Purpose Epithelial ovarian cancer has the highest mortality rate of all gynecological malignancies. We have shown that high RAN expression strongly correlates with high-grade and poor patient survival in epithelial ovarian cancer. However, as RAN is a small GTPase involved in two main biological functions, nucleo-cytoplasmic transport and mitosis, it is still unknown which of these functions associate with poor prognosis. Methods To examine the biomarker value of RAN network components in serous epithelial ovarian cancer, protein expression of six specific RAN partners was analyzed by immunohistochemistry using a tissue microarray representing 143 patients associated with clinical parameters. The RAN GDP/GTP cycle was evaluated by the expression of RANBP1 and RCC1, the mitotic function by TPX2 and IMPβ, and the nucleo-cytoplasmic trafficking function by XPO7, XPOT and IMPβ. Results Based on Kaplan-Meier analyses, RAN, cytoplasmic XPO7 and TPX2 were significantly associated with poor overall patient survival, and RAN and TPX2 were associated with lower disease free survival in patients with high-grade serous carcinoma. Cox regression analysis revealed that RAN and TPX2 expression were independent prognostic factors for both overall and disease free survival, and that cytoplasmic XPO7 expression was a prognostic factor for overall patient survival. Conclusions In this systematic study, we show that RAN and two protein partners involved in its nucleo-cytoplasmic and mitotic functions (XPO7 and TPX2, respectively) can be used as biomarkers to stratify patients based on prognosis. In particular, we reported for the first time the clinical relevance of the exportin XPO7 and showed that TPX2 expression had the strongest prognostic value. These findings suggest that protein partners in each of RAN’s functions can discriminate between different outcomes in high-grade serous epithelial ovarian cancer patients. Furthermore, these proteins point to cellular processes that may ultimately be targeted to improve the survival in serous epithelial ovarian cancer.
References
[1]
Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62: 10–29. doi: 10.3322/caac.20138
[2]
Colombo N, Van Gorp T, Parma G, Amant F, Gatta G, et al. (2006) Ovarian cancer. Crit Rev Oncol Hematol 60: 159–179. doi: 10.1016/j.critrevonc.2006.03.004
Coleman MP, Forman D, Bryant H, Butler J, Rachet B, et al. (2011) Cancer survival in Australia, Canada, Denmark, Norway, Sweden, and the UK, 1995–2007 (the International Cancer Benchmarking Partnership): an analysis of population-based cancer registry data. Lancet 377: 127–138. doi: 10.1016/s0140-6736(10)62231-3
[5]
McGuire WP (2009) Maintenance therapy for ovarian cancer: of Helsinki and Hippocrates. J Clin Oncol 27: 4633–4634. doi: 10.1200/jco.2009.23.6653
[6]
Chen VW, Ruiz B, Killeen JL, Cote TR, Wu XC, et al. (2003) Pathology and classification of ovarian tumors. Cancer 97: 2631–2642. doi: 10.1002/cncr.11345
[7]
Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC (2001) Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr Rev 22: 255–288. doi: 10.1210/edrv.22.2.0422
[8]
de Souza PL, Friedlander ML (1992) Prognostic factors in ovarian cancer. Hematol Oncol Clin North Am 6: 761–782.
[9]
Malpica A (2008) Grading of ovarian cancer: a histotype-specific approach. Int J Gynecol Pathol 27: 175–181. doi: 10.1097/pgp.0b013e31816085e0
[10]
Singer G, Kurman RJ, Chang HW, Cho SK, Shih Ie M (2002) Diverse tumorigenic pathways in ovarian serous carcinoma. Am J Pathol 160: 1223–1228. doi: 10.1016/s0002-9440(10)62549-7
[11]
Seidman JD, Kurman RJ (2000) Ovarian serous borderline tumors: a critical review of the literature with emphasis on prognostic indicators. Hum Pathol 31: 539–557.
[12]
Ouellet V, Provencher DM, Maugard CM, Le Page C, Ren F, et al. (2005) Discrimination between serous low malignant potential and invasive epithelial ovarian tumors using molecular profiling. Oncogene 24: 4672–4687. doi: 10.1038/sj.onc.1208214
[13]
Ouellet V, Guyot MC, Le Page C, Filali-Mouhim A, Lussier C, et al. (2006) Tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer. Int J Cancer 119: 599–607. doi: 10.1002/ijc.21902
[14]
Barres V, Ouellet V, Lafontaine J, Tonin PN, Provencher DM, et al. (2010) An essential role for Ran GTPase in epithelial ovarian cancer cell survival. Mol Cancer 9: 272. doi: 10.1186/1476-4598-9-272
[15]
Fan H, Lu Y, Qin H, Zhou Y, Gu Y, et al.. (2012) High Ran level is correlated with poor prognosis in patients with colorectal cancer. Int J Clin Oncol.
[16]
Woo IS, Jang HS, Eun SY, Kim HJ, Ham SA, et al. (2008) Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells. Apoptosis 13: 1223–1231. doi: 10.1007/s10495-008-0247-0
[17]
Azuma K, Sasada T, Takedatsu H, Shomura H, Koga M, et al. (2004) Ran, a small GTPase gene, encodes cytotoxic T lymphocyte (CTL) epitopes capable of inducing HLA-A33-restricted and tumor-reactive CTLs in cancer patients. Clin Cancer Res 10: 6695–6702. doi: 10.1158/1078-0432.ccr-04-0818
[18]
Xia F, Lee CW, Altieri DC (2008) Tumor cell dependence on Ran-GTP-directed mitosis. Cancer Res 68: 1826–1833. doi: 10.1158/0008-5472.can-07-5279
[19]
Li H, Ren CP, Tan XJ, Yang XY, Zhang HB, et al. (2006) Identification of genes related to nasopharyngeal carcinoma with the help of pathway-based networks. Acta Biochim Biophys Sin (Shanghai) 38: 900–910.
[20]
Sorokin AV, Kim ER, Ovchinnikov LP (2007) Nucleocytoplasmic transport of proteins. Biochemistry (Mosc) 72: 1439–1457. doi: 10.1134/s0006297907130032
[21]
Stewart M (2007) Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8: 195–208. doi: 10.1038/nrm2114
[22]
Clarke PR, Zhang C (2008) Spatial and temporal coordination of mitosis by Ran GTPase. Nat Rev Mol Cell Biol 9: 464–477. doi: 10.1038/nrm2410
[23]
Ohtsubo M, Okazaki H, Nishimoto T (1989) The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. J Cell Biol 109: 1389–1397. doi: 10.1083/jcb.109.4.1389
[24]
Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H (1995) Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J 14: 705–715.
[25]
Matunis MJ, Coutavas E, Blobel G (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135: 1457–1470. doi: 10.1083/jcb.135.6.1457
[26]
Mahajan R, Delphin C, Guan T, Gerace L, Melchior F (1997) A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88: 97–107. doi: 10.1016/s0092-8674(00)81862-0
[27]
Kalab P, Weis K, Heald R (2002) Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295: 2452–2456. doi: 10.1126/science.1068798
[28]
Arts GJ, Fornerod M, Mattaj IW (1998) Identification of a nuclear export receptor for tRNA. Curr Biol 8: 305–314. doi: 10.1016/s0960-9822(98)70130-7
[29]
Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, et al. (1998) Identification of a tRNA-specific nuclear export receptor. Mol Cell 1: 359–369. doi: 10.1016/s1097-2765(00)80036-2
[30]
Koch P, Bohlmann I, Schafer M, Hansen-Hagge TE, Kiyoi H, et al. (2000) Identification of a novel putative Ran-binding protein and its close homologue. Biochem Biophys Res Commun 278: 241–249. doi: 10.1006/bbrc.2000.3788
[31]
Kutay U, Hartmann E, Treichel N, Calado A, Carmo-Fonseca M, et al. (2000) Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily. J Biol Chem 275: 40163–40168. doi: 10.1074/jbc.m006242200
[32]
Macara IG (2001) Transport into and out of the nucleus. Microbiol Mol Biol Rev 65: 570–594, table of contents.
[33]
Strom AC, Weis K (2001) Importin-beta-like nuclear transport receptors. Genome Biol 2: REVIEWS3008. doi: 10.1186/gb-2001-2-6-reviews3008
[34]
Dasso M (2001) Running on Ran: nuclear transport and the mitotic spindle. Cell 104: 321–324. doi: 10.1016/s0092-8674(01)00218-5
[35]
Gruss OJ, Carazo-Salas RE, Schatz CA, Guarguaglini G, Kast J, et al. (2001) Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell 104: 83–93. doi: 10.1016/s0092-8674(01)00193-3
[36]
Rensen WM, Mangiacasale R, Ciciarello M, Lavia P (2008) The GTPase Ran: regulation of cell life and potential roles in cell transformation. Front Biosci 13: 4097–4121. doi: 10.2741/2996
[37]
Dorfman J, Macara IG (2008) STRADalpha regulates LKB1 localization by blocking access to importin-alpha, and by association with Crm1 and exportin-7. Mol Biol Cell 19: 1614–1626. doi: 10.1091/mbc.e07-05-0454
[38]
Mingot JM, Bohnsack MT, Jakle U, Gorlich D (2004) Exportin 7 defines a novel general nuclear export pathway. EMBO J 23: 3227–3236. doi: 10.1038/sj.emboj.7600338
[39]
Aguirre-Portoles C, Bird AW, Hyman A, Canamero M, Perez de Castro I, et al. (2012) Tpx2 controls spindle integrity, genome stability, and tumor development. Cancer Res 72: 1518–1528. doi: 10.1158/0008-5472.can-11-1971
[40]
Winter SF, Lukes L, Walker RC, Welch DR, Hunter KW (2012) Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis. PLoS Genet 8: e1002735. doi: 10.1371/journal.pgen.1002735
[41]
Gruss OJ, Wittmann M, Yokoyama H, Pepperkok R, Kufer T, et al. (2002) Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells. Nat Cell Biol 4: 871–879. doi: 10.1038/ncb870
[42]
Ma Y, Lin D, Sun W, Xiao T, Yuan J, et al. (2006) Expression of targeting protein for xklp2 associated with both malignant transformation of respiratory epithelium and progression of squamous cell lung cancer. Clin Cancer Res 12: 1121–1127. doi: 10.1158/1078-0432.ccr-05-1766
[43]
Vainio P, Mpindi JP, Kohonen P, Fey V, Mirtti T, et al. (2012) High-throughput transcriptomic and RNAi analysis identifies AIM1, ERGIC1, TMED3 and TPX2 as potential drug targets in prostate cancer. PLoS One 7: e39801. doi: 10.1371/journal.pone.0039801
[44]
Etemadmoghadam D, George J, Cowin PA, Cullinane C, Kansara M, et al. (2010) Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer. PLoS One 5: e15498. doi: 10.1371/journal.pone.0015498
[45]
Ramakrishna M, Williams LH, Boyle SE, Bearfoot JL, Sridhar A, et al. (2010) Identification of candidate growth promoting genes in ovarian cancer through integrated copy number and expression analysis. PLoS One 5: e9983. doi: 10.1371/journal.pone.0009983
[46]
Roscioli E, Bolognesi A, Guarguaglini G, Lavia P (2010) Ran control of mitosis in human cells: gradients and local signals. Biochem Soc Trans 38: 1709–1714. doi: 10.1042/bst0381709
[47]
Scharer CD, Laycock N, Osunkoya AO, Logani S, McDonald JF, et al. (2008) Aurora kinase inhibitors synergize with paclitaxel to induce apoptosis in ovarian cancer cells. J Transl Med 6: 79. doi: 10.1186/1479-5876-6-79
[48]
Weaver BA, Cleveland DW (2005) Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death. Cancer Cell 8: 7–12. doi: 10.1016/j.ccr.2005.06.011
[49]
Kurisetty VV, Johnston PG, Johnston N, Erwin P, Crowe P, et al. (2008) RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin. Oncogene 27: 7139–7149. doi: 10.1038/onc.2008.325
[50]
Ly TK, Wang J, Pereira R, Rojas KS, Peng X, et al. (2010) Activation of the Ran GTPase is subject to growth factor regulation and can give rise to cellular transformation. J Biol Chem 285: 5815–5826. doi: 10.1074/jbc.m109.071886
