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

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

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

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Acknowledgments

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).

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Authors and Affiliations

  1. Priority Research Center, Myunggok Medical Research Institute, College of Medicine, Konyang University, Daejeon, 35365, Republic of Korea

    Seong-Lan Yu & Jaeku Kang

  2. Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea

    Han Koo, Young Il Yeom & Dong Chul Lee

  3. Department of Pharmacology, College of Medicine, Konyang University, Daejeon, 35365, Republic of Korea

    Hoi Young Lee & Jaeku Kang

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  1. Seong-Lan Yu
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Correspondence to Dong Chul Lee or Jaeku Kang.

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

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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)

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Yu, SL., Koo, H., Lee, H.Y. et al. Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion. Cell Oncol. 42, 275–285 (2019). https://doi.org/10.1007/s13402-019-00424-4

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