HPV infection, a described cause for human cancer: HPV detection and molecular mechanisms of HPV carcinogenesis (Review).

[en]Human papillomavirus (HPV) belongs to the Papillomaviridae virus family and it is one of the most common sexual transmission infections. HPV genome is composed of eight genes, including two early genes and six late genes. Among these late genes, E6 and E7 code for proteins that trigger cell-cycle re-entry in infected cells, which can lead to cervical cancer development. The IARC (International Agency for Research Cancer) proposed a guideline based on Hill’s criteria to determine whether the relation between HPV infection and cervical cancer is causal or not. Epidemiological studies have demonstrated that HPV infection is a necessary but non-sufficient cause for cervical cancer. Furthermore, HPV infection is considered the first necessary cause described of a human cancer, being HPV16 and 18 carcinogenic to humans and the most studied types. Cervical cancer is the second leading cause of cancer death among women worldwide. Different screening programs are carried out with the aim of preventing cervical cancer; such as cytologies and HPV tests. There are two main methods which are equally usable to detect HPV: the real-time PCR assays and the array assays. Regarding the molecular mechanisms of HPV mediated malignancies, E2, E6 and E7 proteins of HPV16 lead to immune response evasion, inducing IL-10 and TGF-β1 gene expression. Besides, E6 and E7 proteins allow cell-cycle reentry, phosphorylating RB and ubiquitinating p53 respectively. HPV genome integration in host genome leads to the alteration of host and viral genes expression, including oncogenes and tumor suppressor genes. However, the differences of E6 and E7 oncoproteins in different HPV types is poorly known due to the fact that almost the most studied HPV type has been HPV16.

Study of normal and modified nucleosides in serum by RP-HPLC

Modified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined, it was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.

Women's help seeking behaviour and the associated influencing factors on self discovery of a breast symptom

To investigate women’s help seeking behavior (HSB) following self discovery of a breast symptom and determine the associated influencing factors. A descriptive correlation design was used to ascertain the help seeking behavior (HSB) and the associated influencing factors of a sample of women (n = 449) with self discovered breast symptoms. The study was guided by the ‘Help Seeking Behaviour and Influencing Factors” conceptual framework (Facione et al., 2002; Meechan et al., 2003, 2002; Leventhal, Brissette and Leventhal, 2003 and O’Mahony and Hegarty, 2009b). Data was collected using a researcher developed multi-scale questionnaire package to ascertain women’s help seeking behavior on self discovery of a breast symptom and determine the factors most associated with HSB. Factors examined include: socio-demographics, knowledge and beliefs (regarding breast symptom; breast changes associated with breast cancer; use of alternative help seeking behaviours and presence or absence of a family history of breast cancer),emotional responses, social factors, health seeking habits and health service system utilization and help seeking behavior. A convenience sample (n = 449 was obtained by the researcher from amongst women attending the breast clinics of two large urban hospitals within the Republic of Ireland. All participants had self-discovered breast symptoms and no previous history of breast cancer. The study identified that while the majority of women (69.9%; n=314) sought help within one month, 30.1% (n=135) delayed help seeking for more than one month following self discovery of their breast symptom. The factors most significantly associated with HSB were the presenting symptom of ‘nipple indrawn/changes’ (p = 0.005), ‘ignoring the symptom and hoping it would go away’ (p < 0.001), the emotional response of being ‘afraid@ on symptom discovery (p = 0.005) and the perception/belief in longer symptom duration (p = 0.023). It was found that women who presented with an indrawn/changed nipple were more likely to delay (OR = 4.81) as were women who ‘ignored the symptoms and hoped it would go away’ (OR = 10.717). Additionally, the longer women perceived that their symptom would last, they more likely they were to delay (OR = 1.18). Conversely, being afraid following symptom discovery was associated with less delay (OR = 0.37; p=0.005). This study provides further insight into the HSB of women who self discovered breast symptoms. It highlights the complexity of the help seeking process, indicating that is not a linear event but is influenced by multiple factors which can have a significant impact on the outcomes in terms of whether women delay or seek help promptly. The study further demonstrates that delayed HSB persists amongst women with self discovered breast symptoms. This has important implications for continued emphasis on the promotion of breast awareness, prompt help seeking for self discovered breast symptoms and early detection and treatment of breast cancer, amongst women of all ages.

Effects of synbiotics on cancer risk biomarkers

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world and second most common (behind lung cancer) in developed countries. In recent years there has been much interest in the potential use of prebiotics, probiotics and synbiotics in the prevention and treatment of CRC. We have previously shown that synbiotic consumption in Azoxymethane treated rats modulates the immune system, influences the genotoxic potential of caecal contents and reduces the number of colonic tumours compared to control rats who did not receive the synbiotic. The aim of the current study was to identify biomarkers suitable for use as cancer risk markers and as intervention markers. A second aim was to determine the influence of synbiotic consumption on cancer risk biomarkers such as in vivo colonic mucosal proliferation and genotoxic damage along with examining the genotoxic, cytotoxic and tumour promoting potential of faecal water (FW). Synbiotic consumption altered the composition of the gastrointestinal flora and reduced in vivo genotoxic damage and the genotoxic potential of FW in cancer and polyp subjects. Synbiotic consumption also reduced the proliferative activity in the colonic mucosa in polyp subjects. In both cancer and polyp subjects gene expression in the colonic mucosa was modulated in synbiotic consuming subjects. In this and other studies the activity of natural killer cells, the level of PGE2 in FW, IL-12 production by PBMCs, genotoxic damage in the colonic mucosa and the tumour promoting activities of FW have been identified as possible biomarkers of cancer risk. Future large scale studies investigating these parameters in healthy and diseased individuals are needed to confirm the suitability of these markers in assessing cancer risk and the role of synbiotics in modulating them.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1–EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4+CD25+Foxp3+ regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80+ macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Baseline health-related quality-of-life data as prognostic factors in a phase III multicentre study of women with metastatic breast cancer.

The potential value of baseline health-related quality-of-life (HRQOL) and clinical factors in predicting prognosis was examined using data from an international randomised phase III trial which compared doxorubicin and paclitaxel with doxorubicin and cylophosphamide as first line chemotherapy in 275 women with metastatic breast cancer. The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and the related breast module (QLQ-BR23) were used to assess baseline HRQOL data. The Cox proportional-hazards regression model was used for both univariate and multivariate analyses of survival. In the univariate analyses, performance status (P<0.001) and number of sites involved (P=0.001) were the most important clinical prognostic factors. The HRQOL variables at baseline most strongly associated with longer survival were better appetite, physical and role functioning, as well as less fatigue (P<0.001). The final multivariate model retained performance status (P<0.001) and appetite loss (P=0.005) as the variables best predicting survival. Substantial loss of appetite was the only independent HRQOL factor predicting poor survival and was strongly correlated (/r/>0.5) with fatigue, role and physical functioning. In addition to known clinical factors, appetite loss appears to be a significant prognostic factor for survival in women with metastatic breast cancer. However, the mechanism underlying this association remains to be precisely defined in future studies.

Guidelines on the standards for the training of specialised health professionals dealing with breast cancer.

According to EUSOMA position paper 'The requirements of a specialist breast unit', each breast unit should have a core team made up of health professionals who have undergone specialist training in breast cancer. In this paper, on behalf of EUSOMA, authors have identified the standards of training in breast cancer, to harmonise and foster breast care training in Europe. The aim of this paper is to contribute to the increase in the level of care in a breast unit, as the input of qualified health professionals increases the quality of breast cancer patient care.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Towards a field-compatible optical spectroscopic device for cervical cancer screening in resource-limited settings: effects of calibration and pressure.

Quantitative optical spectroscopy has the potential to provide an effective low cost, and portable solution for cervical pre-cancer screening in resource-limited communities. However, clinical studies to validate the use of this technology in resource-limited settings require low power consumption and good quality control that is minimally influenced by the operator or variable environmental conditions in the field. The goal of this study was to evaluate the effects of two sources of potential error: calibration and pressure on the extraction of absorption and scattering properties of normal cervical tissues in a resource-limited setting in Leogane, Haiti. Our results show that self-calibrated measurements improved scattering measurements through real-time correction of system drift, in addition to minimizing the time required for post-calibration. Variations in pressure (tested without the potential confounding effects of calibration error) caused local changes in vasculature and scatterer density that significantly impacted the tissue absorption and scattering properties Future spectroscopic systems intended for clinical use, particularly where operator training is not viable and environmental conditions unpredictable, should incorporate a real-time self-calibration channel and collect diffuse reflectance spectra at a consistent pressure to maximize data integrity.

Associations between Intake of Folate, Methionine, and Vitamins B-12, B-6 and Prostate Cancer Risk in American Veterans.

Prostate cancer (PC) is the second leading cause of cancer death in men. Recent reports suggest that excess of nutrients involved in the one-carbon metabolism pathway increases PC risk; however, empirical data are lacking. Veteran American men (272 controls and 144 PC cases) who attended the Durham Veteran American Medical Center between 2004-2009 were enrolled into a case-control study. Intake of folate, vitamin B12, B6, and methionine were measured using a food frequency questionnaire. Regression models were used to evaluate the association among one-carbon cycle nutrients, MTHFR genetic variants, and prostate cancer. Higher dietary methionine intake was associated with PC risk (OR = 2.1; 95%CI 1.1-3.9) The risk was most pronounced in men with Gleason sum <7 (OR = 2.75; 95%CI 1.32- 5.73). The association of higher methionine intake and PC risk was only apparent in men who carried at least one MTHFR A1298C allele (OR = 6.7; 95%CI = 1.6-27.8), compared to MTHFR A1298A noncarrier men (OR = 0.9; 95%CI = 0.24-3.92) (p-interaction = 0.045). There was no evidence for associations between B vitamins (folate, B12, and B6) and PC risk. Our results suggest that carrying the MTHFR A1298C variants modifies the association between high methionine intake and PC risk. Larger studies are required to validate these findings.

Importance of prostate volume in the European Randomised Study of Screening for Prostate Cancer (ERSPC) risk calculators: results from the prostate biopsy collaborative group.

OBJECTIVES: To compare the predictive performance and potential clinical usefulness of risk calculators of the European Randomized Study of Screening for Prostate Cancer (ERSPC RC) with and without information on prostate volume. METHODS: We studied 6 cohorts (5 European and 1 US) with a total of 15,300 men, all biopsied and with pre-biopsy TRUS measurements of prostate volume. Volume was categorized into 3 categories (25, 40, and 60 cc), to reflect use of digital rectal examination (DRE) for volume assessment. Risks of prostate cancer were calculated according to a ERSPC DRE-based RC (including PSA, DRE, prior biopsy, and prostate volume) and a PSA + DRE model (including PSA, DRE, and prior biopsy). Missing data on prostate volume were completed by single imputation. Risk predictions were evaluated with respect to calibration (graphically), discrimination (AUC curve), and clinical usefulness (net benefit, graphically assessed in decision curves). RESULTS: The AUCs of the ERSPC DRE-based RC ranged from 0.61 to 0.77 and were substantially larger than the AUCs of a model based on only PSA + DRE (ranging from 0.56 to 0.72) in each of the 6 cohorts. The ERSPC DRE-based RC provided net benefit over performing a prostate biopsy on the basis of PSA and DRE outcome in five of the six cohorts. CONCLUSIONS: Identifying men at increased risk for having a biopsy detectable prostate cancer should consider multiple factors, including an estimate of prostate volume.

Evaluation of Altered Kras Codon Bias and NOS Inhibition During Lung Tumorigenesis

The small GTPases HRAS, NRAS and KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in RAS, with 98% of those occurring in the KRAS isoform. While there have been many advances in the understanding of KRAS–driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of Kras–driven tumorigenesis in the lung:

(I) Despite nearly identical protein sequences, the three RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to HRAS and NRAS. To determine the consequences of rare codon bias during de novo tumorigenesis, we created a knock-in Krasex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the Krasex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non–oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in Kras tumorigenesis.

(II) Lung cancer patients exhale higher levels of NO and iNOS-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes Kras–driven lung adenocarcinoma, and NOS inhibition may decrease Kras–driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L–NAME in a genetically engineered mouse model of Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic KrasG12D and dominant-negative Trp53R172H in lung epithelial cells. L–NAME treatment was provided in the water and continued until survival endpoints. In this model, L–NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.

Intermolecular Multiple Quantum Coherences Enable Accurate Thermal Imaging of Red Bone Marrow During Thermal Therapy of Bone Metastases

Prostate and breast cancers are two of the most common types of cancer in the United States, and those cancers metastasize to bone in more than two thirds of patients. Recent evidence suggests that thermal therapy is effective at treating metastatic bone cancer. For example, thermal therapy enables targeted drug delivery to bone, ablation of cancer cells in bone marrow, and palliation of bone pain. Thermal therapy of bone metastases would be greatly improved if it were possible to image the temperature of the tissue surrounding the disease, which is usually red bone marrow (RBM). Unfortunately, current thermal imaging techniques are inaccurate in RBM.

This dissertation shows that many of the difficulties with thermal imaging of RBM can be overcome using a magnetic resonance phenomenon called an intermolecular multiple quantum coherence (iMQC). Herein, iMQCs are detected with a magnetic resonance imaging (MRI) pulse sequence called multi-spin-echo HOMOGENIZED with off resonance transfer (MSE-HOT). Compared to traditional methods, MSE-HOT provided ten-fold more accurate images of temperature change. Furthermore, MSE-HOT was translated to a human MRI scanner, which enabled imaging of RBM temperature during heating with a clinical focused ultrasound applicator. In summary, this dissertation develops a MRI technique that enables thermal imaging of RBM during thermal therapy of bone metastases.

PenPC: A two-step approach to estimate the skeletons of high-dimensional directed acyclic graphs.

Estimation of the skeleton of a directed acyclic graph (DAG) is of great importance for understanding the underlying DAG and causal effects can be assessed from the skeleton when the DAG is not identifiable. We propose a novel method named PenPC to estimate the skeleton of a high-dimensional DAG by a two-step approach. We first estimate the nonzero entries of a concentration matrix using penalized regression, and then fix the difference between the concentration matrix and the skeleton by evaluating a set of conditional independence hypotheses. For high-dimensional problems where the number of vertices p is in polynomial or exponential scale of sample size n, we study the asymptotic property of PenPC on two types of graphs: traditional random graphs where all the vertices have the same expected number of neighbors, and scale-free graphs where a few vertices may have a large number of neighbors. As illustrated by extensive simulations and applications on gene expression data of cancer patients, PenPC has higher sensitivity and specificity than the state-of-the-art method, the PC-stable algorithm.