Progesterone receptor membrane component 1 is involved in oral cancer cell metastasis

Abstract Cancer metastasis is a common cause of failure in cancer therapy. However, over 60% of oral cancer patients present with advanced stage disease, and the five‐year survival rates of these patients decrease from 72.6% to 20% as the stage becomes more advanced. In order to manage oral cancer, identification of metastasis biomarker and mechanism is critical. In this study, we use a pair of oral squamous cell carcinoma lines, OC3, and invasive OC3‐I5 as a model system to examine invasive mechanism and to identify potential therapeutic targets. We used two‐dimensional differential gel electrophoresis (2D‐DIGE) and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF/TOF MS) to examine the global protein expression changes between OC3 and invasive OC3‐I5. A proteomic study reveals that invasive properties alter the expression of 101 proteins in OC3‐I5 cells comparing to OC3 cells. Further studies have used RNA interference technique to monitor the influence of progesterone receptor membrane component 1 (PGRMC1) protein in invasion and evaluate their potency in regulating invasion and the mechanism it involved. The results demonstrated that expression of epithelial‐mesenchymal transition (EMT) markers including Twist, p‐Src, Snail1, SIP1, JAM‐A, vimentin and vinculin was increased in OC3‐I5 compared to OC3 cells, whereas E‐cadherin expression was decreased in the OC3‐I5 cells. Moreover, in mouse model, PGRMC1 is shown to affect not only migration and invasion but also metastasis in vivo. Taken together, the proteomic approach allows us to identify numerous proteins, including PGRMC1, involved in invasion mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of oral cancer invasion.


| INTRODUC TI ON
Oral cancer occurring often in oral cavity belongs to head and neck cancer. Tumours at these sites classically cause drinking and eating difficulties. Alcohol, tobacco and betel quid consumption are well-reported risk factors for more than 90% oral cancer cases. 1 More than nine in ten of cancer deaths are attributable to cancer metastatic disease that has moved to the centre of clinic attention. Metastasis is a multistage process in which cancer cells spread out from primary site to another non-adjacent part. This spread of cancer is formed by a complex and dynamic series of cell-biological events. To comprehend the metastasis mechanism and to find out the main metastasis-regulating factors are the best way to solve this issue.
There are over 60% of oral cancer patients present with advanced stage disease, and the five-year survival rates of these patients decrease from 72.6% to 20% as the stage becomes more advanced. 1 The advanced diseases commonly involve regional lymph nodes metastasis. The distant metastasis of oral cancer cells is rare, arising in 17% of patients, and mostly metastasized to lung following by the mediastinal lymph nodes, liver and bones. Despite the rapid progression in diagnosis and treatment of head and neck cancer during last two decades, only a slight improvement in the 5-year survival rate for oral cancer patients. 3 And at least 50% of patients with regional and distant dis-  [4][5][6] There are also several proteomic studies which reveal the protein expression level of metastasis and non-metastasis oral cancer, and most of those studies used oral cancer patients' tumour tissues as materials. Several metastasis-related protein candidates have been found, such as superoxide dismutase 2 (SOD2) 7 and caldesmon. 8 However, clinical samples exhibit different gene backgrounds from patient to patient and lack of homogeneity, which may shadow the potential metastasis-related proteins and biomarkers. In this study, we used OSCC cell lines OC3 and OC3-I5 with different metastatic potential derived from a single patient as materials to limit the heterogeneity. We analysed the cytosolic and secreted protein abundance of both invasive and non-invasive OSCC by using 2D-DIGE, and identified the proteins with different expression levels by MALDI-TOF MS. A total of 101 differently expressed proteins were identified. Among all the metastasis-related candidates, membrane-associated progesterone receptor component 1 (PGRMC1) was selected for further investigation and show potency as an oral cancer therapeutic targets. By using oral cancer cells carrying a shRNA to inactivate the PGRMC1 gene function, we addressed the role of PGRMC1 in the development of tumour metastasis to the lung in vivo. We demonstrated that the development and metastasis of the oral cancer OC3 cells in vivo depend substantially on PGRMC1 expression.

| Chemicals and reagents
Lipofectamine RNAiMAX reagent and OPTI-MEM were purchased from Life Technologies; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from USB Corporation; CellTiter Blue was purchased from Promega Corporation; Primary antibodies were purchased from Genetex; antimouse and antirabbit secondary antibodies were purchased from GE Healthcare; fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin expression was decreased in the OC3-I5 cells. Moreover, in mouse model, PGRMC1 is shown to affect not only migration and invasion but also metastasis in vivo. Taken together, the proteomic approach allows us to identify numerous proteins, including PGRMC1, involved in invasion mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of oral cancer invasion.

K E Y W O R D S
metastasis, oral cancer, progesterone receptor membrane component 1, proteomics G antibody was purchased from Jackson Immuno Research Laboratories; foetal bovine serum (FBS), trypsin-EDTA, Dulbecco modified Eagle medium (DMEM), and penicillin and streptomycin (P/S) were purchased from Gibco -Invitrogen Corporation; 10-, 15and 4-cm Petri dishes and 96-and 24-well plates were purchased   from Orange Scientific; MTT, Tris base, glycerol, NP-40, sodium dodecyl sulphate (SDS) and tetramethylethylenediamine were purchased from USB Corporation; bovine serum albumin was purchased from Sigma-Aldrich; Horseradish peroxidase-linked antirabbit IgG was purchased from GE Healthcare; Tris HCl (pH 6.8) was purchased from Severn Biotech Ltd; HCl was purchased from Scharlau Chemie; repel solution and cover oil were purchased from GE Healthcare; and an enhanced chemiluminescence substrate kit was purchased from Visual Protein Corporation. All chemicals and biochemicals used in this study were of an analytical grade.

| Cell lines and cell cultures
OC3 and OC3-I5 oral cancer cells were received as a gift from Dr Wang Lu-Hai. The OC3-I5 cells, which were OC3 cells selected from a transwell invasion assay, were reselected every 10 passages to maintain their invasive ability. The detail culture information of OC3 and OC3-I5 cells was reported in our previous publication. 9

| MTT cell viability assay
The OC3 and OC3-I5 cells were seeded into 96-well plates at a density of 7000 cells/well for performing MTT assay. The detail procedure for the assay has been described in our previous publication. 9

| Immunoblotting assay
Immunoblotting was employed to validate the differential expression of mass spectrometry-identified proteins. The detail procedure for the assay has been described in our previous publication. 9

| Enzyme-linked immunosorbent assay (ELISA)
EIA polystyrene microtiter plates were coated with 50 μg of protein lysate sample and incubated at 37°C for 2 hours. The plate was washed three times with phosphate buffered saline with Tween-20 (PBS-T) and three times with PBS. Plates were then blocked with 100 μL of 5% skimmed milk in PBS at 37°C for 2 hours and then washed three times with PBST. Antibody solution was added and incubated at 37°C for 2 hours. After washing with PBST and PBS for 10 times in total, 100 μL of peroxidise-conjugated secondary antibody in PBS was added for incubation at 37°C for 2 hours.
Following 10 washes, 100 μL of 3,3,5,5-tetramethyl benzidine (Pierce) was added. After incubation at room temperature for 30 minutes, 100 μL of 1 M H 2 SO 4 was added to stop the reaction and the absorbance at 450 nm measured using a Stat Fax 2100 microtiter plate reader (Awareness Technology Inc).
The sequences with similar GC content were used as the negative control against PGRMC-1 (Invitrogen). The OC3 and OC3-I5 cells were transfected with 60 nMPGRMC1 small interfering RNA Stable cell lines could be established by puromycin selection after transfection. shPGRMC1 or shLacZtransfectedOC3 and OC3-I5 cells were selected by their resistance to puromycin. One day after subcultured, puromycin was added in transfected OC3 and OC3-I5 cells at a concentration of 1 μg/mL. Cells were incubated with puromycin-containing medium for 48 hours and recovered in serum medium for 48 hours. The puromycin selections were performed every two passages until the cell viability would not affected by puromycin.

| Flow cytometry analysis for apoptosis detection and cell-cycle analysis
For apoptosis detection, the percentage of apoptotic cells stained using the FITC Annexin V Apoptosis Detection kit I (BD Biosciences) was determined. The detail procedure for the assay has been described in our previous publication. 9 Data were further analysed using Cflow Plus analysis software (BD Biosciences).

| Migration and invasion assay
Transwell assay was employed to examine the effect of PGRMC1 on migration and invasion in OC3/ OC3-I5 cells. Transwell cell culture inserts (PET membrane) with a pore size of 8.0 µm (SPL Life Sciences) were coated or not coated with Matrigel™ (BD Biosciences) in an FBS-free DMEM medium. The detail procedure for the assay has been described in our previous publication. 9 The cells that had invaded were visible under the optical microscope and were observed at a magnification of 100X.

| Immunofluorescence staining
For immunofluorescence staining of OC3 and OC3-I5 cells, the detail procedure for the assay has been described in our previous publication. 9 Images were exported in the TIF file format by using Zeiss Axiovision version 4.8 and processed using Adobe Photoshop version 7.0 (Adobe Systems).

| Wound-healing migration assay
Wound-healing assay provides an easy and simple means for monitoring directional cell migration and interaction in vitro. The detail procedure for the assay has been described in our previous publication. 9 The wound areas were calculated using AxioVision version 4.

| MTT cell viability assay
The detail procedure for the assay has been described in our previous publication. 9

| Orthotopic tumour implantation
Five-week-old BALB/cAnN.Cg-Foxnlnu/CrlNarl female mice were housed in a specific-pathogen-free environment. One week after housing, mice were injected with human oral cancer cells. 3 × 10 6 OC3 and OC3-I5 cells stably transfected with shPGRMC1 or shLacZ were mixed with Matrigel™ in PBS (1:7) and inoculated subcutaneously onto the right tight of mice (6 per group). The mice were killed by zoletil/rompun60 days after implantation, and the lungs were removed and fixed in 10% formalin. For further analysis, the lung samples were tissue trimming, tissue processing and embedding, section and H & E staining to examine tissue morphologies, which were performed by National Laboratory Animal Center, Taiwan, ROC All mice were fed ad lib and housed according to the guidelines of the Society of Laboratory Animal Science.

| Statistical analysis
The Student t test and analysis of variance were employed for the statistical analysis, with P < .05 considered statistically significant.

| Invasion properties of OC3-I5 cells compared to OC3 cells
To evaluate the invasion mechanism of oral cancer, we prepared a human oral cancer cell line, OC3, and an invasive oral cancer cell line, OC3-I5. OC3-I5 cells were derived fromOC3 cells and selected through transwell invasion assay. After every ten passages, OC3-I5 cells were re-transwell selected to maintain the invasive ability. Transwell invasion assay is a widely used platform for measuring invasion ability. The transwell inserts create a two-chamber system separated by one layer  Figure 1A). This observation indicated that OC3-I5 cells were at a more mesenchymal cell status than OC3 cells, which confirmed the previous results of invasion assay.
In cells that exhibit movement, a significant part of the migration cycle is the formation of stable attachment points called focal adhesions. Focal adhesions can transduce forces between cytoskeleton and the extracellular matrix (ECM) and execute directional movement via the polarization signalling. OC3-I5 cells presented smaller but much more focal adhesions (actin dots) than OC3 cells did ( Figure 1C) and expressed higher level of focal adhesion proteins JAM-A, paxillin and vinculin ( Figure 1C). These data implied that OC3-I5 cells have better ability of migration than OC3 cells. Moreover, the high level of pSrc ( Figure 1A) in OC3-I5 cells corresponded with focal adhesion data.

| Validation of characterized invasion associated proteins via immunoblotting and ELISA analysis
To further validate the expression trend of identified protein, we performed immunoblotting and ELISA analysis of the differentially F I G U R E 1 The invasive properties and focal adhesion formation of OC3-I5 cells compared to OC3 cells. (A) The expression levels of EMT markers vimentin, E-cadherin and matrix metalloproteinase-3 (MMP3) in OC3 and OC3-I5 cells were presented by immunoblotting. β-actin was used as loading control. (B) The invasion ability of OC3 and OC3-I5 cells was monitored by transwell invasion assay. 10 5 cells in the matrix-coated upper chamber of the transwell inserts, and incubated at 37°C for 18 hours. After incubation, cell was fixed in 4% paraformaldehyde and stained with crystal violet. The crystal violet was dissolved in ethanol/acetic acid solution, and the OD value at 600 nm of the solution was measured. OC3-I5 cells showed 2.7-fold better invasion ability than OC3 cells. Error bars denote mean ± SEM (n = 3). **P < .01. (C) Cells were seeded on 12 mm glass slides. After fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, cells were hybridized with phalloidin. Images were photographed at the magnification of 4000× by microscopy. The actin dots are indicated by yellow arrows. Scale bar, 20 μm. The expression levels of migration markers were monitored by immunoblotting. GAPDH was used as loading control. The actin dots in OC3 and OC3-I5 cells were counted and the quantified. OC3-I5 cells formed more actin dots than OC3 cells at about 3.3-fold. Error bars denote mean ± SEM (n = 3). ***P < .001

| PGRMC1 is required for human oral cancer invasion and migration by regulating EMT via SIP1, Snai1 and Twist transcription factors
Among all the metastasis-related candidates, membrane-associated or without the transfection of siPGRMC1 were also shown in Figure 2D.
Altogether, these results demonstrated that PGRMC1 was required for human oral cancer invasion and migration via regulating EMT-inducing transcription factors SIP1, Snai1 and Twist.
OC3 and OC3-I5 cells transfected with siPGRMC1 showed a great decrease in migratory ability in the absence of a chemotactic gradient. As expected, siPGRMC1 knockdown OC3-I5 cells presented less actin dots than mock cells ( Figure 3). Additionally, we found that knockdown of PGRMC1 reduced the wound-healing ability of OC3-I5 cells in wound-healing assay (Figure 4).

| PGRMC1 is essential for oral cancer proliferation by promoting entry of cells into G2/M phase via p53 down-regulation
To investigate whether PGRMC1 knockdown interferes oral cancer proliferation ability and how PGRMC1 acts, we performed MTT assay. As shown in Figure 5A, OC3-I5 cells showed a 1.2-fold better proliferation ability than OC3 cells. Nevertheless, the proliferation ability of OC3-I5 cells declined when cells were transfected with siPGRMC1. We therefore investigated whether OC3 cells and OC3-I5 cells with siPGRMC1 arrested in G1 phase. We first exanimated the expression levels of cyclin D2 and CDK4 ( Figure 5B).
The cyclin D2-CDK4 complexes release E2F from Rb and E2F can drive G1/S phase transition; however, the immunoblotting analysis showed that OC3-I5 cells expressed lower level of cyclin D2 and CDK4, which did not match the cell-cycle assay. Subsequently, the expression levels of p53 and p27 were validated. Cell-cycle inhibitor p53 and p27 prevents the formation of cyclin D-CDK4 complexes; thus, p53 and p27function as inhibitors of cell-cycle progression at G1to S phase. As shown in Figure 5B Then, we performed cell-cycle analysis using propidium iodide (PI) staining to analyse how PGRMC1 modulates cell proliferation.
The results demonstrated that OC3 cells got more cells stuck in G1 phase but fewer cells in G2/M phase than OC3-I5 cells, and OC5-I5 cells with PGRMC1 knockdown reverted back to OC3 cell-cycle status ( Figure 5C). OC3 and OC3-I5 cells stably transfected with shPGRMC1 or shLacZ were mixed with Matrigel™ in PBS (1:7) and inoculated subcutaneously onto the right tight of mice (6 per group). After 60 days after implantation, the primary tumour sizes were measured ( Figure 7A).

| PGRMC1 knockdown influences lung metastases
The primary tumour growth rates were shown no difference between OC3 shLacZ , OC3-I5 shLacZ and OC3-I5 shPGRMC1 groups, which indicated that OC3 and OC3-I5 cells had similar proliferation ability that were not influenced by PGRMC1 knockdown.
The mice were killed by zoletil/rompun 60 days after implantation, and the lungs were removed and fixed in 10% formalin. The weights of lungs were measured. Lungs of OC3-I5 shLacZ group were heavier than healthy control, OC3 shLacZ and OC3-I5 shPGRMC1 groups ( Figure 7B). These data suggested that OC3-I5 shLacZ might had more severe lung metastasis than OC3 shLacZ group and the knockdown of    In this study, we performed lysine-labelling 2D-DIGE and MALDI-TOF MS analysis for the identification of invasion-related proteins in oral cancer. These altered proteins promoted cell migration, proliferation and anti-apoptosis inside the cell. We classified and arranged the proteins identified in this study and proposed invasion mechanisms that may occur in OC3-I5 cells. The identified up-regulated proteins might serve as indicators for predicting the invasion ability of oral cancer, while the identified altered cytosolic proteins might serve as therapeutic targets for oral cancer patients after surgery to decrease incidence of cancer recurrence.

ACK N OWLED G EM ENTS
This work was supported by the grants from Ditmanson Medical Foundation Chia-Yi Christian Hospital (grant no. R108-026).
Additionally, the authors would like to thank Amber Kao for proofreading and editing of the manuscript.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.