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Simple Summary Tumor microenvironment (TME) is a complex of many cell types and extracellular matrix that play an active role in regulating and sustaining melanoma tumor progression. In this context, the secretion of several molecules, by secretory autophagy or exosome release, stimulates the intercellular communication between the different components of the TME modulating tumor response. Here, we discuss the current awareness around the role of extracellular secretion in melanoma TME and also investigate the molecules related to these secretion pathways in melanoma progression using public databases. Abstract Melanoma is the most aggressive skin cancer characterized by high mutational burden and large heterogeneity. Cancer cells are surrounded by a complex environment, critical to tumor establishment and progression. Thus, tumor-associated stromal components can sustain tumor demands or impair cancer cell progression. One way to manage such processes is through the regulation of autophagy, both in stromal and tumor cells. Autophagy is a catabolic mechanism that provides nutrients and energy, and it eliminates damaged organelles by degradation and recycling of cellular elements. Besides this primary function, autophagy plays multiple roles in the tumor microenvironment capable of affecting cell fate. Evidence demonstrates the existence of novel branches in the autophagy system related to cytoplasmic constituent’s secretion. Hence, autophagy-dependent secretion assembles a tangled network of signaling that potentially contributes to metabolism reprogramming, immune regulation, and tumor progression. Here, we summarize the current awareness regarding secretory autophagy and the intersection with exosome biogenesis and release in melanoma and their role in tumor resistance. In addition, we present and discuss data from public databases concerning autophagy and exosome-related genes as important mediators of melanoma behavior. Finally, we will present the main challenges in the field and strategies to translate most of the pre-clinical findings to clinical practice.

A propagação de notícias falsas sobre o julgamento do ex-presidente Luiz Inácio Lula da Silva, em janeiro de 2018, é aqui examinada conforme a concepção pragmática de verdade proposta por Charles Sanders Peirce (1839-1914). Por meio de coleta em redes sociais on-line, observou-se esforço coletivo para fixação de crenças concorrentes, conforme os métodos peirceanos de tenacidade e autoridade. O processo comunicacional analisado, caracterizado como ativismo transmídia, foi impulsionado pela mediação de hashtags que remetiam à polarização política e pelos posts atribuídos a celebridades. Conclui-se que a disputa pelo valor semiótico da verdade fomentou a expansão transmídia de conteúdos falsos e, paradoxalmente, de notícias verificadas.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20–30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling β1-integrins into large punctate clusters at the leading edge of tumor cells to promote an “adhesive switch,” decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease., Biomarker: Methylated gene promoter increases risk of metastatic disease Epigenetic silencing of a putative tumor suppressor protein confers a metastatic advantage to breast cancer cells and could serve as a mechanistic biomarker of likely disease spread. Researchers in São Paulo, Brazil, led by Érico Costa from the Molecular Oncology Center of the Hospital Sírio-Libanês, showed that NDRG4 (short for N-Myc downstream-regulated gene 4) is actively expressed in normal breast tissue. But in breast tumors and in tumor cell lines, methyl tags cover the promoter region of the NDRG4-encoding gene, resulting in reduced protein expression, increased tumor size, elevated cell mobility and other molecular features indicative of a more aggressive disease state. The researchers experimentally silenced NDRG4 expression in two non-metastatic breast cancer cell lines and observed changes in the adhesive and migratory properties of cells that promote tumor invasiveness and spread.

Intracellular parasites of the genus Leishmania are the causative agents of human leishmaniasis, a widespread emergent tropical disease. The parasite is transmitted by the bite of a hematophagous sandfly vector that inoculates motile flagellated promastigote forms into the dermis of the mammalian host. After inoculation, parasites are ultimately captured by macrophages and multiply as round-shaped amastigote forms. Macrophages seem not to be the first infected cells since parasites were observed invading neutrophils first whose leishmania-containing apoptotic bodies were latter captured by macrophages, thereby becoming infected. The fact that Leishmania spp are able to live and replicate inside immune phagocytic cells and that macrophages are the main cell type found infected in chronicity created the perception that Leishmania spp are passive players waiting to be captured by phagocytes. However, several groups have described the infection of non-phagocytic cells in vivo and in vitro. The objective of this work was to study the cellular mechanisms involved in the invasion of non-professional phagocytes by Leishmania. We show that promastigotes of L.amazonensis actively induces invasion in fibroblasts without cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Inside fibroblasts parasites transformed in amastigotes, remained viable for at least two weeks and re-transformed in promastigotes when returned to insect vector conditions. Similarly to what was observed for T. cruzi, infection involves calcium signaling, recruitment and exocytosis of lysosomes involved in plasma membrane repair and lysosome-triggered endocytosis. Conditions that alter lysosomal function such as cytochalasin-D and brefeldin-A treatment or the knockout of host cell lysosomal proteins LAMP-1 and 2 dramatically affected invasion. Likewise, triggering of lysosomal exocytosis and lysosome-dependent plasma membrane repair by low doses of streptolysin-O dramatically increased parasite entry. Together our results show that L.amazonensis promastigotes are able to take advantage of calcium-dependent lysosomal exocytosis and lysosome-induced endocytosis to invade and persist in non-phagocytic cells.AUTHOR SUMMARYIntracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting but overlooked finding on Leishmania infection is that non-phagocytic cells have also been found infected by amastigotes. Nevertheless, the mechanisms by which Leishmania invades non-phagocytic cells were not studied to date. Here we show that L. amazonensis can actively induce their own entry into fibroblasts independently of actin cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions such as calcium signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair and whose positioning and content interfere in invasion. Parasites were able to replicate and remained viable in fibroblasts, suggesting that cell invasion trough the mechanism demonstrated here could serve as a parasite hideout and reservoir, facilitating infection amplification and persistence.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Background Melanoma is the most lethal type of skin cancer. Since chemoresistance is a significant barrier, identification of regulators affecting chemosensitivity is necessary in order to create new forms of intervention. Prohibitin 1 (PHB1) can act as anti-apoptotic or tumor suppressor molecule, depending on its subcellular localization. Our recent data shown that accumulation of PHB1 protects melanoma cells from chemotherapy-induced cell death. Lacking of post-transcriptional regulation of PHB1 could explain this accumulation. Interestingly, most of melanoma patients have down-regulation of microRNA-195. Here, we investigate the role of miR-195, its impact on PHB1 expression, and on chemosensitivity in melanoma cells. Methods TCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma cell lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by RT-qPCR and western blot, luciferase reporter and genetic rescue experiments. Cell proliferation, cell-cycle analysis and caspase 3/7 assay were performed to investigate the potential action of miR-195 as chemosensitizer in melanoma cells treated with cisplatin and temozolomide. Results Analysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, RT-qPCR data showed that miR-195 is down-regulated while PHB1 is up-regulated in a collection of melanoma cells. We demonstrated that miR-195 regulates PHB1 directly by RT-qPCR and western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as potential anti-proliferative agent, with potential impact in sensitization of melanoma cell death. Conclusions This study support the role of miR-195 as anti-proliferative miRNA via targeting of PHB1 in melanoma cells. Electronic supplementary material The online version of this article (10.1186/s12885-017-3721-7) contains supplementary material, which is available to authorized users.

One approach to protect human skin against the dangerous effects of solar ultraviolet (UV) irradiation is the use of natural products, such as photoprotectors. Phyllanthus orbicularis Kunth (Euphorbiaceae) is a Cuban endemic plant used in popular medicine. Its antigenotoxicity effect against some harmful agents has been investigated. However, the effect in ultraviolet B (UVB)-irradiated human cells has not been previously assessed.The protective effect of a P. orbicularis extract against UVB light-induced damage in human cells was evaluated.DNA repair proficient (MRC5-SV) and deficient (XP4PA, complementation group XPC) cell-lines were used. Damaging effects of UVB light were evaluated by clonogenic assay and apoptosis induction by flow cytometry techniques. The extent of DNA repair itself was determined by the removal of cyclobutane pyrimidine dimers (CPDs). The CPDs were detected and quantified by slot-blot assay.Treatment of UVB-irradiated MRC5-SV cells with P. orbicularis extract increased the percentage of colony-forming cells from 36.03 ± 3.59 and 4.42 ± 1.45 to 53.14 ± 8.8 and 14.52 ± 1.97, for 400 and 600 J/m(2), respectively. A decrease in apoptotic cell population was observed in cells maintained within the extract. The P. orbicularis extract enhanced the removal of CPD from genomic DNA. The CPDs remaining were found to be about 27.7 and 1.1%, while with plant extract, treatment these values decreased to 16.1 and 0.2%, for 3 and 24 h, respectively.P. orbicularis aqueous extract protects human cells against UVB damage. This protective effect is through the modulation of DNA repair effectiveness.

Galectin-3 is a member of the b-galactoside binding lectin family, whose expression is often dysregulated in cancers. While galectin-3 is usually an intracellular protein, found in the nucleus and in the cytoplasm; under certain conditions, galectin-3 can be secreted by an yet unknown mechanism. Under stressing conditions (hypoxia and nutrient deprivation, e.g.) galectin-3 is upregulated, through the activity of transcription factors such as HIF-1a and NF-kB. Here we review evidence that indicates a positive role for galectin-3 in MAPK family signal transduction, leading to cell proliferation and cell survival. Galectin-3 serves as a scaffold protein, which favors the spatial organization of signaling proteins as K-RAS. Upon secretion, extracellular galectin-3 interacts with a variety of cell surface glycoproteins, such as growth factor receptors, integrins, cadherins and members of the Notch family, among other glycoproteins, besides different extracellular matrix molecules. Through its ability to oligomerize, galectin-3 forms lectin lattices that act as scaffolds that sustain the spatial organization of signaling receptors on the cell surface, dictating its maintenance on the plasma membrane or their endocytosis. Galectin-3 induces tumor cell, endothelial cell and leukocyte migration, favoring either the exit of tumor cells from a stressed microenvironment or the entry of endothelial cells and leukocytes, such as monocyte/macrophages into the tumor organoid. Therefore, galectin-3 plays homeostatic roles in tumors, besides its effects in different elements of the immune system, as (i) it favors tumor cell adaptation for survival in stressed conditions; (ii) upon secretion, galectin-3 induces tumor cell detachment and migration; (iii) it attracts endothelial cells and monocytes/macrophages to the tumor mass, inducing both directly and indirectly the process of angiogenesis. These activities are potentially targetable and specific interventions may be designed to counteract the protumoral role of galectin-3.

Melanoma is the most lethal type of skin cancer. Since chemo-resistance is a significant barrier, identification of regulators affecting chemo-sensitivity is necessary in order to create new forms of intervention. Here, we investigate the role of miR-195, its impact on Prohibitin 1 (PHB1) expression, and on chemo-sensitivity in melanoma cells. TCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by qRT-PCR and Western blot, luciferase reporter, and rescue experiments. Cell proliferation, hypodiploid, and caspase 3/7 assays were performed to investigate the potential action of miR-195 as chemo-sensitizer in melanoma cells treated with cisplatin and temozolomide. Analysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, qRT-PCR data showed that miR-195 is downregulated while PHB1 is upregulated in a collection of melanoma lines. We demonstrated that miR-195 regulates PHB1 directly by qRT-PCR and Western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect of miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as a tumor suppressor and potential chemo-sensitivity agent. Citation Format: Priscila D. R. Cirilo, Bruna R. S. Côrrea, Mei Qiao, Luciana N. S. Andrade, Roger Chammas, Luiz O. F. Penalva. MicroRNA-195 acts as a tumor suppressor miRNA in human melanoma cells by targeting Prohibitin 1 [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A06.

Background: The exposure of phosphatidylserine (PS) is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is well described in tumors and is associated with malignancy and poor prognosis. Tumor associated macrophages (TAMs) act suppressing the anticancer immune response. The tumor parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous study we observed that 7-ketocholesterol is capable of inducing autophagy on melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg commercial Phosphatidylserine (PS) and PS associated with 5 mg of 7-ketocholesterol extracted with chloroform/methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and sonicated for 6 minutes. The size and Zeta Pontencial (ZP) of liposomes were evaluated. Antiinflammatory activity of liposomes was evaluated by paw edema induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells, and 4T1 breast cancer cells was assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry with propidium iodide (PI) staining. The presence of acid vacuoles related to autophagy was evaluated by flow cytomery by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma-bearing C57/bl6 mice and 4T1 breast cancer-bearing Balb c mice. Endocytosis efficiency of the liposomes was observed by labeling it with PKH26 fluorescent staining and evaluated in 4T1 cells after 12 h. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG; the solution was homogenized and sonicated for 6 minutes. The samples were centrifuged and part of the supernatant was added to an Amicon® filter (10kD) and concentrated, the concentrated was diluted with 400 uL of PBS and concentrated again. Liposome incorporation was determined by quotient of the radioactivity in the Amicon® by sum of Amicon® and filtrated solutions. Furthermore, lipophilicity (L), hydrophilicity (H), and charge (-/0/+) of the radioactive material were considered in the final analysis. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP. The paw edema was inhibited by both liposomes after 240 min of the carrageenan induction. The concentration of 26 uM/mL of PS and PS/7KC liposomes stimulated cell proliferation. PS/7KC at the concentrations above 84 uM/mL inhibited the cell proliferation. PS/7KC showed intense antiproliferative activity in melanoma cells and breast adenocarcinoma cells, assessed by the MTT method and by flow cytometry with PI. It was observed 10% more autophagic vacuoles on melanoma cells treated with PS/7KC than the control groups. Both in vivo tumor models had the same antiproliferative effect of the PS/7KC liposomes with daily doses. Daily doses of PS liposomes induced a high size of tumors. 99mTc-MIBI was efficiently and strongly incorporated to liposomes than the other proposed formulations. Conclusion: PS liposomes have effects in vivo and in vitro and must be related to phagocyte and autophagy activities. PS/7KC impairs J774 macrophage, B16F10 melanoma, and 4T1 breast adenocarcinoma cell growth. PS/7KC induces the presence of acid vacuoles corresponding to autophagy. The liposomes had a high endocytosis evaluated by PKH 26 labelled particles. PS keeps the tumor proliferation and PS/7KC inhibits tumor growth after ten days of daily doses. Supported by CNPq and Fundação Araucária. Citation Format: Giovani Marino Favero, Tharcisio Citrangulo Tortelli, Jr., Daniel Fernandes, Ana Paula Prestes, Louise N.B. Kmetiuk, Andreia Hanada Otake, Luciana N.S. Andrade, Daniele de Paula Faria, Camila de Godoi Carneiro, Alexandre Teles Garcez, Fabio L.N. Marques, Roger Chammas. 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A50.

β1–6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B>A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor micro-environments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http:/www.jhc.org . Please visit this article online to view these materials. (J Histochem Cytochem 55: 1015–1026, 2007)

Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor- and non-receptor-mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S-91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S-91 melanoma cell proliferation in vitro (EC50 = 10-7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT-1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10-10 m) in S-91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT-1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.

SummaryBackground Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics’ both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However, iPSCs derived from CSB patient’s fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.

Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the NIH/3T3 fibroblast cell line could inhibit renal tumor growth in vivo. NIH/3T3 cells were transduced with retroviral vectors containing the murine endostatin (ES) gene. SCID mice bearing CaKi-1 derived tumors were given a subcutaneous injection of either ES-transduced cells or control cells and were monitored for tumor growth. At the end of the in vivo experiment, the mean tumor volume of treated mice was 51.6 +/- 2.4 mm(3), while the tumor volume of control was 234.5 +/- 14.8 mm(3). Microvascular density was significantly decreased on treatment (control 9.79 vs. ES 2.53%, < 0.001) accompanied by a 23-fold increase in intraturmoral necrotic area and a 2.94-fold increase in the apoptotic index, determined by immunohistochemistry with anti- activated caspase-3. Apoptotic cells were found in foci enriched in infiltrating leukocytes. in conclusion, retroviral endostatin gene transfer led to secretion of functional endostatin that was sufficiently active to inhibit tumor angiogenesis and tumor growth. A second mechanism may also be implied in endostatin-dependent tumor regression, associated with tumor infiltration of leukocytes. Besides its antiangiogenic properties, endostatin may be a promising adjuvant to immunotherapy. IPEN CNEN SP, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, Brazil CEPID FAPESP, Ctr Terapia Celular, São Paulo, Brazil Univ São Paulo, Fac Med, Dept Radiol, Expt Oncol Lab, São Paulo, Brazil Universidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, Brazil Web of Science

High concentrations of certain fatty acids can cause cell death via apoptosis or necrosis. The aim of this study was to investigate the toxicity of saturated and unsaturated fatty acids on melanoma cell lines, which was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. Evidence is presented that saturated and unsaturated fatty acids exert toxic effects on melanoma cells through loss of membrane integrity and/or DNA fragmentation. Arachidonic and linoleic acids were the most effective in decreasing the number of viable S91 murine melanoma cells, causing loss of membrane integrity and DNA fragmentation at 100 microM concentration already after 24 h in culture. In B16F10 murine melanoma cells, palmitic acid was the most toxic, inducing cell death by both apoptosis and necrosis. The human melanoma cell lines were more resistant to the toxic effect of fatty acids. In SK-Mel 23 cells, indications of cytotoxicity were detected only after 48 h treatment with arachidonic, linoleic, palmitic and palmitoleic acids at 200 microM concentration. Linoleic acid was the most toxic for this cell line. In SK-Mel 28 human cells, only palmitic acid caused a significant decrease of the number of viable cells, inducing DNA fragmentation after 24 and 48 h treatments at 200 microM concentration.

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 μg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.

Tumores são definidos como microambientes compostos não só pelas células malignas, mas também por células endoteliais, fibroblastos e leucócitos, que promovem o crescimento tumoral e a angiogênese. Galectina-3, uma proteína que se liga a b- galactosídeos, é abundantemente expressa por monócitos/macrófagos, dentre outros leucócitos. Inúmeras evidências sugerem que galectina-3 atua como uma molécula reguladora da resposta inflamatória. Tendo em vista que o infiltrado inflamatório pode promover a progressão de tumores, o objetivo do presente trabalho foi avaliar se galectina-3, expressa tanto pela célula tumoral como pelas células estromais, modula o crescimento de melanoma. Para tal, células de melanoma murino Tm1 foram transfectadas com o gene de galectina-3. Ambos clones celulares (galectina-3 positivos e negativos) foram injetados na intrafáscia ou no subcutâneo de camundongos (fêmeas) C57BL/6 selvagens e/ou nocautes para o gene de galectina-3 para análise da implantabilidade e crescimento tumoral. Com relação à implantabilidade, não foi observado diferenças no estabelecimento de uma massa tumoral proliferativa em animais selvagens inoculados com células Tm1 transfectadas ou não com o gene de galectina-3 em animais selvagens. Em relação a taxa de crescimento dos tumores, nenhum animal nocaute inoculado com células Tm1 galectina-3 positivas apresentou tumores de dimensões mensuráveis até o 11º dia pós-inóculo. Independente do nível de expressão de galectina- 3 pela célula tumoral, os tumores originados nos animais nocautes apresentavam menor massa em gramas comparados ao grupo selvagem, sugerindo que galectina-3 expressa pelas células estromais promove o crescimento tumoral. Ainda, os tumores originados nos animais nocautes e no grupo selvagem inoculado com células Tm1 galectina-3 positivas apresentavam menor extensão de área necrótica do que os animais selvagens inoculados com células Tm1 galectina-3 negativas. Interessantemente, os animais selvagens e nocautes inoculados com células Tm1 galectina-3 positivas apresentaram tumores com menor área vascular e menor número de estruturas vasculares funcionais quando comparados aos animais selvagens inoculados com células Tm1 galectina-3 negativas. A análise de expressão gênica nos tumores mostrou que os níveis relativos de RNAm de VEFG (fator de crescimento de endotélio vascular) foram menores nos animais inoculados com células Tm1 galectina-3 positivas em relação aos inoculados com células Tm1 galectina-3 negativas, indicando que galectina-3 expressa pelas células tumorais atua como uma molécula anti-angiogênica. Finalizando, o presente trabalho sugere que galectina-3 pode atuar como uma molécula pró- ou anti-tumoral, dependendo do tipo celular que a expressa no microambiente tumoral. Tumors have been described as microenvironments composed not only by malignant cells, but also by endothelial cells, fibroblasts and leukocytes, which can promote tumor growth and angiogenesis. Galectin-3, a b-galactoside binding protein, is expressed by monocytes/macrophages and others leukocytes. In fact, several lines of evidence suggest that galectin-3 act as master regulators of the inflammatory response. Based on the fact that the inflammatory infiltrate can promote tumor progression, the proposal of this study was to evaluate if galectin-3, either from tumor or stromal cells could modulate melanoma growth. Tm1 murine melanoma cell line was transfected with the galectin-3 gene. Both clones (galectin-3 negative and positive) were injected in the foot pad or subcutaneous in female C57BL/6 wild-type (WT) and galectin-3 knock-out (KO) mice to tumor engraftment and growth analysis. There was no difference in the tumor engraftment between animas injected with Tm1 galectin-3 positive or negative cells. In addition, any knock-out mice injected with galectin-3 positive cells had measurable tumors up to day 11 post inoculation. Regardless the galectin-3 expression level in the melanoma cell, tumors from galectin-3 KO mice were smaller than those from WT animals, suggesting that galectin-3 expressed by stromal cells promotes tumor growth. Moreover, tumor necrotic area was smaller in KO mice and in wild-type animals injected with Tm1 galectin-3 positive cells compared to wild type animals injected with Tm1 galectin-3 negative cells. Interestingly, both vascular area and the number of functional vessels in animals injected with galectin-3 positive Tm1 cells were smaller in WT as well as in KO mice compared to the same animals injected with galectin-3 negative Tm1 cells. Gene expression analysis showed that VEGF (vascular endothelial growth factor) mRNA levels were smaller in wild type animals injected with Tm1 galectin-3 positive cells compared to those injected with Tm1 galectin-3 negative cells, indicating that galectin-3 expressed by tumor cells can act as an anti-angiogenic molecule. The present study suggests that galectin-3 can act either as a pro or antitumoral molecule, depending on which type of cell (tumoral or stromal) this lectin is expressed within tumor microenvironment.

Introdução: A despeito da melhora na evolução dos pacientes infectados pelo HIV-1, ainda persistem desafios, como adesão ao tratamento antirretroviral (ART), presença de comorbidades. Nesse estudo, avaliamos a incidência de câncer, mortalidade em pessoas vivendo com HIV/aids (PVHA) em 31 anos, além de analisar os fatores de risco associados à sobrevida e os desfechos clínicos. Metodologia: Trata-se de um estudo observacional retrospectivo de coorte realizado no Ambulatório de Manifestações Dermatológicas do Serviço de Dermatologia Especializada no Instituto Central do Hospital das Clínicas da FMUSP no período de 1989 a 2020 das PVHA que desenvolveram o câncer que foram estudos. Os bancos de dados eletrônicos forneceram as características sociodemográficas, clínicas, terapia antirretroviral, fatores de risco e a sobrevida de PVHA. As análises estatísticas foram realizados testes paramétricos, não paramétricos e curvas de sobrevivência nos softwares estatísticos GraphPad (versão 9) e STATA (versão 14). Resultados: No total, 677 PVHA em seguimento no ambulatório no período de 31 anos, houve 55 casos incidentes de câncer (8,12%). Os diagnósticos mais frequentes foram por neoplasia do colo do útero (66,66%) e a neoplasia anal (40%), sendo 27,27% câncer definidor de aids e 72,73% não definidor de aids. Os fatores de risco mais importantes para o estudo foram o tabagismo (p=0,03) e a presença de oncovírus sendo o HPV (p

O melanoma é classificado como o tipo mais agressivo do câncer de pele. A terapia alvodirigida combinada, com inibidores de BRAF e MEK, considerada a opção terapêutica padrão para o tratamento de pacientes em estágios avançados que possuem mutação em BRAF, resulta em uma taxa de sobrevida em 5 anos de apenas 20%. A transferência de informações via Vesículas Extracelulares (VEs) tem sido descrita como um dos mecanismos capazes de modular os processos de resistência e progressão maligna. No melanoma, a presença de moléculas antitumorais em VEs está associada a maiores taxas de resposta ao tratamento e melhores prognósticos. O miR-195, considerado um supressor tumoral, é down-regulado em melanomas e sua superexpressão resulta na diminuição da proliferação celular e aumento da sensibilidade à quimioterapia com agentes alquilantes e inibidores de BRAF. Neste sentido, o objetivo deste trabalho foi avaliar o efeito da superexpressão de miR-195 na carga de VEs liberadas por células de melanoma e o papel destas partículas na regulação da sensibilidade de células naive a terapia alvo-dirigida combinada. Diferentes linhagens de melanoma humano (A375, SKMel-05, SKMel-28 e UACC-62) foram transfectadas com o miR-195, resultando no aumento da concentração de VEs liberadas, as quais apresentaram menor tamanho em relação ao controle. Análises de expressão gênica por RT-qPCR mostraram que a transfecção de miR-195 induz o aumento da expressão de GTPases envolvidas na biogênese de exosomos, enquanto genes envolvidos na produção de microvesículas têm sua expressão diminuída. VEs secretadas por células transfectadas com este miRNA foram capazes de transferir seu efeito citostático, induzindo a expressão de genes antiproliferativos, como CDKN1A e CDKN3, e diminuindo a Página | 2 expressão de genes pró-proliferativos, como CCND1 e YAP1, em células naive. VEs contendo miR-195 também impactaram o crescimento tumoral in vivo, diminuindo a cinética de crescimento dos tumores. Além disso, o tratamento com VEs miR-195 foi capaz de aumentar a sensibilidade de células naive a terapia alvo-dirigida combinada, resultando em maior expressão de BAD, menor expressão de BCL2L1 e consequentemente maior porcentagem de morte celular, além da diminuição do potencial clonogênico das células. Adicionalmente, com relação ao mecanismo de regulação de miR-195, dados do TCGA mostraram maiores níveis de metilação deste miRNA em melanomas em relação a tecidos saudáveis, entretanto, sua expressão foi restaurada após o tratamento das células com Azacitidina em apenas uma das três linhagens testadas. Análises in silico mostraram que diversos lncRNAs capazes de inibir miR195 encontram-se superexpressos em melanoma, representando outro possível mecanismo de down-regulação. Em resumo, a superexpressão de miR-195 representa uma estratégia promissora para sensibilizar células de melanoma a terapia via transferência horizontal de informação Melanoma is considered the most aggressive form of skin cancer. Combined targeted therapy, with BRAF and MEK inhibitors, considered the standard treatment for BRAF-mutated metastatic melanoma patients, results in 5 years overall survival of 20%. Extracellular Vesicles (EVs)-mediated transfer of information has been reported as a mechanism capable of regulating tumor resistance and progress. The presence of antitumoral molecules within EVs is related to increased response rates and better prognosis for melanoma patients. MiR-195, considered a tumor suppressor miRNA, is down-regulated in melanoma and its overexpression results in decreased cells proliferation and increased sensitivity to chemotherapy with alkylating agents and BRAF inhibitors. We aimed to analyze whether miR-195 overexpression could modulate EVs cargo and the role of these particles in naïve cells response to combined targeted treatment. A375, SKMel-5, SKMel-28 and UACC-62 cells were transfected with miR-195, resulting in increased EVs release and decreased EVs size. Gene expression analysis by RT-qPCR showed that miR-195 mimic induces the expression of GTAPases involved in exosomes biogenesis, while genes involved in microvesicles shedding are down-regulated upon transfection. EVs derived from cells overexpressing miR-195 could transfer its cytostatic effect, inducing the expression of anti-proliferative genes, such as CDKN1A and CDKN3, and inhibiting proproliferative genes, including CCND1 and YAP1, in naïve cells. EVs containing miR-195 also impaired in vivo tumor growth, reducing tumors growth kinetics. Treatment with miR-195 EVs could also sensitize naive cells to combined targeted therapy, resulting in higher expression of BAD, inhibition of BCL2L1 and consequently increased cell death, in addition to decreased Página | 4 clonogenic potential. Additionally, regarding the mechanisms of miR-195 regulation, TCGA data showed higher levels of methylation of this miRNA in melanoma tissues compared to controls, nevertheless, treatment of melanoma cells with Azacitidine only restored miR-195 expression in one out of three cell lines. In silico analysis showed that several lncRNAs capable to sponge miR-195 are overexpressed in melanomas, representing another possible mechanism of down-regulation. In summary, miR-195 overexpression represents a promising strategy to sensitize melanoma cells through horizontal transfer of information

O câncer é uma doença complexa, frequentemente sem cura e que está presente em todas as populações e em todas as idades. O câncer gera enormes perdas econômicas e emocionais, sendo que seus tratamentos atuais são caros e, tipicamente, pouco efetivos em casos avançados. O melanoma é um câncer de pele agressivo na sua evolução e frequentemente mortal para os hospedeiros, que apresentam a doença metastática, essa doença ainda possui poucos tratamentos efetivos. Frente a esta situação, é necessário a criação e desenvolvimento de novas abordagens com maior eficiência no tratamento do câncer. O Interferon beta (IFN) é uma citocina imuno-estimuladora com efeito anti- tumoral conhecido, porém sua aplicação sistêmica é tóxica. Nosso grupo propõe o uso de terapia gênica utilizando vetores adenovirais portadores do transgene terapêutico IFN (Ad-mIFN), assim, proporciona a produção do IFN de modo localizado e transiente, o que pode conferir um escape do efeito tóxico sistêmico. Os dados do grupo mostram que, em modelos murinos, a terapia gênica com Ad-mIFN in vitro resulta em morte celular e no modelo in situ induz retardo na progressão tumoral do melanoma. A investigação nesta tese teve como objetivo estudar os efeitos autócrinos e parácrinos nas células do microambiente e tumorais do melanoma após terapia gênica com IFN e a influência do seu receptor IFNAR1 neste contexto. Para investigar a influência da sinalização do IFN nos efeitos autócrinos e parácrinos, realizou-se o knockout do IFNAR1 em células da B16 e do microambiente (tEnd, NIH3T3) utilizando a tecnologia de CRISPR/Cas9. Os resultados mostraram que células transduzidas pelo Ad-mIFN necessitam da produção e liberação do IFN para a ativação da via do IFN/, pela ligação com os receptores IFNAR1/IFNAR2 e finalmente induzir a célula a morte, mecanismo conhecido como autócrino. As células transduzidas pelo Ad-mIFN também induzem efeito parácrino por inibir as células próximas, pelo mecanismo parecido com o autócrino, mas direcionado a outras células próximas. As células knockout para o IFNAR1 ficaram bloqueadas de receber os efeitos autócrinos e parácrinos do Ad-mIFN e da proteína recombinante do IFN (rIFN), porém mesmo sem o IFNAR1 elas continuam produzindo o efeito parácrino. Esses dados foram observados em células tumorais e células endoteliais e de fibroblastos. Outro estudo in vitro da terapia com Ad-mIFN e Ad-p19 na angiogênese utilizando células endoteliais, descobriu uma linhagem endotelial não responsiva ao tratamento com Ad-mIFN e a proteína IFN sozinha, porém essas células sofriam inibição quando colocadas em ensaios de efeito parácrino pela linhagem de melanoma transduzida com o Ad-mIFN. A observação desse fenômeno sugere a possibilidade da existência de outros fatores além do IFN, produzidos pelas células de melanoma transduzidas com Ad-mIFN, que estariam envolvidos no efeito parácrino. Ensaios de expressão gênica da linhagem de melanoma transduzida pelo Ad-mIFN mostraram aumento significativo de TRAIL, que pode ser um candidato para estar envolvido no efeito parácrino. Juntos, estes resultados indicam que a presença de IFNAR1 não é essencial para produção dos fatores necessários para induzir o efeito parácrino, porem a presença de IFNAR1 é necessário para a célula sofrer este efeito. Futuramente, as linhagens deficientes em IFNAR1 podem ser avaliadas em ensaios in vivo para revelar o papel do IFNAR1 nos efeitos autócrinos e parácrinos entre células tumor e seu microambiente após tratamento, incluindo com Ad- mIFN Cancer is a complex disease, often without cure, which is present in all populations and at all ages. Cancer generates enormous economic and emotional losses, and its current treatments are expensive and, typically, ineffective in advanced cases. Melanoma is an aggressive skin cancer in its evolution and often deadly for the hosts that present metastatic disease since few effective treatments are available. Faced with this situation, it is necessary to create and develop new approaches with greater efficacy in the treatment of cancer. Interferon beta (IFN) is an immunostimulatory cytokine with a known anti-tumor effect, but its systemic application is toxic. Our group proposes the use of gene therapy using adenoviral vectors that carry the therapeutic transgene IFN (Ad-mIFN), thus providing the production of IFN in a localized and transient way, which should avoid the systemic toxic effect. Our studies have shown that, in murine models, gene therapy with Ad-mIFN in vitro results in cell death and, in the in situ model, delays the progression of melanoma tumors. The research performed in this thesis aimed to study the autocrine and paracrine effects in the microenvironment and tumor cells of melanoma after IFN gene therapy and the role of the IFNAR1 receptor. To investigate the influence of IFN signaling on autocrine and paracrine effects, we performed IFNAR1 knockout on B16 and microenvironment cells (tEnd, NIH3T3) using the CRISPR / Cas9 technology. The results showed that cells transduced by Ad- mIFN require the production and release of IFN for activation of the IFN/ pathway, by binding with the IFNAR1/IFNAR2 receptors and finally inducing cell death, a mechanism known as autocrine. The cells transduced by Ad-mIFN also induce a paracrine effect by inhibiting nearby cells, by a mechanism similar to the autocrine but directed at other nearby cells. The cells with IFNAR1 knockout were blocked from receiving the autocrine and paracrine effects of Ad-mIFN and recombinant IFN protein (rIFN), however even without IFNAR1 they continue to produce the paracrine effect. These data were observed in tumor cells and endothelial and fibroblast cells. Another in vitro study of therapy with Ad-mIFN and Ad-p19 in angiogenesis identified an endothelial cell line unresponsive to treatment with Ad-mIFN and the IFN protein alone, however these cells suffered inhibition when tested for the paracrine effect provided by the melanoma cell line transduced with Ad-mIFN. The observation of this phenomenon suggests the existence of factors other than IFN, produced by melanoma cells transduced with Ad-mIFN, which would be involved in the paracrine effect. Gene expression assays of the melanoma lineage transduced by Ad-mIFN showed a significant increase in TRAIL, which may be a candidate for involvement in the paracrine effect. Together, these results indicate that the presence of IFNAR1 is not essential for the production of the factors necessary to induce the paracrine effect, however the presence of IFNAR1 is necessary for the cell to suffer this effect. In the future, IFNAR1-deficient strains can be evaluated in in vivo assays to reveal the role of IFNAR1 in the autocrine and paracrine effects between tumor cells and their microenvironment after treatment, including with Ad-mIFN

O câncer de cervical é causado por uma infecção persistente por algum dos tipos oncogênicos de Papilomavírus Humano (HPV) e continua a ser um problema de saúde pública, especialmente nos países em desenvolvimento. Por Peptide Phage Display, foram selecionadas sequências de peptídeos com afinidade de ligação a linhagens celulares ou a tumores associados ao HPV. Dentre elas, foi possível identificar sequências contidas em moléculas importantes para a progressão de tumores, como a -manosidase e triptase, enzimas que desempenham um papel importante na progressão tumoral. Para caracterizar o efeito da inibição de -manosidase II na terapia de tumores cervicais em camundongos, utilizamos a droga Swainsonina (SW), previamente descrita como inibidor desta enzima. Nós testamos o efeito desta droga tratando animais inoculados com células tumorais que expressam os oncogenes E6 e E7 de HPV16. Observamos que os animais tratados com Swainsonina apresentaram crescimento tumoral significativamente mais rápido do que os animais controle. Investigando os mecanismos por trás desse efeito, descobrimos que embora SW module parcialmente os macrófagos associados aos tumores, o tratamento induz o acúmulo de células com fenótipo mieloderivado supressor no baço dos animais, potencializando o efeito tolerogênico dos tumores sobre o sistema imune. Sendo assim, sugerimos cautela no uso deste fármaco para a terapia de pacientes com tumores HPV&#43. Outro braço do trabalho foi avaliar o papel da triptase, que é produzida por mastócitos, no infiltrado inflamatório. Para isto padronizamos um modelo de co-cultura de esferóides tumorais da linhagem TC-1 com a linhagem de mastócitos murinos PT18. Através deste modelo pudemos observar que a linhagem tumoral consegue induzir a desgranulação dos mastócitos independentemente de anticorpos. Além disso, quando em co-cultura, a linhagem tumoral parece estar aumentando a meia-vida dos mastócitos e estimulando a proliferação destes. Em experimentos in vivo observamos que tumores induzidos com as células PT18 e TC-1 cresceram mais rapidamente do que tumores induzidos apenas com TC-1. Através da imunofenotipagem dos tumores ficou evidenciado um aumento de células CD31+ e no infiltrado inflamatório total de tumores induzidos com o co-cultivo. Justificando o fato destes crescerem mais rapidamente, sugerindo que os mastócitos podem ter efeitos tanto proliferativos quanto no processo de angiogênese tumoral. Cervical cancer is caused by a persistent infection by some of the oncogenic types of Human Papillomavirus (HPV) and continues to be a public health problem, especially in developing countries. By Peptide Phage Display peptide sequences were selected with binding affinity to cell lines or to HPV-associated tumors. Among them, it was possible to identify sequences contained in molecules important for the progression of tumors, such as -mannosidase and tryptase, enzymes that play an important role in tumor progression. To characterize the effect of -mannosidase II inhibition on cervical tumor therapy in mice, we used the drug Swainsonina (SW), previously described as an inhibitor of this enzyme. We tested the effect of this drug by treating animals inoculated with tumor cells expressing HPV16 E6 and E7 oncogenes. We observed that Swainsonine treated animals had significantly faster tumor growth than control animals. Investigating the mechanisms behind this effect, we found that although SW partially modulates tumor-associated macrophages, the treatment induces the accumulation of cells with suppressive myeloderivative phenotype in the animals spleens, enhancing the tolerogenic effect of tumors on the immune system. Therefore, we suggest caution in the use of this drug for the therapy of patients with HPV&#43 tumors. Another arm of the study was to evaluate the role of tryptase, which is produced by mast cells, in the inflammatory infiltrate. For this we standardized a co-culture model of tumor spheroids of the TC-1 lineage with the murine mast cell line PT18. Through this model we could observe that the tumoral lineage can induce mast cell degranulation independently of antibodies. In addition, when co-cultured, the tumoral lineage appears to be increasing the half-life of mast cells and stimulating the proliferation of these. In in vivo experiments we observed that tumors induced with PT18 and TC-1 cells grew faster than tumors induced only with TC-1. Tumor immunophenotyping revealed an increase in CD31&#43 cells and in the total inflammatory infiltrate of tumors induced with co-culture. Justifying the fact that they grow faster, suggesting that mast cells can have both proliferative effects and the process of tumor angiogenesis.

Melanoma é o mais letal tipo de câncer de pele e é originado a partir da transformação de melanócitos e várias alterações gênicas estão relacionadas a esse processo. Mutações e aumento no número de cópias de genes Ras é um dos mais importantes eventos, que ocorre em cerca de 91% dos casos de melanoma. Há vários efetores de Ras com potencial terapêutico. A família RASSF (Ras-association Family) é constituída por 10 membros (RASSF1-10), a maioria deles está relacionada a funções de supressores tumorais. RASSF9 é um membro do grupo N-terminal, que primeiramente foi descrito como P-CIP1, uma proteína envolvida no trafego de vesículas e foi demonstrada interagindo com Ras. A expressão de RASSF9 em camundongo é predominante no epitélio estratificado, onde é crucial para a homeostase tecidual. Além disso, a expressão de RASSF9 é maior em pessoas que tem uma maior exposição solar. Entretanto, não há informações sobre a função de RASSF9 em melanoma. Nós desenvolvemos uma linhagem de melanoma murino não metastática, Tm1.Luc, nocaute de RASSF9 com CRISPR/Cas9. Dados do TCGA demonstraram que RASSF9 está discretamente superexpressa em amostras de melanoma e que não há correlação entre o nível de expressão do mRNA e a fosforilação de seu promotor. Nós observamos uma redução na capacidade proliferativa in vitro e in vivo das células deficientes de RASSF9 que pode ser explicada pela supressão da via Ras/Raf/MERK/ERK, o que foi confirmado pro Western-Blot. Apesar da relevância de RASSF no processo de morte celular descrita em outros sistemas experimentais, nossos dados não permitem concluir, até o momento, se RASSF9 é capaz de modular significativamente esse processo celular em melanoma. Em resumo, nossos dados segurem que RASSF9 atua na regulação de Ras promovendo a proliferação de melanoma. Melanoma is the most lethal skin cancer and is originated from melanocyte transformation. Several genetic changes are related to this process. Mutation or copy-number amplification of Ras is one of the most important events, which occurs in 91% of the cases. Therefore, regulators and/or partners of Ras could potentially be good drug targets for treatment. The Ras-association family (RASSF) comprises ten proteins (RASSF1-10), several of them are related to tumor suppression. RASSF9 is a member of N-terminal RASSF proteins and was first described as P-CIP1, a protein that is involved in vesicle trafficking, and has been shown to bind Ras proteins. In mice RASSF9 is predominantly expressed in the stratified epithelium, where it is crucial for epidermal homeostasis. Moreover, it was confirmed the RASSF9 gene is upregulated in samples of higher sun-exposure people. However, the function and importance of RASSF9 in melanoma is still obscure. Aiming to understand the role of RASSF9 in melanoma growth and metastasis, we have developed RASSF9-knockout TM1-luciferase cells by using CRISPR/Cas9 system. TCGA data analysis shows RASSF9 is upregulated in melanoma samples but without correlation between mRNA expression and promoter phosphorylation. We observed in RASSF9 knockout cells a reduction in proliferative capacity in vitro and in vivo that could be explained by suppression of Ras/Raf1/MEK/ERK pathway, as confirmed by Western-blot. Although previous evidence of the relevancy of RASSF members in cell death pathway, we were not able to conclude whether RASSF9 acts as a modulator in this cellular process in our experimental system. In summary, our results suggest that RASSF9 plays a role in Ras activation leading to proliferative capacity in melanoma cells.

O câncer colorretal é o terceiro em mortalidade, e apesar das classificações moleculares, entre 30% e 40% dos pacientes apresentam recidiva após quimioterapia, o que aponta a necessidade de novas estratégias terapêuticas. É crescente o estudo de vetores virais para a terapia gênica do câncer, e dados do nosso laboratório mostraram que a entrega combinada dos genes Arf (supressor tumoral e parceiro funcional de p53) e interferon-beta (IFNbeta, citocina imunomodulatória), via vetores adenovirais sob o controle do promotor responsivo à p53 causou indução de morte celular massiva e um efeito imunomodulatório importante. Entretanto, esses resultados foram observados em modelos murinos. O presente trabalho avalia como a combinação da modulação das vias de p53 e IFNbeta impacta células de carcinoma colorretal humano em ensaios in vitro. Para isto, utilizamos nosso adenovírus para transferir p53 (p53) e IFN? como uma estratégia combinada para induzir a morte celular na presença de um modulador imunológico. As linhas celulares HCT116, HCT116p53-/- toleraram uma MOI (multiplicidade de infecção) de 25, enquanto uma MOI de 100 foi suportada por HT29 (mutante p53 R273H). O promotor PG, dependente de p53, como esperado, proporcionou níveis de expressão de GFP reduzidos em HCT116 p53 - / - e HT29, mas a expressão foi aumentada em todas as linhas celulares quando co-transduzida com o vetor codificando p53, mas não com IFNbeta. Em geral, HCT116 e HCT116p53-/- foram mais sensíveis à transferência de p53, enquanto o HT29 foi particularmente afetado pela combinação de p53 + IFNbeta. Esta tendência reflectiu-se na viabilidade celular (curva de crescimento, MTT), formação de colónias e acumulação de células hipodiplóides. Os níveis de morte celular correlacionaram-se com a atividade da caspase 3/7 e coloração com o Anexina V. Os marcadores de morte imunogênico ATP e calreticulina também foram aumentados, especialmente para o HT29 tratado com INFbeta sozinho ou em combinação com p53. Além disso, observamos aumento da expressão de TP63, TP73, bem como alvos transcricionais de p53, como p21 e NOXA, enquanto SESTRIN foi reduzido em ambas as linhagens HCT, mas aumentou em HT29. Adicionalmente, testamos a combinação p53 + IFNbeta em associação com quimioterápicos, revelando cooperação entre a transferência gênica e a doxorrubicina ou 5-FU, mesmo nas menores doses testadas. Ensaios em andamento incluem a avaliacao da ativacao de celulas dentriticas humanas expostas para as celulas tumorais tratadas com os vetores portadores de p53 e/ou IFNbeta. Com este projeto, nossa abordagem de transferência gênica revelará a resposta aos transgenes em modelo humano e também abrirá o caminho para estudar o envolvimento do sistema imune humano Colorectal cancer is the third leading cause of cancer death and despite new molecular classifications, 30% to 40% of all patients will relapse after chemotherapy, pointing the need for new and innovative therapeutic strategies. The number of studies about viral vectors for gene therapy is growing, and previous data from our laboratory indicates that the combined delivery of Arf (a tumor suppressor gene and functional partner of p53) and interferon beta (IFNbeta, an immunomodulator cytokine), under a p53 responsive promoter, induced massive cell death and an important immunomodulatory effect. However, these results were only observed in murine models. Here we present an RGD-modified adenovirus whose transgene is under the control of a p53 responsive promoter (PG), used to transfer p53 (p53) and IFNbeta as a combined strategy to induce cell death in the presence of an immune modulator. Cell lines HCT116, HCT116 p53 -/- tolerated a MOI (multiplicity of infection) of 25, while a MOI of 100 was supported by HT29 (mutant p53 R273H). The p53-dependent PG promoter, as expected, provided reduced GFP expression levels in HCT116 p53 -/- and HT29, but the expression was increased in all cell lines when co-transduced with the p53 vector, but not with IFNbeta . In general, HCT116 and HCT116p53-/- were more sensitive to p53 gene transfer while HT29 was particularly affected by the combination of p53 + IFNtbeta. This trend was reflected in cell viability (growth curve, MTT), colony formation and accumulation of hypodiploid cells. Levels of cell death correlated with caspase 3/7 activity and staining with Annexin V. Immunogenic markers were also increased, especially for HT29 treated with INFbeta and p53/IFNbeta, as we detected exposure of calreticulin and release of ATP. Also, we observed increased expression of TP63, TP73 as well as p53 transcriptional targets, such as p21 and NOXA; SESTRIN was reduced in both HCTs but increased in HT29. Additionally, we tested the p53+IFNb combination in association with chemotherapeutics, revealing cooperation between gene transfer and doxorubicin or 5-FU even at the lowest doses tested. Ongoing studies include the evaluation of human dendritic cells exposed to tumor cells treated with the vectors for p53/IFNbeta. In conclusion, our combined gene transfer approach has potentiated the killing of colorectal cancer cell lines and may provide an immunomodulatory effect

NTRODUÇÃO: O melanoma é um câncer de alta mortalidade tanto na medicina quanto na veterinária, devido à baixa resposta às terapias utilizadas e à capacidade de evolução metastática da doença. Avanços no campo da oncologia têm mostrado que os agentes que têm como alvo componentes do sistema imunológico, bem como as terapias molecularmente dirigidas, são muito promissores no tratamento do melanoma. Nosso grupo tem desenvolvido vetores virais para a transferência gênica de fatores antitumorais. O aprimoramento no vetor adenoviral inclui a inserção do tripeptídeo RGD, que permite um amplo tropismo de transdução, e o uso de um promotor responsivo a p53 para controlar expressão do gene terapêutico. Visto que o melanoma canino pode ser um modelo experimental por se tratar de um câncer de ocorrência espontânea e de comportamento biológico semelhante ao melanoma humano, procuramos testar nossa abordagem, que chamamos AdRGD-PG, neste modelo. MÉTODOS: Para isso foram estabelecidas linhagens celulares de melanoma canino para a determinação da capacidade de transdução pelos vetores adenovirais. Em seguida foi realizado o sequenciamento dos exons 4 - 8 do gene TP53 e a avaliação da expressão de genes da via de TP53 induzidos por doxorrubicina e Nutlin-3. RESULTADOS: As quatro linhagens de melanoma canino estabelecidas possuem capacidade tumorigênica e de serem transduzidas pelos vetores adenovirais. Não foram identificadas alterações na sequência do TP53 na região avaliada e a doxorrubicina promoveu aumento da expressão dos transgenes dirigidos pelo promotor PG e a ativação de genes da via de TP53. CONCLUSÕES: A funcionalidade desta plataforma adenoviral aprimorada abre oportunidades para estudos da transferência gênica, incluindo da combinação p19ARF/IFN-beta, nas linhagens estabelecidas. Com o sucesso destas análises, teremos um importante modelo experimental a ser utilizado no desenvolvimento de novas terapias para o melanoma INTRODUCTION: Melanoma is a cancer of high mortality both in human and veterinary medicine due to the poor response to therapies and the metastatic evolution of the disease. Advances in the field of oncology have shown that agents that target immune system components, as well as molecularly targeted therapies, are very promising for the treatment of melanoma. Our group has developed viral vectors for gene transfer of antitumor factors. The improvement in the adenoviral vector includes the insertion of the RGD tripeptide, which allows a broad transduction tropism, and the use of a p53 responsive promoter to control therapeutic gene expression. Canine melanoma may be considered as an experimental model since it is a spontaneous cancer with biological behavior similar to human melanoma, thus we aim to test our approach, which we call AdRGD-PG, in this model. METHODS: For this purpose, canine melanoma cell lines were established before determining the transduction capacity of adenoviral vectors. Next, sequencing of exons 4-8 of the TP53 gene and evaluation of the expression of genes in the TP53 pathway upon induction with doxorubicin and Nutlin-3 were performed. RESULTS: The four established canine melanoma lines have tumorigenic capacity and are transduced by the adenoviral vectors. No changes were identified in the TP53 sequence in the assessed region and doxorubicin promoted increased expression of the transgenes directed by the PG promoter as well as activation of the TP53 pathway genes. CONCLUSIONS: The functionality of this improved adenoviral platform opens up opportunities for gene transfer studies, including the p19ARF / IFN-beta combination, in the established cell lines. With the success of these analyses, we have an important experimental model to be used in the development of new therapies for melanoma

O desenvolvimento do câncer cervical está associado à infecção por alguns tipos de Papilomavírus Humano (HPV). Entre os mecanismos de carcinogênese associados ao HPV incluem-se alterações em moléculas que modulam a manutenção de componentes da matriz extracelular (MEC), como as metaloproteinases de matriz (MMP) e alguns de seus reguladores. A proteína RECK (reversion inducing cysteine rich protein with kazal motifs) apresenta função essencial na remodelação tecidual e na angiogênese fisiológica ou tumoral, através da regulação pós-transcricional da atividade de MMP-2, MMP-9 e MMP-14 (MT1-MMP). Resultados publicados previamente por nosso grupo apontam para a correlação entre a expressão da oncoproteína E7 de HPV16, a alta expressão e atividade de MMP-9 e a baixa expressão de seus reguladores, TIMP-2 e RECK. A expressão de RECK também é baixa em lesões do colo uterino de alto grau e em amostras de câncer cervical, quando comparadas a amostras de pacientes com cervicite. O presente estudo visa determinar o papel de RECK no processo de tumorigênese mediado por HPV. Para isto, estabelecemos linhagens derivadas de tumor de colo de útero (SiHa, SW756 e C33A) que superexpressam RECK a partir de transdução com lentivírus. Os efeitos da superexpressão de RECK sobre o potencial tumorigênico de SiHa, SW756 e C33A foram avaliados em modelos de estudo in vivo e in vitro. De maneira geral, a superexpressão de RECK foi associada com a capacidade reduzida de invasão em câmara de Matrigel® e de formação de colônia independente de ancoragem. Ainda, camundongos nude inoculados s.c. com células tumorais superexpressando RECK apresentaram atraso no estabelecimento e crescimento tumoral e sobrevida global estendida quando comparados aos controles. Ambos tumores derivados de SiHa RECK e SW756 RECK apresentaram redução na frequência de células tumorais e endoteliais, ao passo que mostrarm aumento no infiltrado inflamatório. Esta observação foi acompanhada de redução na população de neutrófilos e potenciais células mieloderivadas supressoras em tumores de ambas as linhagens. Em tempo, analisamos séries de dados de expressão de CIN e carcinomas cervicais do banco de dados GEO e verificamos que a hipermetilação do gene RECK e a inibição da expressão de mRNA de RECK são eventos precoces no desenvolvimento do câncer de colo de útero. Avaliamos que a baixa expressão de RECK foi associada a progressão de lesões CIN3+ e ao aumento de metástases em linfonodo pélvico em pacientes com câncer de colo de útero. Ademais, notamos que o tratamento com quimio radioterapia levou ao aumento dos níveis de mRNA de RECK em um outro grupo de pacientes com câncer de colo de útero. Concluímos que a superexpressão de RECK (i) reduz o potencial tumorigênico de linhagens celulares derivadas de colo de útero independente do status de infecção por HPV e que (ii) o seu efeito sobre as populações intratumorais se mostrou específico para as linhagens infectadas por HPV. Estes resultados apontam para uma possível interação entre as alterações no microambiente tumoral associadas ao HPV e a função de RECK. Finalmente, a regulação negativa da expressão de RECK é um evento precoce na história natural do câncer cervical. Persistent infection with high-risk Human Papillomavirus (HPV) types is the main etiologic factor for the development of cervical cancer. The HPV carcinogenic mechanisms include alterations in extracellular matrix (ECM) components, as matrix metalloproteinases (MMP) and its regulators. The Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) plays a central role on tissue remodeling, tumor angiogenesis and exert inhibitory effects on the transcription, synthesis, activation and activity of MMP-2, MMP-9 e MMP-14 (MT1-MMP). As previously published by our group, it has been observed a correlation between the HPV16 E7 oncoprotein expression, the up-regulation of MMP-9 and the down-regulation of its inhibitors, RECK and tissue inhibitor of metalloproteinases 2 (TIMP-2). Also, RECK expression was downregulated in cervical intraepithelial neoplasias grades 2 and 3 (CIN2/3) and invasive carcinoma samples levels when compared with clinical samples of pacients diagnosed with cervicitis. The present study aims to determine the role of RECK in HPV mediated tumorigenesis. In order to do so, we generated cervical tumors derived cell lines (SiHa, SW756 e C33A) superexpressing RECK by lentiviral transduction followed by FACS. We assessed the effects of RECK superexpression in the tumorigenic potential of SiHa, SW756 e C33A using both in vitro and in vivo protocols. Overall, RECK superexpression is associated with reduced chamber invasion and reduced anchorage independent colony formation. Moreover, nude mice injected s.c. with RECK superexpressing tumor cells presented (i) delayed tumor establishment and (ii) increased overall survival, when compared with controls. Both SiHa and SW756 superexpressing RECK presented decreased frequency of tumor and endothelial cells, whilst showed increase in inflammatory infiltrate population. This observation was followed by a decrease in potential myeloid derived suppressor cell and neutrophil populations in both SiHa RECK and SW756 RECK tumors. Additionally, we observed hipermethylation and premature and consistent downregulation of RECK mRNA expression in CIN and cervical cancer expression datasets from GEO database. We observed that reduced RECK expression was associated with CIN3+ progression and increased pelvic lymph node metastasis in cervical cancer patients. Furthermore, we found that chemo radiotherapy treatment led to increased levels of mRNA in another set of cervical cancer patients. We conclude that RECK superexpression reduces the tumorigenic potential of cervical cancer derived cell lines regardless of HPV infection status. However, we found that the effect of RECK over the intratumoral cells populations is specific to HPV infected tumor cell lines. These results points to a possible interaction between HPV associated tumor microenvironment alterations and RECK. Finally, RECK downregulation is an early event in the natural history of cervical cancer.

O Glioblastoma (GBM) é um astrocitoma grau IV, representando o glioma de maior malignidade e o tumor cerebral primário mais frequente em humanos. A terapia indicada para o GBM é a ressecção cirúrgica, quimioterapia e radioterapia, todas com baixíssima eficiência devido a agressividade e as características do GBM. Consequentemente, a sobrevida dos pacientes indicados aos tratamentos convencionais é pouco mais de um ano. A inflamação é, sabidamente, uma das características que participa de modo decisivo do desenvolvimento tumoral, incluindo do GBM, e as relações entre mediadores inflamatórios e câncer são alvos de pesquisa nos últimos anos. Diversos estudos apontam um papel da via dos eicosanoides na modulação de processos patológicos envolvidos na inflamação e no câncer. Os eicosanoides são mediadores lipídicos bioativos, envolvidos em diversos processos fisiológicos e patológicos, em especial os associados à resposta inflamatória. As principais vias de produção de eicosanoides (ciclooxigenases, lipoxigenases, e citocromo P450), assim como seus produtos, são frequentemente alterados em diversos tipos de tumor, associados ao crescimento e a progressão tumoral. Contudo, o perfil dessas vias é consideravelmente pouco compreendido em GBM. O objetivo deste estudo foi analisar in vitro o perfil e o papel das três vias de eicosanoides e seus produtos (eicosanoides ou não) nas linhagens de GBM (U251-MG, U87-MG, A172, T98G e U138-MG), modulando a atividade de enzimas chaves com drogas especificas para, então, analisar parâmetros de proliferação, migração e morte celular. Nossos resultados mostram, em todas as linhagens analisadas, um perfil heterogêneo das enzimas e receptores chaves das três vias. O perfil lipídico evidencia a produção de 13-HODE, produto de 15-lipoxigenase-1 em todas as linhagens, bem como ausência de leucotrienos e 5-HETE do eixo de 5-lipoxigenase. Os inibidores farmacológicos para 15-LOX e 12-LOX/15-LOX foram capazes de reduzir o crescimento, modular o ciclo celular e a migração celular das linhagens U251-MG, U87-MG e A172. O mesmo é visto com a inibição de mPGES-1. A inibição de 5-LOX por outro lado não afetou nenhum parâmetro nas mesmas linhagens. Todos os resultados apontam, portanto, para um papel do eixo de 15-LOX e COX no crescimento e na migração das células de GBM humano. Glioblastoma (GBM) is a grade IV astrocytoma, the most malignant and the most frequent primary brain tumour in humans. Standard therapies for treating GBM are surgical resection, chemotherapy and radiotherapy, all with low efficiency due to GBM aggressiveness and characteristics. Therefore, patient survival after conventional treatments is about one year. Inflammation is one of the main characteristics that plays a decisive role in tumour development, including in GBM. The relationships between inflammatory mediators and cancer have been the subject of research in recent years. Several studies point to the eicosanoid pathways as modulators of pathological processes between inflammation and cancer. Eicosanoids are bioactive lipid mediators, involved in various physiological and pathological processes, especially those associated with the inflammatory response. The major eicosanoid pathways (cyclooxygenases, lipoxygenases, and cytochrome P450) as well as their products, are frequently altered in several tumours, associated with tumour growth and progression. However, the profile of these pathways is poorly understood in GBM. The objective of this study was to analyse, in vitro, the profile and role of eicosanoid pathways and their products (eicosanoids or not) in GBM cell lines (U251-MG, U87-MG, A172, T98G and U138-MG), modulating the key enzymes with inhibitors, analysing proliferation, migration and cell death. Our results show, in all analysed cell lines, a heterogeneous profile for the key enzymes and receptors of the three pathways. The lipid profile shows the production of 13-HODE, a product of 15-lipoxygenase-1, as well the absence of leukotrienes and 5-HETE of the 5-lipoxygenase axis. The pharmacological inhibitors for 15-LOX and 12-LOX / 15-LOX led to changes in cell cycle, reduced growth, reduced migration of U251-MG, U87-MG and A172 cell lines. The same was seen with the inhibition of mPGES-1. Inhibition of 5-LOX, on the other hand, did not affect any of these parameters in the same cell lines. All results, therefore, point to an important role of 15-LOX and COX pathways in the growth and migration of human GBM cells.

O glioblastoma (GBM) é o tumor mais frequente do sistema nervoso central com um alto grau de malignidade e um prognóstico desfavorável. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e/ou quimioterapia, não há tratamento eficiente disponível para o GBM. Os prostanoide são derivados do ácido araquidônico e estão envolvidas com vários processos do desenvolvimento e progressão do câncer. O objetivo deste estudo foi analisar in vitro o perfil de diferentes prostanoides nas linhagens de GBM. Além de analisar o papel dos prostanoides e dos receptores na migração celular de GBM. Os resultados demostraram um perfil dos prostanoides da série 2 diferente entre as linhagens, além da expressão dos genes envolvidos na biossíntese de PGE2. Nos ensaios de migração os dados demostraram que os tratamentos realizados com os prostanoides exógenos aumentaram a migração celular e os tratamentos com os antagonistas de EP2 e EP4 diminuiram a migração. Em conjunto esses resultados, demonstram o papel importante dos prostanoides, especialmente PGE2, no processo de migração das células de GBM. Glioblastoma (GBM) is the most common tumor of the central nervous system with a high degree of malignancy and poor prognosis. Despite advances in surgical techniques and radiation therapy and/or chemotherapy, there is no effective treatment available for GBM. The prostanoid are derived from arachidonic acid and are involved in many processes of development and progression of cancer. The aim of this study was to analyze in vitro profile of different prostanoids in the lines of GBM. In addition to analyzing the role of prostanoids and receptors on the cell migration of GBM. The results showed a profile of series 2 prostanoids the different between the cell lines, in addition to expression of genes involved in the biosynthesis of PGE2. In migration testing data showed that the treatments performed with exogenous prostanoids increased cell migration and treatment with antagonists of EP2 and EP4 decreased migration. Together these results demonstrate the important role of prostanoids, especially PGE2, in the migration process of the GBM cells.

Neste trabalho investigamos o papel do receptor do Fator ativador de Plaquetas (PAF) em diferentes tumores. Observamos que animais PAFR KO são mais resistentes ao crescimento do melanoma B16F10 e do carcinoma TC-1 e apresentaram maior infiltrado de linfócitos CD4+, neutrófilos e de macrófagos M1 do que animais WT. Células de carcinoma humano (C33/SiHa/HeLa/SSC78/ SSC90) expressam PAFR e tiveram sua proliferação in vitro reduzida por um antagonista de PAFR e aumentada pela adição de PAF. A irradiação gama induziu ligantes PAFR. O bloqueio do PAFR durante a radioterapia aumentou a morte induzida pela irradiação. Em modelo de repopulação tumoral observamos que tumores PAFR+ (KBP) tiveram um crescimento acelerado em relação à tumores PAFR- (KBM). Nossos dados sugerem que durante a irradiação ocorre ativação de PAFR nas células tumorais aumentando sua sobrevivência e proliferação. Ao mesmo tempo que ativa PAFR nos macrófagos reprogramando-os para um perfil pró-tumoral. A associação da radioterapia com antagonistas de PAFR pode ser uma estratégia terapêutica promissora. We investigate the role of the platelet activating factor receptor (PAFR) in tumors. We observed that PAFR KO animals are more resistant to the growth of B16F10 melanoma and TC-1 carcinoma than WT animals. PAFR KO had more infiltration of CD4+ cells, neutrophil and M1 macrophages than WT animals. Human carcinoma cells (C33 / SiHa / HeLa / SSC78 / SSC90) express PAFR and had their in vitro proliferation reduced by a PAFR antagonist and increased by the addition of PAF. Gamma irradiation induced PAFR ligands. Blocking PAFR during radiotherapy increased radiation-induced cell death. In tumor repopulation model, PAFR+ tumors (KBP) had an accelerated growth compared to PAFR- (KBM) tumors. Our data suggest that during irradiation occurs PAFR activation in tumor cells increasing their survival and proliferation. Also, irradiation promote activation of PAFR in macrophages by reprogramming them to a pro-tumor profile. We propose that PAFR represents a possible target for improving the efficacy of radiotherapy through inhibition of tumor repopulation.

O vínculo entre papilomavírus humano de alto risco e câncer cervical está bem estabelecido. Apesar da existência de vacinas profiláticas contra infecções pelos tipos mais comuns de HPV, para infecções e tumores causados por esses vírus as alternativas terapêuticas são restritas. Encontramos alguns motivos com homologias para proteínas do HPV de alto risco durante o imunoscreening de uma biblioteca de phage display com soros de participantes HPV-16-soropositivos da coorte Ludwig-McGill. Após enriquecimento das sequências, os bacteriófagos recombinantes foram purificados e amplificados para uso como imunógenos.Usando uma abordagem profilática, nós vacinamos experimentalmente camundongos imunocompetentes com um dos nossos bacteriófagos recombinantes, usando o bacteriófago sem inserto como controle. Estes camundongos foram então desafiados com células tumorais TC-1 (HPV-16 positivas), tendo-se avaliado as respostas imunes disparadas durante a progressão tumoral. Também usamos uma abordagem terapêutica, aonde os camundongos foram primeiro injetados com as células tumorais e imunizados com o bacteriófago após o estabelecimento do tumor. O crescimento tumoral foi monitorado e os tumores, baço e linfonodos foram avaliados quanto à quantidade e qualidade da resposta imunológica. Os testes de ELISA revelaram que todos os camundongos vacinados responderam à imunização com os diferentes bacteriófagos. O crescimento tumoral foi significativamente reduzido nas imunizações profiláticas e terapêuticas, embora a redução do tumor fosse mínima quando os camundongos foram tratados 9 dias após o enxerto. A redução no crescimento tumoral também se traduziu em uma sobrevivência significativamente maior para os camundongos imunizados. Estudos de infiltração celular não revelaram alterações em diversas sub-populações imunes, mas uma tendência de aumento de linfócitos T citotóxicos foi observada nos camundongos imunizados com PEP1 (bacteriófago contendo inserto). A importância deste aumento de CD8 na redução observada do crescimento tumoral foi confirmada utilizando camundongos CD8-knockout, onde a redução do crescimento tumoral previamente observada foi anulada. Foi observado um aumento de taxa CD8:CD4 nos camundongos imunizados e isto é uma indicação de ambiente tumoral citotóxico. Os ensaios de proliferação celular para testar a especificidade do antígeno dos linfócitos dos camundongos imunizados foram, no entanto, inconclusivos; da mesma forma, não pudemos alterar o padrão observado com o uso de adjuvante CpG. A utilidade da técnica de phage display também foi observada neste trabalho experimental. Trabalhos adicionais para entender o mecanismo de ação desses fagos recombinantes no controle do crescimento de tumores causados por HPV e seu potencial imuno-estimulador são necessários The link between high-risk human papillomavirus and cervical cancer is well established. Despite the existence of prophylactic vaccines against infections by the most common types of HPV, therapeutic alternatives are limited for infections and tumors caused by these viruses. We found some homology motifs for high-risk HPV proteins during the immune-panning of a phage display library with sera from HPV-16- seropositive participants of the Ludwig-McGill cohort. After enrichment of the sequences, the recombinant bacteriophages were purified and amplified for use as immunogens. Using a prophylactic approach, we vaccinated experimentally immunocompetent mice with one of our recombinant bacteriophages using the insertless bacteriophage as a control. These mice were then challenged with TC-1 tumor cells (HPV-16 positive), and the immune responses triggered during tumor progression were evaluated. We also used a therapeutic approach where mice were first injected with tumor cells and immunized with the bacteriophage after tumor establishment. Tumor growth was monitored and tumors, spleen and lymph nodes were evaluated for the quantity and quality of the immune response. ELISA tests revealed that all vaccinated mice responded to immunization with the different bacteriophages. Tumor growth was significantly reduced in prophylactic and therapeutic immunizations, although tumor reduction was minimal when mice were treated 9 days after TC-1 cells grafting. The reduction in tumor growth also translated into a significantly greater survival for the immunized mice. Cell infiltration studies did not reveal changes in several immune subpopulations, but an upward trend in cytotoxic T lymphocytes was observed in mice immunized with PEP1 (insert-containing bacteriophage). The importance of this increase in CD8 in the observed reduction of tumor growth was confirmed using CD8-knockout mice, where the previously observed reduction of tumor growth was abolished. An increase in CD8:CD4 rate was observed in the immunized mice and this is an indication of a cytotoxic tumor environment. Cell proliferation assays to test the antigen specificity of lymphocytes from immunized mice were, however, inconclusive; likewise, we could not change the pattern observed with the use of CpG adjuvant. The usefulness of the phage display technique was also observed in this experimental work. Additional studies to understand the mechanism of action of these recombinant phages in the control of HPV tumor growth and its immunostimulatory potential are warranted

As células que compõem o tumor podem interagir entre si, através da liberação e incorporação de vesículas extracelulares, muitas vezes contribuindo para a progressão tumoral. Dessa maneira, o presente trabalho teve como objetivo observar se as vesículas extracelulares , como as microvesículas e os exossomas liberados pelas células cancerosas em condições de estresse celular, após quimioterapia e indução de hipóxia conferem alguma vantagem adaptativa às células tumorais. Nossos resultados mostram que vesículas liberadas por células de melanoma humano em hipóxia ou normóxia apresentam tamanho médio característico de exossomos e microvesículas e não modulam os processos de proliferação, morte e migração celular. As vesículas liberadas pelas células após tratamento com o quimioterápico temozolamida também apresentam tamanho característico de exossomos e microvesículas; em adição, o tratamento com a temozolamida induziu um aumento na secreção dessas vesículas pelas células de melanoma. A incubação das células tumorais com vesículas oriundas da terapêutica com a temozolamida aumentou a proliferação celular, conferindo vantagem proliferativa às células de melanoma humano Tumor cells can interact with each other by releasing and incorporating extracellular vesicles, contributing to tumor progression. Therefore, the aim of this study was to evaluate if extracellular vesicles, such as microvesicles and exossomes, released by cancer cells under cell stress conditions like chemotherapy and hypoxia, induce an adaptive advantage to tumor cells. Our results show that vesicles shed by human melanoma cells under hypoxia, or normoxia exhibit the characteristic size of exossomes and microvesicles and do not modulate cell proliferation, death or migration. The vesicles released by melanoma cells after temozolomide treatment also showed the average size of exossomes and microvesicles; moreover, temozolomide treatment induced an increase in extracellular vesicles shedding by tumor cells. Incubation of tumor cells with vesicles released under temozolamide therapeutics caused an increase in cell proliferation, providing a proliferative advantage to human melanoma cells

Introducão. A perda da resposta aos mecanismos que regulam o crescimento da população celular em tecidos normais, as alterações em níveis genéticos e epigenéticos, induzem a carcinogênese. Durante o crescimento, seja tumor primário ou metástase, ge- ralmente há uma fase avascular, caracterizada por tumores pequenos onde os recursos necessário para proliferação da população são oriundos dos tecidos adjacentes. Ade- mais, um aspecto marcante é a heterogeneidade intratumoral, que é desencadeada por fatores intrínseco e ambientais às células, desempenhando papeis importante no cresci- mento e resposta ao tratamento. Métodos. Foi proposto um modelo estocástico espacial em termos de Cadeia de Markov para simular a dinâmica de populações de um câncer in situ em fase avascular e o tratamento quimioterapico. As transições de estado das célu- las consideradas foram duplicação, migração, morte, geração de variação na população e quiescência. Assumimos que as taxas de duplicação, migração e morte, dependem do tamanho da população, onde a taxa de duplicação diminui conforme a população au- menta e as taxas de migração e morte aumentam conforme a população cresce. As taxas dependente do tamanho da população foram descritos em termos de funções sigmóidais onde a suavidade da transição entre os valores mínimos e máximos das taxas foi dado pelo parâmetro k, o tamanho da população na qual a taxa de duplicação cai na metade do valor máximo foi dado por N e a taxa de duplicação na qual as taxa de migração/morte atingem a metade do valor máximo por 0\'. A heterogeneidade do tumor foi representada como um conjunto de duas subpopulações (1) mais proliferativa e (2) mais migratória. Em seguida, foi aclopado um modelo de difusão de drogas e um modelo farmacodinâ- mico para avaliar a repopulação no tratamento com uma única droga. Resultados. As simulações acerca do crescimento tumoral mostram que a população atingiu a densidade de saturação de acordo com os valores do parâmetro k. A densidade das variantes prol iferativas e migratórias não foram diretamente relacionadas a taxa de geração de variação, mas sim pelos parâmetros N e a. Para determinado conjunto de valores atribuídos aos parâmetros k e N,espacialmente observou-se crescimento difuso da população. No que tange ao tratamento, houve emergência da resistência, todavia observou-se padrões diferente na repopulação de acordo com os valores da taxa de geração de variação, k e N. Espacialmente, observa-se que as células resistentes vão ao gradiente droga. O modelo no estágio atual consegue recuperar qualitativamente algumas evidências experimentais atreladas coexistência de duas subpopulações onde uma cresce mais do que a outra, crescimento difuso da população e migração da população resistente para o gradiente de droga Introduction. The loss of response to the mechanism which regulates the growth of cell population in normal tissue, the changes at both genetic and epigenetic levei lead to carcinogenesis. During the growth of primary tumor or metastasis often there is an avascular phase which is featured by small tumor where the resource necessary to proli- feration nd survival come from surrounding tissues. Furthermore, a remarkable aspect of carcinogenesis is intratumoral heterogeneity which is fueled by both intrinsic and environ- mental facto r, playing a crucial role in the growth and treatment response. Methods Here we propose a stochastic spatial model in term of Markov Chain to simulate the population dynamic of in situ cancer in avascular phase and the chemotherapeutic treatment. The cells state transition considered was duplication, migration, death, generation of new variants in population and quiescence. We are assuming that duplication, migration and death rates depend on population size, where duplication rates decrease as long as population increase but both migration and death rates, in it turn increase. Theses population size dependent rates were described in terms of sigmoidal function where the smoothness of transition between maximal e minimum values was indicated by k, the size of population at which duplication rates falls on half of it maximal value was indicated by Nand the duplication rate where both migration and death rates goes to half of it maximal value was indicated by a. The heterogeneity was featured as a set of a (1) highly proliferative and (2) highly migratory cells. In doing so, we coupled a drug diffusion drug and a phar- macodynamical model in order to evaluate the repopulation in the single drug treatment. The simulations about tumor growth showed that population reached a saturation density accordingly with values of the parameter k.The density of both proliferative and migratory variants was not directed related to the generation of variants rates but by the parameter Nand a. To some specific set of assigned values to the parameters k and N,spatially was observed that diffuse growth of population. Concerning to the treatment, there was resistance emergence, h oweve r, we observed differents patterns in the repopulation ac- cordingly the generation of variants rates, k and N.Spatially, we observed the resistant cells goes to drug gradient. The model, currently, has recovered qualitatively some ex- perimental evidence linked to the co-existence of two subpopulations where one of them grow more than other, diffuse growth of population and migration of resistant population to drug gradient

INTRODUÇÃO: A maioria dos pacientes com CECP é diagnosticada em estágios avançados da doença, apresentando taxas de sobrevida insatisfatórias. Em carcinomas localmente avançados e irressecáveis, o tratamento padrão na rotina do ICESP é a QRT a base de cisplatina. Entretanto, cerca de dois terços desses pacientes apresentam recidiva local, à distância ou óbito em cinco anos. Entender os mecanismos de resistência a QRT e descobrir marcadores que possam indicar resposta ao tratamento continuam sendo um desafio. Os microRNAs possuem papel chave nos mecanismos de resistência a QRT, sendo possível quantificá-los em saliva. Neste estudo avaliamos a expressão de microRNAs na saliva de pacientes com CEC de cavidade oral e orofaringe e se esses microRNAs estão contidos dentro de microvesículas. MÉTODOS: Por meio de revisão da literatura e análises in sílico, selecionamos 8 microRNAs para serem avaliados: miR-15a-5p, 21-5p, 23a-3p, 125b-5p, 142-3p, 200b-3p, 296-5p e 503-5p. A expressão dos microRNAs foi determinada por PCR quantitativo na saliva livre de células de 70 pacientes portadores de CEC de cavidade oral e orofaringe localmente avançado e irressecável, em diferentes estágios da progressão da doença: antes de iniciar tratamento (G1), com falha do tratamento (G2) e livre da doença há 2 anos (G3) e em 28 voluntários sadios (G0). Microvesículas foram isolados por ultracentrifugação ou exoQuick, analisados por Microscopia Eletrônica de Transmissão (MET), Nanosight e para determinação da expressão do miR-21-5p. RESULTADOS: Os miRs-296-5p e 503-5p foram indetectáveis na maioria das amostras testadas. Quando comparamos os grupos em diferentes situações clínicas, encontramos diferença significativa na expressão do miR-21-5p (p=0.005), miR-23a-3p (p=0,026), miR-125-5p (p=0,013), miR-142-3p (p=0,033) e miR-200b-3p (p=0,031). Observou-se aumento na expressão dos miRs 21-5p (p=0,001), 23a-3p (p=0,004), 125b-5p (p=0,026) e 142-3p (p=0,005) na saliva dos pacientes em G1 em relação ao voluntários sadios (G0). O grupo G3 também apresentou maior expressão do mir-21-5p (p=0,018), 125b-5p (p=0,002) e 200b-3p (p=0,014) comparado ao G0, assim como o grupo G2 teve maior expressão do mir-15a-5p (p=0,023) e 23a-3p (p=0,017) comparado ao grupo G0. O grupo G2 apresentou menor expressão do miR-200b-3p (p= 0,019) e maior expressão do miR-15a-5p (p=0,057) em relação ao grupo G3. Além disso, os pacientes tabagistas e/ou etilistas apresentaram maior expressão relativa do miR-21-5p (p=0,001 e p=0,046, respectivamente) e os etilistas também tiveram maior expressão do miR-200b-3p (p=0,013). Com relação à resposta inicial do paciente ao tratamento avaliada pelo médico bem como, com a resposta a longo prazo (recidiva, status global e prognóstico), a expressão dos miR-15a-5p, miR-125b-5p, miR-23a-3p e miR-142-3p apresentaram associação com sobrevida livre de progressão e sobrevida global, porém não atingiram significância estatística. Microvesículas foram detectadas na saliva tanto na MET como na contagem no nanosight. A expressão do miR-21-5p foi predominantemente detectada dentro de microveículas em relação ao sobrenadante livre de vesículas. CONCLUSÕES: Alguns dos microRNAs analisados foram diferencialmente expressos entre os diferentes grupos estudados, a expressão do miR-21 foi associada ao tabagismo e etilismo e a do miR-200b com etilismo e a expressão de alguns microRNAs podem estar associadas à resposta ao tratamento e ao prognóstico dos pacientes BACKGROUND: Most patients with HNSCC are diagnosed in advanced stages of the disease, with unsatisfactory survival rates. In locally advanced and unresectable carcinoma, the standard treatment in ICESP is cisplatin based QRT. However, about two-thirds of these patients have local recurrence, distance or death within five years. Understanding the QRT resistance mechanisms and discovery of markers that may indicate benefit of treatment is a great challenge. MicroRNAs have key role in the mechanisms of QRT resistance and are detectable in saliva. In the present study we analyzed the expression of microRNAs in the saliva from oral cavity/oropharynx squamous cell carcinoma (OSCC) patients and whether these microRNAs are contained within exosomes. METHODS: Through a review of studies in the literature and by in silico analysis, we choose 8 microRNAs to be evaluated: miR-15a-5p, 21-5p, 23a-3p, 125b-5p, 142-3p, 200b-3p, 296-5p and 503-5p. The expression of microRNAs was determined by quantitative PCR in the cell-free saliva from 70 locally advanced and unresectable OSCC patients at different stages of disease progression: treatment naive (G1), after treatment failure (G2) and disease free for at least 2 years (G3) and 28 healthy volunteers (G0). Exosomes were isolated by ultracentrifugation or exoQuick, analyzed by transmission electron microscopy (MET), Nanosight quantification and by determination of the miR-21-5p expression. RESULTS: The miRs-296-5p and 503-5p were undetectable in almost all saliva samples. Significant differences was observed in the expression of miR-21-5p (p=0.005), miR-23a-3p (p=0.026), miR-125-5p (p=0.013) miR-142-3p (p=0.033) and miR-200b-3p (p=0.031) among the groups analyzed. The expression of miRs 21-5p (p=0.001), 23a-3p (p=0.004), 125b-5p (p=0.026) and 142-3p (p=0.005) were high in saliva of G1 patients as compared to heath volunteers (G0). The G3 group also showed higher expression of mir-21-5p (p=0.018), 125b-5p (p=0.002) and 200b-3p (p=0.014) and G2 presented higher expression of miR-15a-5p (p=0.023) and 23a-3p (p=0.017) as both compared to the G0. The group G2 presented lower expression of the miR-200b-3p (p=0.019) and higher expression of the miR-15a-5p (p=0.057) as compared to the G3 group. In addition, saliva from the smokers and/or drinkers patients showed high relative expression of the miR-21-5p (p=0.001 e p=0.046; respectively) compared to former drinker/smokers and drinkers also showed high expression of the miR-200b-3p (p=0.013). In relation to the initial response to treatment assessed by the physician, as well the long-term response (relapse, global status and prognosis), the expression of the miR-15a-5p, miR-125b-5p, miR-23a-3p e miR-142-3p showed association with progression-free survival and overall survival, however did not reach statistical significance. Microvesicles were detected in saliva by both MET as the count in nanosight. The expression of the miR-21-5p was predominantly detected in microvesicles in relation to the supernatant. CONCLUSIONS: Some of the microRNAs analyzed were differentially expressed between the different groups, the expression of the miR-21 was associated with smoking and drinking habits and of the miR-200b with drinking habits and the expression of some microRNAs may be associated with the treatment response and prognosis of the patients

O lactato, produto do metabolismo alterado das células tumorais, possui papel na modulação de fenótipo supressor em células imunes. Macrófagos são uma das principais células do microambiente tumoral, sendo pró-tumorais em câncer cervical. O objetivo desse trabalho foi avaliar o efeito do lactato no fenótipo de macrófagos associados a células tumorais derivadas de câncer cervical. Linhagens celulares positivas e negativa para HPV foram utilizadas para formação de esferoides tumorais, os quais receberam monócitos de doadores saudáveis para diferenciação em macrófagos. Macrófagos cultivados com linhagens tumorais positivas para HPV apresentaram fenótipo misto e pró-tumoral, enquanto os cultivados com a linhagem negativa, um fenótipo mais anti-tumoral. A inibição da enzima lactato desidrogenase, nas células tumorais, promoveu diferentes efeitos no fenótipo dos macrófagos, dependendo do tipo celular a que estavam associados. Essas alterações diminuíram o aspecto pró-tumoral desses macrófagos, que induziram maior proliferação de linfócitos T e morte de células tumorais. The lactate, product of the altered metabolism of tumor cells, is associated with the induction of a suppressor phenotype in immune cells. Macrophages are one of the major cells of the tumor microenvironment, and are pro-tumor in cervical cancer. The aim of this study was to evaluate the effect of lactate in the phenotype of macrophages associated with tumor cells derived from cervical cancer. HPV positive and negative cell lines were used for formation of tumor spheroids, which received healthy donor monocytes to differentiate into macrophages. Macrophages cultivated with HPV positive tumor cell lines presented a mixed and pro-tumor phenotype, while macrophages cultivated with the HPV negative cell line, a more anti-tumor phenotype. Inhibition of lactate dehydrogenase in the tumor cells promoted different effects on macrophages phenotype, depending on the cell type that they were associated. These changes decreased the pro-tumor property of macrophages, which were able to induced T cell proliferation and tumor cell death.

O câncer de mama é a neoplasia de maior ocorrência na população feminina. É uma doença hormônio-dependente com etiologia complexa e multifatorial. O câncer de mama invasivo é uma das principais causas da morbidade e mortalidade de mulheres no mundo. O desenvolvimento e a progressão de câncer de mama estão associados com acúmulo de várias alterações genéticas e epigenéticas, que resultam na expressão gênica diferencial entre células normais e tumorais. A hipóxia pode ser considerada uma das características principais de tumores sólidos e uma força motriz para a sobrevida das células tumorais e progressão maligna. O gene supressor de tumor PAWR (PKC apoptosis WT1 regulator; também denominado PAR-4, prostate apoptosis response-4) tem importante papel na apoptose tanto pela via intrínseca quanto pela via extrínseca e pode ser considerado um alvo para a terapia seletiva de células tumorais uma vez que a expressão de PAR-4 aumenta a sensibilidade da maioria das células de câncer a um segundo estímulo apoptótico. Alguns estudos, incluindo os de nosso grupo, têm mostrado o envolvimento de PAR-4 em câncer de mama e seu papel como fator prognóstico e preditivo de resposta a quimioterapia. No entanto, pouco é conhecido sobre o papel funcional deste gene ou os mecanismos envolvidos na regulação da expressão de PAR-4 na glândula mamária e no processo de tumorigênese da mama. No presente trabalho investigamos os efeitos da hipóxia na expressão de PAR-4 nas linhagens celulares MCF10A, MCF7 e MDA-MB-231. Para isto, as células foram incubadas por períodos de 30 minutos, 1, 2, 6 ou 24 horas em ambiente controlado com 5 % CO2, 95% N2 e 0.2% O2. A expressão de Par-4 foi avaliada quanto aos transcritos, por PCR em Tempo Real e quanto à expressão protéica, por western blot. Observamos que a baixa concentração de oxigênio diminui a expressão do mRNA de PAR-4, em tempos de 6 e 24 horas, nas linhagens MCF10A e MCF7 e no tempo de 24h nas células MDA-MB-231. Já quando os níveis protéicos de Par-4 foram analisados não foi observada diferença significativa nessas células após exposição à condição de hipóxia. A expressão do gene NDRG1, a qual se encontra aumentada em condições de hipóxia, foi utilizada como controle. Nossos dados sugerem que a expressão dos transcritos do gene supressor de tumor PAR-4 é inibida pelos mecanismos celulares de sobrevivência celular ativados durante a hipóxia Breast cancer has the highest incidence rate of all malignant neoplasias affecting women. It is a hormone dependent disease with complex and multifactorial etiology. Invasive breast cancer is one of the principal causes of women morbidity and mortality in the world. Breast cancer development and progression are associated with accumulation of various genetic and epigenetic alterations, which result in differential gene expression among normal and tumor cells. Hypoxia could be considered one of the principal hallmarks of solid tumors and the driving force for the tumor cell survival and malignant progression. The tumor suppressor gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, prostate apoptosis response-4) has an important role in apoptosis either by intrinsic or extrinsic pathways and could be considered as a target for selective therapy of tumor cells. Some studies, including of our group, have shown the PAR-4 involvement in breast cancer and its role as prognostic and predictive factor in response to chemotherapy. However, little is known about functional role of this gene or the mechanisms involved in PAR-4 expression regulation in mammary gland and in breast tumorigenesis process. In the present study we evaluated the hypoxia effects on PAR-4 expression in MCF10A, MCF7 and MDA-MB-231 cell lines. Therefore, the cells were incubated for time periods of 30 minutes, 1, 2, 6 or 24 hours under controlled environment with 5 % CO2, 95% N2 e 0.2% O2. PAR-4 expression was evaluated for transcripts, by Real Time PCR, and for protein expression, by western blot. We observed that low oxygen concentration decreases PAR-4 mRNA expression, in periods of 6 and 24 hours, in MCF10A and MCF7 cell lines and in the period of 24 hours in MDA-MB-231 cells. However, when the Par-4 protein levels were evaluated, no significant difference was observed in these cells after incubation under hypoxic conditions. NDRG1 gene expression, which is increased under hypoxia, was used as a control. Our data suggest that the expression of tumor suppressor gene PAR-4 transcripts is inhibited by cell survival mechanisms activated during hypoxia

PGC-1 beta é um co-ativador de transcrição gênica responsável pela regulação do metabolismo celular, principalmente na biogênese e função mitocondrial, disponibilidade de substrato e síntese de lipídios. Nos últimos anos, outras isoformas de PGC-1 têm sido descritos como participantes na gênese e manutenção de tumores. Portanto, nosso objetivo foi determinar se o PGC-1beta está relacionado ao aumento da proliferação celular de células de melanoma. Inicialmente, foi demonstrado que os níveis de RNAm e proteína de PGC-1beta são muito mais elevados em linhagens de células de melanoma (Tm1 e Tm5) do que na linhagem parental de melanócitos não tumorais (Melan-a) como detectado por PCR quantitativa e western blotting. A fim de descobrir uma relação causal entre a expressão de PGC-1? e crescimento celular da linhagem Tm5, células de tal linhagem foram transfectadas com um oligonucleotídeo antisense (ASO) contra PGC-1beta. As células tratados com ASO apresentaram níveis mais baixos de RNAm e proteína PGC-1beta, além de redução em sua atividade avaliada pela expressão de genes PGC-1beta dependentes. Além disso, as células transfectadas apresentaram uma taxa de proliferação inferior em comparação com células de controle Tm5. Este fenômeno também foi observado in vivo. Quando injetadas em camundongos, as células Tm5 desenvolvem-se em um tumor que atinge 1,34 ± 0,20 cm3 após nove dias. Tumores tratados com ASO após o mesmo tempo apresentaram volume tumoral de 0,75 ± 0,05 cm3. Este crescimento não estava relacionada à necrose tumoral, mas sim com a proliferação reduzida de células. Finalmente, verificamos se o mesmo fenômeno seria observado em humanos. A expressão PGC-1beta foi muito maior em amostras de melanoma do que em nevos, alterações não-malignas da pele com alto conteúdo de melanina. Por conseguinte, conclui-se que a expressão PGC-1? está aumentada no melanoma, tanto murino e humano, e que o bloqueio da sua atividade leva à diminuição da proliferação celular e crescimento tumoral PGC-1beta is a co-activator of gene transcription primarily responsible for the regulation of cellular metabolism, mainly in mitochondrial biogenesis and function and also substrate and lipid synthesis. In recent years, other isoforms of PGC-1 have been described as participating in the genesis and maintenance of tumors. Therefore, our objective was to determine whether PGC-1beta is related to increased proliferation of melanoma cells. Initially, it was demonstrated that mRNA and protein levels of PGC-1beta are much higher in melanoma cell lines (Tm1 and TM5) than in the non-tumoral parental lineage melanocytes (melan-a) as detected by quantitative PCR and Western blotting. In order to find a causal relationship between the expression of PGC-1beta and cell growth, Tm5 lineage cells were transfected with an antisense oligonucleotide (ASO) against PGC-1beta. The cells treated with ASO had lower levels of PGC-1beta mRNA and protein, as well as reduction in its activity detected by quantitation of PGC-1beta dependent genes expression. Furthermore, transfected cells showed a lower rate of proliferation compared to Tm5control cells. This phenomenon was also observed in vivo. When injected into mice, Tm5 cells develop a tumor which reaches 1.34 ± 0.20 cm3 after nine days. Tumors treated with ASO, after the same time, presented tumor volume of 0.75 ± 0.05 cm 3. This growth was not related to tumor necrosis, but with reduced cell proliferation. Finally, we checked whether the same phenomenon would be observed in humans. The PGC-1beta expression was much higher in melanoma samples than in nevi, a non-malignant skin alteration filled with melanin. Therefore, we concluded that PGC-1beta expression in melanoma is increased, both in murine and human, and that blocking its activity leads to decreased cell proliferation and tumor growth