RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation

RCC2 cDNA coding sequence (NM_001136204.2) was fused with N-terminal eYFP (GeneCopoeia clone# EX-E0423-M15; Rockville, MD, USA). The constitutively activated pRK5-myc-Rac1-Q61L was created by Dr. Hall’s lab [13], and the leucine substitution prevents endogenous and GAP-stimulated GTPase activity of Rac1. Plasmid DNAs were prepared with EndoFree® Plasmid kit (Qiagen, Valencia, CA, USA).

Three RCC2-specific siRNAs were used: siG151029043959 (5’-CCACGAAGTGATTGTGTCT), siG151029044022 (5’-GGAGGTAAAGACTCTGGAT) and siG151029044006 (5′- GCCTGTACCAAACGTGGTT). Ribobio Negative Control siRNA was used as negative control (Ribobio Co. Guangzhou, China).

Plasmids or siRNA were transiently transfected into HeLa cells (ATCC® CCL-2™, American Type Culture Collection (ATCC), Manassas, VA, USA), CRL5800 (ATCC® CRL-5800™, ATCC) and MDA-MB-231 (ATCC® HTB-26™, ATCC) using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA). Stable cell lines expressing YFP or RCC2-YFP were also established by selections with G418 (400 μg/ml) for 3 weeks, and the YFP and RCC2-YFP expression were monitored using an inverted fluorescence microscope.

HeLa cells with stable expression of YFP, RCC2-YFP and parental cells were cultured in 96-well plates. Cell counts were determined daily using trypan blue exclusion method. For soft agar assays, 1 × 10⁴ cells were suspended in 2 ml of soft agar (0.35% Bactoagar in DMEM/F12 with 20% FCS), plated onto 5 ml of solidified agar (0.75% Bactoagar in DMEM/F12) in a 6-well plate, and cultured at 37 °C in 5% CO2 for 10 days. Colonies were fixed with methanol and stained with Giemsa.

HeLa cells were transfected with YFP, RCC2-YFP, and/or Rac1-Q61L for 48 h in 96-well plates. Caspase-Glo® 3/7 Reagent (Promega, Madison, WI, USA) was added to cells at a 1:1 ratio (volume), mixed and luminescence measured in a plate-reading luminometer. Results were averaged between six wells from two separate transfections.

HeLa cells expressing YFP or RCC2-YFP were lysed in 300 μL of lysis buffer and pre-cleared by incubating with 20 μL of protein A–Sepharose (Pharmacia, Piscataway, NJ, USA) for 1 h at 4 °C with gentle rotation. Pre-cleared protein lysate were incubated with rabbit anti-GFP antibody (ab6556, Abcam, Cambridge, MA, USA) overnight at 4 °C and followed by incubating with 10 μl of protein A–Sepharose for 1 h. After three washes in lysis buffer, proteins were eluted at 90 °C in 30 μl of SDS–PAGE loading buffer and resolved by SDS–PAGE under reducing conditions (4%–12% gradient gels). For Western blot analyses, proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Waltham, MA, USA), blocked in PBS containing 0.1% Tween 20 (PBST) and 5% dried milk for 1 h, and detected with monoclonal anti-GFP (ab1218, Abcam), anti-Rac1 (ARC03, Cytoskeleton, Denver, CO, USA), anti-cdc42 (ACD03, Cytoskeleton), or anti-Rho A (ARH04, Cytoskeleton) using a chemiluminescence method (ECL; Amersham, Piscataway, NJ, USA).

Rho GTPase pull down was performed with a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit™ (Cytoskeleton; Denver, CO, USA). Briefly, HeLa cells expressing YFP or RCC2-YFP were cultured in serum-free medium overnight and stimulated by adding 1.3 ml FBS per 5 ml medium for 5 min. Cells were washed with cold PBS and lysed in cold cell lysis buffer with a cell scraper. 600 μg of protein lysate were incubated with 25 μl rhotekin-RBD or PAK-PBD beads at 4 °C for 1 h. The beads were washed once with wash buffer and beads-binding proteins eluted in loading buffer and Western blotted with antibodies to RhoA, Rac1 or Cdc42. For total Rho GTPase, crude protein lyses without pull down were evaluated.

HeLa cells, CRL5800 and MDA-MB-231 expressing YFP or RCC2-YFP were cultured in 96-well plates, treated with vehicles or increasing doses of chemotherapeutic drugs for 48 h, and live cells quantitated by CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Vehicles were 0.9% NaCl (Cisplatin) or DMSO (Taxol, Nocodazole, hydroxyurea, Daunorubicin, CPT, STS, 5-Fluorouracil and Irinotecan) and drugs were dissolved in vehicles at 1,000X stock concentration. The surviving cells were calculated as the fraction of vehicle controls. Results were averaged between six wells per dose in two experiments.

Lung carcinoma progression tissue microarray (LC2083; Biomax; Rockville, MD, USA) and ovarian cancer and normal tissue high density tissue microarray (OV208; Biomax) were de-paraffinized in xylene, antigen-retrieved by heating in 0.01 M sodium citrate buffer (pH 6.0) at 95 °C for 10 min, blocked in 10% normal goat serum for 30 min and incubated with an anti-RCC2 antibody (D14F3; Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Immunohistochemistry staining was performed using a mouse and rabbit specific HRP/AEC (ABC) detection IHC kit (Abcam Ab93705; Boston, MA, USA). RCC2 expression was scored by two experienced researchers. Cases with inconsistent scoring were reviewed by a third pathologist.

RCC2-YFP was transiently expressed in three tumor cell lines including HeLa, lung cancer CRL5800 and breast cancer MDA-MB-231. Approximately 80% of CRL5800 or MDA-MB-231 cells and 90–100% of HeLa cells were YFP-positive 24 h after transfection. RCC2-YFP was localized at nuclei in interphase cells and midbody/midzone region in anaphase cells, similar to those endogenous RCC2 [2]. RCC2-YFP expression was confirmed by a western blot analysis with an anti-RCC1 antibody (Fig. 1a). The RCC2-YFP expression resulted in significant increase in HeLa cell proliferation, but not in CRL5800 and MDA-MB-231 (Fig. 1b). Increased cell growth was also confirmed in RCC2-YFP HeLa cells seeded in soft agar plates (Fig. 1c). By DAPI stain, approximately 10~15% of HeLa cells were spontaneously apoptotic cultured in medium containing either 10% FBS or serum-free medium. By contrast, virtually no apoptotic cells were detected in these cells expressing RCC2-YFP (Fig. 1d & e), suggesting that RCC2-YFP expression blocked the spontaneous apoptosis of HeLa cells. Both CRL5800 and MDA-MB-231 cells showed no spontaneous apoptosis in culture. To study whether the RCC2-YFP expression also confers protection against apoptosis in these cells, STS (10 μM) was added to the cultured cells for 48 h and apoptosis was scored by DAPI stain. Both cell lines with RCC2-YFP expression were significantly more resistant to STS-induced cell death when compared to control cells (Fig. 1f).

All three cell lines, HeLa, CRL5800 and MDA-MB-231, expressed endogenous RCC2 (Fig. 1a). We studied whether a decreased RCC2 in these cells will alter their sensitivity to STS-induced apoptosis. Expression of three sets of RCC-specific siRNA for 24 h, 48 h and 72 h all resulted in significant RCC2 knock down at both mRNA and protein level (Fig. 2a and b). These cells were treated with increasing STS for 24 h and surviving cells were quantitated using a CellTiter-Glo® Luminescent Cell Viability Assay. As shown in Fig. 2c, cells expressing RCC2-specific siRNA were consistently more sensitive to STS at different concentration as compared to the control cells expression a control siRNA. At the highest STS concentration, most cells were killed with or without RCC2 knock down.

RCC2 is capable of directly binding Rac1 [2]. Because multiple studies suggested a role of Rac1 in apoptosis, we explored the possibility that RCC2-YFP attenuates apoptosis by blocking Rac1 activation. We first evaluated the interaction between RCC2-YFP and endogenous Rac1 in HeLa cells expressing YFP or RCC2-YFP. A co-immunoprecipitation assay was performed, which confirmed the presence of Rac1, but not of Rho A or cdc42, in the RCC2-YFP pull down complex (Fig. 3a). We next evaluated the effectiveness of RCC2-YFP expression on blocking the serum-induced Rac1 activation. HeLa cells expressing YFP or RCC2-YFP were serum-starved for 48 h, followed by 20% serum stimulation for 5 min. Activated Rac1 was pulled down by GST-PAK-p21 binding domain (PBD). Serum stimulation led to increased Rac1 activation in YFP cells as expected; however, Rac1 activation was not detected in RCC2-YFP HeLa cells, confirming that the RCC2-YFP expression blocked the serum-stimulated Rac1 activation (Fig. 3b). In addition, YFP HeLa cells had endogenous Rac1 activation in serum-free culture, which was also blocked by the expression of RCC2-YFP (Fig. 3b). Similar pull down assays showed no significant difference for other Rho GTPases including RhoA and cdc42 (data not shown). Rac1 activation leads to C-Jun kinase (JNK) activation, i.e., its phosphorylation at Thr183 and Tyr185. Western blot analysis showed that the RCC2-YFP expression blocked the STS-induced JNK phosphorylation in all three cell lines, consistent with Rac1 inactivation (Fig. 3c). We then co-transfected tumor cells with both RCC2-YFP and a constitutively activated Rac1-Q61L. Apoptosis was induced by adding STS and scored by DAPI stain. The Rac1-Q61L expression largely revoked the apoptosis protection by RCC2-YFP in these cells (Fig. 3d). In addition, the co-expression of Rac1-Q61L neutralized the protection of RCC2-YFP against spontaneous apoptosis in HeLa cells (Fig. 3e). By a Caspase-Glo® 3/7 assay, RCC2-YFP HeLa cells had significantly decreased activity of Caspase 3/7 as compared to control cells (Fig. 3f).

The apoptotic mechanisms for chemotherapeutic drugs are often cell- and/or drug-specific; and Rac1/JNK pathways are critical to drug-induced cell death in several settings [14‐16]. We established a series of cell lines stably expressing YFP or RCC2-YFP. These cells were evaluated for their sensitivity to a panel of nine common chemotherapeutic drugs (Fig. 4a). Tumor cells were treated with drugs or vehicles for 48 h and cells quantitated by a CellTiter-Glo® Luminescent Cell Viability Assay. No significant difference was observed between untreated cells and vehicle-treated cells (0.1% DMSO or 0.1% saline) (Fig. 4b). All cell lines expressing RCC2-YFP were more resistant to Taxol, Nocodazole, Daunorubicin, Cisplatin, STS and 5-Fluorouracil (5-FU) when compared to control YFP cells; however, these cells were more sensitive to Camptothecin (CPT), a DNA topoisomerase I inhibitor. The response to Irinotecan and Hydroxyurea was cell line-specific: the expression of RCC2-YFP in both CRL5800 and MDA-MB-231 cells resulted in increased sensitivity to Irinotecan; however, RCC2-YFP HeLa cells showed decreased sensitivity to Irinotecan. The RCC2-YFP expression in MDA-MB-231 and HeLa cells resulted in increased sensitivity to Hydroxyurea, although RCC2-YFP CRL5800 cells showed decreased sensitivity to Hydroxyurea (Fig. 4c, d & e).

RCC2 expression in lung cancer and ovarian cancer was evaluated by IHC of tissue microarrays. The anti-RCC2 antibody (D14F3, cell signaling) was validated by IHC of cultured HeLa cells, which showed a nuclear localization in interphase cells and spindle midzone/midbody localization in mitosis as expected (Fig. 5a). RCC2 expression on tumor tissue microarrays was studied using a 5-tier scoring system (−, −/+, +, ++, +++) depending on signal intensity. In 120 cases of human lung cancers including various types (LC2083; Biomax), 106 (88.3%) had RCC2 expression at +~+++, and 14 of them had - or −/+ (11.7%). In contrast, none of 23 normal lungs expressed RCC2 (Fig. 5b). Cancer-adjacent normal lung tissues and inflammatory pseudotumors expressed low-level RCC2.

(− or−/+)(P < 0.001). RCC2 was mainly localized in the nuclei of tumor cells, although some had significant cytoplasmic RCC2 in addition to nuclear localization (Fig. 5c). RCC2 expression level was not significantly different among three grades of non-small cell lung cancer (well-, moderately- and poorly differentiated tumors). Detailed clinicopathological characteristics of lung cancers and RCC2 expression level were listed in Table 1. In an ovarian cancer tissue array (OV208), increased RCC2 expression (++ ~ +++) was detected in 92 of 141 cases of ovarian cancers (65.2%); and 62 of them had RCC2 at +++ (44%). Most normal ovaries expressed none or weak RCC2 (−, −/+ or +)(Fig. 5d). RCC2 expression was significantly higher in metastatic ovarian cancers (P < 0.05), higher grade tumors (P < 0.05) and bigger tumor (P < 0.05). Detailed clinicopathological characteristics of ovarian cancers and RCC2 expression level were listed in Table 2.