Identification of SEC62 as a potential marker for 3q amplification and cellular migration in dysplastic cervical lesions

Background Chromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology. Methods Expression levels of Sec62 and vimentin were analyzed in liquid based cytology specimens from 107 women with varying grades of cervical dysplasia ranging from normal cases to cancer by immunofluorescence cytology. Additionally, a subset of 20 representative cases was used for FISH analyses targeting SEC62. To further explore the functional role of Sec62 in cervical cancer, HeLa cells were transfected with a SEC62 plasmid or SEC62 siRNA and analyzed for their proliferation and migration potential using real-time monitoring and trans-well systems as well as changes in the expression of EMT markers. Results FISH analyses of the swabbed cells showed a rising number of SEC62 gains and amplifications correlating to the grade of dysplasia with the highest incidence in high grade squamous intraepithelial lesions and squamous cell carcinomas. When analyzing the expression level of Sec62 and vimentin, we found a gradually increasing expression level of both proteins according to the severity of the dysplasia. In functional analyses, SEC62 silencing inhibited and SEC62 overexpression stimulated the migration of HeLa cells with only marginal effects on cell proliferation, the expression level of EMT markers and the cytoskeleton structure. Conclusions Our study suggests SEC62 as a target gene of 3q26 amplification and a stimulator of cellular migration in dysplastic cervical lesions. Hence, SEC62 could serve as a potential marker for 3q amplification, providing useful information about the dignity and biology of dysplastic cervical lesions. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2739-6) contains supplementary material, which is available to authorized users.


Background
Cervical cancer represents the third most common cancer in women worldwide and accounts for approximately 8 % of all female cancer deaths [1]. Over the past decades, the molecular carcinogenesis of this cancer entity has been intensively studied. This has not only led to a better understanding of cancer cell biology, but also resulted in new therapeutic approaches, e.g., the clinical use of Bevacizumab in advanced and recurrent cases of cervical cancer [2]. An amplification of the long arm of chromosome 3 (3q) has been identified as a characteristic genomic alteration in more than 75 % of cervical cancer cases [3,4] and the smallest amplified region was mapped down to 3q26-27 [5,6]. When screening dysplastic cells of precancerous cervical lesions for this genomic alteration, the frequency of 3q amplification increased with the severity of the dysplasia with an incidence of 8-35 % in severe dysplasia [7] and 32-90 % in invasive squamous cell carcinomas [3,4,8,9]. In normal cervical epithelium as well as mild and moderate dysplasia, 3q amplification was only sporadically found [7]. Thus, 3q amplification designates the transition from intraepithelial cervical neoplasia to invasive cancer [3].
Apart from cervical cancer, 3q amplification was identified as a common genomic alteration in other cancers as well including non-small-cell lung cancer (NSCLC) [10], esophageal cancer [11], ovarian cancer [12] and head and neck squamous cell carcinomas (HNSCC) [13,14]. Consequently, much effort has been spent identifying potential oncogenes encoded in this region. This has led to the identification of SEC62 [15], PIK3CA [16], SOX2 [17], TP63 [18], EIF4G, CLAPM1 and FXR1 [19] as candidate oncogenes, but no functional correlation of potential oncogenic function has been reported for the majority of these genes. However, for SEC62 encoding for an endoplasmic reticulum transmembrane protein involved in intracellular protein transport [20][21][22], we previously reported that overexpression of SEC62 increases the migration ability of different human cancer cells as a basic mechanism of metastasis [15,23]. These data suggest SEC62 as a migrationstimulating oncogene [24]. Nevertheless, the molecular mechanism of migration stimulation by the SEC62 gene remains unknown. In this context, a recent proteomic study demonstrated that stable overexpression of SEC62 in HEK293 cells induced a rise in vimentin expression [25] and a morphological change of the actin cytoskeleton. Consequently, it was proposed that the SEC62-induced stimulation of cell migration could be mediated by the induction of epithelial-mesenchymal transition (EMT).
EMT, a highly conserved biological process leading to the induction of invasive growth and metastasis formation, has intensively been studied and is described for multiple cancers, including cervical cancer [26][27][28]. On the molecular level, EMT is marked by an increased expression of vimentin, a reorganization of the actin cytoskeleton and downregulation of E-cadherin with a switch to higher levels of N-cadherin [29,30]. In cervical cancer, epidermal growth factor (EGF) has been shown to be a potent inducer of EMT and to be associated with tumor invasion and lymph node metastases [31,32].
In this study, we investigated (i) if 3q amplification in precancerous and cancerous cervical lesions targets SEC62 as potential 3q encoded oncogene, (ii) if the dysplastic cervical cells show a corresponding overexpression of the SEC62 gene and (iii) if SEC62 had an oncogenic function in cultured cervical cancer cells through altering cell migration, cell proliferation and EMT induction.

Patient characteristics and liquid-based cytology
In total, 107 female patients were enrolled in this study who presented at the Department of Gynecology, Obstetrics and Reproductive Medicine of the Saarland University Medical Center (Homburg/Saar, Germany) between January 2012 and January 2013 in the context of the national cervical cancer prevention program. From all patients, liquid-based cytological swab material of the uterine cervix was used for further analyses. Thereby, we collected subsamples for cytological negative samples, and each of the histology groups CIN-I (cervical intraepithelial lesion grade I) through CIN-III (cervical intraepithelial lesion grade III; each of size 25) as well as a sample of 7 patients with histologic SCC (squamous cell carcinoma). For 82 patients (82/ 107; 76.6 %), probe excisions of the uterine cervix were also available. For patients with a normal cytological swab, we abstained from an incisional biopsy. Exclusion criteria included a history of surgical or medicinal treatment of dysplastic cervical lesions, an acute or chronic cervicitis or colpitis and non representative cytological or histological material. From each patient, a cytological smear from the uterine cervix was taken using the Cytobrush Plus (Cooper Surgical Inc.; Trumbull, CT, USA) in an ambulatory setting. After wiping off the macroscopically suspect mucosal areas, brushes were shaken out in the PreservCyt solution (Hologic Deutschland GmbH; Wiesbaden, Germany). The cellular suspensions were used for the preparation of microscope slides using the ThinPrep-system (Hologic Deutschland GmbH; Wiesbaden, Germany) according to the manufacturer's instructions. For cytopathological staging, the microscope slides were stained according to Papanicolaou using a standard protocol. The slides were classified by two independent examiners with wide experience in valuing cytological smears of the uterine cervix. The respective cytological diagnoses according to the Bethesda classification system were NILM (negative for intraepithelial lesion/malignancy, n = 25), ASCUS (atypical squamous cells of undetermined significance, n = 9), LSIL (low-grade squamous intraepithelial lesion, n = 25), HSIL (high-grade squamous intraepithelial lesion, n = 38) and SCC (squamous cell carcinoma, n = 10). The Saarland Medical Association ethics review committee approved the scientific use of the patient's tissue and clinical data (index number 207/10). Written informed consent was obtained from all patients.

Fluorescence in situ hybridization (FISH) analysis
Prepared microscope slides were pretreated with RNase A and pepsin, then denatured with 70 % formamide/ 2xSSC at 72°C, dehydrated in a series of cold ethanol washes and air-dried.
The BAC clone RP11-379 K17 encoding SEC62 (Ima-Genes, Berlin, Germany) was biotin labeled using the BioPrime DNA Labeling System (Invitrogen, Life Technologies, Darmstadt, Germany). As internal control, a centromeric probe for chromosome 10 (D10Z3) labeled with digoxigenin by standard nick-translation according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany) was used. After probe hybridization overnight, the slides were washed two times in 2× SSC at 42°C and three times in 50 % formamide/2× SSC at 42°C. Immunofluorescence detection of the biotin signals was carried out using Streptavidin-FITC and -biotinylated anti-Streptavidin antibodies (Vector Laboratories, Burlingame, CA, USA). For the detection of the digoxigenin signals, anti-Dig-Cy3 and goat-anti-mouse-Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. The slides were mounted in an anti-fade solution containing DAPI (4, 6diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA) and analyzed with the BX61 fluorescent microscope equipped with a charge-coupled device camera (Olympus, Hamburg, Germany). In total, 200 nonoverlapping, morphologically well-preserved nuclei per slide were analyzed. Thereby, we selectively evaluated the number of FISH signals in the morphologically conspicuous nuclei in the CIN-I, CIN-II, CIN-III and SCC (histological diagnosis) cases. For the "no CIN" cases, every nucleus was considered. Gains were defined as three or four signals per probe; five or more signals were defined as amplification. The specificity of each probe was determined by hybridizing and enumerating normal human lymphocytes and metaphase spreads, prepared according to standard protocols, for cutoff ranges and an analysis of cross hybridizations by non-stringency of hybridization conditions.

Immunofluorescence cytology (IFC)
To simultaneously analyze Sec62 and vimentin expression in the swabbed cells, prepared microscope slides were dried for 30 min at room temperature. The slides were washed three times in distilled water (aqua dest.) and PBS pH7.2. Epitope unmasking was performed by incubation in Target Retrieval Solution (DAKO, Glostrup, Denmark) at 95°C for 60 min. After cooling to room temperature and three PBS pH 7.2 washes, the slides were incubated with the primary antibody solution (1:100 dilution in 0.1 % BSA/PBS) for 60 min at room temperature. After another three PBS washes, the slides were incubated with the secondary antibody solution (1:100 dilution in 0.1 % BSA/PBS) for 60 min at room temperature, again followed by three PBS washes. The slides were counterstained with Hemalaun (1:4 dilution in aqua dest.) and mounted in DAPI-Fluoroshield -mounting medium (Sigma-Aldrich, St. Louis, MO, USA).
To detect Sec62, we generated a polyclonal affinitypurified rabbit antibody directed against the COOHterminal undecapeptide of the human Sec62 protein as previously described [15,[23][24][25] and detected it with a goat anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate (FITC; Dianova, Hamburg, Germany). The monoclonal Clone 9 vimentin antibody was labeled with Cy3 (Sigma-Aldrich, St. Louis, MO, USA). Slides were imaged with the Nikon Eclipse TE2000-S inverted microscope, the Nikon Digital Sight DS-5Mc camera and the NIS-Elements AR software version 3.0 (Nikon; Tokyo, Japan).
The fluorescent signals for Sec62 and vimentin were quantified in morphologically dysplastic cells in relation to normal cells of the same slide by six independent examiners. The staining intensity was valued as "-1"for a weaker fluorescent signal in dysplastic cells compared with normal cells, "0" for no difference in the staining intensity between dysplastic and normal cells and "+1", "+2" or "+3" for a little stronger, moderately stronger or markedly stronger signals in dysplastic cells compared with normal cells. If no dysplastic cells were found in the slide, the staining intensity of two normal cells was compared to each other. The overall immunoreactive score (IRS) for Sec62 and vimentin was set as a sum of the six single scorings (six separate examiners) with a minimal score of −6 and a maximal score of 18. For all IFC analyses, we referred to the histological diagnosis when grouping the patients into the CIN-I, CIN-II, CIN-III and SCC group. For the "no CIN" cases we had to refer to the cytological diagnosis as no probe excision of the uterine cervix was available for these patients.

Cell culture and transfections
HeLa cells (DSMZ-No. ACC 57) and MCF-7 cells (DSMZ-No. ACC 115) were cultured in DMEM medium (Gibco Invitrogen, Karlsruhe, Germany) containing 10 % FBS (Biochrom, Berlin, Germany) and 1 % penicillin/streptomycin (PAA, Pasching, Austria) at 37°C in a humidified environment with 5 % CO 2 . Both cell lines were characterized by the German Collection of Microorganisms and Cell Culture (DSMZ) using multiplex PCR of minisatellite markers, isoelectric focusing and karyotyping. The cell lines were obtained by the DSMZ in 2015.
For gene silencing, 5.2 × 10 5 HeLa cells were seeded in 6 cm dishes and transfected with SEC62 siRNA directed against the 3′ untranslated region (CGUAAAGUGUAUU-CUGUACtt; Ambion, TX, USA) or control siRNA (All-Stars Neg. control siRNA; Qiagen, Hilden, Germany) using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After 24 h, the medium was changed and the cells were transfected again for additional 24 h.
For overexpression studies, 5.2 × 10 5 HeLa cells were seeded in 6 cm dishes. After 24 h, the medium was changed and the cells were transfected with either the IRES-GFP-SEC62 plasmid (SEC62 plasmid) or the negative control IRES-GFP-LV plasmid (control plasmid) using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. For both plasmids, pcDNA3 served as parent plasmid.

Real-time cell proliferation analysis
The xCELLigence SP and DP systems (Roche Diagnostics GmbH, Mannheim, Germany) were used for the real-time analysis of cell proliferation. These systems measure changes of impendance in special plates with micro electrodes covering the well bottoms (E-plates, Roche Diagnostics GmbH, Mannheim, Germany). The relative changes are recorded as Cell Index, a dimensionless parameter. 2.5 × 10 3 HeLa cells transfected with either siRNA or plasmids were seeded in a 96-or 16well e-plate (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Cells transfected with siRNA were seeded 24 h after the second transfection (48 h after the initial siRNA transfection). Cells transfected with plasmids were seeded 24 h after the plasmid transfection. Cell proliferation was monitored for 96 h and the data was evaluated with RTCA 2.0 software (Roche Diagnostics GmbH, Mannheim, Germany). All cell proliferation experiments were repeated fourfold (n = 4) and a triplicate of every cell population was analyzed in each experiment.

Migration potential analysis
Cell migration was analyzed using CIM-devices and the xCELLigence DP system (Roche Diagnostics GmbH, Mannheim, Germany) as a technique of real-time migration monitoring. 2.0 × 10 4 HeLa cells transfected either with siRNA or plasmids were seeded 24 h after the final transfection in the upper chamber of the CIM-device in culture medium with 5 % FBS. The upper chamber was then placed on the lower part of the CIM-device containing culture medium either supplemented with 10 % FBS as a chemoattractant for cell migration or without FBS (negative control). Cell migration was followed over a time period of 48 h by changes of the impedance signal in the CIM-plate system measured on the backside of the membrane. In parallel, cell proliferation was monitored in a 96well e-plate (xCELLigence SP system) or in a 16-well eplate (xCELLigence DP system) as described above.
The BD Falcon FluoroBlok system (BD, Franklin Lakes, NJ, USA) with 8 μm pore inserts for 24-well plates was also used to assess migration. 5 × 10 4 HeLa cells transfected with either siRNA or plasmids were loaded into the inserts in normal medium containing 5 % FBS. The inserts were then placed in the wells of a 24-well plate in medium with either 10 % FBS as a chemoattractant for migration or without FBS (negative control). After 15 h (39 h after the last transfection), the cells were fixed with methanol, the nuclei counterstained with DAPI and the number of migrated cells was analyzed by a bottom reading fluorescence microscope.
All cell migration experiments were repeated fourfold (n = 4) and a triplicate of every cell population was analyzed in each experiment.
Immunofluorescence of cultured cells 5 × 10 5 HeLa cells either transfected with SEC62 siRNA, a SEC62 plasmid, control siRNA or a control plasmid were seeded onto polylysine coated coverslips. 24 h later, the coverslips were transferred into the wells of a 6-well plate and covered with PBS at 4°C for 3 min. All following steps were performed in a light protected environment. The cells were fixed in paraformaldehyde for 20 min at 4°C. The coverslips were then washed four times in PBS (+0.1 M glycine/4 mM MgCl 2 ) before incubating with PSS (PBS + 5 % FCS/0.1 % saponine/50 μg/ ml RNAse I) for membrane permeabilization and blocking for 1 h. The coverslips were incubated in primary antibody diluted in PSS (1:100 for Sec62-, vimentin-and E-cadherin antibody; 1:250 for Phalloidin-Alexa488 (Life Technologies, Carlsbad, CA USA)) for 1 h, washed twice with PSS, and incubated with secondary antibody diluted 1:1000 in PSS (anti-rabbit Alexa488 and anti-mouse Texas Red; Life Technologies, Carlsbad, CA, USA) before the final three washes in PSS. The coverslips were air-dried and mounted on microscope slides with DAPI-Fluoroshield mounting medium. Imaging was performed as described for IFC.

Statistical analysis
Statistical analysis of IFC and FISH was performed with a two-sided Mann-Whitney-U-test using the Statistical Package for the Social Sciences v. 17.0 (IBM, Chicago, IL, USA) and XLStat Pro (Addinsoft, NY, USA) software. Normality test and statistical analysis of cell proliferation and migration was performed with the D' Agostino & Pearson normality test and a two-sided, paired Student's t-test using GraphPad Prism 6.0 h (GraphPad Software, La Jolla, CA, USA). P-values <0.05 were considered statistically significant (α = 0.05). In the figures, statistically significant results are marked by * (p ≤ 0.05), ** (p ≤ 0.01) or *** (p ≤ 0.001). Statistically non-significant results are marked by "n.s.".

The incidence of SEC62 gains and amplifications rises with the grade of dysplasia
To determine whether the copy number of the 3q26 encoded SEC62 gene changes in dysplastic cells of the uterine cervix, we performed FISH analyses of SEC62 on cytological specimens in a representative subset of 20 patients. Their histological diagnoses were "no CIN" (n = 5), CIN-I (n = 5), CIN-II (n = 5), CIN-III (n = 5) and SCC (n = 5; for the corresponding cytological diagnoses, see Methods). The centromere region of chromosome 10 served as an internal control. Gains of the SEC62 gene were found in 3 % of the counted nuclei in normal cases, 4 % of the nuclei in CIN-I cases, 4 % of the nuclei in CIN-II cases, 9 % of nuclei in CIN-III cases and 23 % of nuclei in SCC cases (Fig. 1). Additionally, amplifications of the SEC62 gene were found in dysplastic nuclei of two CIN-I cases, one CIN-III case and four SCC cases. Overall, we observed a rise of SEC62 gains and amplifications corresponding to the severity of dysplasia with a significantly higher incidence of SEC62 gains in SCC cases compared to all other cases (p = 0.006).

Simultaneous overexpression of Sec62 and vimentin designates higher grades of cervical dysplasia
To evaluate if the detected SEC62 gains and amplifications correlate with increased cellular Sec62 protein levels, we quantified the level of Sec62 in the swabbed cells of all 107 female patients. As an overexpression of the SEC62 gene in HEK293 cells has been reported to induce a rise in vimentin expression, suggesting that SEC62 mediates EMT induction [25], we analyzed Sec62 and vimentin protein levels simultaneously. Therefore, we developed IFC as a new staining method for liquid-based cytological swabs. After imaging the immunostained cells, the slides were used for Papanicolaou staining to evaluate the morphology of the swabbed cells. Figure 2 shows representative images for two patients, whose cervical swabs were staged LSIL (A) and HSIL (B). Figure 3 summarizes the immunoreactive scores (IRS) for Sec62 and vimentin delineated for the different histological and cytological diagnoses for all included patients.
SEC62 and vimentin were overexpressed in dysplastic cells compared with normal cells on the same slide with a gradual increase of expression corresponding to the rising severity of the dysplasia. When comparing the expression level of Sec62 and vimentin in the dysplastic cells, we found a distinct correlation between the Sec62and vimentin-IRS (r 2 = 0.87). To exclude that the dysplastic cells show increased fluorescent signals for all cytoplasmic proteins due to an altered cellular shape instead of a specific overexpression of the respective genes, we performed additional IFC stainings for 10 representative cases targeting Sec62 and β-actin (see Additional file 1). Indeed, there was no relevant change of β-actin expression depending on the severity of dysplasia. Therefore, the rise in Sec62 and vimentin protein levels in the dysplastic cervical cells is likely attributed to a specific overexpression of both genes.

Altering Sec62 protein levels influences HeLa cell migration
The IFC analyses indicated that SEC62 overexpression marks the transition from intraepithelial neoplasia to an invasive phenotype. To evaluate whether SEC62 has potential oncogenic function, we altered Sec62 levels in HeLa cells and evaluated changes in cell migration and proliferation. The experiments were repeated fourfold (n = 4) and a triplicate of every cell population was analyzed in each experiment.
First, the cells were transfected with SEC62 siRNA, resulting in decreased Sec62 protein levels to 22 ± 1 % (mean ± standard error of the mean, SEM) compared with  control siRNA transfected cells. While marginal effects of SEC62 silencing on cell proliferation were observed (86 ± 3 %, mean ± SEM), there was a crucial reduction in cell migration (27 ± 4 %, mean ± SEM) compared to control cells using the xCELLigence DP system and the FluoroBlok system for migration monitoring (Figs. 4 and 5).
Next, SEC62 was overexpressed by transfecting the cells with a SEC62 plasmid resulting in an increase of Sec62 protein levels to 487 ± 50 % (mean ± SEM) compared with control cells. This overexpression of SEC62 led to increased cell migration (171 ± 7 %, mean ± SEM) with no influence on cell proliferation (93 ± 3 %, mean ± SEM; Figs. 4 and 5). In all transfection experiments, the transfection procedure itself led to a slightly reduced cell proliferation without however showing relevant differences between the control siRNA and the SEC62 siRNA transfected cells respectively the control plasmid and the SEC62 plasmid transfected cells.  (1), moderately more intense (2) or much more intense (3). For each case, the quantitation of 6 independent examiners was toted up to an overall IRS ranging from −6 to 18. In (c), the overall IRS for Sec62 was correlated with the overall IRS for vimentin. The strength of squared correlation is indicated by the squared correlation coefficient (R 2 )

SEC62 overexpression cannot induce EMT in HeLa cells
As SEC62 overexpression in HEK293 cells was reported to induce a rise in vimentin expression and a reorganization of the actin cytoskeleton [25], we next investigated if the SEC62-driven stimulation of HeLa cell migration can be attributed to an induction of EMT. SEC62 was either overexpressed by plasmid transfection or downregulated by siRNA transfection and the effects on cellular vimentin and E-cadherin levels were analyzed using western blot and immunofluorescence microscopy. These markers were chosen, because both are known to change their expression level when cancer cells undergo EMT with an upregulation of vimentin and a downregulation of Ecadherin levels [33]. As the subcellular F-actin structure shows structural changes during EMT too [34], we additionally analyzed β-actin as a third EMT marker. There were no changes in the expression level of vimentin, Ecadherin and β-actin and no changes in the subcellular structure of the β-actin cytoskeleton (Fig. 6). All differentially pretreated HeLa cell populations contained a moderate expression of vimentin and β-actin independent of the different treatments and E-cadherin was not detected in HeLa cells agreement with previous reports [35].

Discussion
Cervical cancer represents the third most common cancer in women worldwide, resulting in approximately 275,000 deaths each year [1]. Despite much effort to develop new diagnostic [36,37] and therapeutic strategies [38], the 5-year survival rate has remained at about 70 % with no significant changes over the past 30 years [39]. 3q amplification has been identified as a common genomic alteration in cervical cancer [3,4], marking the transition of intraepithelial neoplasia to invasive cancer [7]. Recently, we observed that SEC62 encoded at 3q26.2 was frequently amplified and overexpressed in NSCLC tissue specimens [15]. Moreover, a high expression of SEC62 predicts a poorer clinical outcome for this cancer entity [24] and crucially influences cell migration, calcium homeostasis and ER stress tolerance of various human tumor cells [23,24,40].
In this study, we investigated the potential role of SEC62 in the carcinogenesis of cervical cancer.
We found (i) that SEC62 is a potential candidate gene of the amplified 3q region in precancerous and earlystage cancerous cervical lesions, (ii) that SEC62 is overexpressed on the protein level in dysplastic cells of the uterine cervix compared to normal cells and (iii) that the ability of cervical cancer cells' to migrate depends on their cellular Sec62 protein level.
FISH analyses of representative uterine cervix samples demonstrated a rise in SEC62 gains and amplifications corresponding to the grade of dysplasia, with the highest incidence in invasive cancer cases. Accordingly, we detected an increase in cellular Sec62 protein level correlating to the severity of dysplasia in IFC analyses. These results agree with previous studies reporting a comparable incidence for the amplification of the entire 3q26 region in precancerous cervical lesions and cervical cancer [41], suggesting that the SEC62 gene harbors an oncogenic function. However, there are other potential 3q26-encoded oncogenes with a similar pattern of amplification and overexpression in dysplastic cervical lesions including hTERC, LAMP3 and PIK3CA [42][43][44][45]. Kuglik et al. reported that gains of the hTERC gene are specific genomic changes in cytological specimens of the uterine cervix associated with the progression to a malignant phenotype [42]. Furthermore, a meta-analysis of 12 studies evaluating the diagnostic value of hTERC in dysplastic cervical lesions found that the detection of hTERC amplification is a valuable marker for high-grade cervical lesions and invasive cancer [43]. However, no functional analyses have been performed to confirm the potential oncogenic function of hTERC or the molecular mechanism behind its oncogenic activity. It is also probable that multiple genes in the 3q26 region are responsible for the transition of precancerous cervical lesions to invasive cancer and that their interplay bridges the gap from 3q amplification to the molecular cell biology of cervical cancer carcinogenesis.
As in the first part of our study FISH-and IFC-analyses indicated a potential oncogenic function of SEC62, we sought to identify a functional correlate in cancer cell biology using HeLa cells an in vitro model. Thereby, SEC62-silencing significantly inhibited cell migration while conversely, SEC62 overexpression stimulated cell migration.
These results confirmed conclusions of previous studies reporting similar effects of SEC62 gene silencing on lung cancer, prostate cancer, fibrosarcoma, glioblastoma and thyroid cancer cell lines [15,23], as well as effects of SEC62 overexpression on human embryonic kidney cells [24]. However, the molecular mechanism of how SEC62 is able to regulate cell migration remains elusive. SEC62 encodes for a transmembrane protein of the endoplasmic reticulum (ER) that is thought to be involved in protein transport across the ER membrane, including the translocation of the C-terminus of membrane proteins [20], the membrane insertion and orientation of moderately hydrophobic signal anchor proteins [21] and the secretion of small proteins independent of the signal recognition particle pathway [22]. Hence, we speculate that Sec62 might influence the intracellular transport of proteins that are involved in cell migration. We previously reported that Sec62 overexpression in HEK293 cells resulted in an increased vimentin expression and observed a structural reorganization of the actin cytoskeleton [25]. As increased vimentin expression is a key marker of EMT [29], SEC62-mediated increase of vimentin expression represents an alternative mechanism of how SEC62 could influence cell migration. In support of this hypothesis, IFC-analyses of cervical brush biopsies demonstrated a distinct correlation between SEC62 and vimentin expression in our study. However, changes in Sec62 protein levels in HeLa cells did neither result in detectable changes of the expression of EMT markers nor a rearrangement of the actin cytoskeleton structure, contrary to our previous findings in HEK293 cells [25]. A possible explanation for these contradictory results could be that different human cell lines have a varying capability for EMT induction [46] and cytoskeleton remodeling [47]. Alternatively, it is possible that SEC62 can induce EMT in vivo but requires unknown accessory factors and thus, loses this function in an artificial cell culture model.
Irrespective of the underlying molecular mechanism, the inhibition of cell migration by SEC62 silencing represents a promising approach for a new targeted therapy, as the molecular effects of SEC62 silencing on cell migration and ER stress tolerance can be mimicked by trifluoperazine [24], an antipsychotic drug used to treat schizophrenia patients [48]. In addition to this potential role of Sec62 as a therapeutic target, the detection of SEC62 overexpression by IFC could serve as a potential indicator for 3q26 amplification. As this genomic alteration has a high predictive value for distinguishing CIN-II/III lesions from normal cases [41] and can predict the further development of precancerous cervical lesions [49], Sec62-IFC may provide useful information for the treatment of women with dysplastic cells in their cervical swab.

Conclusions
Taken together, our study has demonstrated a rising incidence of SEC62 gains and amplifications in dysplastic cervical lesions as well as an increased cellular Sec62 protein levels corresponding to the severity of dysplasia. In functional analyses, we found that SEC62 overexpression promoted an invasive phenotype by stimulating the cervical cancer cells' capability to migrate. Thus, we propose that SEC62 functions as a migration-stimulating oncogene in the carcinogenesis of cervical cancer and constitutes not only a potential marker for 3q26 amplification but also a potential target for anti-cancer treatment.