J Korean Med Sci.  2016 Aug;31(8):1215-1223. 10.3346/jkms.2016.31.8.1215.

OTX1 Contributes to Hepatocellular Carcinoma Progression by Regulation of ERK/MAPK Pathway

• Li H
• Miao Q
• Xu CW
• Huang JH
• Zhou YF
• Wu MJ

• KMID: 2373748
• DOI: http://doi.org/10.3346/jkms.2016.31.8.1215

Abstract

Orthodenticlehomeobox 1 (OTX1) overexpression had previously been associated with the progression of several tumors. The present study aimed to determine the expression and role of OTX1 in human hepatocellular carcinoma (HCC). The expression level of OTX1 was examined by quantitative real-time PCR (qRT-PCR) in 10 samples of HCC and paired adjacent non-cancerous tissues, and by immunohistochemistry (IHC) analysis in 128 HCC samples and matched controls. The relationship between OTX1 expression and the clinicopathological features werealso analyzed. Furthermore, the effects of OTX1 knockdown on cell proliferation and migration were determined in HCC cell lines. Axenograft mouse model was also established to investigate the role of OTX1 in HCC tumor growth. TheqRT-PCR and IHC analyses revealed that OTX1 was significantly elevated in HCC tissues compared with the paired non-cancerous controls. Expression of OTX1 was positively correlated with nodal metastasis status (P = 0.009) and TNM staging (P = 0.001) in HCC tissues. In addition, knockdown of OTX1 by shRNA significantly inhibited the proliferation and migration, and induced cell cycle arrest in S phase in vitro. Tumor growth was markedly inhibited by OTX1 silencing in the xenograft. Moreover, OTX1 silencing was causable for the decreased phosphorylation level of ERK/MAPK signaling. In conclusion, OTX1 contributes to HCC progression possibly by regulation of ERK/MAPK pathway. OTX1 may be a novel target for molecular therapy towards HCC.

Keyword

OTX1; Hepatocellular Carcinoma; Growth; Migration; ERK/MAPK

MeSH Terms

Aged
Animals
Blotting, Western
Carcinoma, Hepatocellular/metabolism/*pathology
Cell Line, Tumor
Cell Movement
Cell Proliferation
Disease Progression
Female
Gene Expression Regulation, Neoplastic
Humans
Immunohistochemistry
Liver/metabolism/pathology
Liver Neoplasms/metabolism/*pathology
Lymphatic Metastasis
MAP Kinase Signaling System
Male
Mice
Mice, Inbred BALB C
Mice, Nude
Middle Aged
Neoplasm Staging
Otx Transcription Factors/antagonists & inhibitors/genetics/*metabolism
Phosphorylation
RNA Interference
Real-Time Polymerase Chain Reaction
S Phase Cell Cycle Checkpoints
Transplantation, Heterologous
Otx Transcription Factors

Figure

• 

  Fig. 1 OTX1 is highly expressed in clinical HCC tissues. (A) qRT-PCR analysis showed that the average mRNA level of OTX1 in the tumor tissues were approximately two-fold increased compared with the paired non-cancerous tissues (P = 0.008). (B) Protein level of OTX1 was analyzed by IHC in a subset of 128 HCC tissues and the paired adjacent non-cancerous tissues. Staining result for each slide was graded as negative (−), weakly positive (+), moderate positive (++), and strongly positive (+++) according to the stain density. OTX1 was shown to be mainly located in the membrane and cytoplasm. The representative images that showed the markedly higher level of OTX1 in tumor tissues than non-cancerous tissues were provided. Magnification 400 ×.

• 

  Fig. 2 Knockdown efficiency of OTX1 shRNA was assessed in vitro. Initially, effects of transfection of the shRNA against OTX1 (shOTX1) on mRNA level of OTX1 were assessed in Hep G2 cells (A) and SMMC-7221 cells (B). shOTX1, instead of the scrambled shRNA significantly knocked the mRNA level of OTX1 down in both cell lines. The protein level of OTX1 was accordingly decreased after transfection of shOTX1 in both cell lines (C). * P < 0.01.

• 

  Fig. 3 Effects of OTX1 knockdown on cell viability. Knockdown of OTX1 by shRNA caused significantly lower proliferation rate in both Hep G2 cells (A) and SMMC-7221 cells (B). The proliferation rate was approximately half of the control cells by day 5. * P < 0.01.

• 

  Fig. 4 Knockdown of OTX1 impairs the colony formation ability in vitro. (A) Fewer colonies were visually observed in shOTX1-transfected Hep G2 cells and SMMC-7221 cells. (B) Quantification of the colony numbers showed that only average 30 colonies were formed in shOTX1-transfected Hep G2 cells (15% of the controls). About 50 colonies were formed in shOTX1-transfected SMMC-7221 cells, accounting for only 19% of the controls. * P < 0.01.

• 

  Fig. 5 Knockdown of OTX1 induces cell cycle arrest in S phase. Flow cytometry was employed to analyze the cell cycle distribution. Cell proportion in S phase was significantly increased, whereas cell proportion in G2/M phase was significantly decreased in shOTX1-transfected Hep G2 cells and SMMC-7221 cells. * P < 0.01.

• 

  Fig. 6 Knockdown of OTX1 inhibits tumor growth in a xenograft mouse model. A mouse model bearing the hepatic cancer was initially established using shOTX1-transfected Hep G2 cells (shRNA group) or negative control shRNA-transfected Hep G2 cells (NC group). (A) Periodic monitor of tumor dimensions in both the NC group and shRNA group. Average tumor volume from shRNA group was only 25% of control group by day 27. (B) Tumors were dissected and presented on day 27. (C) All tumor weights were determined and averaged for each group. The average tumor weight from the shRNA group was approximately 33% of the control group. * P < 0.05, † P < 0.01.

• 

  Fig. 7 Knockdown of OTX1 inhibits cell migration ability in vitro. Both Hep G2 cells and SMMC-7221 cells with distinct treatments were allowed to migrate in the upper chamber of a transwell for 12 hours. (A) After 12 hours, the under-surface of the upper chamber was stained with crystal violet. The stained cells were observed and photographed under a light microscopy. (B) Quantification of the migrated cell numbers showed that shOTX1-transfected cells exhibited significantly lower migration ability in both cell lines. Approximately 78% migration ability was decreased after knockdown of OTX1 in both Hep G2 and SMMC-7221 cells. * P < 0.01.

• 

  Fig. 8 Deregulation of ERK/MAPK pathway is associated with knockdown of OTX1. Knockdown of OTX1 caused the according decreases of phosphorylated MEK, ERK, MAPK and JNK without affecting the total MEK, ERK MAPK and JNK levels. ERK, extracellular signal regulated kinase. MAPK, mitogen-activated protein kinase. MEK, a MAPK kinase, also known as MAP2K, or dual-specificity mitogen-activated protein kinase kinase. JNK, c-Jun N-terminal kinase.

Reference

1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007; 132:2557–2576.

2. El-Serag HB, Kanwal F. Epidemiology of hepatocellular carcinoma in the United States: where are we? Where do we go? Hepatology. 2014; 60:1767–1775.

3. Bosch FX, Ribes J, Díaz M, Cléries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004; 127:S5–16.

4. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003; 362:1907–1917.

5. Shin JW, Chung YH. Molecular targeted therapy for hepatocellular carcinoma: current and future. World J Gastroenterol. 2013; 19:6144–6155.

6. Chan SL, Mok T, Ma BB. Management of hepatocellular carcinoma: beyond sorafenib. Curr Oncol Rep. 2012; 14:257–266.

7. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004; 64:7099–7109.

8. Chang YS, Adnane J, Trail PA, Levy J, Henderson A, Xue D, Bortolon E, Ichetovkin M, Chen C, McNabola A, et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol. 2007; 59:561–574.

9. Klein WH, Li X. Function and evolution of Otx proteins. Biochem Biophys Res Commun. 1999; 258:229–233.

10. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Simeone A. The role of Otx and Otp genes in brain development. Int J Dev Biol. 2000; 44:669–677.

11. Larsen KB, Lutterodt M, Rath MF, Møller M. Expression of the homeobox genes PAX6, OTX2, and OTX1 in the early human fetal retina. Int J Dev Neurosci. 2009; 27:485–492.

12. Pagani IS, Terrinoni A, Marenghi L, Zucchi I, Chiaravalli AM, Serra V, Rovera F, Sirchia S, Dionigi G, Miozzo M, et al. The mammary gland and the homeobox gene Otx1. Breast J. 2010; 16:Suppl 1. S53–6.

13. Omodei D, Acampora D, Russo F, De Filippi R, Severino V, Di Francia R, Frigeri F, Mancuso P, De Chiara A, Pinto A, et al. Expression of the brain transcription factor OTX1 occurs in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma. Am J Pathol. 2009; 175:2609–2617.

14. Zakrzewska M, Grešner SM, Zakrzewski K, Zalewska-Szewczyk B, Liberski PP. Novel gene expression model for outcome prediction in paediatric medulloblastoma. J Mol Neurosci. 2013; 51:371–379.

15. de Haas T, Oussoren E, Grajkowska W, Perek-Polnik M, Popovic M, Zadravec-Zaletel L, Perera M, Corte G, Wirths O, van Sluis P, et al. OTX1 and OTX2 expression correlates with the clinicopathologic classification of medulloblastomas. J Neuropathol Exp Neurol. 2006; 65:176–186.

16. Terrinoni A, Pagani IS, Zucchi I, Chiaravalli AM, Serra V, Rovera F, Sirchia S, Dionigi G, Miozzo M, Frattini A, et al. OTX1 expression in breast cancer is regulated by p53. Oncogene. 2011; 30:3096–3103.

17. Yu K, Cai XY, Li Q, Yang ZB, Xiong W, Shen T, Wang WY, Li YF. OTX1 promotes colorectal cancer progression through epithelial-mesenchymal transition. Biochem Biophys Res Commun. 2014; 444:1–5.

18. Li LL, Xue AM, Li BX, Shen YW, Li YH, Luo CL, Zhang MC, Jiang JQ, Xu ZD, Xie JH, et al. JMJD2A contributes to breast cancer progression through transcriptional repression of the tumor suppressor ARHI. Breast Cancer Res. 2014; 16:R56.

19. Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene. 2010; 29:4989–5005.

20. Wang S, Huang X, Li Y, Lao H, Zhang Y, Dong H, Xu W, Li JL, Li M. RN181 suppresses hepatocellular carcinoma growth by inhibition of the ERK/MAPK pathway. Hepatology. 2011; 53:1932–1942.

21. Pratilas CA, Solit DB. Targeting the mitogen-activated protein kinase pathway: physiological feedback and drug response. Clin Cancer Res. 2010; 16:3329–3334.

22. Villanueva A, Minguez B, Forner A, Reig M, Llovet JM. Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy. Annu Rev Med. 2010; 61:317–328.

23. Thomas MB, Abbruzzese JL. Opportunities for targeted therapies in hepatocellular carcinoma. J Clin Oncol. 2005; 23:8093–8108.

24. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008; 359:378–390.

25. Chen H, Sukumar S. Role of homeobox genes in normal mammary gland development and breast tumorigenesis. J Mammary Gland Biol Neoplasia. 2003; 8:159–175.

26. Jeon K, Lim H, Kim JH, Han D, Lee ER, Yang GM, Song MK, Kim JH, Cho SG. Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities. Biochim Biophys Acta. 2012; 1823:2190–2200.

27. Burotto M, Chiou VL, Lee JM, Kohn EC. The MAPK pathway across different malignancies: a new perspective. Cancer. 2014; 120:3446–3456.

28. Huynh H, Nguyen TT, Chow KH, Tan PH, Soo KC, Tran E. Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: its role in tumor progression and apoptosis. BMC Gastroenterol. 2003; 3:19.

29. Ito Y, Sasaki Y, Horimoto M, Wada S, Tanaka Y, Kasahara A, Ueki T, Hirano T, Yamamoto H, Fujimoto J, et al. Activation of mitogen-activated protein kinases/extracellular signal-regulated kinases in human hepatocellular carcinoma. Hepatology. 1998; 27:951–958.

30. Sun Y, Tang S, Jin X, Zhang C, Zhao W, Xiao X. Opposite effects of JNK and p38 MAPK signaling pathways on furazolidone-stimulated S phase cell cycle arrest of human hepatoblastoma cell line. Mutat Res. 2013; 755:24–29.

31. Sun Y, Tang S, Jin X, Zhang C, Zhao W, Xiao X. Involvement of the p38 MAPK signaling pathway in S-phase cell-cycle arrest induced by Furazolidone in human hepatoma G2 cells. J Appl Toxicol. 2013; 33:1500–1505.

32. Iguchi T, Miyakawa Y, Yamamoto K, Kizaki M, Ikeda Y. Nitrogen-containing bisphosphonates induce S-phase cell cycle arrest and apoptosis of myeloma cells by activating MAPK pathway and inhibiting mevalonate pathway. Cell Signal. 2003; 15:719–727.