Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma

Yuji Hayashi; Shoko Miyoshi; Itaru Watanabe; Nagomi Yano; Kodai Nagashio; Mihiro Kaneko; Teppei Kaminota; Tomoyoshi Sanada; Yuki Hosokawa; Takashi Kitani; Sohei Mitani; Mohammed E Choudhury; Hajime Yano; Junya Tanaka; Naohito Hato

doi:10.1016/j.anl.2024.01.006

Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma

Auris Nasus Larynx. 2024 Mar 22;51(3):472-480. doi: 10.1016/j.anl.2024.01.006. Online ahead of print.

Authors

Affiliations

¹ Department of Otorhinolaryngology, Head and Neck Surgery, Ehime University Medical School, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan.
² Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan.
³ Department of Otorhinolaryngology, Matsuyama Red Cross Hospital, Matsuyama, Ehime, Japan.
⁴ Department of Otorhinolaryngology, Head and Neck Surgery, Uwajima City Hospital, Uwajima, Ehime, Japan.
⁵ Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan. Electronic address: hajime-y@m.ehime-u.ac.jp.

PMID: 38520980
DOI: 10.1016/j.anl.2024.01.006

Abstract

Objective: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na⁺ / H⁺ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells.

Methods: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line.

Results: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells.

Conclusion: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Keywords: Extracellular matrix; Head and neck squamous cellcarcinoma; Lysyl oxidase like 2; Na(+)/H(+) exchanger 1; Primary xenograft lesion; Tumor microenvironment.