Tumor Biol. (2016) 37:2655–2663 DOI 10.1007/s13277-015-4095-6
ORIGINAL ARTICLE
Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells K. D. M. Nakamura 1 & T. M. Tilli 2 & J. L. Wanderley 3 & A. Palumbo Jr. 1 & R. M. Mattos 1 & A. C. Ferreira 4 & C. E. Klumb 4 & L. E. Nasciutti 1 & E. R. Gimba 2,5
Received: 11 June 2015 / Accepted: 13 September 2015 / Published online: 24 September 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc
isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment. Keywords Osteopontin . Splicing isoforms . Prostate cancer . Docetaxel . Cell survival
Introduction * E. R. Gimba
[email protected];
[email protected] 1
Programa de Pós Graduação em Ciências Morfológicas, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
2
Programa de Pós Graduação Stricto Sensu em Oncologia, Coordenação Geral Técnico Científica do Instituto Nacional de Câncer (INCa), Rio de Janeiro, RJ, Brazil
3
Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Macaé, RJ, Brazil
4
Laboratório de Hemato-Oncologia Celular e Molecular. Programa de Pesquisa em Hemato-Oncologia Molecular—CGTC, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil
5
Departamento de Ciências da Natureza (RCN), Instituto de Humanidades e Sáude IHS, Universidade Federal Fluminense, Rua Recife s/n-Bairro Bela Vista, Rio das Ostras, Rio de Janeiro, RJ 28895-532, Brazil
Osteopontin (OPN) is a matricellular phosphoprotein involved in several pathophysiological processes, including cell attachment, migration, invasion, proliferation, tissue remodeling, bone formation, inflammation and cancer [1–3]. OPN is frequently overexpressed in human cancers and contributes to tumor progression [4]. The OPN primary transcript is subject to alternative splicing, generating three isoforms, OPNa, OPNb and OPNc. OPNa corresponds to the full-length isoform [5], while OPNb lacks exon 5 and OPNc lacks exon 4 [6, 7]. OPN splicing isoforms (OPN-SI) are aberrantly expressed specially in tumor cells and have tissue- and tumor-specific roles [7–10]. However, the molecular mechanisms mediating these specificities are currently unknown. Our previous data demonstrated that PI3K signaling mainly mediates the roles of OPNb or OPNc in prostate cancer (PCa) tumor progression features. We also provided some evidence that this pathway mediates OPN-SI pro-survival
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roles in PCa cells. Cells overexpressing OPNb or OPNc proliferate more rapidly than OPNa and empty vector control clones, even under serum starving conditions. However, no difference on cell death rates was observed among PC3 PCa cells overexpressing the three OPN-SI. Besides, PC3 cells overexpressing OPNc, when treated with an anti-OPNc neutralizing antibody, decreased their proliferative potential, and were induced to die [11]. Many molecular mechanisms mediate tumor cell survival activation in order to contribute to tumor progression, including the dysregulation of the epithelial-mesenchymal transition (EMT) program, which contributes to prostate cancer progression [12]. Several EMT markers have been described as involved in PCa progression, as well as promoting resistance to chemotherapeutic drugs, including vimentin [13], Snail [14], N-cadherin [15], and Ecadherin [16] and other molecules, such as clusterin [17], BCL-2 [18], STAT1 [19], interleukin 6 [20], and multidrug resistance (MDR) proteins [16]. The molecular interactions among signaling pathways and mechanisms mediating PCa cancer cell survival, control of proliferation, and resistance to cell death have been the subject of several studies in cancer biology [21]. The gene products involved in these pathways have been broadly investigated as potential cancer therapeutic targets [22]. Several reports have also associated total OPN with drug resistance [23–27]. However, none of these reports have described the specific involvement of each OPN-SI. Chemotherapy with docetaxel (DXT) represents the standard first-line treatment in patients with castration-resistant prostate cancer (CRPC), improving survival by a few months, remaining the standard of care in CRPC patients. However, existing or developing resistance to DXT in patients is the main limitation to the efficacy of this treatment approach [28, 29]. Docetaxel resistance has been associated to several mechanisms, including genetic modifications and alterations in drug transporters [30]. The current study investigated the putative cellular mechanisms that could differentially mediate pro-survival roles in PC3 cells overexpressing OPN splicing isoforms, using an in vitro model of DXT-induced cell death. We demonstrated that PC3 cells overexpressing OPNb or OPNc splice variants are more resistant to DXT-induced cell death. Our data also provided evidence that EMT program could mediate OPNb or OPNc pro-survival roles in DXT-treated cells.
Materials and methods Reagents DXT was obtained from LIBBS Farmacêutica Ltda (São Paulo, SP), which was diluted in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS).
Tumor Biol. (2016) 37:2655–2663
Cell culture We used the PC-3 human prostate cancer cell line (supplied from ATCC) overexpressing each OPN-SI or empty vector control cells, which have been used in a previous publication from our group [11]. Clones stably overexpressing each OPNSI have been designated OPNa1, OPNb4 and OPNc4, and one empty vector control clone (empty vector). OPN isoform overexpression have been validated by real-time PCR and immunoblot in our previous publication, which demonstrated that OPNb and OPNc isoforms activated several aspects of PCa progression [11]. Clones were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 800 μg/ mL of geneticin in a humidified environment with 5 % CO2 at 37 ° C. DXT treatment PC-3 cells overexpressing each OPN-SI or empty vector control were cultured as described above and then treated with DXT (1.2×10−6 μM). For proliferation assay, 4 h after cell plating, culture medium was removed and replaced by culture medium containing DXT dissolved in DMSO. Cell viability and morphology analysis PC-3 cells overexpressing each OPN-SI or empty vector control cells were seeded in 24-well plates (5×104 cells per well) with or without DXT (1.2×10−6 μM) treatment for 72 h. Cell morphology was analyzed by phase contrast microscopy and cell viability by crystal-violet incorporation assay. For these assays, cells were washed twice with PBS and fixed in glutaraldehyde for 10 min, followed by staining with 0.1 % crystal violet and solubilization with 0.2 % Triton X-100. Microtiter plates were read in a spectrophotometer at 550 nm. Quantitative real-time PCR RNA was extracted using the RNeasy kit (QIAGEN), and cDNA synthesis has been performed using SuperScript II First-Strand Synthesis System for RT-PCR, using oligo (dT) primer (Invitrogen) and 1 μg of total RNA. Conditions for amplification of transcripts coding for EMT marker by quantitative real-time PCR (qRT-PCR) were 50 °C for 2 min, 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s and a final melting curve analysis in order to check specific amplification. Oligonucleotides used for qRT-pCR assays are shown in Table 1, including primers sequences for GAPDH, which was used as the constitutive gene. Actin
Tumor Biol. (2016) 37:2655–2663 Table 1
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Oligonucleotide primers used for analysis of qRT-PCR expression of epithelial-mesenchymal transition (EMT) markers
Gene
Forward primers
Reverse primers
E-cadherin
5′-G GAA TGA CAA CAA GCC CGA AAT-3′
5′-GAC CTC CAT CAC AGA GGT TCC-3′
Claudin-4
5′-GGC TGC TTT GCT GCA ACT GTC–3′
5′-GAG CCG TGG CAC CTT ACA CG-3′
Claudin-1 Claudin-3
5′-TTC GTA CCT GGC ATT GAC TGG–3′ 5′-CTG CTC TGC TGC TCG TGT CC-3′
5′-TTC GTA CCT GGC ATT GAC TGG-3′ 5′-TTA GAC GTA GTC CTT GCG GTC GTA G-3′
Cytokeratin 8 Cytokeratin 18
5′-GCTGACCGACGAGATCAACT-3 5′-CGAGAAGGAGACCATGCA-3′
5′-CATGGACAGCACCACAGATG-3′ 5′-GGTGGTCCCGGATTTTGATCT-3′
Snail
5′-TTC CAG CAG CCC TAC GAC CAG-3′
5′-CTT TCC CAC TGT CCT CAT C-3′
Vimentin Twist
5′-GAC AAT GCG TCT CTG GCA CGT CTT-3′ 5′-CCC AAC TCC CAG ACA CCTC-3′
5′-TCC TCC GCC TCC TGC AGG TTC TT–3′ 5′-CAA AAA GAA AGC GCC CAA C–3′
N-cadherin Slug
5′-GGT GGA GGA GAA GAA GAC CAG–3′ 5′-TGG TTG CTT CAA GGA CAC AT–3′ 5′-TGA CCC CTT CAT TGA CCT CA-3′
5′-GCA TCA GGC TCC ACA GT–3′ 5′-GTT GCA GTG AGG GCA AGA A–3′ 5′-AGT CCT TCC ACG ATA CCA AA-3′
GAPDH
served as an internal control to normalize the expression data. All qRT-PCR reactions were conducted using the SYBR Green (Applied Biosystems). Relative gene expression of the target gene was calculated by using the ΔΔCT method.
Immunocytochemical staining For immunocytochemistry, cells were grown for 72 h and then fixed with cold ethanol for 20 min at room temperature, followed by washing with PBS. After permeabilization with 5 % BSA in PBS-T, samples were incubated with primary antibodies anti-cytokeratin (Dako) or anti-vimentin (Sigma) overnight at 4 °C. Detection was performed using goat anti-mouse Alexa 488 secondary antibody (Invitrogen), and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology). Finally, cells were washed in distilled water and mounted on histological slides with N-propyl gallate (Sigma). In all immunostaining-negative controls, reactions were performed by omitting the primary antibody. Images were captured using confocal microscopy (Olympus IX81) and a Hamamatsu ORCA-R2 digital camera.
Data and statistical analysis Results are presented as the mean±standard error of at least three independent experiments. Differences among groups were evaluated by Student’s t test, using GraphPad Prism software (San Diego, CA, USA). A value of p
