Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion

  • Seong-Lan Yu
  • Han Koo
  • Hoi Young Lee
  • Young Il Yeom
  • Dong Chul LeeEmail author
  • Jaeku KangEmail author
Original Paper



Previously, it has been reported that homeobox A9 (HOXA9) protein expression is downregulated in lung cancer cells, and that its expression is inversely correlated with the metastatic potential of lung cancer cells both in vitro and in vivo. As such, HOXA9 shows therapeutic potential. The development of therapeutic strategies based on this protein is, however, limited due to its poor membrane permeability. To overcome this problem, we developed a system to deliver HOXA9 protein into non-small cell lung cancer (NSCLC) cells.


First, we constructed a delivery vector expressing polyarginine, a cell-penetrating peptide, as well as HOXA9. The resulting recombinant R10-HOXA9 protein was effectively introduced into A549 and NCI-H1299 NSCLC cells. Next, we examined the roles and molecular mechanisms of recombinant R10-HOXA9 in processes involved in tumor progression. To investigate the therapeutic efficacy of the delivery system, we performed cell motility assays using both in vitro and in vivo experimental models.


We found that recombinant R10-HOXA9 protein reduced the invasion and migration rate, but not the proliferation rate, of the NSCLC cells tested, both in vitro and in vivo. Treatment of NSCLC cells with recombinant R10-HOXA9 protein led to a significant increase in E-cadherin expression. Conversely, we found that the expression of snail family zinc finger 2 (SLUG), a transcriptional repressor of E-cadherin, was markedly decreased. In an experimental metastatic mouse model, recombinant R10-HOXA9 protein was found to effectively reduce the rate of lung cancer cell motility.


Our data suggest that the developed cell-permeable R10-HOXA9 system may serve as a useful tool to prevent NSCLC cell migration and invasion.


Non-small cell lung cancer Invasion Migration Homeobox A9 Cell-penetrating peptide 



This work was supported by grants from the National Research Foundation of Korea (NRF-2017R1A2B4003684) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A6A1A03015713).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures involving animal experiments were approved by the Animal Research Ethics Committee of the Korea Research Institute of Bioscience and Biotechnology.

Supplementary material

13402_2019_424_MOESM1_ESM.docx (13 kb)
Supplementary Fig. 1 Generation of recombinant cell-permeable R10-HOXA9 protein. (a) Construction of the HOXA9 open reading frame or the fusion of ten arginine residues (R10) and the HOXA9 open reading frame using EcoRI and NotI sites in the pET-41a(+) vector. (b) DNA gel electrophoresis after the NotI and EcoRI digestion of subclones. (c) Identification of recombinant C-HOXA9 and R10-HOXA9 expression by IPTG induction using Coomassie blue-stained SDS-PAGE. (d) Recombinant C-HOXA9 and R10-HOXA9 protein were purified using Ni-NTA beads. The purified proteins were identified by Coomassie blue staining (left panel) and immunoblotting using an anti-HOXA9 antibody (right panel). (DOCX 13 kb)
13402_2019_424_MOESM2_ESM.pdf (342 kb)
ESM 2 (PDF 342 kb)


  1. 1.
    U. Thorsteinsdottir, A. Mamo, E. Kroon, L. Jerome, J. Bijl, H.J. Lawrence, K. Humphries, G. Sauvageau, Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 99, 121–129 (2002)CrossRefGoogle Scholar
  2. 2.
    M. Pojo, C.S. Goncalves, A. Xavier-Magalhaes, A.I. Oliveira, T. Goncalves, S. Correia, A.J. Rodrigues, S. Costa, L. Pinto, A.A. Pinto, J.M. Lopes, R.M. Reis, M. Rocha, N. Sousa, B.M. Costa, A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide. Oncotarget 6, 7657–7674 (2015)CrossRefGoogle Scholar
  3. 3.
    S.Y. Ko, A. Ladanyi, E. Lengyel, H. Naora, Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype. Am. J. Pathol. 184, 271–281 (2014)CrossRefGoogle Scholar
  4. 4.
    P.M. Gilbert, J.K. Mouw, M.A. Unger, J.N. Lakins, M.K. Gbegnon, V.B. Clemmer, M. Benezra, J.D. Licht, N.J. Boudreau, K.K. Tsai, A.L. Welm, M.D. Feldman, B.L. Weber, V.M. Weaver, HOXA9 regulates BRCA1 expression to modulate human breast tumor phenotype. J. Clin. Invest. 120, 1535–1550 (2010)CrossRefGoogle Scholar
  5. 5.
    L. Alvarado-Ruiz, M.G. Martinez-Silva, L.A. Torres-Reyes, P. Pina-Sanchez, P. Ortiz-Lazareno, A. Bravo-Cuellar, A. Aguilar-Lemarroy, L.F. Jave-Suarez, HOXA9 is underexpressed in cervical cancer cells and its restoration decreases proliferation, migration and expression of epithelial-to-mesenchymal transition genes. Asian Pac. J. Cancer Prev. 17, 1037–1047 (2016)CrossRefGoogle Scholar
  6. 6.
    F. Li, L. Dong, X. Qu, K. Li, H. Wang, L. Cheng, H. He, L. Wang, Z. Qi, H. Ren, S. Jin, Down-expression of homeobox A9 promotes cancer cell invasion in human hepatocellular carcinomas. Int. J. Clin. Exp. Pathol. 9, 563–575 (2016)Google Scholar
  7. 7.
    J.A. Hwang, B.B. Lee, Y. Kim, S.H. Hong, Y.H. Kim, J. Han, Y.M. Shim, C.Y. Yoon, Y.S. Lee, D.H. Kim, HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer. Mol. Carcinog. 54, E72–E80 (2015)CrossRefGoogle Scholar
  8. 8.
    J.W. Son, K.J. Jeong, W.S. Jean, S.Y. Park, S. Jheon, H.M. Cho, C.G. Park, H.Y. Lee, J. Kang, Genome-wide combination profiling of DNA copy number and methylation for deciphering biomarkers in non-small cell lung cancer patients. Cancer Lett. 311, 29–37 (2011)CrossRefGoogle Scholar
  9. 9.
    S.H. Hwang, K.U. Kim, J.E. Kim, H.H. Kim, M.K. Lee, C.H. Lee, S.Y. Lee, T. Oh, S. An, Detection of HOXA9 gene methylation in tumor tissues and induced sputum samples from primary lung cancer patients. Clin. Chem. Lab. Med. 49, 699–704 (2011)Google Scholar
  10. 10.
    S.L. Yu, D.C. Lee, H.A. Sohn, S.Y. Lee, H.S. Jeon, J.H. Lee, C.G. Park, H.Y. Lee, Y.I. Yeom, J.W. Son, Y.S. Yoon, J. Kang, Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear factor-kappa B activity in non-small cell lung cancer cells. Mol. Carcinog. 55, 1915–1926 (2016)CrossRefGoogle Scholar
  11. 11.
    V. Gopal, Bioinspired peptides as versatile nucleic acid delivery platforms. J. Control. Release 167, 323–332 (2013)CrossRefGoogle Scholar
  12. 12.
    H.J. Kim, M.H. Kim, J.T. Kim, W.J. Lee, E. Kim, K.S. Lim, J.K. Kim, Y.I. Yang, K.D. Park, Y.H. Kim, Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction. J. Control. Release 215, 55–72 (2015)CrossRefGoogle Scholar
  13. 13.
    H. He, L. Sun, J. Ye, E. Liu, S. Chen, Q. Liang, M.C. Shin, V.C. Yang, Enzyme-triggered, cell penetrating peptide-mediated delivery of anti-tumor agents. J. Control. Release 240, 67–76 (2016)CrossRefGoogle Scholar
  14. 14.
    T. Skotland, T.G. Iversen, M.L. Torgersen, K. Sandvig, Cell-penetrating peptides: possibilities and challenges for drug delivery in vitro and in vivo. Molecules 20, 13313–13323 (2015)CrossRefGoogle Scholar
  15. 15.
    D.A. Mann, A.D. Frankel, Endocytosis and targeting of exogenous HIV-1 tat protein. EMBO J. 10, 1733–1739 (1991)CrossRefGoogle Scholar
  16. 16.
    P.A. Wender, D.J. Mitchell, K. Pattabiraman, E.T. Pelkey, L. Steinman, J.B. Rothbard, The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U. S. A. 97, 13003–13008 (2000)CrossRefGoogle Scholar
  17. 17.
    A. Bolhassani, B.S. Jafarzade, G. Mardani, In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides. Peptides 87, 50–63 (2017)CrossRefGoogle Scholar
  18. 18.
    U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)CrossRefGoogle Scholar
  19. 19.
    J.H. Cho, Y.M. You, Y. Il Yeom, D.C. Lee, B.K. Kim, M. Won, B.C. Cho, M. Kang, S. Park, S.J. Yang, J.S. Kim, J.A. Kim, K.C. Park, RNF25 promotes gefitinib resistance in EGFR-mutant NSCLC cells by inducing NF-kappaB-mediated ERK reactivation. Cell Death Dis. 9, 587 (2018)CrossRefGoogle Scholar
  20. 20.
    Y. Xia, S. Shen, I.M. Verma, NF-kappaB, an active player in human cancers. Cancer Immunol. Res. 2, 823–830 (2014)CrossRefGoogle Scholar
  21. 21.
    G. Guidotti, L. Brambilla, D. Rossi, Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol. Sci. 38, 406–424 (2017)CrossRefGoogle Scholar
  22. 22.
    D.J. Mitchell, D.T. Kim, L. Steinman, C.G. Fathman, J.B. Rothbard, Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56, 318–325 (2000)CrossRefGoogle Scholar
  23. 23.
    G. Tunnemann, G. Ter-Avetisyan, R.M. Martin, M. Stockl, A. Herrmann, M.C. Cardoso, Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J. Pept. Sci. 14, 469–476 (2008)CrossRefGoogle Scholar
  24. 24.
    J.T. Hsieh, J. Zhou, C. Gore, P. Zimmern, R11, a novel cell-permeable peptide, as an intravesical delivery vehicle. BJU Int. 108, 1666–1671 (2011)CrossRefGoogle Scholar
  25. 25.
    T. Zhang, K. Wu, C. Ding, K. Sun, Z. Guan, X. Wang, J.T. Hsieh, D. He, J. Fan, Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus. Oncotarget 6, 37782–37791 (2015)Google Scholar
  26. 26.
    W. Wang, N. Zhang, T. Zhao, M. Liu, T. Zhang, D. Li, Inhibition of tumor growth by polyarginine-fused mutant cytosine deaminase. Appl. Biochem. Biotechnol. 175, 1633–1643 (2015)CrossRefGoogle Scholar
  27. 27.
    Y. Du, L. Wang, W. Wang, T. Guo, M. Zhang, P. Zhang, Y. Zhang, K. Wu, A. Li, X. Wang, J. He, J. Fan, Novel application of cell penetrating R11 peptide for diagnosis of bladder cancer. J. Biomed. Nanotechnol. 14, 161–167 (2018)CrossRefGoogle Scholar
  28. 28.
    M. Yousefi, T. Bahrami, A. Salmaninejad, R. Nosrati, P. Ghaffari, S.H. Ghaffari, Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options. Cell. Oncol. 40, 419–441 (2017)CrossRefGoogle Scholar
  29. 29.
    Q. Zhang, Y. Zhang, K. Li, H. Wang, H. Li, J. Zheng, A novel strategy to improve the therapeutic efficacy of gemcitabine for non-small cell lung cancer by the tumor-penetrating peptide Irgd. PLoS One 10, e0129865 (2015)CrossRefGoogle Scholar
  30. 30.
    Y. Zhang, J. Yang, M. Ding, L. Li, Z. Lu, Q. Zhang, J. Zheng, Tumor-penetration and antitumor efficacy of cetuximab are enhanced by co-administered iRGD in a murine model of human NSCLC. Oncol. Lett. 12, 3241–3249 (2016)CrossRefGoogle Scholar
  31. 31.
    Z. Duan, C. Chen, J. Qin, Q. Liu, Q. Wang, X. Xu, J. Wang, Cell-penetrating peptide conjugates to enhance the antitumor effect of paclitaxel on drug-resistant lung cancer. Drug Deliv. 24, 752–764 (2017)CrossRefGoogle Scholar
  32. 32.
    N. Mohammad, P. Malvi, A.S. Meena, S.V. Singh, B. Chaube, G. Vannuruswamy, M.J. Kulkarni, M.K. Bhat, Cholesterol depletion by methyl-beta-cyclodextrin augments tamoxifen induced cell death by enhancing its uptake in melanoma. Mol. Cancer 13, 204 (2014)CrossRefGoogle Scholar
  33. 33.
    N. Mohammad, S.V. Singh, P. Malvi, B. Chaube, D. Athavale, M. Vanuopadath, S.S. Nair, B. Nair, M.K. Bhat, Strategy to enhance efficacy of doxorubicin in solid tumor cells by methyl-beta-cyclodextrin: Involvement of p53 and Fas receptor ligand complex. Sci. Rep. 5, 11853 (2015)CrossRefGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2019

Authors and Affiliations

  1. 1.Priority Research Center, Myunggok Medical Research InstituteCollege of Medicine, Konyang UniversityDaejeonRepublic of Korea
  2. 2.Biotherapeutics Translational Research CenterKorea Research Institute of Bioscience and BiotechnologyDaejeonRepublic of Korea
  3. 3.Department of Pharmacology, College of MedicineKonyang UniversityDaejeonRepublic of Korea

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