An outlook on ovarian cancer and borderline ovarian tumors: focus on genomic and proteomic findings.

Over the past decade, the power of high-throughput technologies has stimulated their use in many bio-medical applications. In this field, proteomics has become an important methodology in the biomarker identification. Using this method the expression of hundreds of proteins can be studied simultaneously, by taking advantage of their different biochemical properties. This approach has been applied to ovarian cancer leading to the identification of several differentially expressed proteins related to platinum resistance. Among these, five proteins annexin A3, destrin, cofilin 1, GSTO1-1 and IDHc showed the more appreciable results, confirmed also by quantitative PCR and western blot [ 71 ]. Proteomics studies have also been applied to tumor classification by using diseased tissues and serum specimens: Bengtsson et al . used DIGE technology to distinguish between benign and malignant tumor groups leading to the identification of an heterogeneous groups of proteins involved in several cellular processes [ 72 ]. By coupling laser capture microdissection and 2-DE, Brown Jones et al . revealed that the 52 KDa FK506 binding protein, RhoG-protein dissociation inhibitor and glyoxalase I were overexpressed in invasive ovarian cancer when compared to low malignant potential ovarian tumors [ 73 ]. The immune response to tumor associated antigens represents a possible serological tool for the diagnosis of cancer. Autoantibodies direct against tumor associated antigens presented by MHC I molecules have been identified by liquid chromatography - mass spectrometry/mass spectrometry (LC-MS/MS) using two human ovarian adenocarcinoma cell lines and serum sample from twenty patients, showing important implications in cancer detection and prognosis [ 74 ]. Recently, emerging technologies are unravelling the plasma proteome. Surface-enhanced laser desorption and ionization with time-of-flight spectrometry (SELDI-TOF) was employed for the rapid identification of serum proteins in ovarian cancer patients. The strength of SELDI technology is based on the selective binding of proteins on a solid-phase protein chip surface where the sample is ionized by an energy laser impulse. The protein profile of each specimen is then analyzed by software to discovery changes in the protein pattern. Petricoin et al. analyzed the serum of unaffected controls and patients with ovarian cancers and were able to discriminate with a sensibility of 100% and a specificity of 94% all ovarian cancers in the early stage [ 75 ]. Among the possible marker identified with SELDI there was the Haptoglobin, a glycoprotein secreted by liver cells and implicated in the metabolism of hemoglobulin. High levels of Hp-α subunit were found serum of patients with ovarian cancer. The results of the ELISA test showed for Hp-α a 64% sensitivity and 90% specificity alone and 91% sensitivity and 95% specificity if combined with CA125. Expression levels of 169 proteins were studied by Mor et al. by antibody microarray in serum of 28 healthy women, 18 women newly diagnosed with EOC and 40 women with recurrent disease. This analysis led to the identification of four proteins: leptin, prolactin, osteopontin, and insulin-like growth factor-II. Additional validation studies showed that the combination of the four proteins exhibited a sensitivity of 95%, a positive predictive value of 95%, a specificity of 95% and negative predictive value of 94% [ 76 ].

Ovarian cancer is the sixth most frequent female cancer worldwide, with 200,000 new cases diagnosed every year, and the most lethal of all gynaecological cancers. Given that ovarian cancer in 75% of cases is diagnosed in advanced stage, the disease has a fairly poor prognosis with an overall five-year survival of around 40%, ranging from 10% for patients with tumors with distant metastases to 80% for patients with localized tumors [ 1 ]. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian neoplasms display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline and invasive tumors. Borderline ovarian tumors (BOTs) account for 10%-20% of ovarian epithelial tumors and are typically diagnosed before 40 years of age, raising issues regarding the use of conservative surgery to preserve fertility [ 2 ]. So, despite enormous progress in cancer research, this disease represents one of the principal causes of death worldwide; therefore, there is a need for the identification of molecules which can act as indicators of an early malignant process, when disease burden is localized. However, the possibility to use many of these molecules in the clinical practice remains, unfortunately, still elusive. Evaluation and validation of biomarkers need rigorous procedures to ensure clinical validity. The process follows different steps to assist researchers in developing biomarker, these phases include: preclinical exploratory studies, clinical assay and validation, retrospective longitudinal, prospective screening and cancer control [ 3 ]. In addition, genomic and proteomics technologies have increased the number of potential biomarker entering clinical development, hence the validation of these candidate biomarkers require an integrated effort among government, academic and pharmaceutical groups to coordinate the activity of many laboratories [ 4 ].

High-throughput technologies have the potential to address this task and in particular, recent advances in mass spectrometry imaging may stimulate new research in this area. The direct analysis of ovarian cancer tissue sections by matrix-assisted laser desorption/ionization (MALDI) Imaging led to the identification of an 11S proteasome fragment as an ovarian cancer marker with a different cellular localization in benign and malign tissues [ 99 ]. So, MALDI-Imaging could bypass the limitations of many current technologies allowing the visualization of proteins, peptides, lipids and small molecules directly on thin sections cut from fresh frozen or paraffin embedded tissues (FFPE) [ 100 , 101 ] without long and tedious steps of separation and extraction. In our laboratory we are also exploring the possibility to probe mechanical properties of cancer cells by an atomic force microscopy (AFM) in order to obtain a nanomechanical classification of BOTs. AFM is a versatile tool for imaging and measuring the elastic properties of living and fixed cells [ 102 ]. The great advantage of AFM lies in the unique possibility to work with living cells in physiological conditions at 37°C. Basically, cells seeded into Petri dishes are scanned by a tip, which is mounted to a cantilever spring to obtain a topographic image of the sample at high spatial resolution.

Widely asymptomatic at the moment of the diagnosis, more than 70% of the patients with ovarian cancer are already in an advanced disease state [ 5 ]. On the contrary, BOTs are more common in women of younger age, do not share a similar epidemiological risk profile than women with invasive disease and appear to have a distinct genetic and protein background respect to high grade tumors. The serum CA125 in combination with transvaginal or transabdominal ultrasonography (US) have been helpful for clinicians in the diagnosis and monitoring of a suspicious ovarian formation, leading to their acceptance for clinical screening [ 6 ], since US is a non-invasive acceptable procedure with a large diffusion in world women screening programs and without complications. The CA125, identified in 1981, is the only serum molecules significantly associated with ovarian cancer, but also in endometriosis, pelvic inflammatory disease and other conditions. Normally, serum levels are less than 35 U/ml and are increased in about 90% of patients with advanced ovarian cancer (stage III-IV) but only in 50% of women with early stage disease. This protein belongs to the protein family of mucins, a high molecular weight protein that normally coats the epithelium [ 7 , 8 ]. In virtue of their localization, mucine proteins have extremely important functions in cellular physiology, like, for example, cellular adhesion. Alterations in their oligosaccharide structure have been noticed in different cancer forms [ 9 ]. CA 125 is a useful marker for the assessment of response to chemotherapy, predicting relapses and for discrimination between benign and malignant masses [ 10 , 11 ]. The development of an algorithm that calculates risk of ovarian cancer, based on serial CA125 values, has shown that 20% of ovarian cancers have little or no expression of CA125 and has pointed out the importance of additional serum markers identification, as well as of new molecules capable of completing or replacing CA125 altogether, to facilitate earlier detection of ovarian cancer [ 12 ]. US in combination with CA125 has also been suggested as a potential means for early detection of ovarian cancer [ 13 ]; others studies confirmed the efficacy of this combination for the screening of ovarian cancer, but a recent study from the American National Cancer Institute (NCI), shows that these two screening methods, alone or in combination, can also produce many false-positive test results, causing needless surgery [ 14 ]. For this reason, evaluation of a cystic formation with US characteristic of benignity, should lead to a normal echographic follow-up [ 13 - 15 ]. However, additional studies performed with US screening have not demonstrated adequate sensitivity and sensibility for an early diagnosis of ovarian cancer [ 16 ]. Hereby, in ovarian cancer types detecting, good results were obtained by the combination of CA125 with others serum markers; serum total inhibin levels are sensitive and specific markers of epithelial ovarian cancer in postmenopausal women; the production of inhibin by the ovaries almost stops after the menopause, but it continues from ovarian tumors. After menopause, circular inhibin levels are undetectable; on the other hand, they are elevated in women with ovarian cancer [ 17 ]. Inhibin is a glycoprotein that exists as a dimeric form of two subunits (α e βA o βB), to form inhibin A and inhibin B (αβA) e B (αβB); moreover, inhibin seems to be complementary to CA125 and both markers have a total 95% of sensitivity with 95% of specificity. Studies are directed toward establishing whether there are cancer specific inhibin forms, which may be suitable during the reproductive years, when these proteins show substantial fluctuations that would seriously compromise a preoperative assessment of any ovarian cancer [ 18 ].

An extensive analysis of the ovarian cellular pathways provided better insights in the molecular distinction between ovarian surface epithelium (OSE), BOTs and high grade tumors. A key feature is the modification in E-cadherin and N-cadherin levels, integral membrane glycoproteins that mediate calcium-dependent cell adhesions: a role for E-cadherin in the early events leading to cellular transformation has been proposed for two principal reasons. The protein is expressed in inclusion cysts, in benign, borderline and malignant tumors of all stages but not in normal ovarian surface epithelium (OSE) [ 77 ]. Moreover, N-cadherin expression was detected in most benign and borderline tumors but was absent or heterogeneous in most carcinomas [ 78 ]. E-cadherin induces the activation of Akt and MAPK pathways and in this way increases proliferation and survival of ovarian cancer cells [ 79 ]. To corroborate these hypotheses, the expression of exogenous E-cadherin in mesenchymal IOSE-29 (immortalized-OSE) has been associated with the development of ovarian cancer [ 80 ]. The role of E-cadherin in advanced tumor and metastasis is still debated and not strictly related to the aim of this review. In general, the absence or the lower E-cadherin expression in ovarian cancers predicts poor patient survival when compared with ovarian tumors that express E-cadherin and correlates with high tumor grade and low overall survival rate [ 81 ]. Transforming growth factor-β (TGF- β) and epidermal growth factor (EGF) are known to promote EMT in ovarian cancer [ 82 , 83 ]. Smad3 is required to sustain TGF-β induced EMT. Analysis of Smad3 expression in microarrays of serous cystadenocarcinoma displayed lower levels of Smad3 nuclear expression than benign and borderline tumors and a correlation with N-cadherin expression and poor survival [ 84 ]. Activin A (βA), a member of the TGF- β family, was found to regulate the activation status of Akt and GSK. In ovarian cancer, βA expression was elevated in stromal cells from carcinomas. Moreover, a different GSK-β phosphorylation status was identified in benign cystadenomas, borderline tumors, and carcinomas [ 85 ]. EMT can be induced by EGF through the activation of JAK2/STAT3 signaling pathway as demonstrated by Colomiere et al. [ 86 , 87 ]. In patients, both phosphorylated JAK2 and activated STAT3 showed higher expression in high grade tumors respect to normal ovaries and borderline tumors [ 86 ]. The two cancer cell lines OVCA 433 and SKOV3 treated with EGF down-regulated the expression of epithelial-restricted E-cadherin and increased the expression of vimentin and α2, α6 β1 integrin subunits [ 87 ]. EGF decreased also the expression of Neutrophil gelatinase-associated lipocalin (NGAL) in the transition to mesenchymal like cells. In vivo , NGAL was not expressed in normal tissues while both BOT and grade I tumors showed an increased staining compared to grade 2 and 3 tumors [ 83 ]. Key cellular processes including adhesion and migration depend also largely on remodelling of cytoskeleton, a complex network of molecules whose organization influences not only cellular shape but also the interaction between cells and the ECM microenvironment. Both actins and microtubules have a role in these processes; however, ovarian cancer cell invasion and migration seemed to be principally affected targeting actin dynamics [ 88 ]. Actin is one of the most abundant intracellular proteins; it is represented by three distinct isoforms, α, β and γ. β and γ actin isoforms are expressed principally in non-muscle cells, α isoform in muscle cells. The organization of actin is driven by a plethora of proteins also known as Actin Binding Proteins under the control of specific signaling pathways [ 89 ]. Disorganisation of actin cytoskeleton contributes to alterations in growth control and tumorigenesis [ 90 ]. Furthermore, actin remodelling is involved in lamellipodia and pseudopodium formation during cancer cells invasion and metastasis [ 91 ]. The actin-binding protein Gelsolin regulates actin filaments length and severing. It is downregulated in a large variety of human cancers including BOT and ovarian carcinomas [ 92 ]. Gelsolin showed suppressive activity in ovarian cancer cells growth and was correlated with tumor grade [ 93 ]. Fascin is implied in the organization of F-actin in parallel bundles (actin-structures with a role in cell architecture) and dynamic structures including filopodia and lamellipodia [ 94 ]. Fascin was not expressed in normal epithelial ovarian tissue [ 95 ], but up-regulated in cystadenomas, BOTs and carcinomas, suggesting that fascin may play an important role in tumor progression [ 96 ]. In addition, its stromal expression was significantly higher in malign epithelial ovarian tumors and BOT, compared to normal ovaries and benign epithelial ovarian tumors [ 97 ]. Also other cytoskeletal components, like intermediate filaments (IFs), have been reported to be involved in ovarian cancer. By two dimensional electrophoresis (2-DE) and mass spectrometry analysis, Bengtsson et al. identified several IFs differentially expressed in benign and malignant tumors when compared to BOTs. The proteins identified were Vimentin, Keratin I cytoskeletal 19 and Lamin A/C respectively [ 98 ].

Because of a lack of specific symptoms in the early phase and the limitations of existing markers for diagnostic methods, the ovarian cancer still represent, despite its low incidence, one of the leading cause of death worldwide. The complete understanding of mechanisms controlling ovarian progression represents an important step toward the development of a future appropriate therapy based on genomic and proteomics findings. Nevertheless, the study of low-abundant proteins and the need to analyze a large body of sample to extrapolate significant results, make the proteomics approach labour-intensive and the potential of a gene therapy for ovarian cancer, that involves blocking expression of specific ovarian oncogenes, seems to be far to be applied. Currently, many genomic tests used to diagnose ovarian cancer or guide its therapy do not decrease women's risk of dying from this disease or do not improve their quality of life (QoL). Therefore, for their rapid investigations of biological specimens, mass spectrometry and protein arrays are evolving as alternative methodologies to the “ classic proteomics approach ” facilitating their use at many points of the disease management and the expanding our knowledge on biomarkers is likely to lead to more individualization of therapy and improvements in ovarian cancer prognosis. Several works has been published on observing the impact of this technology in biomedical research [ 103 ]. We believe that the translation of all these information into the clinical practice could guide the development of appropriate targeted clinical therapy, overtaking the current invasive surgical approach and avoiding the uncertainty of a long term clinical and instrumental follow up of a possible recurrence.

In the case of ovarian cancer specific subtypes, it is often reported the evidence that different histological subtypes of ovarian cancer can be distinguished by some microarray profiling, and that these subtypes might be partly reflected by a different aetiology through the deregulation and activation of different pathways. This neoplasm heterogeneity could also account for different ovarian tumor behaviours and it could be revealed by specific biomarkers; for this evidence, it is reported by authors that gene expression profiling could be a useful prognostic tool, predicting chemo sensitivity to the standard treatment combination of paclitaxel and platinum chemotherapy for advanced ovarian cancer, but more knowledge about chemo sensitivity could eventually lead to a more tailored cancer therapy [ 3 - 5 ]. The purpose of this study is to summarize current dates regarding the use of novel protein biomarkers, based on genomics and proteomics, in the diagnosis, prognosis and response to therapy in ovarian cancer and in borderline ovarian tumors. Our attention will be focus not only on serum markers but also on intracellular proteins. Indeed, the expression of these proteins can reflect cellular and molecular mechanisms that control onset and neoplastic progression. Proteins included in this group belong to different cellular pathways including cellular metabolism and signal transduction. Of particular interest, it is the role of signaling molecules that regulate interaction pathways between tumor cells and the microenvironment.