Korean J Physiol Pharmacol.  2023 Sep;27(5):457-470. 10.4196/kjpp.2023.27.5.457.

Kinesin superfamily member 15 knockdown inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma

• Cai Y
• Lai Q
• Zhang X
• Zhang Y
• Zhang M
• Gu S
• Qin Y
• Hou J
• Zhao L

• KMID: 2545536
• DOI: http://doi.org/10.4196/kjpp.2023.27.5.457

Abstract

The aim of this study was to investigate the role of kinesin superfamily member 15 (KIF15) in nasopharyngeal carcinogenesis (NPC) and explore its underlying mechanisms. We employed various assays, including the CCK-8 assay, flow cytometry, the Transwell and scratch assay, Western blotting, and nude mice transplantation tumor, to investigate the impact of KIF15 on NPC. Our findings demonstrate that KIF15 plays a critical role in the proliferation, apoptosis, migration, and invasion of NPC cells. Furthermore, we discovered that silencing KIF15 inhibits cell proliferation, migration, and invasion while promoting apoptosis, and that KIF15's effect on NPC cell growth is mediated through the PI3K/AKT and P53 signaling pathways. Additionally, we showed that KIF15 promotes nasopharyngeal cancer cell growth in vivo. Our study sheds light on the significance of KIF15 in NPC by revealing that KIF15 knockdown inhibits NPC cell growth through the regulation of AKT-related signaling pathways. These findings suggest that KIF15 represents a promising therapeutic target for the prevention and treatment of NPC.

Keyword

Cell proliferation; Kinesins; Nasopharyngeal carcinoma

Figure

• 

  Fig. 1 Expression of KIF15 in normal nasopharyngeal epithelial cells and NPC cell lines. The mRNA (A) and protein (B) expression of KIF15 were detected in different cell lines (NP69, C666-1, CNE-2, SUNE-1, HONE-1, and 5-8F). Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma. *p < 0.05 vs. the NP69 group, n = 5.

• 

  Fig. 2 Effect of KIF15 knockdown on the proliferation of NPC cell lines. The mRNA (A) and protein expression (C, D) of KIF15 were detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. The mRNA (B) and protein expression (C, E) of KIF15 were detected in KIF15-shRNA/5-8F and ScrshRNA/5-8F cell lines. The cell viability (F) and relative colony formation (stained with 0.5% crystal violet staining solution) (H, I) was detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. The cell viability (G) and relative colony formation (stained with 0.5% crystal violet staining solution) (J, K) was detected in KIF15-shRNA/5-8F and ScrshRNA/5-8F cell lines. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma. **p < 0.01 vs. the shcontrol group, n = 5.

• 

  Fig. 3 KIF15 knockdown induces apoptosis in NPC cells. (A, B) The flow cytometric analysis was detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. (C, D) The flow cytometric analysis was detected in KIF15-shRNA/5-8F and ScrshRNA/5-8F cell lines. The mRNA expression of Bax (E), Bcl-2 (G), and Bax/Bcl-2 (I) were detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. The mRNA expression of Bax (F), Bcl-2 (H), and Bax/Bcl-2 (J) were detected in KIF15-shRNA/5-8F and ScrshRNA/5-8F cell lines. The protein expression of cleaved-caspase 3 was detected in CNE-2 (K) and 5-8F (L) cells. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma; ns, no significance. *p < 0.05 and **p < 0.01 vs. the shcontrol group, n = 5.

• 

  Fig. 4 Effect of KIF15 knockdown on migration and invasion of NPC cell lines. Representative images of transwell-migration assays of CNE-2 (A) and 5-8F (B) cells and (C, D) quantification of cell migration expressed by cell counting. Representative images of scratch assay performed in CNE-2 (E) and 5-8F (F) cells. (G, H) The mobility (%) was measured 24 h after cells were scratched. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma; transwell assays: stained with 0.1% crystal violet staining solution; original magnification ×200. *p < 0.05 and **p < 0.01 vs. the shcontrol group, n = 5.

• 

  Fig. 5 Effect of KIF15 knockdown on AKT and p53-dependent pathway of NPC cell lines. (A) The protein expression of p-AKT, AKT (B), P53 (C), MMP2 (E), and MMP9 (G) were detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. (H) The protein expression of p-AKT, AKT (I), P53 (J), MMP2 (L), and MMP9 (N) were detected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines. The mRNA expression of MMP2 (D, F) and MMP9 (K, M) were detected. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma. *p < 0.05 and **p < 0.01 vs. the shcontrol group, n = 5.

• 

  Fig. 6 KIF15 knockdown induces apoptosis in NPC cells through activation of p53. CNE-2 (A) or 5-8F (B) cells were transfected with sip53 or negative control (NC) for 48 h, the mRNA expression of p53 was detected. KIF15-shRNA/CNE-2 (C, E) or KIF15-shRNA/5-8F (D, F) cells were transfected with sip53 or NC. The flow cytometric analysis was detected. The mRNA expression of bax (G, H), bcl-2 (I, J) and bax/bcl-2 (K, L) were detected. The protein expression of cleaved-caspase 3 was detected in CNE-2 (M) and 5-8F (N) cells. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma. *p < 0.05 and **p < 0.01 vs. the KIF15-shRNA-NC group, n = 5.

• 

  Fig. 7 Effect of KIF15 knockdown on NPC cell growth in vivo. (A, B) Mice and tumors in the different groups were shown. Tumor volume (C) and body weight (D) were recorded. (E, F) Representative in vivo images of luminescence shown in the livers of the live nude mouse was injected in KIF15-shRNA/CNE-2 and ScrshRNA/CNE-2 cell lines, respectively. Values are presented as mean ± SD. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma. **p < 0.01 vs. the shcontrol group, n = 5.

• 

  Fig. 8 Molecular mechanism of KIF15 on NPC development. KIF15, kinesin superfamily member 15; NPC, nasopharyngeal carcinoma.

Reference

1. Tang SQ, Mao YP, Xu C, Guo R, Li WF, Tang LL, Sun Y, Ma J. 2020; The evolution of the nasopharyngeal carcinoma staging system over a 10-year period: implications for future revisions. Chin Med J (Engl). 133:2044–2053. DOI: 10.1097/CM9.0000000000000978. PMID: 32810045. PMCID: PMC7478675. PMID: 45b20d7da1104cda981fe16dd2ef952e.

2. Hung CC, Tu MY, Chien TW, Lin CY, Chow JC, Chou W. 2023; The model of descriptive, diagnostic, predictive, and prescriptive analytics on 100 top-cited articles of nasopharyngeal carcinoma from 2013 to 2022: bibliometric analysis. Medicine (Baltimore). 102:e32824. DOI: 10.1097/MD.0000000000032824. PMID: 36820592. PMCID: PMC9907932.

3. Pontoriero F, Silverman AM, Pascasio JM, Bajaj R. 2020; Nonkeratinizing nasopharyngeal carcinoma, undifferentiated type with trisomy 2: a case report and short review of cytogenetic and molecular literature. Pediatr Dev Pathol. 23:448–452. DOI: 10.1177/1093526620945861. PMID: 32755442.

4. Lam WKJ, Chan JYK. 2018; Recent advances in the management of nasopharyngeal carcinoma. F1000Res. 7:F1000 Faculty Rev-1829. DOI: 10.12688/f1000research.15066.1. PMID: 30519454. PMCID: PMC6249636. PMID: 8086e0a2c60c441698858e8bc2691c39.

5. Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. 2019; Nasopharyngeal carcinoma. Lancet. 394:64–80. DOI: 10.1016/S0140-6736(19)30956-0. PMID: 31178151.

6. Lee HM, Okuda KS, González FE, Patel V. 2019; Current perspectives on nasopharyngeal carcinoma. Adv Exp Med Biol. 1164:11–34. DOI: 10.1007/978-3-030-22254-3_2. PMID: 31576537.

7. Lee YT, Tan YJ, Oon CE. 2018; Molecular targeted therapy: treating cancer with specificity. Eur J Pharmacol. 834:188–196. DOI: 10.1016/j.ejphar.2018.07.034. PMID: 30031797.

8. Hirokawa N, Noda Y, Tanaka Y, Niwa S. 2009; Kinesin superfamily motor proteins and intracellular transport. Nat Rev Mol Cell Biol. 10:682–696. DOI: 10.1038/nrm2774. PMID: 19773780.

9. Huo D, Yang H, Huang JD, Cai JP, Cui J. 2021; Roles of kinesin superfamily proteins in colorectal cancer carcinogenesis (Review). Oncol Rep. 46:121. DOI: 10.3892/or.2021.8072. PMID: 33955521.

10. Niwa S. 2015; Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis. Anat Sci Int. 90:1–6. DOI: 10.1007/s12565-014-0259-5. PMID: 25347970.

11. Wang Z, Chen M, Fang X, Hong H, Yao Y, Huang H. 2021; KIF15 is involved in development and progression of Burkitt lymphoma. Cancer Cell Int. 21:261. DOI: 10.1186/s12935-021-01967-z. PMID: 33985517. PMCID: PMC8117549. PMID: dbca50a309e04f1389cba3241fd2e9fd.

12. Sun RF, He N, Zhang GY, Yu ZY, Li LS, Ma ZJ, Jiao ZY. 2023; Combined inhibition of KIF11 and KIF15 as an effective therapeutic strategy for gastric cancer. Curr Cancer Drug Targets. 23:293–306. DOI: 10.2174/1568009622666220616122846. PMID: 35713129.

13. Klejnot M, Falnikar A, Ulaganathan V, Cross RA, Baas PW, Kozielski F. 2014; The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development. Acta Crystallogr D Biol Crystallogr. 70(Pt 1):123–133. DOI: 10.1107/S1399004713028721. PMID: 24419385. PMCID: PMC3919264.

14. Gao X, Zhu L, Lu X, Wang Y, Li R, Jiang G. 2020; KIF15 contributes to cell proliferation and migration in breast cancer. Hum Cell. 33:1218–1228. DOI: 10.1007/s13577-020-00392-0. PMID: 32578050.

15. Sun YF, Wu HL, Shi RF, Chen L, Meng C. 2020; KIF15 promotes proliferation and growth of hepatocellular carcinoma. Anal Cell Pathol (Amst). 2020:6403012. DOI: 10.1155/2020/6403012. PMID: 32318326. PMCID: PMC7157793. PMID: 56c6e1dbeba04ef68252ee05e62e5a77.

16. Gao L, Zhang W, Zhang J, Liu J, Sun F, Liu H, Hu J, Wang X, Wang X, Su P, Chen S, Qu S, Shi B, Xiong X, Chen W, Dong X, Han B. 2021; KIF15-mediated stabilization of AR and AR-V7 contributes to enzalutamide resistance in prostate cancer. Cancer Res. 81:1026–1039. DOI: 10.1158/0008-5472.CAN-20-1965. PMID: 33277366.

17. Perri F, Della Vittoria Scarpati G, Caponigro F, Ionna F, Longo F, Buonopane S, Muto P, Di Marzo M, Pisconti S, Solla R. 2019; Management of recurrent nasopharyngeal carcinoma: current perspectives. Onco Targets Ther. 12:1583–1591. DOI: 10.2147/OTT.S188148. PMID: 30881013. PMCID: PMC6396653.

18. Vasudevan HN, Yom SS. 2021; Nasopharyngeal carcinoma and its association with Epstein-Barr virus. Hematol Oncol Clin North Am. 35:963–971. DOI: 10.1016/j.hoc.2021.05.007. PMID: 34187713.

19. Yu Y, Feng YM. 2010; The role of kinesin family proteins in tumorigenesis and progression: potential biomarkers and molecular targets for cancer therapy. Cancer. 116:5150–5160. DOI: 10.1002/cncr.25461. PMID: 20661912.

20. Singel SM, Cornelius C, Zaganjor E, Batten K, Sarode VR, Buckley DL, Peng Y, John GB, Li HC, Sadeghi N, Wright WE, Lum L, Corson TW, Shay JW. 2014; KIF14 promotes AKT phosphorylation and contributes to chemoresistance in triple-negative breast cancer. Neoplasia. 16:247–256.e2. DOI: 10.1016/j.neo.2014.03.008. PMID: 24784001. PMCID: PMC4094827. PMID: 6c64beef889741d38f469bb388b70ed5.

21. Shimo A, Tanikawa C, Nishidate T, Lin ML, Matsuda K, Park JH, Ueki T, Ohta T, Hirata K, Fukuda M, Nakamura Y, Katagiri T. 2008; Involvement of kinesin family member 2C/mitotic centromere-associated kinesin overexpression in mammary carcinogenesis. Cancer Sci. 99:62–70. DOI: 10.1111/j.1349-7006.2007.00635.x. PMID: 17944972.

22. Nagahara M, Nishida N, Iwatsuki M, Ishimaru S, Mimori K, Tanaka F, Nakagawa T, Sato T, Sugihara K, Hoon DS, Mori M. 2011; Kinesin 18A expression: clinical relevance to colorectal cancer progression. Int J Cancer. 129:2543–2552. DOI: 10.1002/ijc.25916. PMID: 21213216.

23. Ding L, Li B, Yu X, Li Z, Li X, Dang S, Lv Q, Wei J, Sun H, Chen H, Liu M, Li G. 2020; KIF15 facilitates gastric cancer via enhancing proliferation, inhibiting apoptosis, and predict poor prognosis. Cancer Cell Int. 20:125. DOI: 10.1186/s12935-020-01199-7. PMID: 32322172. PMCID: PMC7160940. PMID: 477878391acb4aaebcaecb0a53c96a89.

24. Zheng S, Tang D, Wang X, Liu C, Zuo N, Yan R, Wu C, Ma J, Wang C, Xu H, He Y, Liu D, Liu S. 2022; Kif15 is required in the development of auditory system using zebrafish as a model. Front Mol Neurosci. 15:844568. DOI: 10.3389/fnmol.2022.844568. PMID: 35370541. PMCID: PMC8971910. PMID: 459e99e04b934a66bd00f63a2bbe352f.

25. Xu M, Liu D, Dong Z, Wang X, Wang X, Liu Y, Baas PW, Liu M. 2014; Kinesin-12 influences axonal growth during zebrafish neural development. Cytoskeleton (Hoboken). 71:555–563. DOI: 10.1002/cm.21193. PMID: 25250533. PMCID: PMC4236235.

26. Wang J, Wang D, Fei Z, Feng D, Zhang B, Gao P, Hu G, Li W, Huang X, Chen D, Ding X, Wu W. 2021; KIF15 knockdown suppresses gallbladder cancer development. Eur J Cell Biol. 100:151182. DOI: 10.1016/j.ejcb.2021.151182. PMID: 34781077.

27. Wade M, Li YC, Wahl GM. 2013; MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer. 13:83–96. DOI: 10.1038/nrc3430. PMID: 23303139. PMCID: PMC4161369.

28. Blagih J, Buck MD, Vousden KH. 2020; p53, cancer and the immune response. J Cell Sci. 133:jcs237453. DOI: 10.1242/jcs.237453. PMID: 32144194.

29. Kale J, Osterlund EJ, Andrews DW. 2018; BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ. 25:65–80. DOI: 10.1038/cdd.2017.186. PMID: 29149100. PMCID: PMC5729540.

30. Weng C, Chen Y, Wu Y, Liu X, Mao H, Fang X, Li B, Wang L, Guan M, Liu G, Lu L, Yuan Y. 2019; Silencing UBE4B induces nasopharyngeal carcinoma apoptosis through the activation of caspase3 and p53. Onco Targets Ther. 12:2553–2561. DOI: 10.2147/OTT.S196132. PMID: 31040698. PMCID: PMC6459139.

31. Wang Z, Liao K, Zuo W, Liu X, Qiu Z, Gong Z, Liu C, Zeng Q, Qian Y, Jiang L, Bu Y, Hong S, Hu G. 2017; Depletion of NFBD1/MDC1 induces apoptosis in nasopharyngeal carcinoma cells through the p53-ROS-mitochondrial pathway. Oncol Res. 25:123–136. DOI: 10.3727/096504016X14732772150226. PMID: 28081741. PMCID: PMC7840771.

32. Jiang S, Huang C, Zheng G, Yi W, Wu B, Tang J, Liu X, Huang B, Wu D, Yan T, Li M, Wan C, Cai Y. 2022; EGCG inhibits proliferation and induces apoptosis through downregulation of SIRT1 in nasopharyngeal carcinoma cells. Front Nutr. 9:851972. DOI: 10.3389/fnut.2022.851972. PMID: 35548580. PMCID: PMC9084317. PMID: 6b11df6f0e6347b89f84c58a0682b4e7.

33. Hernández Borrero LJ, El-Deiry WS. 2021; Tumor suppressor p53: biology, signaling pathways, and therapeutic targeting. Biochim Biophys Acta Rev Cancer. 1876:188556. DOI: 10.1016/j.bbcan.2021.188556. PMID: 33932560. PMCID: PMC8730328.

34. Junttila MR, Evan GI. 2009; p53--a Jack of all trades but master of none. Nat Rev Cancer. 9:821–829. DOI: 10.1038/nrc2728. PMID: 19776747.

35. Moreira J, Almeida J, Saraiva L, Cidade H, Pinto M. 2021; Chalcones as promising antitumor agents by targeting the p53 pathway: an overview and new insights in drug-likeness. Molecules. 26:3737. DOI: 10.3390/molecules26123737. PMID: 34205272. PMCID: PMC8233907. PMID: 6b9d17e5acf149adb7d23cb2cbf99477.

36. Kruse JP, Gu W. 2009; Modes of p53 regulation. Cell. 137:609–622. DOI: 10.1016/j.cell.2009.04.050. PMID: 19450511. PMCID: PMC3737742.

37. Mayo LD, Donner DB. 2001; A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A. 98:11598–11603. DOI: 10.1073/pnas.181181198. PMID: 11504915. PMCID: PMC58775.

38. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC. 2001; HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol. 3:973–982. Erratum in: Nat Cell Biol. 2002; 4:736. DOI: 10.1038/ncb1101-973. PMID: 11715018.

39. Zanotelli MR, Zhang J, Reinhart-King CA. 2021; Mechanoresponsive metabolism in cancer cell migration and metastasis. Cell Metab. 33:1307–1321. DOI: 10.1016/j.cmet.2021.04.002. PMID: 33915111. PMCID: PMC9015673.

40. Liang W, Shi J, Xia H, Wei X. 2021; A novel ruthenium-fluvastatin complex downregulates SNCG expression to modulate breast carcinoma cell proliferation and apoptosis via activating the PI3K/Akt/mTOR/VEGF/MMP9 pathway. Oxid Med Cell Longev. 2021:5537737. DOI: 10.1155/2021/5537737. PMID: 34221232. PMCID: PMC8221895.

41. Zhu W, Wu X, Yang B, Yao X, Cui X, Xu P, Chen X. 2019; miR-188-5p regulates proliferation and invasion via PI3K/Akt/MMP-2/9 signaling in keloids. Acta Biochim Biophys Sin (Shanghai). 51:185–196. Erratum in: Acta Biochim Biophys Sin (Shanghai). 2019;51:980. DOI: 10.1093/abbs/gmy165. PMID: 30668826.

42. Zhu Y, Yan L, Zhu W, Song X, Yang G, Wang S. 2019; MMP2/3 promote the growth and migration of laryngeal squamous cell carcinoma via PI3K/Akt-NF-κB-mediated epithelial-mesenchymal transformation. J Cell Physiol. 234:15847–15855. DOI: 10.1002/jcp.28242. PMID: 30714134.

43. Li HL, Deng NH, He XS, Li YH. 2022; Small biomarkers with massive impacts: PI3K/AKT/mTOR signalling and microRNA crosstalk regulate nasopharyngeal carcinoma. Biomark Res. 10:52. DOI: 10.1186/s40364-022-00397-x. PMID: 35883139. PMCID: PMC9327212. PMID: 5e5616c222d647368dacc772e1aca5c1.

44. Xie X, Xiong G, Chen W, Fu H, Li M, Cui X. 2020; FOXD3 inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma through regulation of the PI3K-Akt pathway. Biochem Cell Biol. 98:653–660. DOI: 10.1139/bcb-2020-0011. PMID: 32459973.

45. Fan X, Xie X, Yang M, Wang Y, Wu H, Deng T, Weng X, Wen W, Nie G. 2021; YBX3 mediates the metastasis of nasopharyngeal carcinoma via PI3K/AKT signaling. Front Oncol. 11:617621. DOI: 10.3389/fonc.2021.617621. PMID: 33816248. PMCID: PMC8010247. PMID: 358fd794b7514d329b00141c1817b77b.

46. Ho HC, Huang CC, Lu YT, Yeh CM, Ho YT, Yang SF, Hsin CH, Lin CW. 2019; Epigallocatechin-3-gallate inhibits migration of human nasopharyngeal carcinoma cells by repressing MMP-2 expression. J Cell Physiol. 234:20915–20924. DOI: 10.1002/jcp.28696. PMID: 31012106.

47. Luo Y, Zhang B, Xu L, Li M, Wu J, Zhou Y, Li Y. 2022; Downregulation of KIF15 inhibits the tumorigenesis of non-small-cell lung cancer via inactivating Raf/MEK/ERK signaling. Histol Histopathol. 37:269–285. DOI: 10.14670/HH-18-408. PMID: 34908156.

48. Ge W, Chen Y, Guo Y, Zhao D, Mu L, Zhang K, Zhuo W. 2021; KIF15 upregulation promotes leiomyosarcoma cell growth via promoting USP15-mediated DEK deubiquitylation. Biochem Biophys Res Commun. 570:117–124. DOI: 10.1016/j.bbrc.2021.07.042. PMID: 34280614.