1.      Introduction 

Epithelial Ovarian Cancer (EOC) is the most deadly gynecological cancer. Specifically targeting cancer stem cells (CSC) represents a major challenge in EOC treatment. Novel and more specific and effective treatments are urgently needed. Identification of critical regulators in EOC CSC properties is pivotally important. 

Sex-determining region Y (SRY)-box 9 (SOX9) is a member of the SOX transcription factor family. It plays an important role in sex determination and bone development [1]. In recent years; deregulation of SOX9 has been implicated in various diseases, including fibrosis and cancer. SOX9 plays a tumor-promoting role and is associated with CSC in lung, pancreatic, breast, oral, liver, colon, and other cancers [2-8]. Regarding to ovary, the role of SOX9 has mainly been studied in Sertoli-Leydig cell tumors and granulose cell tumors [9-11]. Recently, SOX9 has been shown to allow the survival of EOC cells upon hypoxic condition and its aberrant activation and high expression inhuman EOC tissues is prominent in patients with aggressive EOC [12]. However, the potential involvement of SOX9 in EOC CSC is totally unknown. 

As a critical gene in the development of bones and testes, SOX9 expression is regulated by related factors [13]. However, the regulation of aberrant expression of SOX9 in cancer is much less known and the regulatory factors of SOX9 in EOC cells are essentially unknown [14]. We tested the potential regulatory effects on SOX9 expression exerted by LPA. LPAis a proven and validated oncolipid and target for EOC [15-20]. LPA regulates many known oncogenes [16-18, 21]. However, whether it can regulate SOX9 is unknown in any cells. We tested the regulatory roles of LPA in SOX9 expression and the role of SOX9 pertinent to CSC related cellular properties in mouse and human EOC. Genetic, biochemical, and cell biological approaches are utilized in the investigation. 

2.      Materials and Methods 

3.1. Reagents, Cell Lines and Culture 

Oleoyl-LPA was from Avanti Polar Lipids (Birmingham, AL). The following reagents were used: BrP-LPA (EBI, Salt Lake City, UT); Y27632 (Biovision, Milpitas, CA)); GW9662 (EMD Corp; Billerica, MA); pertussis toxin (PTX; Invitrogen,Grand Island, NY); H89 and actinomycin D (ActD; Sigma-Aldrich, St. Louis, MO).Anti-SOX9 antibody (Cat. Log # AB5535) was from EMD Millipore (Billerica, MA). The pair of PE01/PE04 cell lines were from Dr. Daniela Matei (Northwestern University); the OVCAR3 cells were obtained from ATCC (Manassas, VA).The ID8, T29, and OVCA433 cell lines were kind gifts from Dr. R. Bast (M.D Anderson), Dr. Jinsong Liu ((M.D Anderson), and Dr. Paul F Terranova (University of Kansas Medical Center), respectively. These cell lines were authenticated by ATCC. All cell lines were maintained in a humidified atmosphere at 37°C with 5% CO2. ID8 cells (mouse epithelial ovarian cancer cell line) were maintained in high glucose DMEM containing 5% FBS (ATCC, Manassas, VA) and 100 μg/mL Penicillin/Streptomycin/ Amphotericin B (PSA).OVCA433 cells and PE01/PE04 cells were cultured in RPMI 1640 with glutamine, 10% FBS (ATCC, Manassas, VA), and 100 μg/mL Penicillin/Streptomycin/Amphotericin B (PSA).OVCAR3 cells were maintained in RPMI-1640 supplemented with 20% FBS, 0.01 mg/mL insulin and 100 μg/mL PSA. PE01/PE04 cells were cultured in RPMI 1640 with glutamine, 10% FBS, and 100 μg/mL penicillin / streptomycin (P/S). For serum starvation, cells were incubated in the basal medium without FBS or antibiotics. LPA treatment was performed in cells starved from serum for 16-24 hr.

3.2. Stable Cell lines 

SOX9 CRISPR lentiVirus HCP217635-LvSG03 and Cas9 pCRISPR-LvSG03 vectors (GeneCopoeia, Rockville, MD) were co-transfected with the delta 8.9 packaging plasmid and the pCMV-VSVG plasmid into 293T cells for virus packaging, using Fugene6 (Promega, Madison, WI). Cell medium was changed to DMEM supplemented with 30% FBS following overnight incubation. After 48 hrs, cell media were harvested and filtered using 0.45 μm filter syringes. PE04 and OVCAR3 cells were transduced by packaged viruses in the presence of Polybrene (8 μg/mL) for 48 hrs, followed by selection with puromycin (0.5 μg/mL) for at least 7 days. 

3.3. Western Blot Analysis 

Western blot analyses were conducted using standard procedures and proteins were detected using primary antibodies and fluorescent secondary antibodies (IRDye 800CW-conjugated or IRDye 680-conjugated anti-species IgG, Li-Cor Biosciences, Lincoln, NE) as we described previously [22]. The fluorescent signals were captured on an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE) with both 700- and 800-nm channels. Boxes were manually placed around each band of interest, and the software returned near-infrared fluorescent values of raw intensity with background subtraction (Odyssey 3.0 analytical software, Li-Cor Biosciences, Lincoln, NE). The protein MW marker used was the Pre-stained SDS-PAGE Standards, broad range (BIO_RAD, Cat. Log # 161-0318). 

3.4. Cell Proliferation, Anoikis-Resistance, Colony- and Spheroid-Formation Assays 

Cell proliferation was analyzed based on MTT hydrolysis using Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MA). Anoikis-resistance and soft agar colony assays were described in detail previously [22]. Single cells were re-suspended at 1×10³ to 1×10⁵cells/mL in serum-free DMEM/F12 supplemented with 5 μg/mL insulin (Sigma), 20 ng/mL human recombinant epidermal growth factor (EGF; Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 0.4% bovine serum albumin (BSA; Sigma), followed by culturing in 24-or 96-well Ultra Low Attachment plates (Corning, NY).Spheroids were photographed after seven days in culture. 

3.5. Immunofluorescence Assay 

To assess the expression level of CD44 in EOC cells, immune fluorescence was performed using antibody against CD44 (Abcam, ab6124; Biotechnology company, Cambridge, MA). Cells were fixed with 4% Para formaldehyde and permeabilized using a blocking solution consisting of 5% Normal Goat Serum and 0.1% Triton X-100 in PBS. The primary antibodies against CD44 were diluted 1:200 in the same blocking solution. 

3.6. Statistical Analyses 

The Student’s t-test was utilized to assess the statistical significance of the difference between two treatments. The asterisk rating system as well as quoting the P value in this study was * P< 0.05; ** P< 0.01; and *** P< 0.001. AP value of less than 0.05 was considered significant.

3.Results

 4.1. LPA-dose and -time dependently up-regulated SOX9in mouse and human EOC cells 

We tested the potential effect of LPA on SOX9 expression and found that LPA up-regulated SOX9 in PE01 cells in a dose-and time-dependent manner, with the optimal dose and time being 5-10 µM and 6 hrs, respectively (Figures. 1A and 1B). LPA also up-regulated SOX9 in OVCAR3, another HGSOC cell line, and in OVCA433 EOC cell line, but not in a human ovarian surface epithelial cell (HOSE) line T29 (Figure. 1C).

. SOX9 expressed at higher levels in more aggressive EOC cells and LPA-induced SOX9 expression was PPARγ-dependent 

We have developed a highly aggressive EOC cell line ID8-P1 through in vivo passage of ID8-P0 cells in C57BL6 mice [22]. The tumor/ascites formation time is reduced from ~90 days for ID8-P0 cells to ~30 days in different P1 cell lines isolated from tumors in different organs or from ascites [22]. We found that SOX9 was expressed at higher levels in the more aggressive ID8-P1 cells than in ID8-P0 cells. In addition, LPA induced further increases in SOX9 expression in these cells (Figure. 2A). Similarly, in the paired human HGSOC

prior to LPA treatment (10 μM, 6 hrs). Reproducible results from independent experiments were shown.

PE01/PE04 cell lines, SOX9 was expressed at much higher levels in the drug-resistant PE04 cells than in PE01 cells [23]. (Figure. 2B) 

The majority of known cellular effects of LPA are mediated by membrane G protein-coupled receptors (GPCRs; LPAR_1-6) [24, 25,21, 26]. To determine which LPA receptors are involved in LPA-SOX9 up-regulation, we used BrP-LPA, a pan-LPA receptor [27] Surprisingly, this inhibitor did not significantly block the effect (Figure. 2C). We then employed several selective inhibitors mediated by LPA GPCRs in EOC cells as we and others shown previously [28-32], including pertussis toxin (PTX), a G_i inhibitor; Y27632, a G_12/23/Rho-Rock kinase pathway inhibitor; and H89, a G_s-protein kinase A inhibitor.Consistent with the receptor inhibitor BrP-LPA, these inhibitors had insignificant or only weak effects on LPA-induced SOX9 expression (Figurs. 2B, 2C). On the other hand, the PPARγ selective inhibitor GW9662 completely blocked the effect; strongly suggest that LPA-induced SOX9 was mediated by PPARγ, but not its GPCR receptors. LPA-induced SOX9 expression was sensitive to Actinomycin D (ActD), a transcription inhibitor, suggesting that transcription is involved (Figure. 2D). 

4.3. SOX9was Functionally Involved In CSC Related Activities in EOC Cells 

To investigate the role of SOX9 in EOC, we generated SOX9-knockout (KO) clones using the CRISPR/Cas9 system in PE04 and OVCAR3 cells (Figure. 3). We found that SOX9-KO did not affect cell proliferation when cells were cultured in 2D dishes, but significantly reduced anoikis-resistance when cells were cultured in suspension in both PE04 and OVCAR3 cells (Figure. 4). This is very similar to what we have observed in ID8-P1 and -P0 cell [22]. Even though time to tumor/ascites formation is reduced from 90 days to 22-45 days in ID8-P1 vs. -P0 cells, the P1 cells do not gain a proliferation advantage when cultured in 2D dishes, but have greatly enhanced anoikis-resistanc.²² This anchorage-independent growth is related to transformation and CSC properties. 

formation being consistent markers for EOC CSC [33]. Spheroids are present in the malignant ascites of essentially all EOC patients and represent a significant impediment to efficacious treatment due to their roles in progression, metastasis, and drug-resistance [34,35]. Spheroids, in general, have high SP, drug-resistance, and CSC activity [36-38]. LPA has been shown recently to be a potent spheroid inducer in EOC cells [39]. We tested whether SOX9 KO affect spheroid formation in EOC cells. As shown in Figure. 5, the spheroid- formation was dependent on the cell density used and under the same conditions, KO of SOX9 essentially diminished spheroid-formation in HGSOC cells. 

CD44 is one of the CSC markers identified in EOC. CD44 expression in OVCAR3 cells was examined by immune staining. SOX9 KO essentially blocked CD44 expression in these cells (Figure. 6). Taken together, the data showed here support that SOX9 is regulated by the EOC oncolipid LPA and plays an important role in CSC-related activities in EOC cells.

Immunostaining of CD44 in OVCAR3 and OVCAR3-SOX9-KO cells with or without LPA (10 µM, 24 hrs) treatment.

4.Discussion 

Compelling evidence has been accumulated in recent years to support the concept that stem cell populations within each individual tumor are key contributors of therapy failure. Thus, it is becoming increasingly important to develop effective CSC targeting strategies. One of the major obstacles in development of therapeutic strategies targeting CSCs is the inherited high diversity and plasticity of CSC cells [40]. Hence, a much better understanding of these features and identification of multiple targets for co-targeting are critical in making progression in this field.             

The presented data in this work support this notion. While LPA is a confirmed oncolipid and target in EOC [15- 20], and at least three compounds blocking LPA GPCR receptors have passed phase I and phase II clinical trials for different diseases [25], our study suggests that certain important LPA tumor promoting actions are mediated by PPAR

Figure 1: LPA induced SOX9 up-regulation in human HGSOCand T29 cells

Figure 2: Endogenous and LPA-induced SOX9 expression in EOC cells and PPARγ-dependent LPA induction.

Figure 3: SOX9-KO clones were generated in PE04 and OVCAR3 cells.

Figure 4: SOX 9 did not affect cell proliferation in EOC cells.

Figure 5: SOX9-KO blocked spheroid-formation in EOC cells.

Figure 6: SOX9-KO inhibited CD44 expression spheroid-formation in EOC cells.

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