Human papillomavirus and genome instability: from productive infection to cancer

HPVInfection with high oncogenic risk human papillomavirus types is the etiological factor of cervical cancer and a major cause of other epithelial malignancies, including vulvar, vaginal, anal, penile and head and neck carcinomas. These agents affect epithelial homeostasis through the expression of specific proteins that deregulate important cellular signaling pathways to achieve efficient viral replication. Among the major targets of viral proteins are components of the DNA damage detection and repair machinery. The activation of many of these cellular factors is critical to process viral genome replication intermediates and, consequently, to sustain faithful viral progeny production. In addition to the important role of cellular DNA repair machinery in the infective human papillomavirus cycle, alterations in the expression and activity of many of its components are observed in human papillomavirus-related tumors. Several studies from different laboratories have reported the impact of the expression of human papillomavirus oncogenes, mainly E6 and E7, on proteins in almost all the main cellular DNA repair mechanisms. This has direct consequences on cellular transformation since it causes the accumulation of point mutations, insertions and deletions of short nucleotide stretches, as well as numerical and structural chromosomal alterations characteristic of tumor cells. On the other hand, it is clear that human papillomavirus-transformed cells depend on the preservation of a basal cellular DNA repair activity level to maintain tumor cell viability. In this review, we summarize the data concerning the effect of human papillomavirus infection on DNA repair mechanisms. In addition, we discuss the potential of exploiting human papillomavirus-transformed cell dependency on DNA repair pathways as effective antitumoral therapies

Alterations in the expression and activity of extracellular matrix components in HPV-associated infections and diseases

Infection with human papillomaviruses is associated with a series of benign and malignant hyperproliferative diseases that impose a heavy burden on human populations. A subgroup of mucosal human papillomavirus types are associated with the majority of cervical cancers and a relevant fraction of vulvar, vaginal, anal, penile and head and neck carcinomas. Human papillomaviruses mediate cell transformation by the expression of two pleiotropic oncoproteins that alter major cellular regulatory pathways. However, these viruses are not complete carcinogens, and further alterations within the infected cells and in their microenvironment are necessary for tumor establishment and progression. Alterations in components of the extracellular matrix for instance, matrix metalloproteinases and some of their regulators such as tissue inhibitors of metalloproteinases, have been consistently reported in human papillomaviruses-associated diseases. Matrix metalloproteinases function by remodeling the extracellular matrix and alterations in their expression levels and/or activity are associated with pathological processes and clinical variables including local tumor invasion, metastasis, tumor relapse and overall patient prognosis and survival. In this review we present a summarized discussion on the current data concerning the impact of human papillomavirus infection on the activity and expression of extracellular matrix components. We further comment on the possibility of targeting extracellular matrix molecules in experimental treatment protocols

Human polyomaviruses and cancer: an overview

The name of the family Polyomaviridae, derives from the early observation that cells infected with murine polyomavirus induced multiple (poly) tumors (omas) in immunocompromised mice. Subsequent studies showed that many members of this family exhibit the capacity of mediating cell transformation and tumorigenesis in different experimental models. The transformation process mediated by these viruses is driven by viral pleiotropic regulatory proteins called T (tumor) antigens. Similar to other viral oncoproteins T antigens target cellular regulatory factors to favor cell proliferation, immune evasion and downregulation of apoptosis. The first two human polyomaviruses were isolated over 45 years ago. However, recent advances in the DNA sequencing technologies led to the rapid identification of additional twelve new polyomaviruses in different human samples. Many of these viruses establish chronic infections and have been associated with conditions in immunosuppressed individuals, particularly in organ transplant recipients. This has been associated to viral reactivation due to the immunosuppressant therapy applied to these patients. Four polyomaviruses namely, Merkel cell polyomavirus (MCPyV), Trichodysplasia spinulosa polyomavirus (TSPyV), John Cunningham Polyomavirus (JCPyV) and BK polyomavirus (BKPyV) have been associated with the development of specific malignant tumors. However, present evidence only supports the role of MCPyV as a carcinogen to humans. In the present review we present a summarized discussion on the current knowledge concerning the role of MCPyV, TSPyV, JCPyV and BKPyV in human cancers

Oxidative stress: therapeutic approaches for cervical cancer treatment

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

ATM Pathway Is Essential for HPV-Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation.

Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors

Lactate transporters and vascular factors in HPV-induced squamous cell carcinoma of the uterine cervix

Lactate transporters and vascular factors in HPV-induced squamous cell carcinoma of the uterine cervixBACKGROUND: Tumour microenvironment is a fundamental aspect of tumour behaviour, modulating important events as cancer cell migration and invasion, as well as angiogenesis and metastisation. Among other microenvironment features, hypoxia and acidity play important roles in this modulation. As the metabolic reprogramming of cancer cells induces extracellular acidity, which in turn induces angiogenesis, and hypoxia induces both the metabolic reprogramming and angiogenesis, the present study aims to evaluate the immunohistochemical expression of a variety of metabolic and vascular markers as common targets of the hypoxic microenvironment in a series of cervical squamous cells carcinoma, as well as using an in vitro 3D culture model. METHODS: Immunohistochemical expression of MCT1, MCT4, CD147, GLUT1 and CAIX was assessed in a series of 28 chronic cervicitis, 34 LSIL, 29 HSIL, 38 cases of squamous cells carcinoma (SCC), as well as in in vitro 3D culture of keratinocytes expressing HPV genes. Furthermore, VEGF family members' expression was assessed in the SCC cases. The expression profiles were associated with patients' clinicopathological parameters. RESULTS: We found an increase of MCT4 expression along progression to malignancy in cervical samples. Also, MCT4 was associated with CD147 and CAIX expression. VEGF-A expression was more frequently found in cases without MCT1 expression. Both MCT4 and CD147 were more frequently expressed in younger patients at diagnosis while no associations were found between VEGF family and clinicopathological parameters. Finally, we show evidence for the upregulation of MCT4, as well as CD147 and CAIX, after HPV transfection. CONCLUSIONS: The results herein presented point at MCT4 as a promising therapeutic target in squamous cells carcinoma of the uterine cervix. Importantly, we show a possible association between lactate transport and angiogenesis, which should be further explored.CP received a post-doctoral fellowship (SFRH/BPD/69479/2010) and FM-S received a doctoral fellowship (SFRH/BD/87139/2012) from FCT (Portuguese Foundation for Science and Technology). This work was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of "Programa Operacional Tematico Factores de Competitividade" (COMPETE) of "Quadro Comunitario de Apoio III" and co-financed by Fundo Comunitario Europeu FEDER and also by FAPESP 2008/03232-1 to LLV

MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"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)

Tobacco Exposure Enhances Human Papillomavirus 16 Oncogene Expression via EGFR/PI3K/Akt/c-Jun Signaling Pathway in Cervical Cancer Cells

High-risk human papillomavirus (HR-HPV) infection is not a sufficient condition for cervical cancer development because most infections are benign and naturally cleared. Epidemiological studies revealed that tobacco smoking is a cofactor with HR-HPV for cervical cancer initiation and progression, even though the mechanism by which tobacco smoke cooperates with HR-HPV in this malignancy is poorly understood. As HR-HPV E6/E7 oncoproteins overexpressed in cervical carcinomas colocalize with cigarette smoke components (CSC), in this study we addressed the signaling pathways involved in a potential interaction between both carcinogenic agents. Cervical cancer-derived cell lines, CaSki (HPV16; 500 copies per cell) and SiHa (HPV16; 2 copies per cell), were acutely exposed to CSC at various non-toxic concentrations and we found that E6 and E7 levels were significantly increased in a dose-dependent manner. Using a reporter construct containing the luciferase gene under the control of the full HPV16 long control region (LCR), we also found that p97 promoter activity is dependent on CSC. Non-synonymous mutations in the LCR-resident TPA (12-O-tetradecanoylphorbol 13-acetate)-response elements (TRE) had significantly decreased p97 promoter activation. Phosphoproteomic arrays and specific inhibitors revealed that CSC-mediated E6/E7 overexpression is at least in part reliant on EGFR phosphorylation. In addition, we showed that the PI3K/Akt pathway is crucial for CSC-induced E6/E7 overexpression. Finally, we demonstrated that HPV16 E6/E7 overexpression is mediated by JUN. overexpression, c-Jun phosphorylation and recruitment of this transcription factor to TRE sites in the HPV16 LCR. We conclude that acute exposure to tobacco smoke activates the transcription of HPV16 E6 and E7 oncogenes through p97 promoter activation, which involves the EGFR/PI3K/Akt/C-Jun signaling pathway activation in cervical cancer cells