In vitro effects of osteopontin on endometrial cancer cells and signaling pathways in epithelial-mesenchymal transition

This study aims to demonstrate in vitro effects of osteopontin (OPN), an extracellular matrix molecule, on proliferation and migration in human umbilical vein endothelial cells (HUVEC) and Ishikawa human endometrial adenocarcinoma cells (Ishikawa) cultures. In addition, the effects of OPN on phosphatidylinositol 3 kinase (PI3K) and extracellular signal regulatory kinase 1/2 (ERK1/2), which are involved signaling pathways in cell cycle, were investigated. This research was carried out as a prospective cell culture study in Aydın Adnan Menderes University Biochemistry Laboratory. Two types of cell culture were used, HUVEC representing endothelial cells and Ishikawa representing tumour cells. The HUVEC and Ishikawa were extracted from liquid nitrogen and seeded in culture medium. With the increasing concentrations of recombinant human OPN (rhOPN) in both cell lines, proliferation and migration of cells and PI3K and ERK1/2 levels in the medium were investigated. Detection of cell proliferation in these two cell types with MTT test, and migration assay were performed. Proliferation was found to increase in both cell types with increasing levels of rhOPN (p < 0.01). In both cell cultures, rhOPN induced mesenchymal transformation, indicating migration at the highest (400 ng/mL) concentrations. Increasing levels of PI3K and ERK1/2 were measured with the increasing concentrations of rhOPN (p < 0.01 and p < 0.05, respectively). Proliferation, migration, and PI3K and ERK1/2 molecules are increased with rhOPN in both cell cultures studied. This suggests that OPN could be a candidate prognostic marker in endometrial cancer.

Endometrial cancer; Extracellular signal regulatory kinase 1/2; Human umbilical vein endothelial cells; Ishikawa cells; Osteopontin; Phosphatidylinositol 3 kinase

[1] Braun MM, Overbeek-Wager EA, Grumbo RJ. Diagnosis and management of endometrial cancer. American Family Physician. 2016; 93: 468–474.

[2] Du X, Jiang T, Sheng X, Gao R, Li Q. Inhibition of osteopontin suppresses in vitro and in vivo angiogenesis in endometrial cancer. Gynecologic Oncology. 2009; 115: 371–376.

[3] Kerenidi T, Kazakou AP, Lada M, Tsilioni I, Daniil Z, Gourgoulianis KI. Clinical significance of circulating osteopontin levels in patients with lung cancer and correlation with VEGF and MMP-9. Cancer Investigation. 2016; 34: 385–392.

[4] Li Y, Xie Y, Cui D, Ma Y, Sui L, Zhu C, et al. Osteopontin promotes invasion, migration and epithelial-mesenchymal transition of human endometrial carcinoma cell HEC-1A through AKT and ERK1/2 signaling. Cellular Physiology and Biochemistry. 2015; 37: 1503–1512.

[5] Ramachandran S, Kwon K, Shin S, Kwon S, Cha S, Lee H, et al. Regulatory role of osteopontin in malignant transformation of endometrial cancer. Molecular Biology Reports. 2013; 40: 3623–3629.

[6] Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis assays: a critical overview. Clinical Chemistry. 2003; 49: 32–40.

[7] Grant DS, Yenisey C, Rose RW, Tootell M, Santra M, Iozzo RV. Decorin suppresses tumor cell-mediated angiogenesis. Oncogene. 2002; 21: 4765–4777.

[8] Norrby K. Angiogenesis: new aspects relating to its initiation and control. APMIS. 1997; 105: 417–437.

[9] Brooks PC. Cell adhesion molecules in angiogenesis. Cancer and Metastasis Reviews. 1996; 15: 187–194.

[10] Güllüoğlu MG, Dizdaroğlu F. Tumor angiogenesis. Turkish Journal of Surgery. 2000; 16: 279–290.

[11] Cho H, Kang ES, Kim YT, Kim J. Diagnostic and prognostic impact of osteopontin expression in endometrial cancer. Cancer Investigation. 2009; 27: 313–323.

[12] Wai PY, Kuo PC. The role of Osteopontin in tumor metastasis. Journal of Surgical Research. 2004; 121: 228–241.

[13] Yang M, Jiang C, Chen H, Nian Y, Bai Z, Ha C. The involvement of osteopontin and matrix metalloproteinase-9 in the migration of endometrial epithelial cells in patients with endometriosis. Reproductive Biology and Endocrinology. 2015; 13: 95.

[14] Kang Y, Forbes K, Carver J, Aplin JD. The role of the osteopontin-integrin αvβ3 interaction at implantation: functional analysis using three different in vitro models. Human Reproduction. 2014; 29: 739–749.

[15] Hahne JC, Meyer SR, Kranke P, Dietl J, Guckenberger M, Polat B, et al. Studies on the role of osteopontin-1 in endometrial cancer cell lines. Strahlentherapie Und Onkologie. 2013; 189: 1040–1048.

[16] Wang J, Wei Q, Wang X, Tang S, Liu H, Zhang F, et al. Transition to resistance: an unexpected role of the EMT in cancer chemoresistance. Genes & Diseases. 2016; 3: 3–6.

[17] Sun T, Qin Y, Zhong W. Epithelial-mesenchymal transition and its regulation in tumor metastasis. In Xu K (ed.) Tumor metastasis (pp. 217–239). 1st edn. IntechOpen: Rijeka. 2016.

[18] Fedarko NS, Jain A, Karadag A, Van Eman MR, Fisher LW. Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clinical Cancer Research. 2001; 7: 4060–4066.

[19] Carvalho MS, Cabral JM, da Silva CL, Vashishth D. Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties. Journal of Cellular Biochemistry. 2019; 120: 6555–6569.

[20] Bast RC, Raamanathan A, Gentry-Maharaj A, Skates S, Baggerly KA, Fourkala R, et al. Validation of a multi-marker panel for early detection of ovarian cancer. Journal of Clinical Oncology. 2016; 34: 5570–5570.

[21] Guo J, Yang WL, Pak D, Celestino J, Lu KH, Ning J, et al. Osteopontin, macrophage migration inhibitory factor and anti-interleukin-8 autoantibodies complement CA125 for detection of early stage ovarian cancer. Cancers. 2019; 11: 596.

[22] Rani S, Sehgal A, Kaur J, Pandher D, Punia R. Osteopontin as a tumor marker in ovarian cancer. Journal of Mid-Life Health. 2022; 13: 200.

[23] Al-Maghrabi H, Gomaa W, Al-Maghrabi J. Increased osteopontin expression in endometrial carcinoma is associated with better survival outcome: Ginekologia Polska. 2020; 91: 73–78.

[24] Shi L, Hou J, Wang L, Fu H, Zhang Y, Song Y, et al. Regulatory roles of osteopontin in human lung cancer cell epithelial-to-mesenchymal transitions and responses. Clinical and Translational Medicine. 2021; 11: e486.

[25] Qin S, Yi L, Liang Y, Chen Y, Wang W, Liao Y, et al. Biological and clinicopathological characteristics of OPN in cervical cancers. Frontiers in Genetics. 2022; 13: 836509.

[26] Poleboyina PK, Alagumuthu M, Pasha A, Ravinder D, Pasumarthi D, Pawar SC. Entrectinib a plausible inhibitor for osteopontin (SPP1) in cervical cancer—integrated bioinformatic approach. Applied Biochemistry and Biotechnology. 2023; 195: 7766–7795.

[27] Kariya Y, Kariya Y. Osteopontin in cancer: mechanisms and therapeutic targets. International Journal of Translational Medicine. 2022; 2: 419–447.