Application of RNA-Seq transcriptome analysis: CD151 is an Invasion/Migration target in all stages of epithelial ovarian cancer
© Mosig et al; licensee BioMed Central Ltd. 2012
Received: 21 November 2011
Accepted: 24 January 2012
Published: 24 January 2012
RNA-Seq allows a theoretically unbiased analysis of both genome-wide transcription levels and mutation status of a tumor. Using this technique we sought to identify novel candidate therapeutic targets expressed in epithelial ovarian cancer (EOC).
Specifically, we sought candidate invasion/migration targets based on expression levels across all tumors, novelty of expression in EOC, and known function. RNA-Seq analysis revealed the high expression of CD151, a transmembrane protein, across all stages of EOC. Expression was confirmed at both the mRNA and protein levels using RT-PCR and immunohistochemical staining, respectively.
In both EOC tumors and normal ovarian surface epithelial cells we demonstrated CD151 to be localized to the membrane and cell-cell junctions in patient-derived and established EOC cell lines. We next evaluated its role in EOC dissemination using two ovarian cancer-derived cell lines with differential levels of CD151 expression. Targeted antibody-mediated and siRNA inhibition or loss of CD151 in SKOV3 and OVCAR5 cell lines effectively inhibited their migration and invasion.
Taken together, these findings provide the first proof-of-principle demonstration for a next generation sequencing approach to identifying candidate therapeutic targets and reveal CD151 to play a role in EOC dissemination.
KeywordsCD151 Epithelial Ovarian Cancer Invasion Migration Metastasis RNA-Seq
Epithelial ovarian cancer (EOC) is the most common cause of gynecologic cancer death and the fifth most lethal cancer among women . Despite a relatively low occurrence rate (1 in 72) compared to other female cancers, the low 5-year survival rate of ~40% translates to greater than 14,000 yearly deaths from ovarian cancer in the United States . One main contributor to the low survival rate is the late stage at which EOC is usually detected: upwards of 80% of EOC is discovered after localized spread. When detected early, the EOC 5-year survival rate is ~90% .
Beyond earlier diagnosis and detection, the identification of novel therapeutic targets or approaches to overcome chemoresistance is necessary to treat late stage or recurrent disease that will occur even with more sensitive and specific screening and detection methods. Since the introduction of platinum-based drugs as first line chemotherapy in the early 1980's followed by the addition of taxane containing agents in the mid-1990's, there has been little change to the first line treatment of EOC . Novel administration methods, such as intraperitoneal therapy, and dosing, such as dose-dense taxol, have yielded slight improvements in progression-free survival and overall survival . Molecularly targeted therapies to treat recurrent and/or chemoresistant disease show some promise but large conclusive trials have not been completed . Therefore, the need for new targets and drugs remains high .
Next Generation Sequencing technology is now allowing for the thorough and unbiased profiling of a number of cancer genomes and transcriptomes [4–7]. Analysis of mutational profiles, copy number variations, and expression profiles have yielded insights into commonly affected genes and pathways important for carcinogenesis in a number of cancers including melanoma, pancreatic, lung, and breast cancers [4–7]. Theoretically, by applying RNA-Seq technology to ovarian cancers relevant pathways and molecules for therapeutic intervention should be identifiable.
CD151 is an integral membrane protein and member of the tetraspanin family. It associates with integrins and other transmembrane proteins tetraspanin-enriched microdomains, thereby playing a role in cell-matrix or cell- cell attachment [8, 9]. Migration signaling pathways including PI3K, FAK, and Rho/Src mediate cell behavior in response to CD151 interactions [10–18]. Functionally, a CD151-targeting antibody was shown to inhibit cell migration in an in vivo breast cancer xenograft model and contain breast cancer cells within a single contiguous tumor .
Using a highly clinically annotated sample set of EOC, representing both early and late stage tumors, we interrogated global expression patterns by RNA-Seq. We identified the transmembrane protein CD151 as being overexpressed in all tumor samples and then demonstrated it to be a potential target to inhibit metastasis and dissemination of ovarian cancer.
Patients and Specimen Collection
EOC tumor samples and ascites cells were collected from MSSM and San Gerardo Hospital patients at the time of surgery under their respective IRB-approved protocols, as previously described . Samples were divided in the operating room and a portion sent for pathology confirmation and staging. A portion was flash frozen for subsequent RNA and protein analysis, and another portion used for generating patient-derived cell lines. For the RNA-Seq "discovery set", 16 papillary serous tumor samples representative of all stages of the disease (3 stage I/II, 8 stage III, 1 stage IV, 2 peritoneal metastatic lesions, and 2 recurrent tumors) and two borderline serous tumors were collected and analyzed. An additional set of 25 tumors (6 stage I/II, 7 stage III/IV, 8 peritoneal metastatic lesions, and 4 recurrent tumors) were used as a "validation set".
RNA was extracted from frozen tissue using QIAzol according to manufacturer's instructions (Qiagen, Valencia, California). Briefly, tissue was homogenized in QIAzol on ice. Chloroform was added, mixed and centrifuged to allow for separation and removal of the aqueous layer. RNA was precipitated in isopropanol overnight at -20°C. The suspension was centrifuged to pellet the RNA, washed with 75% ethanol and then resuspended in RNAase-free water. RNA integrity numbers (RINs) were analyzed using the Agilent Bioanalyzer and only RNA with a RIN of > 8.0 was submitted for next-generation sequencing.
Epithelial ovarian cancer transcriptomes were prepared for paired-end sequencing on the Illumina GAII platform using the manufacturer's protocols and with a second size selection step to reduce ligation artifacts. Reads were aligned using Eland32 (provided with the Illumina sequencing platform). Expression levels were quantified by running ERANGE v. 3.0.2. . For each gene, ERANGE reported the number of mapped reads per kilobase of exon per million mapped reads (RPKM).
Quantitative Real-time Reverse Transcription PCR
RNA-Seq data was confirmed by quantitative real-time PCR. One microgram of RNA was reverse transcribed to cDNA using the BioRad Iscript system (Biorad, Hercules, California). Quantitative real-time PCR was performed on an ABI PRISM 7900 HT sequence detection system (Applied Biosystems, Carlsbad, California). Cycle number values were normalized against two housekeeping genes, B2 M and GAPDH. Data shown is the average of three separate experiments, each performed in triplicate. The CD151 primers used were CD151 Fwd: 5'- AGACAGCTGCTGCAAGAC-3' and CD151 Rev: 5'-TGGATGAAGGTCTCCAACT-3'.
Immunohistochemistry and Fluorescent Immunocytochemistry
Four-micrometer thick tumor sections were stained with α-CD151 antibody (Cat # NCL-CD151, Leica, Wetzlar, Germany) and the R&D mouse cell and tissue DAB staining kit and counterstained with Hematoxylin. A murine IgG1 isotype control antibody (Clone 11711, R&D systems MAB002) was used as a negative experimental control.
CD151 expression levels and subcellular localization were examined in patient-derived and commercially available tumor cell lines using the α-CD151 antibody (Cat #NCL-CD151, Leica, Wetzlar, Germany). Alexa fluor- 548 goat anti-mouse secondary (Cat #A21137, Invitrogen, Carlsbad, California) was used and nuclear counterstaining was performed with Vectashield mounting medium for fluorescence with Dapi (Cat #H1200, Vector Laboratories, Burlingame, California).
Generation of Low Passage Number Ascites Cell Lines and Cell Culture
Ascites fluid was centrifuged for 10 minutes at low speed at 4°C to pellet the cellular fraction. Cells were resuspended in DMEM containing 10% FBS and Penicillin-Streptomycin and allowed to adhere. Media was changed daily until cells reached confluence at which time they were passaged. RNA and immunostaining procedures were performed using only 2nd or 3rd passage cells.
SMARTPool siRNA targeting human CD151 (Dharmacon, Lafayette, Colorado) was transfected into SKOV3 or OVCAR5 cells using Lipofectamine as described previously . Knockdown was confirmed at the RNA level using qRT-PCR and at the protein level using IHC as described above.
Migration and Invasion Assays
Migration and invasion experiments were performed according to the manufacturer's recommended protocol (BD Biosciences, Franklin Lakes, New Jersey). Briefly, SKOV3 or OVCAR5 cells were resuspended in serum free media with or without mouse α-CD151 or control mouse α-V5 antibody (Invitrogen, Carlsbad, California) in the upper chamber of modified Boyden chamber transwells. Bottom chambers were filled with media containing 10% FBS as a chemoattractant. Cells were allowed to migrate or invade through matrigel for 24 hours followed by calcein dye staining and visualization using a fluorescent detector. Results shown are the averages of 3 separate experiments performed in triplicate. Statistical significance was measured using the Student's T test with p < 0.05 considered significant.
CD151 is expressed across all stages of EOC
RNA-Seq tumor coverage values
qRT-PCR tumor expression values
Overall, CD151 was expressed across all stages and no differences were noted with either increasing stage or a particular subtype (Table 1). One trend of note, given the relatively small sample set in this study, was that primary tumors, regardless of stage, on average expressed higher levels of CD151 than either recurrent tumors or metastatic lesions presenting at time of primary debulking surgery (27.4 v 18.4, respectively, p = 0.11). Borderline tumors possessed the lowest average coverage values (14.0).
CD151 is expressed in EOC tumors and normal ovary surface epithelial cells
CD151 is expressed in ascites-derived and EOC cell lines and immortalized ovarian surface epithelial cells (IOSE) and is localized to cell-cell junctions
Migration and invasion of ovarian cancer cell lines is blocked by inhibition of CD151
A second method of CD151 inhibition, antibody-mediated, also significantly inhibited the ability of SKOV3 cells to invade and migrate (Figure 4d-e). In the CD151 higher expressing OVCAR5 cells, antibody-mediated blockade, at the concentrations used, had only an ~ 20% decrease in invasion but no measurable effect on migration (Figure 4d-e).
These studies are the first to demonstrate that the tetraspanin CD151 is expressed in epithelial ovarian cancers and that its expression is independent of stage or histological subtype. We analyzed EOC tumor expression levels of all RNA transcripts in an unbiased manner through the use of RNA-Seq with the goal of identifying possible novel therapeutic targets. From this analysis of 18 whole transcriptomes we identified high CD151 expression across all stages and subtypes of EOC, including borderline ovarian tumors. We then confirmed expression of CD151 in this discovery set and a second set of tumor tissues using qRT-PCR and immunohistochemistry. The previously described role of CD151 as a possible anti-metastatic target led us to further examine the role of CD151 in ovarian cancer and its migration and invasion in culture.
Immunohistochemical staining of EOC tumors and normal ovary showed CD151 expression in tumor cells as well as normal OSE cells. Immunocytochemistry of cells from patient ascites fluid showed CD151 to be localized to the cell periphery and highly concentrated at cell-cell junction points and along outstretched cellular elongations. Finally, functional studies showed that SKOV3 and OVCAR5 cell invasion and migration are differentially inhibited by α-CD151 antibody or siRNA-mediated knockdown of CD151. It appears from these experiments that the level of CD151 expression may affect our ability to inhibit invasion and migration and therefore CD151 blockade may need to be titrated to achieve the most robust inhibition.
CD151 has previously been postulated to play important roles in a number of cancers including colorectal and breast cancers but no studies had previously identified or examined its expression and function in ovarian cancer. In colorectal cancer CD151 is differentially expressed between normal (high expression), primary (low expression), and metastatic (high expression) tissue . It is proposed that these changes are part of a hypoxic response that drives cell detachment and migration . In our own discovery sample set of EOC tumors, while not reaching statistical significance, we did find an interesting trend of higher CD151 expression in primary ovarian tumors and relatively lower expression in disseminated metastatic or recurrent disease (Tables 1 and 2). It is possible that this is due to different environmental effects in secondary sites, such as hypoxia or surrounding cell types. This hypothesis will need to be expanded upon by examination of a larger collection of samples.
The expression of tetraspanin CD151 on the cell surface of EOC cells may play a role in the spread of these cells to other organs in the peritoneal cavity. In xenograft tumor models, antibody-mediated inhibition of CD151 has been shown to hinder the spread of metastatic tumors, reinforcing the idea that CD151 is functionally important for either cell detachment from a tumor or migration away from that tumor [19, 32]. Intriguingly, in breast cancer models comparing CD151-expressing cells against CD151-ablated cells, tumor growth was delayed in the absense of CD151 . In immunohistochemical studies of breast cancer, staining varied greatly in both intensity and cellular localization, which may reflect the heterogeneity of the tumor cells' environment or activity . CD151 staining intensity and localization in normal OSE and ovarian tumor cells also varies. CD151 protein was localized to both the membrane and the cytoplasm of cancer cells (Figure 1). In contrast, in normal ovarian surface CD151 localized mostly to the cell membrane. It is possible that the internalization of CD151 into cytoplasmic endosomes may reduce its ability to cooperate with other binding partners and cells, allowing detachment from the primary tumor . On ascites-derived patient cell lines, IOSE, and EOC cell lines, CD151 not only localized to the membrane but more specifically to the cell-cell junctions and along cell membrane extensions (Figure 3). Although in vitro cellular localization suggests no difference between cancerous cell lines and OSE cells, this may due to distinct conditions in cell culture such as attachment to plastic and tightly controlled O2/CO2 balance.
Expression of CD151 in EOC tumors, the known involvement of CD151 in cell migration and invasion, and the in vivo ability of αCD151 antibody to contain breast cancer tumors to single nodules and eliminate tumor spread suggested that CD151 may also represent a promising and relevant candidate for therapeutic targeting in ovarian cancer. We show that in the ovarian cancer cell lines SKOV3 and OVCAR5, CD151 is a functionally important molecule whose silencing or blockade impeded cell migration or invasion to different levels dependent on level of CD151 expression. A role for CD151 in cell migration and invasion is consistent through many cancer types including breast, prostate, colorectal, and pancreatic cancers [15, 19, 32, 34–36]. Ultimately, the value of CD151 as a therapeutic target in EOC will need to be demonstrated in in vivo studies.
epithelial ovarian cancer
RNA integrity number
immortalized ovarian surface epithelial.
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