Altered Salivary Protein Profiles among Individuals Diagnosed with Cervical Dysplasia and Cervical Carcinoma in Situ

Objective: The objective of this study was to determine if protein-by-products secondary to cervical cancer oncogenes appear in the saliva. Methods: Four pooled (n=10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, while other pooled specimens were from women diagnosed with CIN 2 moderate cervical dysplasia (n=10), CIN 3 severe cervical dysplasia (n=10) and the other pooled group from women diagnosed with cervical carcinoma in situ (n=10). Differential expression of proteins was measured by isotopically tagging proteins in each groups and comparing them to the healthy control group. Saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. Results: The results of the salivary analyses yielded approximately 133 proteins in the saliva specimens. Forty eight proteins were differentially expressed between the healthy control pool, precancerous and cancerous conditions. Conclusions: The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to precancerous and cancerous conditions and that these proteins may be useful in the study of cervical cancer progression, treatment efficacy and the tailoring of individualized patient care.


Introduction
Invasive cervical cancer is the second most common cause of death from cancer among women (Bosch et al., 1995).In the United States, it is estimated that the number of new cases of cervix uteri cancer is approximately 7.7 per 100,000 women per year.The number of deaths was 2.3 per 100,000 women per year.These rates are age-adjusted and based on 2008-2012 cases and deaths as reported by the NCI Surveillance, 2014, Epidemiology and End Results Program.
Cervical lesions are divided into different stages of aberrant cell growth.The initial stages are the precancerous conditions designated as cervical intraepithelial neoplasia (CIN) which exhibit alteration of the cervical epithelial lining with the presence of abnormal cell growth (Garbett et al., 2014).CIN status is determined by the extent in which the cervical epithelial lining and is divided into three stages based on the histological features, nuclear changes and the extent of epithelial involvement.The grades are mild, moderate and severe with the moderate and severe conditions having a higher likelihood to progress into carcinoma in situ (CIS) or invasive carcinoma (Garbett et al., 2014).
Collectively, it is the hypothesis of this preliminary investigation to determine if there are alterations in salivary proteins as a consequence of CIN and CIS.This information could be used as a tool to further the understanding of cervical cancer pathogenesis and clinical decision making.

Proteomic Design
The investigators analyzed pooled, stimulated whole saliva specimens.Each pooled specimen within a cohort consisted of ten individual patient saliva specimens from a bank of control and cancer specimens frozen at -80°C.One pooled saliva specimen consisted of saliva from 10 healthy subjects; another pooled specimen was a pooled saliva specimen from 10 subjects diagnosed with moderate cervical dysplasia, severe dysplasia and with cervical carcinoma in situ.In other words each pooled saliva specimen corresponded to 10 patients from each cervical lesion considered in this study.Except for the control group, the dysplasia and cancer cohorts were as determined by the pathology report.All subjects were closely matched for age and race and were non-tobacco users.All disease categories were HPV positive.
All participating volunteers were explained their participation rights and signed an IRB consent form.The saliva specimens and related patient data are non-linked and bar coded in order to protect patient confidentiality.This study was performed under the UTHSC IRB approved protocol# HSC-DB-05-0394.

Saliva Collection and Sample Preparation
Stimulated whole salivary gland secretion is a reflex response occurring during the mastication of a bolus of food.Usually, a standardized bolus (1 gram) of paraffin or a gum base (generously provided by the Wrigley Co., Peoria, IL) is given to the subject to chew at a regular rate.The individual, upon sufficient accumulation of saliva in the oral cavity, expectorates periodically into a preweighed disposable plastic cup.This procedure is continued for a period of five minutes.The volume and flow rate is then recorded along with a brief description of the specimen's physical appearance (Streckfus et al., 2008).The cup with the saliva specimen is reweighed and the flow rate determined gravimetrically.The authors recommend this salivary collection method with the following modifications for consistent protein analyses (Streckfus et al., 2008).A protease inhibitor cocktail from Sigma Co (St. Louis, MI, USA) is added along with enough dithiothreitol from a 1 M stock solution to bring its concentration 1 mM.The treated samples were centrifuged for 10 minutes at top speed in a table top centrifuge.The supernatant was divided into 1 ml aliquots and frozen at -80°C.

Bottom-up Mass Spectrometry Using iTRAQ Labeling
A thorough explanation for the top-down mass spectrometry using iTRAQ labeling can be found in detail in previous publications (Streckfus et al., 2008).Briefly, the saliva samples were thawed and immediately centrifuged to remove insoluble materials.The supernatant was assayed for protein using the Bio-Rad protein assay (Hercules, CA, USA) and an aliquot containing 100 µg of each specimen was precipitated with six volumes of -20ºC acetone.The precipitate was resuspended and treated according to the manufacturer's instructions.Protein digestion and reaction with iTRAQ labels was carried out as previously described and according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).Briefly, the acetone precipitable protein was centrifuged in a tabletop centrifuge at 15,000 x g for 20 minutes.The acetone supernatant was removed and the pellet resuspended in 20μL dissolution buffer.The soluble fraction was denatured and disulfides reduced by incubation in the presence of 0.1% SDS and 5 mM TCEP (tris-(2-carboxyethyl) phosphine) at 60ºC for one hour.Cysteine residues were blocked by incubation at room temperature for 10 minutes with MMTS (methyl methane-thiosulfonate).
Afterwards, trypsin was added to the mixture to a protein: trypsin ratio of 10:1.The mixture was incubated overnight at 37ºC.The protein digests were labeled by mixing with the appropriate iTRAQ reagent and incubating at room temperature for one hour.On completion of the labeling reaction, the four separate iTRAQ reaction mixtures were combined.Since there are a number of components that can interfere with the LC-MS/MS analysis, the labeled peptides are partially purified by a combination of strong cation exchange followed by reverse phase chromatography on preparative columns.The combined peptide mixture is diluted 10 fold with loading buffer (10 mM KH 2 PO 4 in 25% acetonitrile at pH 3.0) and applied by syringe to an ICAT Cartridge-Cation Exchange column (Applied Biosystems, Foster City, CA) column that has been equilibrated with the same buffer.The column is washed with 1 ml loading buffer to remove contaminants.
To improve the resolution of peptides during LC-MS/MS analysis, the peptide mixture is partially purified by elution from the cation exchange column in three fractions.Stepwise elution from the column is achieved with sequential 0.5 ml aliquots of 10 mM KH 2 PO 4 at pH 3.0 in 25% acetonitrile containing 116 mM, 233 mM and 350 mM KCl respectively.The fractions are evaporated by Speed Vac to about 30% of their volume to remove the acetonitrile and then slowly applied to an Opti-Lynx Trap C18 100 μL reverse phase column (Alltech, Deerfield, IL) with a syringe.The column was washed with 1 ml of 2% acetonitrile in 0.1% formic acid and eluted in one fraction with 0.3 ml of 30% acetonitrile in 0.1% formic acid.The fractions were dried by lyophilization and resuspended in 10 μL 0.1% formic acid in 20% acetonitrile solution.Each of the three fractions was analyzed by reverse phase nano-LCMS/MS on an API QSTAR XL mass spectrometer (ABS Sciex Instruments).

Bioinformatics & Statistical Analysis
The Swiss-Prot database was employed for protein identification while the PathwayStudio ® bioinformatics software package was used to determine Venn diagrams were also constructed using the Venn Diagram Plotter NIH software program (https://omics.pnl.gov/software/venn-diagram-plotter).Pathways were retrieved from three databases: DAVID (Huang et al., 2009), KEGG (Kanehisa et al., 2014), and the NCI's Protein Interaction Database (Schaefer et al., 2009).Gene ontologies were determined by employing the GO and AmiGO databases (Schaefer et al., 2009).
The accumulated MSMS spectra were analyzed by ProQuant and ProGroup software packages (Applied Biosystems) using the SwissProt fasta database for protein identification.The ProQuant analysis was carried out with a 75% confidence cutoff with a mass deviation of 0.15 Da for the precursor and 0.1 Da for the fragment ions.The ProGroup reports were generated with a 95% confidence level for protein identification.Additionally, univariate t-test were used to calculate p-values for the protein comparisons.

Results
The bottom-up salivary proteomic analysis revealed a total of 133 proteins 48 (36%) of which were differentially expressed in at least one of the three groups of women.The results are provided in Table 1 giving both the p-value and the ratio for each protein.In this study, the ratio represents the protein value of the disease state divided by the healthy control protein value.Among the three groups 31 (65%) proteins were up-regulated and 17 (35%) were down-regulated.Note: CIN-2 = moderate cervical dysplasia; CIN-3 severe cervical dysplasia; CIS cervical carcinoma in situ.
Figure 1 and Table 2 illustrate the overlap of proteins among the three groups of women.Fourteen proteins (29%) were common to all three cohorts.CIN-2 and CIN-3 had only two unique significantly differentiated proteins while the CIS cohort exhibited 10 unique salivary proteins.As presented in Table 1, the CIS group expressed nine unique proteins that were significant.The proteins that were unique to CIS are ENOA, COBA1, SPRR3, CYTC, HPT, TRFL, ALBU, THIO and TRFE. Figure 1.Overlap of salivary proteins among the three cohorts Table 3 lists the fourteen proteins that are common to all three groups with the majority of the salivary proteins (74%) being up-regulated.The information listed in the "Trends" column in Table 3 is a graphical representation of the protein ratios across CIN-2, CIN-3 and CIS.For example, protein annexin A-1 has a CIN-2 ratio of 2.0351, a CIN-3 ratio of 2.0638 and a CIS ratio of 1.3091.Across the three cohorts for Annexin A-1 the CIN-2 and CIN-3 are approximately the same whereas the CIS ratio is approximately 47% lower than the CIN-2 and CIN-3 protein ratios; therefore, the trend appears as "¯¯\" in that column.A heat map was generated from data reflecting protein expression of p-values across the three categories of women exhibited in Table 4. Spaces that are white are not statistically significant while the varying shades of gray represent significant p-values.The darker the shade of gray among data the more statistically significant is the p-value.Note: White equals no statistical significance; light gray is slightly significant p>0.001; medium gray is p>0.0001;dark grey is highly significant p<0.0001.
Table 5 illustrates the basic functional categories of the proteins along with their biochemical activity respectively.Collectively, cellular function is categorized as follows: 1) metabolic proteins (44%), anti-inflammatory and immunoresponse proteins (27%), cytoskeletal proteins (19%) and a small number of miscellaneous proteins (10%).Additionally, Table 5 categorizes the proteins according to their biochemical activity.The major biochemical functions are binding, protein binding, cellular structure and catalytic activity.

Discussion
The literature did not reveal any manuscripts using saliva as a fluid for studying cervical cancer progression.As a consequence, the author will compare the results of this investigation to studies analyzing cell lines, tissues and other body fluids (Garbett et al., 2014;Thiede et al., 2013).
The results of this study support the hypothesis that salivary proteins are altered secondary to the presence of neoplastic lesions of the cervix.The entire list of proteins is too long to discuss in this manuscript so the author has selected a few examples to illustrate how the findings support the hypothesis.For example, salivary proteins such as ENOA (Capello et al., 2011), ANXA1 (Wang et al., 2008;Bae et al., 2005), K1C13 (Carrilho et al., 2004), K1C14 (Southern et al., 2001), S100A9 (Qin et al., 2010) and ZA2G (Hassan et al., 2008) to name just a few of the proteins have been reported in the cervical cancer literature and they appear altered in saliva secondary to CIS.
In Table 1 and Table 4, ENOA was up-regulated and significantly differentiated at the p<0.004 level which corresponds to the findings of Bae et al., 2005 and was further reported by Capello, Ferri-Borgogno, Cappello & Novelli (2011).ENOA is a glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate and is the frequently deregulated in various types of cancers (Capello et al., 2011).The overexpression of ENOA is associated with the Warburg effect which is a "hallmark" of many malignancies.Interestingly, ENOA was unchanged among the CIN-2 and CIN-3 cohorts, but was up-regulated among the CIS grouping.This suggests a demarcation between dysplastic and cancerous tissues that warrants further investigation.
ANXA1 is a member of the annexin superfamily which binds to phospholipid membranes producing anti-inflammatory and anti-proliferative effects (Wang et al., 2008;Bae et al., 2005).In the presence of cancer, the protein is found to be down-regulated.As illustrated in Table 1 and Table 4, ANXA decreases in up-regulation across cohorts with tumor progression and is marginally significant among the CIS cohort.This finding is supported by Wang et al. (2008) where they found up-regulation with increasing cancer progression.
Of particular interest is cytokeratin proteins K1C13 and K1C14 across the three cohorts of women.Among the CIN-2 and CIN-3 cohorts K1C13 and K1C14 are highly up-regulated and significant at the p<0.0001 level; however, among the CIS cohort these cytoskeletal protein are not significant.Table 1 illustrates the relative decrease in ratios across the three cohorts from the dysplasia to carcinoma in situ to the point where the proteins are not significant among the CIS group.Research by Carrilho (2004) and Southern (2001) support this finding as they found a deregulation of these genes among individuals diagnosed with invasive cervical carcinoma.This finding, albeit preliminary may help our understanding of cervical tumorigenesis.
S100A8 (Jin et al., 2012) and S100A9 (Qin et al., 2010) proteins are known as a damage-associated molecular pattern molecules due to their pro-inflammatory actions.Moreover, these chemotaxic proteins are involved in various processes including calcium homeostasis, cellular migration, and energy metabolism.S100A9 reportedly is overexpressed in a variety of cancers (Yong & Moon., 2007) and are implicated in the metastatic process; however, our findings tend to agree more with the clinicopathological data published by Zhu et al. (2013) which showed that the expression of S100A9 gradually decreased during the development of cervical squamous carcinoma.The S100A8 & S100 A9, as shown in Table 1 are, indeed, significantly over-expressed for the CIS cohort, but the ratios are approximately three-times less than those shown for the CIN-2 & CIN-3 groups.As suggested by Zhu et al. (2013), S100A9 may play a dual role in tumor progression, depending on the cancer type.
Zinc-alpha2-glycoprotein is a 40-kDa single chain polypeptide assigned to the chromosome 7q22.1.And is involved in carcinogenesis and differentiation.Similar to the S100 A8 and S100 A9 proteins, recent reports showed ZA2G as overexpressed in some tumors, but lost or reduced in other tumors (Abdul-Rahman et al., 2007).In this study, ZA2G is overexpressed and is contrary to our findings in the salivary profile of in situ breast cancer patients where ZA2G was down regulated (Streckfus et al., 2008).One can only speculate, but the difference may be attributed to the two different types of carcinoma.In this study the carcinomas are squamous cell where as in the breast cancer study the tumors were adenocarcinomas (Streckfus et al., 2008).The differences are not clear and require further research.
This is a preliminary study and has its limitations.For example, the results produced a panel of high abundance proteins found in saliva.Low abundance proteins along with those bound to albumin and other carrier proteins warrant investigation.The salivary marker panel also needs to be validated among a larger cohort of subjects taking into account socioeconomic background, HPV status, tumor staging and other potential confounding variables.Analysis of a larger number of cervical cancer specimens and the correlation with biological phenotypes with gene expression patterns may also identify clinically meaningful characteristics.As a footnote, the investigator protein profiled squamous cell carcinomas located in the head and neck.In that study there were 29 differentiated salivary proteins.When comparing the head & neck salivary protein profiles with the CIS salivary pattern there was a 69% percent protein overlap of the differentiated proteins between the two sites i.e. cervix and head/neck demonstrating the heterogeneity of carcinoma from a molecular perspective.
In conclusion, the author has identified differences in protein expression between CIN2, CIN3 and CIS saliva specimens.We have identified a number of aberrantly regulated genes that were previously described in cervical cancers, and a list of genes that were not previously associated with cervical cancer development.What has been demonstrated is the importance in analyzing carcinomas as the tumors progress in staging.In the proceeding paragraphs, examples have been presented showing the changes in protein amplification as the tumor progresses.Some proteins increased during tumorigeneses while others decreased in value across stages and may no longer be significantly differentiated.Taken together the results suggest that alterations in salivary protein expression in cervical tumors may yield clues to their pathogenesis.This could provide important clues to develop novel markers as well as strategies for efficient prevention and therapy for cervical cancer.

Table 1 .
Ratios and p-values for differentiated salivary proteins across the three cohorts of individuals

Table 2 .
Overlap of proteins among the three groups of women

Table 3 .
Lists the fourteen proteins that are common to all three disease groups

Table 4 .
Is a graphical representation (heat map) of the data where the individual values contained in a matrix are represented as shades of white and gray

Table 5 .
Functional analysis of the proteins associated with the varying disease states