14-3-3σ attenuates RhoGDI2-induced cisplatin resistance through activation of Erk and p38 in gastric cancer cells

Rho GDP dissociation inhibitor 2 (RhoGDI2) promotes tumor growth and malignant progression and enhances chemoresistance of gastric cancer. Recently, we noted an inverse correlation between RhoGDI2 and 14-3-3σ expression, which suggests that 14-3-3σ is a target of gastric cancer metastasis and the chemoresistance-promoting effect of RhoGDI2. Herein, we evaluated whether 14-3-3σ is regulated by RhoGDI2 and is functionally important for the RhoGDI2-induced cisplatin resistance of gastric cancer cells. We used highly metastatic and cisplatin-resistant RhoGDI2-overexpressing SNU-484 cells and observed decreased 14-3-3σ mRNA and protein expression. Depletion of 14-3-3σ in SNU-484 control cells enhanced cisplatin resistance, whereas restoration of 14-3-3σ in RhoGDI2-overexpressing SNU-484 cells impaired cisplatin resistance in vitro and in vivo. We also found that the phosphorylation levels of Erk and p38 kinases significantly decreased in RhoGDI2-overexpressing SNU-484 cells and recovered after 14-3-3σ expression, and that decreased activities of these kinases were critical for RhoGDI2-induced cisplatin resistance. In conclusion, 14-3-3σ is a RhoGDI2-regulated gene that appears to be important for suppressing the chemoresistance of gastric cancer cells.


INTRODUCTION
Although the incidence and mortality of gastric cancer have steadily declined in recent decades, it remains the fourth most common type of cancer and the second leading cause of cancer mortality worldwide [1]. Surgery is an effective treatment for gastric cancer. Recent research has also shown that chemotherapy following radical surgery is an effective adjuvant therapy for East Asian patients [2]. Cisplatin is one of the most widely used drugs for chemotherapy and improves the overall survival for cancer patients [3][4][5][6][7]. However, cancer treatment is limited by the different efficacies of chemotherapeutic regimens, diverse disease states of patients and response rate to drugs, drug-related side-effects, acquisition of drug resistance, and cancer recurrence with metastasis [8][9][10][11]. To this end, the targeted approaches that focus on drug resistance-associated molecules are required to improve the efficacy of chemotherapy against advanced gastric cancers.
RhoGDI2 belongs to a family of Rho GTPase dissociate inhibitors (RhoGDIs). They are pivotal regulators of the function of Rho GTPase and typically exert their effects by forming a complex with Rho GTPase, and thereby modulating their nucleotide exchange and membrane association. Therefore, RhoGDIs play significant roles in regulating the actin cytoskeleton, cell polarity, microtubule dynamics, membrane transport pathways, and transcription factor activities [12,13]. Unlike other members of the family (such as RhoGDI1 and RhoGDI3), RhoGDI2 is preferentially expressed in hematopoietic cells, and appears to have a narrow selectivity and lower binding affinity for Rho GTPases [14]. RhoGDI2 associates with and negatively regulates Rac1 and Rac3 in breast cancer cells, but not RhoA, Cdc42, and RhoC [15]. In contrast, it positively regulates Rac1 in human bladder cancer cells [16]. The significant role of RhoGDI2 in cancer has been previously noted in several lines of study. RhoGDI2 expression is inversely correlated with invasive capacity in bladder cancer cell lines [17]. Furthermore, reduced RhoGDI2 protein expression has been associated with poor prognosis for patients with advanced bladder cancer [18]. In contrast, RhoGDI2 mRNA expression is significantly higher in ovarian adenocarcinomas than in benign adenomas [19]. Consistent with this finding, RhoGDI2 is overexpressed in human breast cancer cell lines, and it increases cancer cell invasiveness and motility in vitro [20]. We also demonstrated that RhoGDI2 expression is positively correlated with tumor progression and metastatic potential in gastric cancer [21]. In addition, our recent work demonstrated that RhoGDI2 is associated with the acquisition of resistance to chemotherapeutic agents (such as cisplatin), which is a hallmark of aggressive cancers, in gastric cancer cells [22].
To delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance, we performed two-dimensional gel electrophoresis (2-DE) on proteins that were derived from a RhoGDI2overexpressing SNU-484 human gastric cancer cell line and control cells, and noted that levels of 14-3-3σ, which is a member of the multifunctional 14-3-3 protein family, were significantly reduced [23]. In this study, we demonstrated that the downregulation of 14-3-3σ is largely correlated with the cisplatin-resistant phenotype of RhoGDI2-overexpessing gastric cancer cells. Of note, the restoration of 14-3-3σ is associated with impaired RhoGDI2-induced chemoresistance of gastric cancer cells through the activation of p38 and Erk.

RhoGDI2 downregulates 14-3-3σ expression
Previously, we identified 14 downregulated proteins in RhoGDI2-overexpressing SNU-484(GDI2-4) gastric cancer cells compared with control SNU-484(Mock) cells by using comparative 2-DE [23]. For further analysis, we selected 14-3-3σ that was previously implicated in cancer cell proliferation, metastasis, and apoptosis. To validate our mass spectrometry results, we performed reverse transcription-polymerase chain reaction and western blot analyses to determine the mRNA and protein expression levels of 14-3-3σ in RhoGDI2-overexpressing SNU-484 cells and RhoGDI2-depleted MKN-28 cells. Consistent with the results of 2-DE and imaging analysis (Fig.  1A), the mRNA and protein expression of 14-3-3σ were significantly downregulated in RhoGDI2-overexpressing SNU-484 cells and upregulated in RhoGDI2-depleted MKN-28 cells, compared to its expression in control cells (Fig. 1B). We also examined the mRNA expression levels of the other 14-3-3 isoforms (β, γ, ε, ζ, η, and τ) in RhoGDI2-overexpressing SNU-484 cells, but could not find any significant differences between these cells and the control cells (Fig. 1C). To further elucidate whether the decreased expression of 14-3-3σ is associated with RhoGDI2 expression, we observed 14-3-3σ expression levels in HeLa cells and MCF-7 cells after transient transfection with a Flag-tagged RhoGDI2 expression vector. As shown in Fig. 1D, transient expression of RhoGDI2 caused the markedly reduced expression of 14-3-3σ compared with the expression level in the vectortransfected control cells, which suggests that 14-3-3σ is a direct target of RhoGDI2.

Suppression of Erk and p38 activities enhances cisplatin resistance of gastric cancer cells
Since mitogen-activated protein kinase (MAPK) pathways are implicated in the execution of apoptosis by different cytotoxic agents and p38/JNK kinase activities have been known to be increased in RhoGDI2-depleted breast cancer cells [15], we first assessed whether the activities of these apoptosis-related kinases are suppressed in RhoGDI2-overexpressing gastric cancer cells. To this end, we examined MAPK activation in RhoGDI2-overexpressing SNU-484 cells by assessing their phosphorylation states by using antibodies specific to the phosphorylated species of each enzyme. As shown in Fig 4A, the phosphorylation levels of Erk and p38, but not JNK, were significantly decreased in RhoGDI2overexpressing SNU-484(GDI2-4 and GDI2-7) cells compared with control cells (Mock) under normal culture condition. We next examined whether suppression of Erk or p38 activity affects cisplatin-induced apoptosis in gastric cancer cells. Suppression of Erk and p38 activity by U0126 and SB203580, respectively, significantly inhibited cisplatin-induced apoptosis ( Fig. 4B and C) and PARP cleavage (Fig. 4D) in SNU-484(Mock) cells. These results suggest that suppression of Erk and p38 activity is critical for the cisplatin resistance of gastric cancer cells. Ectopic expression of 14-3-3σ attenuates RhoGDI2-induced cisplatin resistance of gastric cancer cells through Erk and p38 activation Since 14-3-3σ regulates MAPK activity and the activities of Erk and p38 were repressed in RhoGDI2overexpressing SNU-484(GDI2-4 and GDI2-7) cells (Fig. 4A), we examined whether the downregulation of 14-3-3σ expression is critical for the repression of Erk and p38 activities in RhoGDI2-overexpressing SNU-484(GDI2-7) cells. As we expected, the levels of phospho-Erk and phospho-p38 markedly increased in 14-3-3σ-overexpressing SNU-484(GDI2-7) cells (14-3-3σ-1 and 14-3-3σ-2) compared with control (Mock) cells (Fig. 5A). As shown in Figure 3, the overexpression of 14-3-3σ significantly increased cisplatin-induced apoptosis in RhoGDI2-overexpressing SNU-484(GDI2-7) cells. We next examined whether 14-3-3σ-mediated activation of Erk and p38 activities is critical for the recovery of cisplatin sensitivity in RhoGDI2-overexpressing SNU-484(GDI2-7) cells. Suppression of Erk and p38 activities by their specific inhibitors, respectively, markedly inhibited cisplatin-induced apoptosis ( Fig. 5B and C) and PARP cleavage (Fig. 5D) in 14-3-3σ-overexpressing SNU-484(GDI2-7) cells (14-3-3σ-1 and 14-3-3σ-2). All of these results suggest that the suppression of 14-3-3σ-mediated Erk and p38 activation is critical for the cisplatin resistance
In an effort to exclude the possibility that the effect of 14-3-3σ on the migration and invasion of RhoGDI2overexpressing gastric cancer cells was attributable to different proliferation rates, we compared the growth rates of 14-3-3σ-overexpressing SNU-484(GDI2-7) cells (14-3-3σ-1 and 14-3-3σ-2) with those of control  Figure 6D), thereby indicating that decreased tumor cell migration and invasion via the expression of 14-3-3σ in the respective cells was not associated with their proliferation rates.

DISCUSSION
Several studies over the last decade have linked RhoGDI1 expression to apoptosis and chemoresistance of various human cancer cells. For example, the reduction of RhoGDI1 expression is associated with astrocytoma cell protection and tamoxifen resistance of breast cancer cells [28,29]. In addition, Ronneburg et al. demonstrated that RhoGDI1 may sensitize invasive breast cancer to treatment with CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), and higher RhoGDI1 expression tends to be correlated with a better clinical outcome [30]. However, RhoGDI1 was also described as an antiapoptotic protein in breast, lymphoma, fibrosarcoma, and lung cancer cells [31][32][33]. In contrast to RhoGDI1, several groups have focused their research on elucidating the explicit mechanisms by which RhoGDI2 regulates aggressive features of cancer cells, particularly motility, invasiveness, and metastasis. However, we recently suggested that RhoGDI2 enhances the chemoresistance of gastric cancer through the upregulation of Bcl-2 expression as well as promotes tumor growth and malignant progression. 22 Consistent with our results, Zheng et al. suggested that the knockdown of RhoGDI2 expression significantly increases the sensi tivity of colon cancer cells to 5-FU [34], and that the ectopic expression of RhoGDI2 in gastric cancer cells induces resistance to 5-FU and reverses 5-FU-induced G2/M phase arrest [35].
RhoGDI1 protects breast cancer cells from druginduced apoptosis through the inhibition of Rac1 cleavage that is mediated by capase-3 [31]. Although RhoGDI1 is completely resistant to degradation during apoptosis, RhoGDI2 is well characterized as being a substrate for caspases and is cleaved in various cells during apoptosis [36]. Therefore, RhoGDI2 may act as an antiapoptotic molecule via a mechanism that is distinct from RhoGDI1 and may do so prior to caspase activation during druginduced apoptosis. To delineate the mechanism by which RhoGDI2 contributes to chemoresistance and tumor metastasis, we performed 2-DE on proteins that were derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. We found that the expression levels of 14-3-3σ were significantly downregulated in the RhoGDI2-overexpressing gastric cancer cells [23]. The results of this study indicate that 14-3-3σ is a direct target of RhoGDI2 and that the downregulation of 14-3-3σ is important for enhancing the chemoresistance of RhoGDI2-expressing gastric cancer cells. 14-3-3σ was first identified as being a human mammary epithelium-specific marker 1 (HME1) and participates in the regulation of subcellular localization, protein stability, cell apoptosis, proliferation, and the cell cycle [37][38][39]. However, 14-3-3σ is directly associated with human cancers, and the downregulation of 14-3-3σ expression has been observed in various human cancers, including those of the lung, prostate, breast, and liver [40,41]. Consistent with our findings, several previous findings have shown that 14-3-3σ expression is elevated in response to different cellular stresses, and that it enhances the chemosensitivity of human colorectal, breast, and nasopharyngeal cancer [24][25][26][27].
We also suggested that the ectopic expression of 14-3-3σ could activate Erk and p38 MAPK, and that suppression of 14-3-3σ-mediated Erk and p38 activation is critical for the cisplatin resistance of RhoGDI2overexpressing gastric cancer cells. We showed that the phosphorylation levels of Erk and p38 kinase are markedly downregulated in RhoGDI2-overexpressing (14-3-3σ downregulated) gastric cancer cells. However, the restoration of 14-3-3σ expression reverses this phenomenon. It is believed that MAPK activation is a major component that decides the fate of a cell in response to cisplatin. The pro-death or pro-survival roles of MAPKs in response to cisplatin could depend on the type of activated MAPK. While the activation of p38 plays only a pro-death role, the induction of Erk can take on both roles (survival or cell death) as a consequence [42]. Consistent with our results, the knockdown of RhoGDI2 expression significantly increases the phosphorylation levels of p38 kinase, and pretreatment with p38 kinase inhibitor significantly inhibits the apoptosis of RhoGDI2-depleted breast cancer cells [15]. Benzinger et al. also showed that the ectopic expression of 14-3-3σ enhances Erk1/2 activity in colon cancer cells [43]. The increase in MAPK activity that was observed after 14-3-3σ expression may be due to interactions with multiple ligands. Among others, A-RAF, B-RAF, and c-RAF seem to be good candidates because previous studies on the interaction between RAF proteins and 14-3-3 isoforms have shown that 14-3-3 proteins are critical modulators of RAF activity [44].
We also examined the possible pathways that lead to RhoGDI2-induced 14-3-3σ downregulation. Recently, we demonstrated that phospholipase C-gamma (PLCγ) is activated in RhoGDI2-overexpressing SNU-484 cells, and that it is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer [45]. To determine whether PLC is required for RhoGDI2induced 14-3-3σ downregulation, we examined the expression levels of 14-3-3σ in PLCγ-depleted SNU-484(GDI2) and control cells, but did not observe any difference (data not shown). Alternatively, ongoing studies in our laboratory have revealed that Rac1, but not RhoA or Cdc42, is activated in RhoGDI2-overexpressing gastric cancer cells. Therefore, we are now examining whether the activation of Rac1 is involved in RhoGDI2-induced 14-3-3σ downregulation. www.impactjournals.com/oncotarget

Reverse Transcription-PCR analysis
Total RNA was isolated using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RT-PCR was performed using a Maxime RT-PCR PreMix kit (Intron, Taejon, Korea). 200 ng of total RNA and specific primer were added into the Maxime RT-PCR PreMix tubes and RNase-free water was added to a total volume of 20 µl. RT-PCR was performed using a Thermo Electron PCR thermal cycler.

Apoptosis detection
Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit, Fluorescein or TMR red (Roche Applied Science, Germany) following the manufacturer's instruction. Cisplatin-treated or nontreated cells were washed with cold PBS and fixed with 4% paraformaldehyde. Fixed cells were permeabilized and stained using the TUNEL reaction mixture in the dark. The cells were then stained with 1 µg/ml DAPI solution for 5 min at room temperature in the dark and observed under a fluorescence microscope. The apoptosis rate was quantified by the TUNEL-positive rate.

Tumorigenicity in nude mice
For tumorigenicity experiments, six-weekold female BALB/cSlc-nude mice were injected subcutaneously with 5.5×10 6 SNU-484(GDI2-7/14-3-3σ-2) or SNU-484(GDI2-7/Mock) cells. When tumors measured an average volume of 50 mm 3 , the mice (12 per group) were treated with cisplatin (5mg/ kg, 2 times a week) or physiological saline for three weeks. Tumors were measured with calipers to estimate volume(0.5×width 2 ×length). All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of Gyeongsang National University and followed National Research Council Guidelines.

Invasion and migration assay
The invasion ability of cancer cells was assessed using a matrigel-based transwell system. Briefly, 24well cell culture plate inserts with 8-μm pore size polycarbonate membrane (Corning, NY, USA) were precoated with 100 μl matrigel/DMEM solution (2.2 mg/ ml, BD Bioscience, Bedford, MA) and incubated at 37 o c for 2 h or overnight at 4 o c. All the cells were preincubated in serum-free media with or without inhibitors for 24 h. 2.5 × 10 5 cells in 250 µl of medium (no serum) were placed in the insert and allowed to invade for 48 h. The lower chamber was filled with 750 µl of appropriated media containing 20% FBS. After incubation, medium remaining on top of the insert were removed by pipetting and non-invading cells on the upper surface of the insert membrane were removed with cotton swab. After washing twice with PBS, the insert membranes were fixed for 10 min with MeOH/Acetic acid (3:1) at -20 o c and stained with 50 µg/ml propidium iodide (SIGMA) for 20 min at 37 o c. The upper surface of the insert membrane was gently scrubbed with cotton swab again and washed with distilled water. Membranes were cut and mount on slide glass and the number of invaded cells was counted microscopically at 100-200 × magnification. For wound healing assays, 4.9 × 10 4 cells in 70 µl of medium were seeded into Culture-Insert (Ibidi, Munich, Germany). After the cells were confluent, to inhibit the effect of cell proliferation, the cells were pretreated with 10 µg/ml mitomycin C (SIGMA) for 2 h, and washed with culture medium. After removal of Culture-Insert, cells were incubated with fresh media and photographs of the migration assay were taken at 0, 5, 15, 20, 25 and 30 h using a phase-contrast microscope with digital camera. The cell migration was quantified by calculating the cell-covered area using WimScratch software (Wimasis, Munich, Germany).

Proliferation assay
The cells were placed in a 6-well plate at a concentration of 3 × 10 4 cells per well. After incubation for 1 to 4 days, cells were trypsinized and resuspended in 3 ml of appropriate medium. Cell suspensions were centrifuged at 1000 rpm for 5 min. Cell pellets were resuspended in 1 ml of appropriate medium. The viable cells were counted with a hemocytometer after trypan blue staining.