, and highlights its potential as an alternative therapeutic approach in cancer.

DNA Repair Proteins as Therapeutic Targets in Ovarian Cancer.

Epithelial ovarian cancer is a serious public health problem worldwide with the highest mortality rate of all gynecologic cancers. The current standard-of-care for the treatment of ovarian cancer is based on chemotherapy based on adjuvant cisplatin/carboplatin and taxane regimens that represent the first-line agents for patients with advanced disease. The DNA repair activity of cancer cells determines the efficacy of anticancer drugs. These features make DNA repair mechanisms a promising target for novel cancer treatments. In this context a better understanding of the DNA damage response caused by antitumor agents has provided the basis for the use of DNA repair inhibitors to improve the therapeutic use of DNA-damaging drugs. In this review, we will discuss the functions of DNA repair proteins and the advances in targeting DNA repair pathways with special emphasis in the inhibition of HRR and BER in ovarian cancer. We focused in the actual efforts in the development and clinical use of poly (ADPribose) polymerase (PARP) inhibitors for the intervention of BRCA1/BRCA2-deficient ovarian tumors. The clinical development of PARP inhibitors in ovarian cancer patients with germline BRCA1/2 mutations and sporadic high-grade serous ovarian cancer is ongoing. Some phase II and phase III trials have been completed with promising results for ovarian cancer patients.

AngiomiRs: MicroRNAs driving angiogenesis in cancer (Review).

Angiogenesis is an important hallmark of cancer serving a key role in tumor growth and metastasis. Therefore, tumor angiogenesis has become an attractive target for development of novel drug therapies. An increased amount of anti­angiogenic compounds is currently in preclinical and clinical development for personalized therapies. However, resistance to current angiogenesis inhibitors is emerging, indicating that there is a need to identify novel anti­angiogenic agents. In the last decade, the field of microRNA biology has exploded revealing unsuspected functions in tumor angiogenesis. These small non­coding RNAs, which have been dubbed as angiomiRs, may target regulatory molecules driving angiogenesis, such as cytokines, metalloproteinases and growth factors, including vascular endothelial growth factor, platelet­derived growth factor, fibroblast growth factor, epidermal growth factor, hypoxia inducible factor­1, as well as mitogen­activated protein kinase, phosphoinositide 3­kinase and transforming growth factor signaling pathways. The present review discusses the current progress towards understanding the functions of miRNAs in tumor angiogenesis regulation in diverse types of human cancer. Furthermore, the potential clinical application of angiomiRs towards anti­angiogenic tumor therapy was explored.

Frequency of BRAF V600E Mutation in the Mexican Population of Patients With Metastatic Melanoma.

PURPOSE: The BRAF V600E mutation has been described in melanomas occurring in the Caucasian, European, and Asian populations. However, in the Mexican population, the status and clinical significance of BRAF mutation has not been researched on a large scale. METHODS: Consecutive BRAF-tested Mexican patients with metastatic melanoma (n = 127) were analyzed for mutations in exon 15 of the BRAF gene in genomic DNA by real-time polymerase chain reaction technology for amplification and detection. The results were correlated with the clinical-pathologic features and the prognosis of the patients. RESULTS: The frequency of somatic mutation V600E within the BRAF gene was 54.6% (43 of 127 patients). Nodular melanoma was the most prevalent subtype in our population, with BRAF mutations in 37.2% (16 of 55 patients). In contrast, superficial spread had a frequency of 18.6% BRAF mutation (eight of 24). Other clinicopathologic features were assessed to correlate with the mutation status. CONCLUSION: This study searched for the most prevalent BRAF V600E mutation type in melanoma in a heterogeneous population from Mexico. Nodular melanoma was found to be the most prevalent in metastatic presentation and the presence of BRAF V600E mutation, perhaps related to the mixed ancestry; in the north, ancestry is predominantly European and in the south, it is predominantly Asian. The outcomes of the mutation correlations were similar to those found in other populations.

let-7d-3p is associated with apoptosis and response to neoadjuvant chemotherapy in ovarian cancer.

Altered expression of microRNAs contributes to the heterogeneous biological behavior of human malignancies and it may correlate with the clinical pathological features of patients. The let-7 microRNA family is frequently downregulated in human cancers and its aberrant expression may be a useful marker for prediction of the clinical response to therapy in patients. In the present study, we analyzed the expression of three members of the let-7 family (let-7a-3p, let-7d-3p and let-7f), which remains largely uncharacterized in ovarian cancer tissues. We also investigated the function of let-7d-3p in the apoptosis and sensitization to chemotherapy in ovarian cancer cells. Our data from stem-loop quantitative RT-PCR showed that expression of let-7a-3p and let-7d-3p, but not let-7f, was significantly (P<0.04) upregulated in ovarian tumors relative to that noted in normal ovarian tissues. Markedly, an increased expression of let­7d-3p (also known as let-7d-3*) was associated with positive response to carboplatin/paclitaxel treatment in ovarian cancer patients. To investigate the biological relevance of let­7d-3p, we knocked down its expression in SKOV-3 ovarian cancer cell line using antagomiRs. Loss of function analysis showed that inhibition of let-7d-3p significantly (P<0.05) impaired cell proliferation and activated apoptosis. In contrast, scratch/wound healing and Transwell chamber assays showed that migration and invasion abilities were not affected in the let-7d-3p-deficient SKOV-3 cancer cells. Notably, Annexin V assays showed a significant (P<0.05) increase in cell death of cancer cells treated with the let-7d-3p inhibitor plus carboplatin indicating a synergistic effect of the drug with antagomiR therapy. Gene ontology classification of predicted targets of let-7d-3p identified a number of genes involved in cellular pathways associated with therapy resistance such as ABC transporters, HIF-1, RAS and ErbB signaling. In summary, our findings showed that inhibition of let-7d-3 activates apoptosis and that its upregulation is associated with a positive response of ovarian cancer patients to carboplatin/paclitaxel chemotherapy.

MicroRNAs driving invasion and metastasis in ovarian cancer: Opportunities for translational medicine (Review).

Epithelial ovarian cancer is the fifth most frequent cause of cancer death in women. In spite of the advantages in early detection and treatment options, overall survival rates have improved only slightly in the last decades. Therefore, alternative therapeutic approaches need to overcome resistance and improve the patient survival and outcome. MicroRNAs are evolutionary conserved small non-coding RNAs that function as negative regulators of gene expression by inhibiting translation or inducing degradation of messenger RNAs. In cancer, microRNAs are aberrantly expressed thus representing potential prognostic biomarkers and novel therapeutic targets. The knowledge of novel and unexpected functions of microRNAs is rapidly evolving and the advance in the elucidation of potential clinical applications deserves attention. Recently, a specific set of microRNAs dubbed as metastamiRs have been shown to initiate invasion and metastasis in diverse types of cancer. We reviewed the current status of microRNAs in development and progression of ovarian cancer with a special emphasis on tumor cells invasion and metastasis. Also, we show an update of microRNA functions in oncogenic pathways and discuss the current scenario for potential applications in clinical and translational research in ovarian cancer.

Search of the p.M918T Mutation in the RET Oncogene in Mexican Adult Patients with Medullary Thyroid Carcinoma.

Inherited mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, predispose individuals to the multiple endocrine neoplasia type 2 (MEN 2) cancer syndromes. The major component tumor of these syndromes is medullary thyroid carcinoma (MTC). To date, somatic mutations in RET have been identified in tumors from individuals with MEN 2 finding. RET M918T mutation is present in 95% of the MEN2B cases, and approximately 50% of sporadic MTCs harbor this mutation. We performed a mutational analysis in 17 cases of Medullary thyroid carcinoma, the somatic missense mutation at codon 918 of RET was found in 2 of the 17 MTCs, and one case presented MEN2 phenotype including MTC. The percentage of RET M918T mutation is similar in Mexican MTC patients to other series, although other mutations could be implicated in our population.

Reduction in Hepatocyte Growth Factor Serum Levels is Associated with Improved Prognosis in Advanced Lung Adenocarcinoma Patients Treated with Afatinib: a Phase II Trial.

BACKGROUND: C-met and its ligand, hepatocyte growth factor (HGF) have been associated with the resistance mechanism of EGFR-TKIs. HGF was evaluated as a clinical-marker of response in NSCLC patients treated with afatinib. METHODS: Sixty-six patients with stage IIIB/IV lung adenocarcinoma and progression to any-line chemotherapy received afatinib 40 mg/day. Mutational EGFR and HER2 status were assessed by RT-PCR. HER2 amplification was evaluated by FISH. Serum HGF content was measured by ELISA before and 2 months after the start of treatment. HGF levels were assessed with the objective response rate (ORR), progression-free-survival (PFS), and overall survival (OS). This trial was registered on ClinicalTrials.gov: NCT01542437. RESULTS: Fifty patients (75 %) were EGFR mutation positive. Response was achieved in 59 % of all patients and 78 % of EGFR mutated patients. Median PFS was 10 [95 % CI 6.8-13.1] and 14.5 months [10.9-18.9] for all and EGFR mutated patients, respectively. Median OS was 22.8 [17.5-28.1] and 32.4 months [18.3-46.6] for all and EGFR mutated patients, respectively. Patients with reduced serum HGF levels had improved ORR (75 % vs 44 %; p = 0.011), PFS (15.1 [2.9-27.3] vs 6.5 months [3.9-9.1]; p = 0.005) and OS (NR vs 14.5 months [7.8 - 21.3] p = 0.007). A reduction in serum HGF levels was an independent factor associated with longer PFS (HR 0.40; p = 0.021) and OS (HR 0.31; p = 0.006) in all and EGFR mutated patients. CONCLUSIONS: A reduction in serum HGF levels was associated with improved outcomes in patients treated with afatinib. These results suggest HGF might have a role as a mechanism of resistance to EGFR-TKIs. HGF could represent a potential therapeutic target to prevent or reverse resistance particularly in EGFR mutated patients.

Differential proteomic analysis reveals that EGCG inhibits HDGF and activates apoptosis to increase the sensitivity of non-small cells lung cancer to chemotherapy.

PURPOSE: To search for regulated proteins in response to green tea (-)-epigallocatechin-3-gallate (EGCG) in A549 lung cancer cells. EXPERIMENTAL DESIGN: 2DE and ESI/multistage MS (ESI-MS/MS) were performed to identify modulated proteins in A549 cells treated with EGCG. Cell migration was evaluated by transwell assays. RNA interference was used to silence the hepatoma-derived growth factor (HDGF). Caspase-3, caspase-9, and HDGF were immunodetected by Western blot assays. Flow cytometry was used for detection of mitochondrial membrane potential and apoptosis. RESULTS: We found that HDGF expression was threefold suppressed by EGCG treatment. Downregulation of HDGF by EGCG was confirmed using anti-HDGF antibodies in three lung cancer cell lines. EGCG treatment and HDGF abrogation by RNA interference resulted in a decreased migration of A549 cells. In addition, EGCG induced a marked synergistic effect with cisplatin in cell death. Consistently, an enhanced cytotoxicity in HDGF-silenced cells was also found. Cell death was associated to increased apoptosis, disruption of the mitochondrial membrane potential, and activation of caspase-3 and caspase-9. CONCLUSION AND CLINICAL RELEVANCE: Our data suggest for the first time that abrogation of HDGF by EGCG enhances cisplatin-induced apoptosis and sensitize A549 cells to chemotherapy. Therefore, we propose that decreasing the HDGF levels by using EGCG may represent a novel strategy in lung cancer therapy.

Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase.

In higher eukaryotic cells mRNA degradation initiates by poly(A) tail shortening catalyzed by deadenylases CAF1 and CCR4. In spite of the key role of mRNA turnover in gene expression regulation, the underlying mechanisms remain poorly understood in parasites. Here, we aimed to study the function of EhCAF1 and identify associated proteins in Entamoeba histolytica. By biochemical assays, we evidenced that EhCAF1 has both RNA binding and deadenylase activities in vitro. EhCAF1 was located in cytoplasmic P-bodies that increased in number and size after cellular stress induced by DNA damage, heat shock, and nitric oxide. Using pull-down assays and ESI-MS/MS mass spectrometry, we identified 15 potential EhCAF1-interacting proteins, including the endoribonuclease EhL-PSP. Remarkably, EhCAF1 colocalized with EhL-PSP in cytoplasmic P-bodies in trophozoites. Bioinformatic analysis of EhL-PSP network proteins predicts a potential interaction with EhRRP41 exosome protein. Consistently, we evidenced that EhL-PSP colocalizes and physically interacts with EhRRP41. Strikingly, EhRRP41 did not coimmunoprecipitate EhCAF1, suggesting the existence of two EhL-PSP-containing complexes. In conclusion, our results showed novel interactions between mRNA degradation proteins and evidenced for the first time that EhCAF1 is a functional deadenylase that interacts with EhL-PSP endoribonuclease in P-bodies, while EhL-PSP interacts with EhRRP41 exosome protein in this early-branched eukaryote. BIOLOGICAL SIGNIFICANCE: This study provides evidences for the functional deadenylase activity of EhCAF1 and shows a link between different mRNA degradation proteins in E. histolytica. By proteomic tools and pull down assays, we evidenced that EhCAF1 interacts with the putative endoribonuclease EhL-PSP, which in turn interacts with exosome EhRRP41 protein. Our data suggest for the first time the presence of two complexes, one containing the endoribonuclease EhL-PSP and the deadenylase EhCAF1 in P-bodies; and another containing the endoribonuclease EhL-PSP and the exosome EhRRP41 exoribonuclease. Overall, these results provide novel data that may help to understand mRNA decay mechanisms in this parasite.

Proteomic profiling reveals that EhPC4 transcription factor induces cell migration through up-regulation of the 16-kDa actin-binding protein EhABP16 in Entamoeba histolytica.

Actin cytoskeleton is an essential structure involved in cell migration and invasion in parasites. In Entamoeba histolytica, the protozoan parasite causing human amoebiasis, the mechanisms underlying the expression of migration-related genes are poorly understood. Here, we investigated the biological effects of ectopic overexpression of EhPC4 (positive coactivator 4) in cell migration of E. histolytica trophozoites. Using differential in gel two-dimensional electrophoresis, 33 modulated proteins were detected in EhPC4-overexpressing cells. By electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis, 16 of these proteins were identified. Interestingly, four up-regulated proteins involved in cytoskeleton organization and cell migration were identified. Particularly, we found the up-regulation of a 16-kDa actin-binding protein (EhABP16) which is a putative member of the cofilin/tropomyosin family involved in actin polymerization. EhPC4 overexpression induced a significant increase in migration of trophozoites and in the destruction of human SW480 colon cells. Consistently, silencing of gene expression by RNA interference of EhABP16 significantly impairs cell migration. These changes were associated to alterations in the organization of actin cytoskeleton, and suppression of uropod-like structure formation in EhABP16-deficient cells. In summary, we have uncovered novel proteins modulated by EhPC4, including EhABP16, with a potential role in cell migration, cytopathogenicity and virulence in E. histolytica. BIOLOGICAL SIGNIFICANCE: The human pathogen Entamoeba histolytica infects around 50million people worldwide resulting in 40,000-100,000 deaths annually. Cell motility is a complex trait that is critical for parasites adaptation, spread and invasion processes into host tissues; it has been associated with virulence. In this study, we used a differential proteomic approach to demonstrate that E. histolytica EhPC4 induces changes in the expression of actin cytoskeleton proteins, including EhABP16, promoting a significant increase in cell motility and destruction of intestinal human cells. Particularly, we demonstrated for the first time that abrogation of EhABP16 impairs cell migration by altering the actin cytoskeleton dynamics and uropod-like structure formation in trophozoites. These data contribute to the understanding of molecular mechanisms that regulate virulence properties in this neglected protozoan parasite.

Comparative proteomic profiling of triple-negative breast cancer reveals that up-regulation of RhoGDI-2 is associated to the inhibition of caspase 3 and caspase 9.

There are no targeted therapeutic modalities for triple-negative breast cancer (TNBC), thus it is associated with poor prognosis and worst clinical outcome. Here, our aim was to identify deregulated proteins in TNBC with potential therapeutic applications. Proteomics profiling of TNBC and normal breast tissues through two-dimensional electrophoresis and ESI-MS/MS mass spectrometry revealed the existence of 16 proteins (RhoGDI-2, HSP27, SOD1, DJ1, UBE2N, PSME1, FTL, SH3BGRL, and eIF5A-1) with increased abundance in carcinomas. We also evidenced for the first time the deregulation of COX5, MTPN and DB1 proteins in TNBC that may represent novel tumor markers. Particularly, we confirmed the overexpression of the Rho-GDP dissociation inhibitor 2 (RhoGDI-2) in distinct breast cancer subtypes, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Remarkably, targeted disruption of RhoGDI-2 by RNA interference induced mitochondrial dysfunction, and facilitated caspase-3 and -9 activation in two breast cancer cell lines. Moreover, suppression of RhoGDI-2 resulted in a robust sensitization of breast cancer cells to cisplatin therapy. In conclusion, we identified novel proteins deregulated in TNBC, and confirmed the overexpression of RhoGDI-2. We propose that RhoGDI-2 inhibition may be exploited as a potential therapeutic strategy along cisplatin-based chemotherapy in breast cancer. BIOLOGICAL SIGNIFICANCE: There are no useful biomarkers neither targeted therapeutic modalities for triple-negative breast cancer, which highly contributes to the poor prognosis of this breast cancer subtype. In this work, we used two-dimensional electrophoresis and ESI-MS/MS spectrometry to identify novel deregulated proteins in breast cancer tissues. Particularly, our results showed that RhoGDI-2, a protein that has been associated to metastasis and poor survival in human cancers, is overexpressed in different subtypes of breast tumors, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Our data also provided novel insights about the role of RhoGDI-2 in apoptosis through intrinsic pathway inhibition. Importantly, they suggested that targeted modulation of RhoGDI-2 levels might be a useful strategy for breast cancer therapy.

Treatment of malignant, non-resectable, epithelial origin esophageal tumours with the humanized anti-epidermal growth factor antibody nimotuzumab combined with radiation therapy and chemotherapy.

BACKGROUND: Over-expression of epidermal growth factor receptor in esophageal cancer is associated with poor prognosis. The present study was conducted to evaluate safety and preliminary efficacy of nimotuzumab, a humanized anti-EGFR antibody in combination with radiation and chemotherapy in advanced esophageal tumours. PATIENTS AND METHODS: A Phase II clinical trial was conducted, where patients received cisplatin, 5-fluorouracil, and radiotherapy, either alone or combined with six weekly infusions of nimotuzumab at the dose of 200 mg. Safety was the primary endpoint. The antitumoral objective response rate was the secondary endpoint. Epidermal growth factor receptor expression, KRAS mutation status and anti-idiotypic response were also evaluated. RESULTS: Sixty-three patients were included in the study. Thirty patients were entered into the control group, and thirty-three patients received the treatment with nimotuzumab. The antibody was very well tolerated. Objective response rate was 47.8 % (nimotuzumab group) and 15.4 % (control group). Disease control rate was 60.9 % (nimotuzumab group) and 26.9 % (control group). Response and disease control rate were higher in patients with EGFR overexpressing tumors. CONCLUSION: Nimotuzumab plus chemoradiotherapy was safe and provided statistically significant objective response. A Phase III in patients with similar characteristics will be launched.