ANTICANCER RESEARCH 37 : 1805-1818 (2017) doi:10.21873/anticanres.11515 PI3K Catalytic Isoform Alteration Promotes the LIMK1-related Metastasis Through the PAK1 or ROCK1/2 Activation in Cigarette Smoke-exposed Ovarian Cancer Cells GA BIN PARK 1 and DAEJIN KIM 2 1Department of Biochemistry, Kosin University College of Medicine, Busan, Republic of Korea; 2Department of Anatomy, Inje University College of Medicine, Busan, Republic of Korea Abstract. Aim: To investigate the molecular mechanisms Several studies have shown a strong correlation between by which long-term exposure to cigarette smoke extract cigarette smoke (CS) and cancer metastasis through the (CSE) contributes to ovarian cancer metastasis. Materials induction of numerous factors involved in migration activity and Methods: Western blot analysis for diverse p110 (1-3). The exposure to CS induces the epithelial- isoforms of phosphoinositide 3-kinase (PI3K)-related mesenchymal transition (EMT) process and up-regulates the signaling pathway and epithelial-mesenchymal transition expression of EMT markers, including N-cadherin and (EMT) markers was performed to analyze the underlying vimentin (4, 5). Cigarette smoke extract (CSE) treatment mechanisms. Migratory activity of CSE-exposed ovarian significantly induces interleukin-8 (IL-8) and transforming cancer cells was determined by transendothelial migration growth factor-beta 1 (TGF- β1 ) production and profoundly and invasion assay. Results: After exposure to CSE for four suppresses the proliferation and growth of erythroid and weeks, CaOV3 (primary) and SKOV3 (metastatic) ovarian granulocyte-macrophage progenitors (6). Stimulation with cancer cells showed enhanced mesenchymal characteristics CSE in human lung fibroblast cells induces the expression and produced EMT-related cytokines [intwerleukin-8 (IL-8), of phosphorylated Smad3, a main downstream target of the vascular endothelial growth factor (VEGF) and TGF- β1 receptor, which results in the secretion of vascular transforming growth factor-beta 1 (TGF- β1 )]. CSE exposure endothelial growth factor (VEGF) (7). CSE increases the activated the Src-p110 δ- p21 protein-activated kinase 1 migratory and invasive activity of ovarian cancer cells and (PAK1) in CaOV3 cells and the Lyn-p110 β- Rho-associated induces the expression of the EMT-related transcriptional kinases 1/2 (ROCK1/2) in SKOV3 cells, which led to the factors Snail and Slug (8). Nonetheless, the mechanisms stimulation of LIM kinase 1 (LIMK1) phosphorylation and underlying the induction of EMT by CSEs in ovarian cancer TGF- β1 release. LIMK1 knockdown efficiently blocked the remain unclear. migratory activity and suppressed the mesenchymal TGF- β, a well-established EMT mediator, plays a critical phenotypes of CSE-treated ovarian cancer cells. Reactive role in tumor progression (9, 10). TGF- β1 activates the oxygen species (ROS) initiated the CSE-mediated EMT phosphorylation of Akt, which, in turn, induces the processes in ovarian cancer cells. Conclusion: delocalization of E-cadherin and the acquisition of a spindle Characterization of the p110 isotypes of PI3K is critical for cell morphology (11). The activation of the phosphoinositide regulating cancer metastasis; LIMK1 could be a common 3-kinase (PI3K)/Akt pathway increases Twist1 expression and therapeutic target of ovarian cancer metastasis. sequentially up-regulates TGF- β2 secretion and TGF- β- mediated signaling. In addition, enhanced TGF- β receptor signaling maintains hyperactive PI3K/Akt signaling (12). The precise mechanism by which the PI3K/Akt signaling pathway reinforces TGF- β1 production remains controversial. Correspondence to: Daejin Kim, MD, Ph.D., Department of PI3K and Akt have a wide influence on several cellular Anatomy, Inje University College of Medicine, 75, Bokji-ro, processes, such as cell growth, survival, migration and Busanjin-gu, Busan 47392, Republic of Korea. Tel: +82 518906637, metabolic control (13). Class I PI3Ks are the best- Fax: +82 518966634, e-mail: [email protected] characterized enzymes, including class IA (p110 α, p110 β and Key Words: Phosphoinositide 3-kinase, LIM kinase 1, ovarian p110 δ) and class IB (p110 γ), and the aberrant activation of cancer, p21 protein-activated kinase 1, rho-associated kinases 1/2, this pathway has been widely reported in many human epithelial-mesenchymal transition, cigarette smoke extract. cancers, including ovarian cancer (14). Overexpression and 1805 ANTICANCER RESEARCH 37 : 1805-1818 (2017) mutations in the PIK3CA gene encoding the p110 α catalytic Each cigarette contained 0.726 mg of nicotine, 9.4 mg of tar and subunit of PI3K, which is frequently activated, have been 10.9 mg of total particulate matter. CSE was an extract of frequently detected in various cancers, including breast, mainstream cigarette smoke. Briefly, the smoke was bubbled through 25 ml of DMEM and RPMI-1640 medium without FBS for ovarian, colorectal and liver (15-17). In addition, the 2 min and this solution was used as the stock (100%) for further expression of PI3K p110 β isoforms is related to dilutions. After adjusting the pH to 7.2, the obtained CSE was chemoresistance in ovarian cancer cell lines (18). However, filtered through a 0.22 μm pore filter (Millipore, Bedford, MA, the role of the specific isoforms of PI3K is currently unknown USA) for sterilization. CSE was standardized by measuring the in CSE-mediated EMT processes and TGF- β1 production in absorbance at a wavelength of 320 nm and was used within 30 min ovarian cancer cells. Based on these results, targeting the after the preparation (27). particular PI3K isoforms in ovarian cancer may provide experimental information for effective therapeutic treatments. Western blotting. The cells were washed in PBS and lysed in NP- 40 buffer (Elpis Biotech, Daejeon, Korea) supplemented with a The aberrant expression of Rho and downstream targets, protease inhibitor cocktail (Sigma-Aldrich). Protein phosphorylation Rho-associated protein kinase 1 (ROCK1) and ROCK2, is states were preserved through the addition of phosphatase inhibitors commonly observed in human cancers and often associated with (Cocktail II, Sigma-Aldrich) to the NP-40 buffer. The protein more invasive and metastatic phenotypes (19). Rho is one of concentrations were determined using a bicinchoninic acid (BCA) the downstream targets of PI3K and treatment with a PI3K assay kit (Pierce, Rockford, IL, USA). The proteins (10 μg/sample) inhibitor or ROCK inhibitor inhibits the transendothelial were resolved through sodium dodecyl sulfate-polyacrylamide gel migration of mesenchymal stem cells (20, 21). Treatment with electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). The membranes inhibitors of RhoA, ROCK1/2 and PI3K attenuates TGF- β1 were blocked with 5% skim milk prior to Western blot analysis. expression (22). LIM kinase 1 (LIMK1), which is activated by Chemiluminescence was detected using an enhanced p21-activated kinase (PAK) and ROCK, also contributes to chemiluminescence (ECL) kit (Advansta Corp., Menlo Park, CA, cancer cell migration and invasion by regulating actin dynamics USA) and the multiple gel documentation (Gel DOC) system (23-25). TGF- β1 activates cytoskeletal actin rearrangement (Fujifilm). The following primary antibodies were used: E-cadherin, through Rho/ROCK-mediated LIMK1 activation causing a N-cadherin, vimentin, ZO-1, snail, β- actin, p110 α, p110 β, p110 γ, 144 474 fibrotic response in retinal pigment epithelial cells (26). p110 δ, phospho-PAK1 (Ser ), PAK1, phospho-PAK4 (Ser ), PAK4, RhoA, Fyn, phospho-Lyn (Tyr 507 ), Lyn, phospho-Src Meanwhile, the regulatory effect of LIMK1 on TGF- β1 (Tyr 416 ) and Src (Cell Signaling Technology, Beverly, MA, USA); production in ovarian cancer cells remains unclear. alpha( α)-smooth muscle actin ( α- SMA) (Bioss, Woburn, MA, In this study, we chose primary (CaOV3) and metastatic USA); ROCK1 and ROCK2 (Abcam, Cambridge, UK); and (SKOV3) epithelial ovarian cancer cell lines to characterize phospho-LIMK1 (Thr 505 ), LIMK1 and phospho-Fyn (Thr 12 ) (Santa the altered expression of the p110 isoforms and define the Cruz Biotechnology, Santa Cruz, CA, USA). target signaling molecules. We investigated the controlling mechanism of PI3K-mediated LIMK1 activation to enhance Small interfering RNA (siRNA) transfection. An experimentally TGF- β1 production and EMT processes in CSE-exposed verified human LIMK1 small interfering RNA (siRNA) duplex and ovarian cancer cells. a negative control siRNA were obtained from Bioneer Corporation (Daejeon, Korea). The cells were seeded at a concentration of 1×10 5 per well in a 6-well plate and grown overnight. The cells were then Materials and Methods transfected with 200 nM siRNA using LipofectAMINE RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the Cell lines and chemicals. The human ovarian cancer cell lines manufacturer’s instructions. The cells were used 48 h after CaOV3 and SKOV3 were purchased from the ATCC (Manassas, transfection for further experiments. VA, USA). CaOV3 cells were maintained in DMEM medium (Corning Incorporated, Corning, NY, USA) supplemented with Quantification of human cytokines by ELISA. The conditioned 10% FBS (RMBIO, Missoula, MT, USA), penicillin, streptomycin media were collected and active TGF- β1, VEGF and IL-8 were and glutamine at 37˚C in 5% CO 2. SKOV3 cells were maintained quantified using a single cytokine ELISA assay kit (R&D Systems, in RPMI-1640 medium (Corning Incorporated) supplemented