RESUMO
Together with an anti-tumor immune response, oncolysis using a recombinant viral vector promises to eliminate cancer cells by both gene transfer and host-mediated functions. In this study we explore oncolysis induced by nonreplicating adenoviral vectors used for p14ARF and interferon-ß (hIFNß) gene transfer in human melanoma cell lines, revealing an unexpected role for p14ARF in promoting cellular responses predictive of immune stimulation. Oncolysis was confirmed when UACC-62 (p53 wild-type) cells succumbed upon p14ARF gene transfer in vitro, whereas SK-Mel-29 (p53-mutant) benefitted from its combination with hIFNß. In the case of UACC-62, in situ gene therapy in nude mice yielded reduced tumor progression in response to the p14ARF and hIFNß combination. Potential for immune stimulation was revealed where p14ARF gene transfer in vitro was sufficient to induce emission of immunogenic cell death factors in UACC-62 and upregulate pro-immune genes, including IRF1, IRF7, IRF9, ISG15, TAP-1, and B2M. In SK-Mel-29, p14ARF gene transfer induced a subset of these factors. hIFNß was, as expected, sufficient to induce these immune-stimulating genes in both cell lines. This work is a significant advancement for our melanoma gene therapy strategy because we revealed not only the induction of oncolysis, but also the potential contribution of p14ARF to immune stimulation.
Assuntos
Melanoma , Proteína Supressora de Tumor p14ARF , Camundongos , Animais , Humanos , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Camundongos Nus , Apoptose/fisiologia , Linhagem Celular , Melanoma/genética , Melanoma/terapiaRESUMO
Immune evasion is an important cancer hallmark and the understanding of its mechanisms has generated successful therapeutic approaches. Induction of immunogenic cell death (ICD) is expected to attract immune cell populations that promote innate and adaptive immune responses. Here, we present a critical advance for our adenovirus-mediated gene therapy approach, where the combined p14ARF and human interferon-ß (IFNß) gene transfer to human melanoma cells led to oncolysis, ICD and subsequent activation of immune cells. Our results indicate that IFNß alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. In situ gene therapy of s.c. SK-MEL-147 tumors in Nod-Scid mice revealed inhibition of tumor growth and increased survival in response to IFNß alone or in combination with p14ARF. Emission of critical markers of ICD (exposition of calreticulin, secretion of ATP and IFNß) was stronger when cells were treated with combined p14ARF and IFNß gene transfer. Co-culture of previously transduced SK-MEL-147 cells with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors resulted in increased levels of activation markers HLA-DR, CD80, and CD86. Activated Mo-DCs were able to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. Preliminary data showed that T cells primed by Mo-DCs activated with p14ARF+IFNß-transduced SK-MEL-147 cells were able to induce the loss of viability of fresh non-transduced SK-MEL-147 cells, suggesting the induction of a specific cytotoxic population that recognized and killed SK-MEL-147 cells. Collectively, our results indicate that p14ARF and IFNß delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.
Assuntos
Adenoviridae/fisiologia , Imunoterapia/métodos , Interferon beta/genética , Melanoma/terapia , Linfócitos T/imunologia , Proteína Supressora de Tumor p14ARF/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Terapia Genética , Humanos , Ativação Linfocitária , Melanoma/genética , Camundongos , Camundongos SCID , Terapia Viral Oncolítica , Carga Tumoral , Evasão TumoralRESUMO
BACKGROUND AND OBJECTIVE: Gas station attendants are occupationally exposed to benzene, toluene, ethylbenzene and xylene (BTEX) compounds and thus more susceptible to the biological effects of this mixture present in gasoline, especially due to the carcinogenicity of benzene. Furthermore, the harmful effects of BTEX exposure may be potentiated by genetic and epigenetic inactivation of critical genes. The objective was to evaluate such gene-BTEX interactions accessing the promoter methylation status of p14ARF, p16INK4A and GSTP1 in peripheral blood leukocyte samples. MATERIALS AND METHODS: The 59 exposed and 68 unexposed participants from Rio de Janeiro, Brazil, were included. The promoter methylation status was accessed by methylation-specific PCR (MSP) and GSTP1 Ile105Val polymorphism was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS: Both p14ARF and p16INK4A were significantly hypermethylated in exposed subjects compared to unexposed (p = 0.004 and p<0.001, respectively). Additionally, p16INK4A hypermethylation in the exposed group was correlated with chromosomal abnormalities (CAs) (p = 0.018), thus highlighting the influence of the gene-environment interactions on genome instability. Noteworthy, p16INK4A methylation was significantly associated with miscarriage among female attendants (p = 0.047), in which those who reported miscarriage exhibited hypermethylation in at least 2 of the 3 genes analyzed. The GSTP1 heterozygote genotype, which could affect the metabolism of benzene detoxification, was found in both groups but was more frequent in those occupationally exposed. No significant association was observed between GSTP1 genotypes and methylation status. CONCLUSION: Together, these findings indicate that gas station attendants with the aforementioned epigenetic and genetic profiles may be at greater risk of occupational BTEX exposure-induced genome instability, which could require concerted efforts to establish more preventive actions and constant biomonitoring in gas station attendants.
Assuntos
Derivados de Benzeno/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Gasolina/efeitos adversos , Regiões Promotoras Genéticas/efeitos dos fármacos , Tolueno/efeitos adversos , Xilenos/efeitos adversos , Adulto , Brasil , Estudos de Casos e Controles , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Instabilidade Genômica , Glutationa S-Transferase pi/genética , Humanos , Exposição por Inalação/efeitos adversos , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Saúde Ocupacional , Polimorfismo Genético , Medição de Risco , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-ß cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Imunoterapia/métodos , Interferon beta/uso terapêutico , Neoplasias/terapia , Fases de Leitura/genética , Morte Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Neoplasias/imunologia , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.
Assuntos
Humanos , Terapia Genética/métodos , Fases de Leitura/genética , Interferon beta/uso terapêutico , Técnicas de Transferência de Genes , Imunoterapia/métodos , Neoplasias/terapia , Morte Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína Supressora de Tumor p14ARF/genética , Neoplasias/imunologiaRESUMO
AIM: To explore methylation of DAPK, THBS1, CDH-1, and p14 genes, and Helicobacter pylori (H. pylori) status in individuals harboring esophageal columnar metaplasia. METHODS: Distal esophageal mucosal samples obtained by endoscopy and histologically diagnosed as gastric-type (non-specialized) columnar metaplasia, were studied thoroughly. DNA was extracted from paraffin blocks, and methylation status of death-associated protein kinase (DAPK), thrombospondin-1 (THBS1), cadherin-1 (CDH1), and p14 genes, was examined using a methyl-sensitive polymerase chain reaction (MS-PCR) and sodium bisulfite modification protocol. H. pylori cagA status was determined by PCR. RESULTS: In total, 68 subjects (33 females and 35 males), with a mean age of 52 years, were included. H. pylori cagA positive was present in the esophageal gastric-type metaplastic mucosa of 18 individuals. DAPK, THSB1, CDH1, and p14 gene promoters were methylated by MS-PCR in 40 (58.8%), 33 (48.5%), 46 (67.6%), and 23 (33.8%) cases of the 68 esophageal samples. H. pylori status was associated with methylation of DAPK (P = 0.003) and THBS1 (P = 0.019). CONCLUSION: DNA methylation occurs in cases of gastric-type (non-specialized) columnar metaplasia of the esophagus, and this modification is associated with H. pylori cagA positive infection.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Esofágicas/genética , Trombospondina 1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Antígenos CD , Proteínas de Bactérias/genética , Caderinas/genética , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/microbiologia , Neoplasias Esofágicas/patologia , Feminino , Helicobacter pylori/genética , Humanos , Masculino , México , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estudos Retrospectivos , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
BACKGROUND: Recent diagnostic procedure advances have greatly improved early lung cancer detection. However, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their application. There is a great need of novel non-invasive biomarkers for early lung cancer diagnosis. In the present study, we intend to determine whether the blood signatures of p14ARF promoter methylation are suitable for early detection of lung cancer. METHODS: The study aimed to assess the probability of p14ARF promoter methylation in plasma samples to detect early lung cancer using nested methylation-specific PCR in the training set consisted of tumor tissues and paired blood. Besides, we were further to discuss the difference in time to progression between methylation and unmethylation of p14ARF promoter using univariate and multivariate analysis. RESULTS: The methylation of p14ARF promoter was detected in 33.6 % of tumor tissues, and 12.1 and 25.2 % in distant-cancer mucosa and matched plasma, respectively, and our study has also demonstrated the positive correlation between them by Pearson's test (r = 0.300). The tumor-free survival time of the unmethylation of p14ARF promoter is significantly longer than that of the methylation of p14ARF promoter in tumor tissues (χ (2) = 7.149, P = 0.008). CONCLUSION: The methylation of p14ARF promoter in plasma samples has strong potential as a novel non-invasive biomarker for early detection of lung cancer, and the methylation of p14ARF promoter was considered as prognostic factor in our study.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas/genética , Proteína Supressora de Tumor p14ARF/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Detecção Precoce de Câncer , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Taxa de Sobrevida , Proteína Supressora de Tumor p14ARF/sangueRESUMO
A common causative agent for uterine cervical cancer is the human papillomavirus type 18 (HPV-18) which has three phylogenic variants: Asian-Amerindian, European, and African. Each variant shows significant molecular differences in the E6 gene. E6 oncoprotein is a negative regulator of tumor suppressor protein p53, hence, this oncoprotein indirectly regulates the expression of tumor-suppressor p14(ARF) . p14(ARF) and p16(INK4A) genes are overexpressed in--and have been proposed as markers for--HPV-related cervical cancer. In order to dissect the role of E6 on the regulation of p14(ARF) expression, separating it from that of other intervening factors, transfection of E6 variants to MCF-7 cells was performed, assessing cDNA transcript levels by RT-PCR, whereas p14(ARF) and p53 expression were evaluated by immunocytochemistry and Western blot. E6 transfected cells differentially expressed transcripts of two molecular forms: E6 and E6*. The ratio of these two forms varied with the transfected E6 variant. With the Asian-Amerindian variant, the ratio was E6 > E6*, whereas with the European and the African the ratio was E6* > E6. As expected with the E6* construct, E6* transcripts were solely observed. In addition, when E6 > E6* and p53 expression was low, p14(ARF) was high and when E6* > E6 and p53 expression was high, p14(ARF) was low. In conclusion, each E6 variant distinctively affects p53 levels and consequently p14(ARF) expression, finding that could be related with the differences in oncogenic effect of infection with the diverse high-risk HPV variants.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 18/fisiologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p14ARF/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Western Blotting , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: This study was designed to determine the prognostic role of p14ARF in vulvar squamous cell carcinoma (VSCC). METHODS: Immunohistochemistry for p14ARF and p53 and fluorescent in situ hybridization (FISH) for TP53 were performed in 139 cases of VSCC. Human papillomavirus (HPV) genotyping by hybridization was employed in 100 cases. qRT-PCR for p14ARF and p53 transcript assessment was performed in 16 cases. All results were correlated with clinicopathological variables. RESULTS: Immunohistochemistry analysis showed p14ARF and p53 positivity in 16.4% and 53% cases respectively. Positive p14ARF expression was significantly associated with the following variables: shorter cancer-specific survival (P=0.04) and shorter disease-free survival (P=0.02), presence of perineural invasion (P=0.037), vascular invasion (P=0.047), and node metastasis (P=0.031). Also, p14ARF-positive HPV-negative cases had the shortest cancer-specific survival (P=0.03) and disease-free survival (P=0.04). HPV infection was detected in 32.8% of the cases; HPV16 was the most prevalent type. Viral infection was more common in poorly differentiated tumors (P=0.032). qRT-PCR demonstrated that CDKN2A (p14ARF) had higher expression in tumor samples compared with paired noncancerous samples (P<0.001). The opposite relationship was seen in TP53 expression evaluation (P<0.001). FISH demonstrated 4 cases with deleted TP53 (6.3%). CONCLUSIONS: p14ARF represents an important marker of poor prognosis in VSCC. p53 and HPV infection did not show any prognostic importance. Further clinical trials concerning p14ARF positivity may result in important contributions due to its relationship with poor outcome. Mainly due to the relationship of p14ARF with lymph node metastasis, the immunohistochemistry evaluation of this marker may help to identify a subset of patients more suitable to less radical procedures.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Papillomavirus Humano 16 , Infecções por Papillomavirus/complicações , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Vulvares/metabolismo , Neoplasias Vulvares/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Vasos Sanguíneos/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Nervos Periféricos/patologia , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Vulvares/genética , Adulto JovemRESUMO
The Papanicolaou test (Pap) has been responsible for a significant reduction of cervical cancer-related morbimortality. In order to increase its sensitivity and specificity new markers have been studied and incorporated to cytological and histological methods for diagnosis for cervical cancer, such as p16INK4A that has been considered the immunocytochemical marker of choice for detection of HPV related cancers. We considered that p14ARF could be a complementary marker in order to improve the accuracy of cytological diagnosis because its genetic proximity to p16INK4A. We performed a systematic analysis of several putative cervical cancer markers in order to evaluate their performance in the detection of malignancy, in comparison with p16INK4A and p14ARF, using immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses. Most markers were non-specific and could not discriminate HPV infected cancer cell lines from other non HPV malignant. In contrast, nuclear co-expression of p16INK4A and p14ARF was observed only in HPV-transformed cancer cell lines. Notably, in C-33A cervical cancer cells (HPV negative), p14ARF was present in the nucleoli, but p16INK4A was conspicuously absent from the nuclei of these cells. We conclude that both markers; p16INK4A and p14ARF are complementary and should be evaluated jointly in order to improve the accuracy of cytological diagnosis of cervical cancer.