Proteomic profiling of CYP11A1 overexpressed kidney cancer cell lines Hien Thi My Ong¹ ², Min-Jung Kang¹ ² * , , , ¹Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea ²Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and technology, Seoul 02792, Republic of Korea ABSTRACT METHODS Our study focused on the comparison between proteomic profiling of CYP11A1 overexpressed normal and cancer kidney cell lines. According to the International Agency for Research on
Cancer, kidney cancer is the 12th cause of death from cancer 1. Cell lines: 2. Experiment workflow: worldwide which is currently showed a poor prognosis [1]. CYP11A1 is a mitochondrial side-chain cleavage enzyme catalyze the initial step in the conversion of cholesterol to pregnenolone. The expression of CYP11A1 was rapidly downregulated in six The HEK293T, A498 and Caki-1 cells were cultured in cancer types including colon adenocarcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, lung Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1% squamous cell carcinoma, prostate adenocarcinoma and uterine corpus endometrial carcinoma [2]. Our study indicated overexpression of CYP11A1 can inhibited cancer cells migration and invasion. To explain how CYP11A1 can inhibit cell penicillin/streptomycin (Gibco, USA) and maintained in a humidified atmosphere of 95% air/5% CO2 at 37˚C. migration and invasion, we compared proteomic profiling of CYP11A1-transfection and non-transfection in cancer cell lines. A total of 218 and 161 proteins were identified with high confidence (Score Sequest HT >1, p-value<0.05 and Exp. q- value<0.005)
entries in A498 and Caki-1 respectively. Among these proteins, 78 proteins were overlapped between CYP11A1- overexpressed and non-transfected kidney cancer cell lines. Ingenuity Pathway Analysis (IPA) showed enrichment of many cancer-related biological processes and pathways such as EIF2, mTOR, EIF4 and p70S6K signaling. These results hypothesized that activating steroid biosynthesis may be useful to block cancer progression and targeting of CYP11A1 catalyzes the first step of steroidogenesis as a potential therapeutic target for kidney cancer. RESULTS 1. CYP11A1 transfection in normal vs
cancer cell lines 2. Identification of differentially proteins with CYP11A1 overexpressed in cancer cell lines D. The proteomic analysis identified total 10048 and 8918 proteins known database entries in A498 and Caki-1 respectively. Among these proteins, 218 and 161 were screened reproducibly using strict filtration (High in Protein FDR Confidence <5%, unique peptide ≥2, coverage ≥5%, Score Sequest HT >1, p-value<0.05, Exp. q-value<0.005) these candidate proteins were found in at least 6/9 ratio of combination samples. Figure 2. (A) Veen diagram showed up and down-regulated proteins in
control compared with CYP11A1 overexpressed cancer cell lines (A498 and Caki-1). In (B) 33 Figure 1. (A) Western blot analysis indicated successful establishment of a proteins were significantly CYP11A1-overexpression model in normal HEK293T and cancer cell lines upregulated while (C) showed 45 (A498 and Caki-1). Scratch-wound healing assay (B) Images of scratch-wound down-regulated proteins in both healing respectively at 0, 5 and 24h of non-transfection as control model CYP11A1-overexpressed cancer following (C) images after transfection with CYP11A1. (D) Quantitative groups (Ranked by Log 2
abundance measurement of wound closure ability are shown by % of cell free region, ratio >1.3 or <0.8 respectively) CYP11A1 impaired cell migration in two cancer cell lines after transfection . 3. IPA analysis comparing the top enriched signaling pathways Table 1. Differently expressed proteins in CYP11A1- Table 2. Differently expressed proteins in CYP11A1- overexpressed compared with normal A498 cell line overexpressed compared with normal Caki-1 cell (Ranked by fold change) line (Ranked by fold change) Score Fold Score Fold Accession Description Exp. q- Sum PEP Coverag Sequest Change
Accession Description Exp. q- value: Sum PEP Coverage Sequest Change Score value Score e [%] [%] HT (Log2) P54886 Delta-1-pyrroline-5-carboxylate synthase GN=ALDH18A1 0 5.95 13 2 5.261272 Combined HT (Log2) Q99832 T-complex protein 1 subunit eta GN=CCT7 0 6.784 33 2.75 5.172434 P61204 ADP-ribosylation factor 3 GN=ARF3 0 5.39 10 9.9 6.643856 P63313 Thymosin beta-10 GN=TMSB10 0 5.755 75 5.34 4.695012 P31946 14-3-3 protein beta/alpha GN=YWHAB 0 3.219 44 2.17 5.844188 P53675 Clathrin heavy chain 2 GN=CLTCL1 0 26.389 16 11.9 4.693919 O14818 Proteasome subunit alpha type-7 GN=PSMA7 0 4.61 21 2.19
5.841064 P46776 60S ribosomal protein L27a GN=RPL27A 0 6.659 44 10.07 4.679072 O00299 Chloride intracellular channel protein 1 GN=CLIC1 0 9.079 23 4 5.430237 Q14697-2 Isoform 2 of Neutral alpha-glucosidase AB GN=GANAB 0 4.981 8 2.19 4.676416 P61313 60S ribosomal protein L15 GN=RPL15 0 4.574 16 5.8 5.102854 Q9NR31 GTP-binding protein SAR1a GN=SAR1A 0.007 3.22 30 4.4 4.672404 P11413-2 Isoform Long of Glucose-6-phosphate 1-dehydrogenase GN=G6PD 0 10.128 24 4.62 5.093044 P52815 39S ribosomal protein L12, mitochondrial GN=MRPL12 0.007 3.24 23 2.46 4.668715 Q9Y678 Coatomer subunit gamma-1 GN=COPG1 0
7.245 13 6.91 4.66853 P18621-3 Isoform 3 of 60S ribosomal protein L17 GN=RPL17 0.001 2.194 32 2.21 5.09295 Q92945 Far upstream element-binding protein 2 GN=KHSRP 0 4.872 5 4.52 4.665485 P00918 Carbonic anhydrase 2 GN=CA2 0 5.703 20 3.42 5.08336 P60866-2 Isoform 2 of 40S ribosomal protein S20 GN=RPS20 0 11.076 33 4.17 4.662412 Q00341 Vigilin GN=HDLBP 0 3.908 24 1.65 5.078069 P83731 60S ribosomal protein L24 GN=RPL24 0.007 3.226 13 4.18 4.662149 P35232 Prohibitin GN=PHB 0 10.761 28 8.79 5.077817 P39023 60S ribosomal protein L3 GN=RPL3 0 11.336 19 2 3.347555 P47756 F-actin-capping protein subunit
beta GN=CAPZB 0.001 2.205 26 1.64 5.077746 P18085 ADP-ribosylation factor 4 GN=ARF4 0 10.35 66 13.56 3.123703 P37837 Transaldolase GN=TALDO1 0 27.272 42 12.65 2.469915 P53621-2 Isoform 2 of Coatomer subunit alpha GN=COPA 0 4.986 14 1.61 5.07692 P46778 60S ribosomal protein L21 GN=RPL21 0 9.2 26 2.26 1.829985 P63220 40S ribosomal protein S21 GN=RPS21 0 3.206 64 3.23 5.075975 P30048 Thioredoxin-dependent peroxide reductase, mitochondrial GN=PRDX3 0 13.508 35 15.75 1.824202 P27348 14-3-3 protein theta GN=YWHAQ 0 5.093 47 2.17 5.075 P49448 Glutamate dehydrogenase 2, mitochondrial GN=GLUD2 0.001
4.037 18 4.33 1.755885 P62249 40S ribosomal protein S16 GN=RPS16 0 6.433 33 7.76 5.074729 Q86VP6 Cullin-associated NEDD8-dissociated protein 1 GN=CAND1 0 14.315 14 7.72 1.713507 P62701 40S ribosomal protein S4, X isoform GN=RPS4X 0 7.299 34 5.26 5.07461 P40925-3 Isoform 3 of Malate dehydrogenase, cytoplasmic GN=MDH1 0 9.893 27 4.55 1.707279 P49327 Fatty acid synthase GN=FASN 0 4.028 6 1.83 5.074072 P45974 Ubiquitin carboxyl-terminal hydrolase 5 GN=USP5 0 6.451 18 3.78 1.693518 P0DP25 Calmodulin-3 GN=CALM3 0 20.086 32 9.35 1.667463 P08708 40S ribosomal protein S17 GN=RPS17 0 4.94 16 8.5
5.072873 P09429 High mobility group protein B1 GN=HMGB1 0 17.922 28 10.89 1.639747 P05556 Integrin beta-1 GN=ITGB1 0 7.014 11 3.97 5.072735 P50990 T-complex protein 1 subunit theta GN=CCT8 0 20.577 29 9.6 1.336773 P40227 T-complex protein 1 subunit zeta GN=CCT6A 0 5.421 19 3.81 5.070089 Q13247 Serine/arginine-rich splicing factor 6 GN=SRSF6 0.007 3.156 10 2.71 1.295462 P61353 60S ribosomal protein L27 GN=RPL27 0 8.722 29 10.15 4.705384 P04075-2 Isoform 2 of Fructose-bisphosphate aldolase A GN=ALDOA 0 33.715 48 17.34 1.264637 P39019 40S ribosomal protein S19 GN=RPS19 0 4.638 21 4.95 4.689886
P63000-2 Isoform B of Ras-related C3 botulinum toxin substrate 1 GN=RAC1 0 4.258 33 5.92 1.257614 P62424 60S ribosomal protein L7a GN=RPL7A 0 16.668 38 6.11 1.22993 P62913 60S ribosomal protein L11 GN=RPL11 0 3.423 13 8.04 4.687103 P42224 Signal transducer and activator of transcription 1-alpha/beta GN=STAT1 0 16.547 18 7.97 1.202183 P25398 40S ribosomal protein S12 GN=RPS12 0 5.06 33 6.64 4.686045 Q09666 Neuroblast differentiation-associated protein AHNAK GN=AHNAK 0 32.821 18 12.35 1.192194 P51148-2 Isoform 2 of Ras-related protein Rab-5C GN=RAB5C 0.001 2.532 10 3.84 4.68519 P78417
Glutathione S-transferase omega-1 GN=GSTO1 0.007 3.177 37 7.32 1.189746 Q13200 26S proteasome non-ATPase regulatory subunit 2 GN=PSMD2 0 3.947 12 1.61 4.683437 P49411 Elongation factor Tu, mitochondrial GN=TUFM 0 12.914 19 11.82 1.187908 O43390-2 Isoform 2 of Heterogeneous nuclear ribonucleoprotein R GN=HNRNPR 0 3.615 7 1.6 4.682468 Q14195-2 Isoform LCRMP-4 of Dihydropyrimidinase-related protein 3 GN=DPYSL3 0 18.459 30 12.88 1.185021 P07237 Protein disulfide-isomerase GN=P4HB 0 41.695 42 35.12 1.174548 P61026 Ras-related protein Rab-10 GN=RAB10 0 5.518 36 1.73 4.682068 P27348 14-3-3 protein
theta GN=YWHAQ 0 28.815 63 25.16 1.159127 P31949 Protein S100-A11 GN=S100A11 0 5.357 68 1.76 4.681084 P26641-2 Isoform 2 of Elongation factor 1-gamma GN=EEF1G 0 13.603 36 7.33 1.153373 P61019 Ras-related protein Rab-2A GN=RAB2A 0.002 2.602 23 2.35 4.680507 P25705 ATP synthase subunit alpha, mitochondrial GN=ATP5F1A 0 20.127 25 15.33 1.147234 O75347 Tubulin-specific chaperone A GN=TBCA 0 3.898 27 11.13 4.676951 P63220 40S ribosomal protein S21 GN=RPS21 0.007 3.281 42 1.87 1.1468 Q9Y490 Talin-1 GN=TLN1 0 7.576 12 2.31 4.67535 P11142 Heat shock cognate 71 kDa protein GN=HSPA8 0 100.152 40 91.97
1.13641 P26641-2 Isoform 2 of Elongation factor 1-gamma GN=EEF1G 0 9.265 31 3.74 4.675329 P12004 Proliferating cell nuclear antigen GN=PCNA 0 21.005 18 14.43 1.110215 Q15907 Ras-related protein Rab-11B GN=RAB11B 0 5.513 23 2.63 1.097161 P05388 60S acidic ribosomal protein P0 GN=RPLP0 0 3.406 11 3.68 4.675096 P13489 Ribonuclease inhibitor GN=RNH1 0 25.812 28 12.5 1.083232 P50395 Rab GDP dissociation inhibitor beta GN=GDI2 0 9.868 24 2.02 4.674312 P0DMV9 Heat shock 70 kDa protein 1B GN=HSPA1B 0 49.362 31 37.31 1.065496 Q07960 Rho GTPase-activating protein 1 GN=ARHGAP1 0 4.797 18 1.76 4.673662
P22392-2 Isoform 3 of Nucleoside diphosphate kinase B GN=NME2 0 29.364 76 36.93 1.043189 P61160 Actin-related protein 2 GN=ACTR2 0 3.738 18 1.7 4.67227 Q9UHD8 Septin-9 GN=SEPTIN9 0 6.462 19 6.61 1.039138 P62277 40S ribosomal protein S13 GN=RPS13 0 6.596 38 6.58 4.665286 P12956 X-ray repair cross-complementing protein 6 GN=XRCC6 0 21.138 32 9.36 1.038202 Q96AG4 Leucine-rich repeat-containing protein 59 GN=LRRC59 0 13.444 31 10.11 1.035389 Q13247 Serine/arginine-rich splicing factor 6 GN=SRSF6 0 4.554 10 6.27 4.660588 P05388 60S acidic ribosomal protein P0 GN=RPLP0 0 16.069 22 9.66 1.012529
P42224 Signal transducer and activator of transcription 1-alpha/beta GN=STAT1 0 26.259 37 12.8 3.195033 P08238 Heat shock protein HSP 90-beta GN=HSP90AB1 0 71.977 38 44.86 1.012052 P63104 14-3-3 protein zeta/delta GN=YWHAZ 0 16.884 40 9.44 1.87578 P07195 L-lactate dehydrogenase B chain GN=LDHB 0 33.086 37 40.88 1.010143 O43707 Alpha-actinin-4 GN=ACTN4 0 7.047 23 2.04 1.852348 P62913 60S ribosomal protein L11 GN=RPL11 0 11.64 31 11.18 1.009904 P26038 Moesin GN=MSN 0 18.01 40 8.28 1.839646 Figure 3. The enrichment of signaling pathways in the differentially expressed proteins identified by IPA
software are shown. P08758 Annexin A5 GN=ANXA5 0 37.713 44 36.83 1.00879 P22392-2 Isoform 3 of Nucleoside diphosphate kinase B GN=NME2 0 13.921 42 19.68 1.748414 P21333 Filamin-A GN=FLNA 0 106.342 27 56.83 1.007275 (A) (C) Comparative analysis between normal (HEK293T) and two cancer cell lines (A498 and Caki-1) respectively. P27797 Calreticulin GN=CALR 0 20.426 28 7.43 1.000961 P21796 Voltage-dependent anion-selective channel protein 1 GN=VDAC1 0 5.994 29 5.97 1.721251 P61204 ADP-ribosylation factor 3 GN=ARF3 0 10.72 41 10.94 0.978521 P07237 Protein disulfide-isomerase GN=P4HB 0 27.214 60
17.65 1.638769 P62277 40S ribosomal protein S13 GN=RPS13
