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.

Low RECK Expression Is Part of the Cervical Carcinogenesis Mechanisms.

Human papillomavirus (HPV)-induced carcinogenesis comprises alterations in the expression and activity of matrix metalloproteinases (MMP) and their regulators. Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) inhibits the activation of specific metalloproteinases and its expression is frequently lost in human cancers. Here we analyzed the role of RECK in cervical carcinogenesis. Cervical cancer derived cell lines over expressing RECK were used to determine tumor kinetics as well as, cellular, immune and molecular properties in vivo. Besides, we analyzed RECK expression in cervical cancer samples. RECK over expression (RECK+) delayed tumor growth and increased overall survival in vivo. RECK+ tumors displayed an increase in lymphoid-like inflammatory infiltrating cells, reduced number and viability of tumor and endothelial cells and lower collagenase activity. RECK+ tumors exhibited an enrichment of cell adhesion processes both in the mouse model and cervical cancer clinical samples. Finally, we found that lower RECK mRNA levels were associated with cervical lesions progression and worse response to chemotherapy in cervical cancer patients. Altogether, we show that increased RECK expression reduced the tumorigenic potential of HPV-transformed cells both in vitro and in vivo, and that RECK down regulation is a consistent and clinically relevant event in the natural history of cervical cancer.

Evaluation of Elafin Immunohistochemical Expression as Marker of Cervical Cancer Severity.

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.

TP53 mutation profile of esophageal squamous cell carcinomas of patients from Southeastern Brazil.

Esophageal cancer (EC) is among the 10 most common and fatal malignacies in the world, presenting a marked geographic variation in incidence rates between and within different countries. The TP53 tumor suppressor gene is highly mutated in esophageal tumors and its mutation pattern can offer clues to the etiopathology of the tumor. As Brazil presents one of the highest incidence areas in the West, a deeper knowledge of the molecular mechanisms related to EC development in the Brazilian population is needed. We analyzed the mutation profile of 110 esophageal squamous cell carcinomas (ESCC) of patients from Southeastern Brazil (Rio de Janeiro and São Paulo) and collected data regarding alcohol intake and tobacco smoking. We detected 41 mutations in tumor samples from 38 patients. There was no association between mutation frequency and tobacco smoking or alcohol drinking. The most frequently mutated codons were 179, 214, 220 and 248. Codons 179, 220 and 248 are hot-spots for ESCC, but codon 214 presents only 0.7% of the mutations registered in the IARC database. The mutation profile revealed a high percentage of mutations at A:T base pairs (34.1%) followed by deletions (17.1%). We concluded that the mutation profile detected in this study is different from that of patients from Southern Brazil but very similar to that previously seen in French patients, being characterized by a high frequency of mutations at A:T base pairs, which may be associated with acetaldehyde, the metabolic product of ethanol.

Three Prime Repair Exonuclease 1 (TREX1) expression correlates with cervical cancer cells growth in vitro and disease progression in vivo.

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.

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.

Platelet-Activating Factor Receptor Ligands Protect Tumor Cells from Radiation-Induced Cell Death.

Irradiation generates oxidized phospholipids that activate platelet-activating factor receptor (PAFR) associated with pro-tumorigenic effects. Here, we investigated the involvement of PAFR in tumor cell survival after irradiation. Cervical cancer samples presented higher levels of PAF-receptor gene (PTAFR) when compared with normal cervical tissue. In cervical cancer patients submitted to radiotherapy (RT), the expression of PTAFR was significantly increased. Cervical cancer-derived cell lines (C33, SiHa, and HeLa) and squamous carcinoma cell lines (SCC90 and SCC78) express higher levels of PAFR mRNA and protein than immortalized keratinocytes. Gamma radiation increased PAFR expression and induced PAFR ligands and prostaglandin E2 (PGE2) in these tumor cells. The blocking of PAFR with the antagonist CV3938 before irradiation inhibited PGE2 and increased tumor cells death. Similarly, human carcinoma cells transfected with PAFR (KBP) were more resistant to radiation compared to those lacking the receptor (KBM). PGE2 production by irradiated KBP cells was also inhibited by CV3988. These results show that irradiation of carcinoma cells generates PAFR ligands that protect tumor cells from death and suggests that the combination of RT with a PAFR antagonist could be a promising strategy for cancer treatment.

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.

HPV infection in Brazilian patients with esophageal squamous cell carcinoma: interpopulational differences, lack of correlation with surrogate markers and clinicopathological parameters.

The role of HPV in esophageal squamous cell carcinoma (ESCCs) is controversial. Therefore, we determined, through different methodologies, the prevalence of HPV in 264 ESCC samples from Brazil, and correlated it with the presence of surrogate markers and clinicopathological characteristics. HPV is present in 13% of ESCC, and with a 3-fold variation between high and medium incidence areas. Most HPV positive tumors were infected with HPV16, but this was not associated with p16 expression, TP53 mutation status, patient age, amount of tobacco or alcohol consumption, or overall survival. We conclude that HPV infection may not have a role in ESCC.