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Background The development of efficient strategies for managing high-risk human papillomavirus (HR-HPV)-positive women is a major challenge when human papillomavirus-based primary screening is being performed. The objectives of this study were to evaluate the comparative effectiveness of HR-HPV testing based on self-collection (SC) and HR-HPV testing based on collection by a health professional (HP) and to assess the potential usefulness of HR-HPV testing combined with testing with the biomarkers p16/Ki-67, α-mannosidase, and superoxide dismutase 2 (SOD2). Methods This was a cross-sectional study of 232 women admitted for colposcopy because of an abnormal Papanicolaou smear. The collected material underwent liquid-based cytology, HR-HPV detection, and immunocytochemical testing (p16/Ki-67, α-mannosidase, and SOD2). The gold standard was the histopathological result; the positive reference was CIN2+. Results The overall accuracy of HR-HPV testing was 76.6%; the results for the SC group (78.1%) and the HP group (75.2%) were similar. The positive predictive values (HP, 76.5%; SC, 80.0%), the negative predictive values (HP, 66.7%; SC, 64.3%), the positive likelihood values (HP, 1.35; SC, 1.36), and the negative likelihood values (HP, 0.21; SC, 0.19) were also similar. p16/Ki-67 showed higher sensitivity than the other 2 biomarkers: 78.1% versus 45.8% for α-mannosidase and 44.5% for SOD2. The specificities of the biomarkers were equivalent: 71.4% for p16/Ki-67, 77.8% for α-mannosidase, and 71.2% for SOD2. In the HP group, accuracy also leaned more heavily toward the final score (using α-mannosidase and SOD2) without statistical significance (80.8% vs 77.9%). The contrast with the SC group yielded the same level of accuracy. Conclusions SC, when associated with testing with biomarkers, is as accurate as collection by HPs in the detection of women at risk for cervical cancer.

Persistent infection with some types of mucosal human papillomavirus (HPV) is the etiological factor for the development of cervical cancer and its precursor lesions. Besides, several cofactors are known to play a role in cervical disease onset and progression either by favoring or by preventing HPV infection and persistence. The microbiome of a healthy female genital tract is characterized by the presence of 1 or few varieties of lactobacilli. However, high-throughput studies addressing the bacterial diversity and abundance in the female genital tract have shown that several factors, including hormonal levels, hygiene habits, and sexually transmitted diseases may disrupt the natural balance, favoring the outgrowth of some groups of bacteria, which in turn may favor some pathological states. Recently, the vaginal microbiome has emerged as a new variable that could greatly influence the natural history of HPV infections and their clinical impact. In this context, changes in the vaginal microbiome have been detected in women infected with HPV and women with HPV-associated lesions and cancer. However, the role of specific bacteria groups in the development/progression or prevention/regression of HPV-associated pathologies is not well understood. In this review we summarize the current knowledge concerning changes in vaginal microbiome and cervical disease. We discuss the potential functional interplay between specific bacterial groups and HPV infection outcomes.

Because of its role in preserving DNA/RNA stability, there is an increasing number of studies addressing the relationship between Fused in Sarcoma (FUS) expression and cancer. However, the disparity in FUS/FUS oncogenic vs. tumor-suppressive roles may be attributed to the complex molecular pathways associated with FUS regulation in different cancer types, and its role in cervical carcinogenesis remains largely unexplored. Methods: We determined FUS protein expression in specimens of 61 patients with advanced cervical cancer. Long-term (> 10 years) clinical follow-up data for these patients were available, and we determined disease-free, cancer-related and overall survival as related to FUS expression. Results: There were no significant associations between FUS expression and patients’ age, tumor grade, and acute/late toxicity events related to treatment (either radiation alone or chemoradiation). However, multivariate Cox regression analysis for disease-free survival (recurrence), overall survival (death) and cancer-related survival showed that patients with high average FUS expression fared significantly better than their counterparts with low average FUS expression, both in terms of disease-free survival (HR = 0.31; 95%CI 0.12 to 0.77; p = 0.01) and cancer-related survival (HR = 0.41; 95%CI 0.17 to 0.98; p = 0.04). Conclusions: Our study shows that high FUS protein expression in advanced cervical cancer specimens is a potent harbinger of better prognosis, and can as such be used in clinical practice to help characterize patients and, possibly, plan treatment and follow-up strategies.

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants’ functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.

Introduction: The main risk factor for the development of cervical cancer (CC) is persistent infection by human papillomavirus (HPV) oncogenic types. In order to persist, HPV exhibits a plethora of immune evasion mechanisms. PI3/Elafin (Peptidase Inhibitor 3) is an endogenous serine protease inhibitor involved in epithelial protection against pathogens. PI3/Elafin’s role in CC is still poorly understood. Materials and Methods: In the present study, we addressed PI3/Elafin protein detection in 123 CC samples by immunohistochemistry and mRNA expression in several datasets available at Gene Expression Omnibus and The Cancer Genome Atlas platforms. Results: We observed that PI3/Elafin is consistently downregulated in CC samples when compared to normal tissue. Most of PI3/Elafin-positive samples exhibited this protein at the plasma membrane. Besides, high PI3/Elafin expression at the cellular membrane was more frequent in in situ stages I + II than in invasive cervical tumor stages III + IV. This indicates that PI3/Elafin expression is gradually lost during the CC progression. Of note, advanced stages of CC were more frequently associated with a more intense PI3/Elafin reaction in the nuclei and cytoplasm. Conclusion: Our results suggest that PI3/Elafin levels and subcellular localization may be used as a biomarker for CC severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 19 (COVID-19), was declared pandemic by the World Health Organization in March 2020. SARS-CoV-2 binds its host cell receptor, angiotensin-converting enzyme 2 (ACE2), through the viral spike (S) protein. The mortality related to severe acute respiratory distress syndrome (ARDS) and multi-organ failure in COVID-19 patients has been suggested to be connected with cytokine storm syndrome (CSS), an excessive immune response that severely damages healthy lung tissue. In addition, cardiac symptoms, including fulminant myocarditis, are frequent in patients in a severe state of illness. Diacerein (DAR) is an anthraquinone derivative drug whose active metabolite is rhein. Different studies have shown that this compound inhibits the IL-1, IL-2, IL-6, IL-8, IL-12, IL-18, TNF-α, NF-κB and NALP3 inflammasome pathways. The antiviral activity of rhein has also been documented. This metabolite prevents hepatitis B virus (HBV) replication and influenza A virus (IAV) adsorption and replication through mechanisms involving regulation of oxidative stress and alterations of the TLR4, Akt, MAPK, and NF-κB signalling pathways. Importantly, rhein inhibits the interaction between the SARS-CoV S protein and ACE2 in a dose-dependent manner, suggesting rhein as a potential therapeutic agent for the treatment of SARS-CoV infection. Based on these findings, we hypothesize that DAR is a multi-target drug useful for COVID-19 treatment. This anthraquinone may control hyperinflammatory conditions by multi-faceted cytokine inhibition and by reducing viral infection.

Breast, cervical and ovarian cancers are highly prevalent in women worldwide. Environmental, hormonal and viral-related factors are especially relevant in the development of these tumors. These factors are strongly related to oxidative stress (OS) through the generation of reactive oxygen species (ROS). The OS is caused by an imbalance in the redox status of the organism and is literally defined as "an imbalance between ROS generation and its detoxification by biological system leading to impairment of damage repair by cell/tissue". The multistep progression of cancer suggests that OS is involved in cancer initiation, promotion and progression. In this review, we described the role of OS and the interplay with environmental, host and viral factors related to breast, cervical and ovarian cancers initiation, promotion and progression. In addition, the role of the natural antioxidant compound curcumin and other compounds for breast, cervical and ovarian cancers prevention/treatment is discussed.

It is well known that persistent infection with high-risk HPV (hr-HPV), mostly HPV-16 and 18, is the main cause of cervical cancer development. Manganese superoxide dismutase (MnSOD or SOD2) are highly expressed in different neoplasia. The present study investigated SOD2 protein expression and the presence of hr-HPV types in 297 cervical samples including non-neoplastic tissue, cervical intraepithelial neoplasia grade 3 (CIN3), squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Strong SOD2 expression was significantly higher in ADC (82%) than CIN3 (52%) or SCC (64%). There was no association between SOD2 expression and HPV 16 and/or 18 detection for every lesion analyzed. Binary Logist Regression revealed that strong SOD2 expression (OR: 27.50, 6.16-122.81) and HPV 16 and/or HPV 18 (OR: 12.67, 4.04-39.74) were independently more associated with CIN3 than non-neoplastic cervix. Strong SOD2 expression (OR: 3.30, 1.23-8.86) and HPV 16 and/or HPV 18 (OR: 3.51, 1.03-11.87) were independently more associated with ADC than SCC. Similar findings for SOD2 expression were observed by the Cochran Mantel-Haenszel test, controlling for HPV-16 and/or HPV 18. In conclusion, the expression of SOD2 was increased in CIN3 and SCC, and more increased in cervical ADC than in SCC. Strong SOD2 expression was statistically independent of the presence of HPV 16 and/or 18. These findings suggest that the mitochondrial antioxidant system and HPV infection could follow independent pathways in the carcinogenesis of cervical epithelium and in the differentiation to SCC or ADC of the cervix., Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Grants No. 2008/57889-1, 2010/07739-3 and 2012/16059-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/Instituto Nacional de Ciência e Tecnologia das Doenças Associadas ao HPV (INCT-HPV) (CNPq Grant No. 573799/2008-3), info:eu-repo/semantics/publishedVersion

To determine whether age is a barrier against acceptability of cervicovaginal self-sampling in screening for cervical cancer at two gynecology outpatient clinics., Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). This is a governmental institution that provides funding for research based on an evaluation of the design of the study, its relevance and potential contributions to medical care and public health

Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression., This research was supported by grants of FAPESP (2010/20002-0; 2008/57889-1) and CNPq (480552/2011-8); and fellowships for BP (CNPq 573799/2008-3; CAPES 1524553), WSA (FAPESP 2012/16512-8), MM (FAPESP 2011/14416-9), SH (2013/27006-9).

Persistent infection with high-risk human papilloma virus (HR-HPV) is the main risk factor for the development of invasive cervical cancer although is not sufficient to cause cervical cancer. Several host and environmental factors play a key role in cancer initiation/progression, including cytokines and other immune-response mediators. Here, we characterized the response to the individual and combined action of the pro-inflammatory cytokines tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) on HPV-transformed cells and human keratinocytes ectopically expressing E6 and E7 early proteins from different HPV types. We showed that keratinocytes expressing HPV early proteins exhibited global alterations in the expression of proteins involved in apoptosis regulation/execution, including TNF and TRAIL receptors. Besides, we provided evidence that TNF receptor 1 (TNFR1) was down-regulated and may be retained in the cytoplasm of keratinocytes expressing HPV16 oncoproteins. Finally, fluorescence analysis demonstrated that cytokine treatment induced the production and release of reactive oxygen and nitrogen species (ROS/RNS) in cells expressing HPV oncogenes. Alterations in ROS/RNS production and apoptosis regulatory factors expression in response to inflammatory mediators may favor the accumulation of genetic alterations in HPV-infected cells. Altogether, our results suggested that these events may contribute to lesion progression and cancer onset.

OBJECTIVES: Oxidative stress results from an imbalance between the generation and elimination of oxidant species. This condition may result in DNA, RNA and protein damage, leading to the accumulation of genetic alterations that can favor malignant transformation. Persistent infection with high-risk human papillomavirus types is associated with inflammatory responses and reactive oxygen species production. In this context, oxidative stress, chronic inflammation and high-risk human papillomavirus can act in a synergistic manner. To counteract the harmful effects of oxidant species, protective molecules, known as antioxidant defenses, are produced by cells to maintain redox homeostasis. In recent years, the use of natural antioxidants as therapeutic strategies for cancer treatment has attracted the attention of the scientific community. This review discusses specific molecules and mechanisms that can act against or together with oxidative stress, presenting alternatives for cervical cancer prevention and treatment.

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.

Made available in DSpace on 2018-12-11T17:08:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-03 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: High-risk human papillomaviruses (HPVs) are strongly associated with the development of some malignancies. The E6 and E7 viral oncoproteins are the primary proteins responsible for cell homeostasis alteration and immortalization. Furthermore, the E6 protein from high-risk HPVs can interact with the PDZ (PSD-90/Dlg/ZO-1) domains of cellular proteins, triggering cell transformation. One protein that is associated with pathological conditions and has a PDZ domain is the protease HTRA1 (high temperature requirement 1). This protein is poorly expressed in some cancers, suggesting a tumor suppressor role. The aim of this study was to evaluate the effect of HTRA1 overexpression in HPV16-positive (CasKi) and HPV-negative (C33) cervical cell lines. Methods: The cells were transfected with a vector containing the HTRA1 ORF or an empty vector. HTRA1 overexpression was confirmed by qRT-PCR. The cells were subjected to cell proliferation, colony formation, apoptosis and cell cycle assays. Results: C33 cells expressing HTRA1 grew significantly fewer colonies and showed less proliferation than cells without HTRA1 expression. In contrast, in the CasKi cells overexpressing HTRA1, there was an increase in the cell growth rate and in the colonies density compared to cells expressing low levels of HTRA1. An apoptosis assay showed that HTRA1 does not interfere with the apoptosis rate in these cells. A cell cycle immunofluorescence assay revealed more CasKi cells overexpressing HTRA1 in the S phase and more C33 HTRA1-transfected cells in the G0/G1 phase, suggesting that HTRA1 plays different roles in the cell cycle progression of these cells. Conclusions: HTRA1 overexpression prevents cell proliferation in the HPV-negative cell line and increases cell proliferation in the HPV-positive cell line. Although the E6/HTRA1 interaction has already been described in the literature, more studies are required to confirm whether the present functional findings are a result of this interaction. Instituto de Bioci�ncias Letras e Ci�ncias Exatas - IBILCE/UNESP Department of Biology, Rua Crist�v�o Colombo n 2265, Jardim Nazareth Hospital das Cl�nicas da Faculdade de Medicina da Universidade de S�o Paulo Center for Translational Investigation in Oncology Instituto do C�ncer do Estado de S�o Paulo, Av. Dr. Arnaldo, 251, 8 andar Universidade de S�o Paulo Department of Radiology and Oncology Faculdade de Medicina, Av. Dr. Arnaldo, 251, 8 andar Instituto de Bioci�ncias Letras e Ci�ncias Exatas - IBILCE/UNESP Department of Biology, Rua Crist�v�o Colombo n 2265, Jardim Nazareth FAPESP: 2012/11126-2 CNPq: 478800/2013-4

Background Human papillomavirus (HPV) prevalence in head and neck squamous cell carcinomas (HNSCC) diverges geographically. The reliability of using p16INK4a expression as a marker of viral infection is controversial in HNSCC. We evaluated HPV types and HPV-16 variants prevalence, and p16INK4a expression in HNSCC specimens provided by two different Institutions in São Paulo. Methods HPV DNA from formalin-fixed specimens was accessed by Inno-LiPA, HPV-16 variants by PCR-sequencing, and p16INK4a protein levels by immunohistochemistry. Results Overall, HPV DNA was detected among 19.4 % of the specimens (36/186). Viral prevalence was higher in the oral cavity (25.0 %, 23/92) then in other anatomical sites (oropharynx 14,3 %, larynx 13.7 %) when samples from both Institutions were analyzed together. HPV prevalence was also higher in the oral cavity when samples from both Institutions were analyzed separately. HPV-16 was the most prevalent type identified in 69.5 % of the HPV positive smaples and specimens were assigned into Asian-American (57.2 %) or European (42.8 %) phylogenetic branches. High expression of p16INK4a was more common among HPV positive tumors. Conclusion Our results support a role for HPV-16 in a subset of HNSCC.

We are grateful to Carlos Ferreira do Nascimento and Severino Ferreira from the Hospital A.C. Camargo for technical assistance. We would like to thank Dr. Enrique Boccardo, Dr. Ismar Haga and Dr. Ana Paula Lepique for critical review of the manuscript and Dr. Erico Tosoni Costa for assistance in figure preparation., Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease., Dr. Lara Termini was a Post-Doctoral fellow from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant 05/57274-9).

Oxidative stress reflects an imbalance in the maintenance of the intracellular redox state resulting in the accumulation of oxidant species that may contribute to tumorigenesis. The superoxide dismutase 2 protein (SOD2) contributes to cell homeostasis by catalyzing the dismutation of superoxide anion radicals in oxygen and hydrogen peroxide, preventing the direct inactivation of biomolecules. SOD2 protein can be induced by the activation of the NF-kB pathway in several cell types following treatment with interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNFα), both proinflammatory cytokines. In addition, previous studies from the group have shown a direct correlation between increased levels of SOD2 protein and severity of uterine cervix lesions. The aim of this study is to elucidate the involvement of SOD2 in the pathogenesis associated with HPV and in the effect of proinflammatory cytokines. Methods: Primary human keratinocytes transduced with HPV16 oncogenes and cervical cancer derived cell lines were treated with the proinflammatory cytokines TNFα, and IL-1beta. The expression of SOD2 protein involved in IL-1 β and TNFα signaling pathways was determined by Western blot and ELISA. Results: Preliminary results suggest the presence of higher levels of SOD2 expression in uterine cervix-derived tumor cell lines when compared to normal primary keratinocytes. Treatment with IL-1β and TNFα induces SOD2 protein accumulation in in the HPV16 positive cervical-derived tumor line, SiHa. On the other hand, no SOD2 induction was observed in the HPV-negative cervical cancer derived cell line C33. Further replications and additional experiments are needed for conclusions. Citation Format: Gabriela Ávila Fernandes Silva, Rafaella Almeida Lima Nunes, Enrique Boccardo, Luisa Lina Villa, Lara Termini. Study of superoxide dismutase-2 protein in HPV-mediated cell transformation [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 B69.

BACKGROUND: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme in lung, gastric, colorectal, breast cancer and cervical cancer malignant tumors has been observed. Its relationship with inguinal lymph node metastasis in penile cancer is unknown. METHODS: SOD2 protein expression levels were determined by immunohistochemistry in 125 usual type squamous cell carcinomas of the penis from a Brazilian cancer center. The casuistic has been characterized by means of descriptive statistics. An exploratory logistic regression has been proposed to evaluate the independent predictive factors of lymph node metastasis. RESULTS: SOD2 expression in more than 50% of cells was observed in 44.8% of primary penile carcinomas of the usual type. This expression pattern was associated with lymph node metastasis both in the uni and multivariate analysis. CONCLUSIONS: Our results indicate that SOD2 expression predicts regional lymph node metastasis. The potential clinical implication of this observation warrants further studies., Dr. Lara Termini (FAPESP 2005/57274-9); Dr. Luisa Lina Villa (FAPESP 2008/57889-1 and CNPq 573799/2008-3).

"Manuscript", BACKGROUND: Matrix metalloproteinases (MMP) are important enzymes in the tumor microenvironment associated with progression of cervical intraepithelial neoplasia (CIN) toward squamous cell carcinoma (SCC) of the cervix. However, the role of MMPs in the inflammatory process associated with Chlamydia trachomatis infection concomitant with the carcinogenic process driven by HPV has not yet been addressed. In the present study, we analyzed the state of the MMP-9-RECK axis in cervical carcinogenesis. METHODS: The levels of MMP-9 and RECK expression were analyzed by immunocytochemistry in liquid-based cytology samples from 136 women with high-grade cervical lesions (CIN2/CIN3) and cervical SCC diagnosed by LLETZ, and in 196 women without cervical neoplasia or CIN1. Real-time qPCR was performed to analyze expression of MMP-9 and RECK in 15 cervical samples. The presence of HPV-DNA and other genital pathogens was evaluated by PCR. RESULTS: We found a higher expression of MMP-9 [OR, 4.2; 95% confidence interval (CI), 2.2-7.8] and lower expression of RECK (OR, 0.4; 95% CI, 0.2-0.7) in women with CIN2/CIN3/SCC when compared with women from the control group (no neoplasia/CIN1). A statistically significant association was also found between MMP-9/RECK imbalance and infection by alpha-9 HPV and C. trachomatis. The prevalence of C. trachomatis infection was significantly higher in women with high-grade cervical disease (OR, 3.7; 95% CI, 1.3-11.3). CONCLUSIONS: MMP-9/RECK imbalance in cervical smears is significantly associated with high-grade cervical diseases and infection by alpha-9 HPV and C. trachomatis. IMPACT: MMP-9/RECK imbalance during cervical inflammation induced by C. trachomatis might play a role in HPV-mediated cervical carcinogenesis., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), numbers 2008/03232-1 (to L.L. Villa) and 2012/09746-2 (to M.G. Discacciati and S.S. Maria-Engler) and National Counsel of Technological and Scientific Development pharmaceutical innovation (CNPQ-INCT-if; to S.S. Maria-Engler).

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

Purpose. To describe the acquisition, persistence, and clearance of HPV infection in women with CIN 2 followed up for 12 months.Methods. Thirty-seven women with CIN 2 biopsy, who have proven referral to cervical smear showing low-grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance and tested for HPV, were followed up for one year with cervical smear, colposcopy, and HPV test every three months. HPV DNA was detected by the polymerase chain reaction and genotyping by reverse line blot hybridization assay.Results. CIN 2 regression rate was 49% (18/37), persistence as CIN 1 or CIN 2 was 22% (8/37), and progression to CIN 3 was 29% (11/37). Multiple HPV types were observed at admission in 41% (15/37) of cases. HPV 16 was detected at admission in 58% (11/19) of the cases that persisted/progressed and in 39% (7/18) of the cases that regressed. HPV 16 was considered possibly causal in 67% (10/15) of the cases that persisted or progressed and in 10% (1/10) of the cases that regressed (P=0.01).Conclusion. Multiple HPV infections were frequently detected among women with CIN 2 at admission and during the followup. The CIN 2 associated with HPV 16 was more likely to persist or to progress to CIN 3.

Polymorphism in the Toll-like receptor (TLR) 9 gene has been shown to have a significant role in some diseases; however, little is known about its possible role in the natural history of human papillomavirus (HPV) infections. We investigated the association between a single-nucleotide polymorphism (SNP) (rs5743836) in the promoter region of TLR9 (T1237C) and type-specific HPV infections. Specimens were derived from a cohort of 2462 women enrolled in the Ludwig–McGill Cohort Study. We randomly selected 500 women who had a cervical HPV infection detected at least once during the study as cases. We defined two control groups: (i) a random sample of 300 women who always tested HPV negative, and (ii) a sample of 234 women who were always HPV negative but had a minimum of ten visits during the study. TLR9 genotyping was performed using bidirectional PCR amplification of specific alleles. Irrespective of group, the WT homozygous TLR9 genotype (TT) was the most common form, followed by the heterozygous (TC) and the mutant homozygous (CC) forms. There were no consistent associations between polymorphism and infection risk, either overall or by type or species. Likewise, there were no consistently significant associations between polymorphism and HPV clearance or persistence. We concluded that this polymorphism in the promoter region of TLR9 gene does not seem to have a mediating role in the natural history of the HPV infection.

We thank Dr. Alda Wakamatsu, Dr. Venâncio Avancini and Dr. Alexandre Ab'Saber from Faculty of Medicine, University of São Paulo, for technical assistance and selection of tissues samples, and João Simão Pereira Sobrinho for help in protein sample processing., Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome., This research was funded by FAPESP-São Paulo Research Foundation: 2005/58885-1 (to LBSC), 2008/58817-4 (to SSME) and 2008/03232-1 (to LLV); and by PRPG-USP: Pró-Reitoria de Pós-Graduação da Universidade de São Paulo and CNPq: Conselho Nacional de Pesquisa e Desenvolvimento (to SSME).

Overexpression of kallikrein 7, a proteolytic enzyme important for epithelial cell shedding, may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion. In this study, we have evaluated hK7 (human kallikrein 7) protein levels by immunohistochemistry in 367 cervical histological samples including 35 cases of cervicitis, 31 low-grade cervical intraepithelial neoplasia, 51 high-grade cervical intraepithelial neoplasia (H-SIL), 197 squamous cervical carcinomas (SCC) and 53 cervical adenocarcinomas. We have observed that hK7 staining increased with the severity of cervical disease. Intense hK7 staining was found in 15.2% of cervicitis samples, in contrast to 55% of H-SIL and 68% of SCC. Moreover, 92.5% of adenocarcinomas also exhibited intense hK7 staining. Differences in the expression of hK7 could potentially be used as a biomarker for the characterization of different stages of cervical disease., Grant sponsor: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo; Grant number: 05/57274-9

Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

BACKGROUND: Penile squamous cell carcinoma (PSCC) is a rare tumor. In specific regions of developing countries, however, it may represent one the most frequent male tumors. Presence of lymph node metastasis (LNM) is the main factor significantly associated with reduced survival, and molecular markers to predict it have been extensively pursued. HER2 is an oncogene part of the EGFR family, often overexpressed in PSCC. HER2 overexpression is associated with poor prognosis and presence of LNM in breast tumors, and it is generally coamplified with topoisomerase IIα (TOP2A), a neighboring gene that is a target for anthracyclines. MATERIAL AND METHODS: We evaluated the frequency of HPV infection in 84 PSCC samples.The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Genotyping of positive samples was done by dot blot hybridization using radioactive probes. Expression of HER2 and TOP2a was evaluated through immunohistochemistry and automatedly quantified using ScanScope XT, Aperio. Sixty cases were also submitted to a triple-color fluorescence in situ hybridization (FISH) assay using probes for the genes of HER2 and TOP2a, and also for the chromosome 17 centromere. Cases showing two signals of each probe were considered non-altered, those showing more than two signals of each probe were considered polysomy and those showing more HER2 or TOP2a signals compared to centromere signals were considered amplified. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 22.6% (19) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18, and 2.7% by both subtypes concomitantly. In agreement with studies that showed better prognosis of tumors with HPV infection, 5-year and 10-year survival of HPV-positive patients was, respectively, 79.1% and 70.8%, whereas of HPV-negative patients was 60.2% and 38.7% (p=0,018). Immunohistochemistry showed nuclear positivity for TOP2a in 74.2% (106) cases and cytoplasmic expression of HER2 was seen in 20,3% (29) of cases, which was associated with higher histologic grade (p Citation Format: Alice M. Silva, Lara Termini, Jose Ivanildo Neves, Isabela Cunha, Ademar Lopes, Gustavo Guimaraes, Fernando A. Soares. Evaluation of HER2 and topoisomera II alpha (TOP2a) expression and gene number in penile carcinoma according to HPV status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2013-5296

INTRODUCTION: Penile carcinoma is an infrequent tumor. However, in developing countries it is a common disease and in specific regions it may represents the most frequent tumor among men. The most important prognostic clinical factor is the presence of lymph node metastasis, which is associated with significant reduced survival to the penile carcinoma patients. Nevertheless, until the present date it was not possible to establish clinical or molecular prognostic markers. Various factors are associated with increased risk for de development of this disease, such as poor hygienic habits, lack of circumcision and HPV infection. HPV DNA can be detected in 20% to 80% of penile squamous cell carcinomas (PSCC). However, the role of HPV on patients’ prognosis is still uncertain. METHODS: We evaluated the frequency of HPV infection in 189 PSCC samples. The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Specimens positive for HPV DNA were genotyped by dot blot hybridization using radioactive probes. We also evaluated the expression levels of the proteins p16 and MGMT through immunohistochemistry. These two proteins show aberrant levels in a wide range of tumors. Protein expression was quantified using an automated image system (ScanScope XT - Aperio), which determined the percentage and intensity of positive cells. The quantification was used to determine if there was loss of expression or normal (positive) expression. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 19.6% (37) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18 and 2.7% by both HPV16 and 18 concomitantly. The presence of HPV infection was more frequent among tumors with thickness lower than 5mm, suggesting that PSCC with HPV are less aggressive. This finding is in agreement with some studies that showed better prognosis of tumors with HPV infection. The immunohistochemistry assays showed reduced p16 expression in 35.4% (63) and loss of MGMT in 90.1% (146) cases. None association was observed between HPV infection and p16 protein expression. It is well established that the presence of HPV is associated with p16 positive expression. One interesting observation was that MGMT positive expression was associated with positive viral infection. Until this moment, there is no study describing this association in PSCC neither in other tumor types. It was observed an association between p16 reduced levels and poor differentiated tumors, this pathologic variable is associated with bad prognosis. MGMT loss of expression was associated with the presence of lymph node metastasis, suggesting that expression status of this protein can be a molecular marker for PSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1165. doi:1538-7445.AM2012-1165

Penile carcinoma is an uncommon disease and the most frequent subtype is squamous cell carcinoma. It represents less than 1% of all cases in developed countries. However, in developing countries it may represent 20% of all male cancers. The main risk factors associated to this tumor are poor hygienic habits, phimosis, HPV infections and high number of sexual partners. Very little is known about penile carcinoma pathogenesis, etiology and molecular biology features. In this study, we evaluated the hypermethylation frequency of two genes that present high frequencies of methylation in various tumor types: CDKN2A e MGMT. CDKN2A plays an important role in cell cycle control, encoding a protein (p16) that inhibits pRB phosphorilation through inactivation of the CDK-cyclin D complex, so blocking cell cycle progression. MGMT encodes a DNA repair protein that removes alkyl and methyl groups from guanines without DNA double strand breaks. Hypermethylation of these genes leads to transcriptional repression and has an important role in tumoregenesis. Unfortunately, scarce data is available about the role of hypermethylation in penile carcinomas. We selected 128 patients with penile squamous cell carcinoma surgically treated in a Brazilian cancer center. DNA from the formalin fixed paraffin embedded samples was obtained using a phenol-chloroform standard protocol. The DNA samples were submitted to bisulfite conversion, amplification and sequencing by the pyrosequencing approach, a quantitative methodology that allows the evaluation of the methylation levels in each CpG site of the target sequence. We also analyzed the expression levels by immunohistochemistry of the proteins p16 and MGMT to verify if the presence of hypermethylation is leading to gene silencing. We observed absence of CDKN2A hypermethylation in all samples and MGMT was hypermethylated in 57.4% of the cases, with levels of methylation varying from 16% to 86%. It was observed p16 positive expression in 66.8% of samples and Mgmt was present in only 10.9%. The decrease in MGMT expression was observed more frequent in tumors thicker than 5 millimeters and in patients with palpable nodes, suggesting a more aggressive tumor behavior in the absence of this protein. MGMT hypermethylation was not associated with reduced protein expression. Among samples with loss of MGMT expression 55.7% were methylated suggesting that only the presence of methyl radicals is not enough to induce gene silencing, it is probable that other mechanisms such as chromatin condensation are also required to repress gene transcription. The high frequency of p16 positivity could be explained by the presence of HPV infection, detected by others in about 30% of penile cancers. It also suggests that other members of the pRb pathway would be altered, such as cyclin D and Cdk amplifications or pRb loss. It cannot be discarded that other pathways of cell cycle control may also be deregulated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C17.

A etiologia do câncer do colo do útero, assim como a de uma grande maioria de tumores da região anogenital e alguns cânceres de cabeça e pescoço, está associada à infecção persistente pelo Papilomavírus Humano (HPV). O desequilíbrio do sistema redox celular, caracterizado pela alteração dos níveis de espécies oxidantes e da atividade do sistema antioxidante das células infectadas, é um fator que está diretamente relacionado ao desenvolvimento do câncer do colo do útero, juntamente com outras alterações induzidas por esse vírus. Nesse sentido, a enzima antioxidante SOD2 (Superóxido Dismutase 2), possui um papel fundamental nesse processo. Essa proteína é capaz de prevenir os danos causados por essas espécies reativas às biomoléculas celulares, como DNA, proteínas, carboidratos e lipídeos, uma vez que catalisa a reação de dismutação dos radicais superóxido em peróxido de hidrogênio e oxigênio molecular para detoxificação celular. Estudos anteriores do grupo revelaram que o aumento da expressão da proteína SOD2 está diretamente relacionado com a gravidade das lesões do colo uterino e mais ainda nos cânceres invasores desta região anatômica. Portanto, este estudo teve como objetivo determinar o papel de SOD2 na patogênese associada ao HPV em linhagens celulares derivadas de câncer da cérvice uterina. Para isso, a expressão de SOD2 foi suprimida utilizando-se as técnicas shRNA e CRISPR/Cas9 em HeLa e ativada com o sistema CRISPR/Cas9, enquanto SiHa teve essa enzima silenciada apenas com a abordagem shRNA. Em seguida, essas células foram submetidas à avaliação funcional, incluindo os ensaios de crescimento, viabilidade, migração, invasão, formação de colônias e angiogênese. As análises revelaram que o silenciamento de SOD2 resultou na redução da capacidade de crescimento, viabilidade, migração, invasão e potencial clonogênico de HeLa, e na diminuição apenas do crescimento e migração de SiHa. Interessantemente, a ativação de SOD2 em HeLa culminou no mesmo fenótipo encontrado no silenciamento. Dessa forma, com base nesses resultados, pode-se sugerir que embora a enzima SOD2 pareça desempenhar um papel importante na carcinogênese do colo do útero mediada pelo HPV, essa função é dependente do contexto da célula e provavelmente do microambiente tumoral, já que o silenciamento e ativação dessa enzima revelaram características semelhantes. Assim, análises adicionais são necessárias para uma melhor compreensão dos múltiplos papéis de SOD2 no contexto tumoral, sobretudo do câncer do colo do útero Cervical cancer etiology, as well as that of a vast majority of anogenital tumors and some head and neck cancers, is associated with Human Papillomavirus (HPV) persistent infection. The imbalance of the cellular redox system, characterized by alterations in the levels of oxidant species and the activity of the antioxidant system of infected cells, is a crucial factor that is directly related to the development of cervical cancer, along with other changes induced by this virus. In this sense, the antioxidant enzyme SOD2 (Superoxide Dismutase 2) has a fundamental role in this process. This protein acts to prevent damage caused by these reactive species to cellular biomolecules, such as DNA, proteins, carbohydrates, and lipids, since it catalyzes the dismutation reaction of superoxide radicals into hydrogen peroxide and molecular oxygen for cellular detoxification. Previous studies of our group revealed that high expression of SOD2 protein is directly related to the severity of lesions of the cervix and even more in the invasive cancers in this anatomical region. Therefore, this study aimed to determine the role of SOD2 in the pathogenesis associated with HPV in cell lines derived from cervical cancer. For this, the expression of SOD2 was suppressed using the shRNA and CRISPR/Cas9 techniques in HeLa and was activated by the CRISPR/Cas9 system, while SiHa had this enzyme silenced only by the shRNA approach. Then, these cells were submitted to the functional evaluation, including growth curve, viability, migration, invasion, colony formation, and angiogenesis assays. The analyses revealed that SOD2 silencing resulted in reduced growth capacity, viability, migration, invasion, and clonogenic potential of HeLa, and a decrease only in growth and migration of SiHa. Surprisingly, SOD2 activation in HeLa culminated in the same phenotype observed in SOD2-silenced cells. Thus, based on these results, it can be suggested that although the SOD2 enzyme appears to play an important role in HPV-mediated cervical carcinogenesis, this function is dependent on the cell context and probably on the tumor microenvironment, as silencing and activation of this enzyme revealed similar characteristics. Additional analyzes are needed for a better understanding of the multiple roles of SOD2 in the tumor context, especially in cervical cancer

Introdução: A contribuição do inflammasoma e das suas citocinas (IL-1beta, IL-18) na infecção pelo Papilomavírus Humano (HPV) e no Câncer Cervical(CC) associado ao HPV (HPV-CC) não esta ainda totalmente esclarecida. A infecção in vitro do HPV leva a inibição da produção de IL-1beta em queratinocitos primários. Por outro lado, estudos de associação genética e da produção de IL-1beta e/ou IL-18 mostram uma correlação positiva com à progressão do CC. A partir dos dados obtidos em um estudo de associação genética em uma coorte de mulheres com HPV-CC que apontava para IL- 1beta como fator de risco para o desenvolvimento do CC, propomos aqui aprofundar este achado. Objetivo: Investigar a contribuição do inflamassoma no microambiente tumoral do HPV-CC. Materiais e métodos: Um estudo de associação genética para as variantes do inflamassoma foi replicado em uma nova coorte de 107 mulheres com HPV-CC utilizando sondas alelo-espcificas e qPCR. Uma análise de expressão gênica foi realizada utilizando dados de tecido de cérvix uterino tumoral e normal procedentes de bancos públicos (TCGA, GTEX). Linhagens celulares de tumor cervical (C33-A, SiHa e HeLa) e monócitos derivados de sangue periférico de 39 doadores saudáveis foram cultivados isoladamente e em conjunto (ensaio de co-cultivo). A ativação do inflamassoma foi avaliada de modo indireto através da produção de IL-1beta e IL-18 medida no sobrenadante de cultura por ELISA. Inibidores específicos do inflamassoma foram utilizados em alguns ensaios. Resultados: A análise genética demonstrou mais uma vez a associação entre a variante rs16944 (que leva a aumento de IL-1beta) assim como uma maior expressão do gene IL1B e um maior risco de desenvolver HPV-CC e de um pior prognostico, respectivamente. Os dados obtidos nos ensaios de co-cultivo revelaram que, apenas no modelo de cultivo das linhagens celulares de CC em esferoides (não em monocamada), as células tumorais não produzem IL-1beta, mas induzem a ativação do inflamassoma e a liberação de IL-1beta em monócitos. Esta ativação é pelo menos parcialmente dependente do receptor do inflamassoma NLRP3. Conclusão: Pela primeira vez, ao nosso conhecimento, demonstramos que o aumento de IL-1beta observado na progressão do CC, é possivelmente devido a indução da ativação do NLRP3 inflamassoma das células tumorais nos monócitos do microambiente. Portanto, tanto NLRP3 quanto IL-1beta podem ser considerados como possíveis alvos para novas abordagens terapêuticas Introduction: The contribution of inflammasoma and its cytokines (IL-1beta, IL-18) to Human Papillomavirus (HPV) infection and HPV-associated cervical cancer (CC) (HPV-CC) is not yet fully understood. In vitro HPV infection leads to inhibition of IL-1beta production in primary keratinocytes. On the other hand, studies of genetic association and the production of IL-1beta and / or IL-18 show a positive correlation with the progression of CC. Based on the data obtained in a study of genetic association in a cohort of women with HPV-CC that pointed to IL-1beta as a risk factor for the development of CC, we propose to deepen this finding here. Objective: To investigate the contribution of inflammasome in the tumor microenvironment of HPV-CC. Materials and methods: A genetic association study for inflammasome variants was replicated in a new cohort of 107 women with HPV-CC using allele-specific probes and qPCR. A gene expression analysis was performed using tumor and normal uterine cervix tissue data from public databases (TCGA, GTEX). Cervical tumor cell lines (C33-A, SiHa and HeLa) and monocytes derived from peripheral blood from 39 healthy donors were cultured alone and together (co-culture assay). The activation of the inflammasome was evaluated indirectly through the production of IL-1beta and IL-18 measured in the culture supernatant by ELISA. Specific inflammasome inhibitors have been used in some trials. Results: Genetic analysis demonstrated once again the association between the rs16944 variant (which leads to an increase in IL-1beta) as well as a greater expression of the IL1B gene and a greater risk of developing HPV-CC and a worse prognosis, respectively. The data obtained in the co-culture assays revealed that, only in the spheroid CC cell culture model (not in monolayer), tumor cells do not produce IL-1beta, but induce the activation of the inflammasome and the release of IL -1beta in monocytes. This activation is at least partially dependent on the NLRP3 inflammasome receptor. Conclusion: For the first time, to our knowledge, we demonstrated that the increase in IL-1beta observed in the progression of CC is possibly due to the induction of NLRP3 activation of inflammasome tumor cells in the monocytes of the microenvironment. Therefore, both NLRP3 and IL-1beta can be considered as possible targets for new therapeutic approaches

Introdução: A resposta imune desempenha um papel importante na eliminação das infecções causadas pelo HPV. Como estratégia de evasão do sistema imunológico, o vírus pode modular a produção e secreção de citocinas pelas células infectadas. Além disso, a resposta imune pode contribuir para a criação de um quadro de inflamação crônica, que favorece a progressão das lesões relacionadas ao vírus e o desenvolvimento do câncer. Objetivos: O objetivo deste estudo foi avaliar o papel da via da interleucina-1, mais especificamente IL-1alfa, IL-1beta e IL-18, importantes citocinas pró-inflamatórias, no estabelecimento e progressão dos tumores cervicais. Material e métodos: Queratinócitos normais e transduzidos com os oncogenes E6 e/ou E7 de HPV16, além das linhagens tumorais de origem cervical C33, SiHa e HeLa, foram avaliados quanto à expressão (transcritos e proteínas) de IL-1alfa, IL-1beta, IL-18, seus receptores e antagonistas naturais. O gene codificador de IL-18 foi editado por meio do sistema CRISPR-Cas9, e as linhagens celulares silenciadas foram avaliadas quanto à capacidade de invasão, migração, proliferação, viabilidade e indução da angiogênese. Além disso, 62 biópsias provenientes de pacientes diagnosticadas com câncer cervical avançado foram analisadas por imuno-histoquímica visando à avaliação da expressão de IL-18 nos tecidos tumorais e sua relação com o prognóstico. Resultados: Foi verificado que, embora presente nos queratinócitos normais e transduzidos com os oncogenes do HPV, a expressão de IL-1alfa e IL-1beta encontra-se drasticamente reduzida nas linhagens tumorais de origem cervical, ao contrário de IL-18, que permanece sendo expressa de forma significativa. O silenciamento de IL-18 por CRISPR-Cas9 reduziu a capacidade proliferativa e de formação de colônias das linhagens tumorais cervicais HPV positivas, mas associou-se à tendência de maior capacidade invasiva e de indução da angiogênese. A análise imuno-histoquímica das biópsias mostrou que a expressão de IL-18 na borda dos tumores está associada à resposta completa ao tratamento e menor risco de recorrência linfática (p=0.027 e p=0.034, respectivamente). Embora não se tenha atingido significância estatística, observou-se clara tendência a maiores taxas de sobrevida global e sobrevida livre de doença nos tumores em cuja localização do infiltrado inflamatório era peritumoral (p=0.056 e p=0.165, respectivamente). Da mesma forma, a maior expressão de IL-18 no núcleo e no citoplasma das células tumorais foi associada à tendência de melhor prognóstico. Conclusões: Considerando-se os resultados acima expostos, verificou-se que, embora presentes nos queratinócitos normais e transduzidos com os oncogenes de HPV16, IL-1alfa, IL-1beta, seu antagonista natural e seus receptores são modulados negativamente de forma importante nas linhagens tumorais de origem cervical C33, SiHa e HeLa. Ao contrário, IL-18, seu antagonista natural e seus receptores continuam sendo expressos nas mesmas células. A análise conjunta dos resultados envolvendo as linhagens silenciadas para IL-18 e dos dados obtidos por imunohistoquímica sugere um papel protetor de IL-18, que deve ser confirmado por meio da ampliação da amostra estudada. Deve ser considerada a interação entre as células tumorais e o microambiente, mais especificamente, neste caso, o microambiente imune tumoral, no qual IL-18 constitui um importante mediador. Pretende-se que esta seja a próxima etapa deste estudo Introduction: The immune response plays an important role in clearing HPV infections. As a strategy to evade the immune system, the virus can modulate cytokine production and secretion by infected cells. In addition, the immune response can contribute to create a chronic inflammatory condition, which can favor the progression of virus related lesions and cancer development. Objectives: The objective of this study was to evaluate the role of the interleukin-1 pathway, more specifically IL-1alpha, IL-1beta and IL-18, important pro-inflammatory cytokines, in the establishment and progression of cervical tumors. Material and methods: Normal keratinocytes and keratinocytes transduced with HPV16 E6 and/or E7 oncogenes, in addition to the cervical tumor cell lines C33, SiHa and HeLa, were evaluated for the expression (transcripts and proteins) of IL-1alpha, IL-1beta, IL-18, their natural antagonists and receptors. The gene encoding IL-18 was edited using CRISPRCas9 system, and silenced cell lines were evaluated for invasiveness, migration, proliferation, colony-forming capacity and angiogenesis induction. In addition, 62 biopsies from patients diagnosed with advanced cervical cancer were assessed by immunohistochemistry to assess IL-18 expression in tumor tissues and its relationship with the patients\' prognosis. Results: It was found that, although present in normal and transduced keratinocytes, the expression of IL-1alpha and IL-1beta is drastically reduced in cervical tumor cell lines, in contrast to IL-18, which remains expressed in a meaningful way. IL-18 silencing by CRISPR-Cas9 reduced the proliferative and colony-forming capacity of HPV-positive cervical tumor cell lines, but was associated with the tendency towards greater invasive capacity and angiogenesis induction. The biopsies immunohistochemical analysis showed that IL-18 expression at the edge of the tumor is associated with a complete response to the treatment and a lower risk of lymphatic recurrence (p=0.027 and p=0.034, respectively). Although no statistical significance was reached, there is a clear tendency towards higher rates of overall survival and disease free survival in tumors showing peritumoral location of the inflammatory infiltrate (p=0.056 and p=0.165, respectively). Likewise, the increased IL-18 expression in tumor cells nucleus and cytoplasm was associated with a trend towards better prognosis. Conclusions: Considering the above results, it was found that, although present in normal and transduced keratinocytes, IL-1alpha, IL-1beta, their natural antagonist and receptors are significantly negatively modulated in cervical tumor cell lines C33, SiHa and HeLa. In contrast, IL-18, its natural antagonist and receptors continue to be expressed in the same cells. Together, IL-18 silenced cells analysis and immunohistochemistry data suggest a protective role for IL-18, which should be confirmed by expanding the studied sample. The interaction between tumor cells and the microenvironment must be considered, more specifically, in this case, the tumor immune microenvironment, in which IL-18 is an important mediator. This is intended to be the next stage of this study

A patogênese e o estadiamento do melanoma são clinicamente bem caracterizados, entretanto, muitas variações são observadas no que diz respeito à progressão e respostas terapêuticas. Mecanismos intrínsecos das células de melanoma contribuem para esse fenômeno, como a plasticidade fenotípica e a expressão de determinadas moléculas, como a galectina-3. Galectina-3 tem papel fundamental para o crescimento do tumor, metástase e montagem de uma rede vascular funcional. Dessa forma, o melanoma possui mecanismos alternativos de vascularização, onde células endoteliais e células tumorais formam a superfície luminal dos vasos e são expostas às forças fluídicas do sangue, o shear stress. Nós hipotetizamos que o shear promove a adaptação celular e células de melanoma se comunicam com (1) outras células tumorais para sustentar a progressão do tumor, ou (2) com células endoteliais para garantir a manutenção vascular. Para isso, comparamos dois modelos de geração do shear denominados pela agitação de placas concêntricas e rotação de um cone sobre placa, que é o modelo padrão dos estudos em células endoteliais. O modelo de placas concêntricas gerou resultados mais robustos, pois garantiu a viabilidade e a adesão celular em até 24h. Também investigamos o potencial angiogênico das células de melanoma após monitorar a ativação de células endoteliais em contato com o meio condicionado (MC) do shear. O shear aumentou significativamente a secreção de Angiopoietina-2, IL-8 e PLGF num contexto independente de galectina-3. Além disso, o shear diminuiu a taxa de crescimento das células que expressam galectina-3, o que não foi observado no tratamento com o MC. Já a capacidade de sobreviver em longo prazo foi maior quando as células tumorais são expostas ao MC derivado do shear, sem influência da expressão de galectina-3. Em relação ao efeito do MC liberado após shear para o remodelamento vascular, nós não observamos alteração na proliferação das células endoteliais. Entretanto, o MC do shear parece acelerar a formação de túbulos e favorecer a desorganização das fibras de actina, além de diminuir a fosforilação de ERK nas células endoteliais. Juntos, esses resultados mostram que o shear induz a secreção de moléculas pró-angiogênicas e pró-inflamatórias que promovem a sobrevivência e proliferação a longo prazo das células tumorais naive. Nas células endoteliais, o MC derivado do shear altera a sinalização de ERK e o rearranjo do citoesqueleto Melanoma pathogenesis and staging are clinically well characterized, although a large variation is observed in progression and therapeutic responses. Intrinsic mechanisms of melanoma cells contribute to this phenomenon, such as phenotypic plasticity and the expression of certain molecules as galectin-3. Galectin-3 plays a prominent role in tumor growth, metastasis, and to build a functional vascular network. In this way, melanoma has alternative mechanisms of vascularization, in which both endothelial cells and tumor cells form the luminal surface of vessels and are in contact with flowing blood. Here, we postulated that shear stress promotes cell adaptation and melanoma cells communicate with (1) other tumor cells in order to support tumor progression; or (2) with endothelial cells to ensure vascular maintenance. We compared two methods for generating shear stress, namely revolution of concentric plates and stirring of a suspended cone on a plate, which is the gold standard method for studies on endothelial cells. The concentric plates model yielded more reliable results, since it ensured the greater viability and adhesion of cells up to 24h, as compared to the cone and plate system. We then investigated the angiogenic potential of melanoma cells by monitoring the activation of endothelial cells upon exposure to the conditioned medium (CM) from cells exposed to shear stress. Shear stress significantly increased the secretion of Angiopoietin-2, IL-8 and PLGF, in a galectin-3 independent manner. Also, shear stress decreased the growth rate of galectin-3 positive melanoma cells, but not when galectin-3 was down regulated. No such effect was observed with the shear-derived CM. Instead, we did observed long-term melanoma survival under low density upon treatment with the shear-derived CM. In this scenario, the expression of galectin-3 did not change the results. Regarding the effect of shear-derived CM to vascular remodeling, endothelial cells did not show any change in cell proliferation. However, the CM of melanoma cells upon shear accelerated the endothelial cell tube formation and disorganized the actin stress fibers. CM also decreased the phosphorylation of ERK. Taken together, these data indicate that the production of proangiogenic and proinflammatory molecules by melanoma cells under shear enables long-term survival and proliferation of naïve melanoma cells. To endothelial cells, shear-derived CM led to changes in ERK signaling and cytoskeletal rearrangement

O estresse oxidativo reflete um desequilíbrio na manutenção do estado redox intracelular que pode resultar no acúmulo de espécies oxidantes. Nestas condições, estas espécies podem gerar danos na estrutura/função do DNA, disfunções mitocondriais, alterações no enovelamento de proteínas, peroxidação de lipídeos, dentre outros danos. Assim, o estresse oxidativo pode contribuir para diversas condições patológicas, como por exemplo, câncer, desordens neurológicas, aterosclerose, diabetes e asma. A infecção persistente por papilomavírus humano (HPV) de alto risco está etiologicamente associada ao câncer de colo uterino, uma das principais causas de morte por câncer em mulheres no mundo todo. Além disso, esses vírus estão associados a uma porcentagem relevante de cânceres de pênis, vulva, ânus e cabeça e pescoço. Em células infectadas por HPV de alto risco oncogênico, o estresse oxidativo resultante do metabolismo anormal dos queratinócitos e a resposta inflamatória crônica não efetiva, podem contribuir no processo de transformação celular. Além disso, a presença das oncoproteínas E6 e E7 interfere nos mecanismos de reparo de lesões em DNA, favorecendo o acúmulo de mutações. Ainda mais, a instabilidade genômica promovida pelo estresse oxidativo, pode favorecer a integração do HPV no genoma das células infectadas, principal causa da persistência viral e progressão de lesões ao câncer. A proteína superóxido dismutase 2 (SOD2) contribui com a homeostase celular ao catalisar a dismutação de radicais ânion superóxido em oxigênio e peróxido de hidrogênio, prevenindo a inativação direta de biomoléculas. Em estudos anteriores, observamos que o aumento do transcrito de SOD2 está associado à resistência ao efeito antiproliferativo do fator de necrose tumoral (TNF) em células imortalizadas por HPV. Além disso, mostramos que existe uma correlação direta entre o aumento dos níveis da proteína SOD2 e a severidade de lesões da cérvice uterina. Finalmente, identificamos SOD2 como um marcador preditivo independente de metástases linfonodais inguinais em pacientes com carcinoma de pênis. No entanto, o papel desta proteína na patogênese associada ao HPV não tem sido estudado em profundidade. O presente estudo visa determinar o envolvimento de SOD2 no processo de transformação celular mediado por HPV e seu valor como marcador preditivo de patologias associadas a este vírus Oxidative stress reflects a redox imbalance in the cell in favor of oxidant species, which may result in DNA damage, mitochondrial dysfunction, protein misfolding and lipid peroxidation, among others. As a result, oxidative stress is thought to contribute to diverse pathologies such as cancer, neurological disorders, atherosclerosis, diabetes and asthma. Persistent infection with high risk human papillomavirus (HPV) types is etiologically associated with cervical cancer, one of the leading causes of cancer related female death worldwide. Moreover, HPV infection is associated with a significant percentage of penile, vulvar, anal and head and neck carcinomas. In cells infected with high-oncogenic risk HPV types the oxidative stress generated by the abnormal keratinocytes metabolism and non-efficient chronic inflammatory response may contribute to the cellular transformation process. Furthermore, expression of E6 and E7 oncoproteins interferes with several DNA repair mechanisms, favoring the accumulation of mutations. Moreover, genomic instability promoted by oxidative stress may favor HPV integration in host cell genome, the main cause of viral persistence and of precursor lesions progression to cancer. The protein superoxide dismutase 2 (SOD2) contributes to cellular homeostasis by catalyzing radical super anion dismutation in oxygen and oxygen peroxide preventing the direct inactivation of biomolecules. In previous studies we observed that SOD2 mRNA upregulation is associated with the resistance to tumor necrosis factor (TNF) antiproliferative effect in HPV-immortalized cells. Besides, we showed the existence of a direct correlation between increased SOD2 protein expression and cervical lesions severity. Finally, we identified SOD2 as an independent predictive marker of inguinal lymph node metastases in patients with penile carcinomas. However, the role of this protein in HPV-associated pathologies has not been investigated in depth. The goal of this study is to determine the involvement of SOD2 in HPV-mediated cell transformation and its value as a predictive marker in pathologies caused by this virus

O melanoma representa menos de 5% de todos os cânceres de pele, porém, quando em estádio metastático possui prognóstico ruim. Entretanto, o genótipo dos melanomas pode prover uma oportunidade para intervenção terapêutica pelo fato de 90% dos casos de melanoma possuem p53 selvagem e grande parte destes possuem deleção na região cromossômica codificadora de interferon beta. Em prévios estudos, desenvolvemos o vetor adenoviral AdRGD-PG que fornece expressão do transgene em resposta à p53 através do promotor PG e ainda o tripeptídeo RGD, que possibilita que o adenovírus transduza uma maior gama de células pela alteração de seu mecanismo de entrada. Temos utilizado este vetor para entrega da versão murina de interferon beta em modelos de terapia gênica e imunoterapia de melanoma murino, revelando uma significativa habilidade do interferon beta em inibir a proliferação celular in vitro e in vivo e promover resposta imune antitumoral. No presente trabalho, os esforços se aplicam em adaptar essa estratégia em modelo de melanoma humano para observar se a mesma interação é encontrada. O vetor AdRGD-PGhIbeta, portador do cDNA de interferon beta humano (hIbeta) foi construído e expressão do transgene observada após transdução das linhagens estabelecidas de melanoma humano SK-MEL-05 e SK-MEL-147 (ambas p53 selvagem). Foi observado um robusto efeito antitumoral in vitro onde transferência de hIbeta promoveu acumulo de células hipodiploides (mais que 80% da população celular 96 horas após transdução) e evidências de morte por apoptose (exposição de fosfatidilserina e atividade de caspases 3/7) em ambas as linhagens. Nas duas linhagens, o efeito bystander foi demonstrado quando a presença de poucas células transduzidas (ex., 10%) foi suficiente para promover o acumulo significativo de células hipodiploides (mais que 40% neste exemplo). Em modelo de terapia gênica in situ utilizando células SK-MEL-147, também foi observado forte efeito antitumoral da hIbeta com total remissão do tumor de todos os animais tratados sem recidiva durante noventa dias. A presença de hIbeta na circulação dos animais foi confirmada 48h após o tratamento com AdRGD-PG hbeta mas presente em somente dois de sete animais 90 dias após o tratamento, sugerindo que o tratamento inicial e não um efeito off target foi responsável pela resposta. Com a finalidade de investigar efeitos colaterais do sequestro do vetor adenoviral pelo fígado, observamos a concentração circulante das enzimas aminotransferase de aspartate e aminotransferase de alanine (AST e ALT, respectivamente), que se mostrou não alterada quando comparadas entre animais que receberam injeção do vetor tratamento, vetor controle e solução salina. Com nossos resultados concluímos que vetores adenovirais carreando interferon-beta humano são capazes de transduzir a linhagem de melanoma SK-MEL-147 in vitro e in vivo, promovendo efeito bystander e remissão tumoral sem indução de efeitos adversos Melanoma represents less than 5% of all cases of skin cancer, although, when metastatic, prognosis is dire. However, the genotype of melanomas might provide an opportunity for therapeutic intervention since 90% of melanoma cases possess wild-type p53 and a great portion of these possess deletion of the chromosomal region encoding interferon beta. In previous studies, we developed the adenoviral vector AdRGD-PG that supplies expression of the transgene in response to p53 through the PG promoter and that utilizes the RGD tripeptide, allowing the adenovirus to transduce a wider range of cells due to the alterated mechanism of entrance. We have used this vector to deliver the murine version of interferon beta in murine models of melanoma gene therapy and immunotherapy, revealing a significant ability of interferon beta to inhibit cellular proliferation in vitro and in vivo and promote an anti-tumor immune response. In the present project, we aimed to adapt this strategy for a human melanoma model in order to reveal if the same impact will be observed. The AdRGD-PGhIbeta vector encoding the human interferon beta (hIbeta) cDNA was constructed and expression of the transgene confirmed after transduction of the established human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild-type p53). A striking anti-tumor effect was observed in vitro where the transfer of hIbeta promoted an accumulation of hypodiploid cells (over 80% of the cellular population 96 hours after transduction) and evidence of death by apoptosis (exposure of phosphatidylserine and activity of caspases 3/7) in both cell lines. In these cell lines, a bystander effect was demonstrated when the presence of few transduced cells (ex., 10%) was enough to promote significant accumulation of hypodiploid cells (over 40% in this example). In a model of in situ gene therapy using SK-MEL-147 cells, hIbeta induced a strong anti-tumor effect including total tumor remission in all treated animals without relapse during ninety days. The presence of hIbeta in the circulation of the animals was confirmed 48h after treatment with AdRGD-PGhIbeta, but was present in only two of the seven animals 90 days post-treatment, suggesting that the initial treatment, not off target effects, was responsible for the response. With the goal of investigating collateral effects of adenoviral sequestration by the liver, we assayed the circulating concentration of aspartate aminotransferase and alanine aminotransferase (AST and ALT, respectively), which showed no alteration when compared with animals that received the treatment with a control vector or saline solution. We conclude that our adenoviral vector carrying human interferon-beta is capable of transducing the human melanoma cell line SK-MEL-147 in vitro and in vivo, promoting a bystander effect and tumor remission without inducing adverse effects

Os papilomavírus humanos são vírus de DNA que infectam epitélios em regiões anatômicas específicas. Alguns tipos de HPV, coletivamente denominados de alto risco oncogênico, têm associação etiológica com o câncer do colo do útero. Estes vírus expressam dois oncogenes, E6 e E7, que alteram o ciclo celular e o programa de diferenciação das células. Isto promove o acúmulo de defeitos mitóticos e instabilidade genômica, contribuindo à transformação maligna. Alterações nos sistemas de reparo de dano ao DNA associadas à presença de HPV têm sido descritas em diferentes modelos experimentais, no entanto, não tem sido analisadas de maneira sistemática. No presente estudo, avaliamos a expressão de 135 genes envolvidos nas vias de reparo de dano ao DNA em queratinócitos primários humanos e em linhagens derivadas de carcinomas do colo do útero positivas e negativas para HPV. Nossos resultados indicam a presença de alterações importantes na expressão de genes envolvidos nas vias de reparo de dano ao DNA em linhagens derivadas de tumores do colo do útero. Human papillomaviruses are DNA viruses that infect epithelia in specific anatomical regions. Some HPV types, collectively known as high-risk types are etiologically associated with cervical cancer. These viruses express two oncogenes E6 and E7 that alter the cell cycle and cell differentiation. Besides, they promote genomic instability and the accumulation of mitotic defects contributing to malignant transformation. Alterations in the DNA damage repair systems associated with HPV presence have been described in various experimental model systems. However, they have not been systematically analyzed. In this study, we evaluated the expression of 135 genes involved in the DNA damage repair pathways in primary human keratinocytes and cervical cancer derived cell lines. Our results show the presence of important alterations in the expression of DNA damage repair genes in cervical cancer derived cell lines.