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Integrin α6 overexpression promotes lymphangiogenesis and lymphatic metastasis via activating the NF-κB signaling pathway in lung adenocarcinoma

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Abstract

Objective

It has been reported that tumor-associated lymphangiogenesis plays an important role in lymph node metastasis and contributes to the poor survival of lung adenocarcinoma (LUAD) patients. As yet, however, the molecular mechanism underlying LUAD-associated lymphangiogenesis has remained elusive.

Methods

Immunohistochemistry (IHC) was used to determine the expression of integrin subunit alpha 6 (ITGA6) and the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) in clinicopathologically characterized LUAD specimens. The effect of ITGA6 overexpression on lymphangiogenesis and lymphatic metastasis was examined by tube formation, scratch wound-healing, and cell migration assays in vitro and a popliteal lymph node metastasis model in vivo. Mechanistically, overexpression of ITGA6 and activation of NF-κB signaling were examined by real-time PCR, ubiquitination and dual-luciferase reporter assays. Finally, high ITGA6 expression in LUAD tissue samples was related to copy number variation (CNV) using the TCGA database.

Results

We found that ITGA6 overexpression correlated with microlymphatic vessel density in LUAD specimens (p < 0.01). Importantly, by using a popliteal lymph node metastasis model, we found that ITGA6 upregulation significantly enhanced lymphangiogenesis and lymphatic metastasis in vivo (p < 0.05). In addition, we found that ITGA6 overexpression enhanced the capability of A549 and H1299 LUAD cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs). Mechanistically, we found that ITGA6 sustained NF-κB activity via binding and promoting K63 polyubiquitination of TNF receptor-associated factor 2 (TRAF2). Finally, CNV analysis revealed ITGA6 amplification of 27.5% in the LUAD tissue samples in the TCGA database.

Conclusions

Taken together, our results uncover a plausible role for ITGA6 in mediating lymphangiogenesis and lymphatic metastasis and may provide a basis for targeting ITGA6 to treat LUAD lymphatic metastasis.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Abbreviations

ALL:

Acute lymphoblastic leukemia

ANGPTL4:

Angiopoietin-like 4

CNV:

Copy number variation

CXCL16:

CXC chemokine ligand 16

CRC:

Colorectal cancer

DAB:

Diaminobenzidine

DFS:

Disease-free survival

ECM:

Extracellular matrix

ESCC:

Esophageal squamous cell carcinoma

GALT:

Gut-associated lymphoid tissue

GBC:

Gallbladder carcinoma

GSEA:

Gene Set Enrichment Analysis

HLEC:

Human lymphatic endothelial cell

IHC:

Immunohistochemical

ITGA6:

Integrin subunit alpha 6

LUAD:

Lung adenocarcinoma

LYVE-1:

Lymphatic vessel endothelial hyaluronan receptor 1

MBC:

Metastatic breast cancer

NF-κB:

Nuclear factor-κB

NSCLC:

Non-small cell lung cancer

OS:

Overall survival

qRT-PCR:

Quantitative real-time polymerase chain reaction

RIP1:

Receptor-interacting protein 1

TCGA:

The Cancer Genome Atlas

TNF-α:

Tumor necrosis factor alpha

TRAF2:

TNF receptor associated factor 2

TSPAN15:

Tetraspanin 15

VEGF:

Vascular endothelial growth factor

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Acknowledgements

The authors thank Taizhou Hospital of Zhejiang University for providing the research environment.

Funding

This study was supported by grants from the National Nature Science Foundation in China (NSFC) (grant number: 82002400), the Natural Science Foundation of Zhejiang Provincial (grant numbers: Y19H160116, Q18H160119) and the Science and Technology Department of Guangdong Province (grant number: 2019A1515110740) and Health Commission of Guangdong Province (A2020100).

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Author notes

  1. Sijia Ren, Jing Wang and Anyi Xu have been contributed equally to this work.

Authors and Affiliations

  1. Taizhou Hospital, Zhejiang University, Taizhou, 317000, China

    Sijia Ren & Jianfei Shen

  2. Taizhou Hospital of Zhejiang Province affiliated to Wenzhou Medical University, Taizhou, China

    Jing Wang, Anyi Xu & Jiaqian Bao

  3. Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong, China

    William C. Cho

  4. Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China

    Jinrong Zhu

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  1. Sijia Ren
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Sijia Ren, Jinrong Zhu and Jianfei Shen. The first draft of the manuscript was written by Sijia Ren and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jinrong Zhu or Jianfei Shen.

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The animal experiments were approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province (approval no: tzy-2020160).

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Ren, S., Wang, J., Xu, A. et al. Integrin α6 overexpression promotes lymphangiogenesis and lymphatic metastasis via activating the NF-κB signaling pathway in lung adenocarcinoma. Cell Oncol. 45, 57–67 (2022). https://doi.org/10.1007/s13402-021-00648-3

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