Proteomic analysis reveals LRPAP1 as a key player in the micropapillary pattern metastasis of lung adenocarcinoma

Abstract

Objectives: Lung adenocarcinomas have different prognoses depending on their histological growth patterns. Micropapillary growth within lung adenocarcinoma, particularly metastasis, is related to dismal prognostic outcome. Metastasis accounts for a major factor leading to mortality among lung cancer patients. Understanding the mechanisms underlying early stage metastasis can help develop novel treatments for improving patient survival.

Methods: Here, quantitative mass spectrometry was conducted for comparing protein expression profiles among various histological subtypes, including adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive adenocarcinoma (including acinar and micropapillary [MIP] types). To determine the mechanism of MIP-associated metastasis, we identified a protein that was highly expressed in MIP. The expression of the selected highly expressed MIP protein was verified via immunohistochemical (IHC) analysis and its function was validated by an in vitro migration assay.

Results: Proteomic data revealed that low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) was highly expressed in MIP group, which was confirmed by IHC. The co-expressed proteins in this study, PSMD1 and HSP90AB1, have been reported to be highly expressed in different cancers and play an essential role in metastasis. We observed that LRPAP1 promoted lung cancer progression, including metastasis, invasion and proliferation in vitro and in vivo.

Conclusion: LRPAP1 is necessary for MIP-associated metastasis and is the candidate novel anti-metastasis therapeutic target.

Keywords: LRPAP1; Lung adenocarcinomas; Metastasis; Micropapillary; Proteomics.

Fig. 1

Study design for the proteome analysis of different lung adenocarcinoma histologic subtypes. The flowchart was created on BioRender.com.

Fig. 2

Heat map of gene enrichment analysis. (A) Significantly altered proteins were classified into eight clusters (right panel). Normalized abundance in each row was calculated using the Z-score method. Temporal patterns (middle panel) were used to visualize the relevant changes in each cluster. The core functional entries were labeled in the right panel of the figure after a manual review. u, up; d, down. (B–C) Representative immunostaining images and corresponding statistical comparison histograms (right) for MDC1 (B) and AKT1 (C) in the tumor tissues from each group. Scale bars, 250 μm. Data were presented as mean ± SD. Statistical significance between groups was determined using Kruskal-Wallis nonparametric analysis. *P < 0.05; ***P < 0.001; ns, not significant.

Fig. 3

Lung cell type-specific enrichment analysis. Significantly altered proteins were subdivided into 12 categories. Z-score approach was applied in calculating normalized abundances of diverse rows. Tissue cell type column indicates the cell types in each cluster, and the protein column indicates the identified protein for the corresponding cell types.

Fig. 4

Low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) highly expressed in micropapillary (MIP) adenocarcinoma. Representative immunostaining images and corresponding statistical comparison histograms (right) for LRPAP1 in the tumor tissues from each group. Scale bars = 100 μm. Data were presented as mean ± SD. Statistical significance between groups was determined using Kruskal-Wallis nonparametric analysis. *P < 0.05, **P < 0.01.

Fig. 5

Protein co-expression network (PCN) of LRPAP1. (A) All proteins with a Pearson’s correlation >0.8 with LRPAP1 were defined as co-expressed proteins and illustrated as a network. The node indicates a protein, and the edge indicates the corresponding correlation value between proteins. (B) Abundance comparison of co-expressed proteins in lung cancer data from Clinical Proteomic Tumor Analysis Consortium (CPTAC).

Fig. 6

LRPAP1 promotes cancer cell migration and metastasis. (A) Representative images (top, 24 h after seeding) and histograms (bottom) of the Transwell assay on A549 and H1975 cells overexpressing LRPAP1. (n = 3). Scale bar, 250 μm. (B) Typical images (top) and histograms (bottom) showing the wound-healing assay within A549 and H1975 cells with overexpressed LRPAP1. (n = 3). Scale bar, 150 μm. (C) Representative images (24 h after seeding) and corresponding histograms of the Transwell assay on A549 cells with knockdown of LRPAP1. (n = 3). Scale bar, 250 μm. (D) Representative images and corresponding histograms illustrating the wound-healing assay in A549 cells with knockdown of LRPAP1. (n = 3). Scale bar, 150 μm. (E) Subcutaneous injection of 5 × 10⁶ cells into BALB/c nude mice led to tumor formation after a 19-day injection period (n = 3 per group). Scale bar, 1 cm. (F) Summary of tumor volume measurements obtained from mice. (G) Summary of tumor volume change in mice. (H) Histogram depicting the terminal weights of tumors. All data were presented as mean ± SD. Statistical significance between groups was determined using Student’s t-test (*P < 0 0.05, **P < 0 0.01).