From Bowen disease to cutaneous squamous cell carcinoma: eight markers were verified from transcriptomic and proteomic analyses
Authors: Biao Tang , Cai-feng He , Xiao-hong Lu , Xiao-li Chang , Wen-bei Liu , Jun Wang , Chao Ci , Tao Yuan 
Published: September 2022

Abstract
Background
Bowen's disease is a cutaneous squamous cell carcinoma (CSCC) in situ. If left untreated, BD may progress to invasive CSCC. CSCC is one of the most common cutaneous carcinoma in the elderly and the advanced, metastasis CSCC usually have a poor outcomes. However, the mechanisms of invasion and metastasis from Bowen’s disease to CSCC is complicated and still unclear.

Objectives
The aim of this study was to explore the biomarkers and molecular alterations in Bowen’s disease development process via analyzing the proteomics changes in tissues of CSCC, Bowen disease and healthy skin.

Methods
A total of 7 individuals with CSCC (5 for proteomics study and 2 for validation), 7 individuals with Bowen disease (5 for proteomics study and 2 for validation) and 7 healthy controls (5 for proteomics study and 2 for validation) presented to the Department of Dermatology, Yijishan Hospital, the First Affiliated Hospital of Wannan Medical College between January 2021 and December 2021 were enrolled. The proteomics analysis was performed to screen differentially expressed proteins/gens (DEPs/DEGs) in the lesions of CSCC, Bowen disease and healthy skin tissues. The transcriptomic data (GSE32628) of CSCC was selected and downloaded from the GEO database. The common DEGs in our proteomics results and GSE32628 between CSCC and healthy skin tissues were selected. And then, the common DEGs which significantly up or down-regulated between CSCC and Bowen disease in our proteomics results were further screened to identify using Western blot methods in the validation group. CSCC A431 cells were transfected with SERPINB1 small interfering RNA (si-SERPINB1) or small interfering RNA negative control (si-NC). To explore the effect of SERPINB1 silencing on migration and invasion ability of A431 cells.

Results
A total of 501 proteins were differentially expressed between the CSCC and healthy skin tissues, with 332 up-regulated and 169 down-regulated at least 1.5-fold with a P value < 0.05. These DEPs involved multiple biological functions such as protein binding process, immune, inflammation, ribosome, protein digestion and absorption, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway and others. A total of 20 common DEGs (COL3A1, LUM, TNC, COL1A1, ALDH3A2, FSCN1, SERPINB4, SERPINB1, CD36, COL4A1, CSTB, GPX3, S100A7, ACTN1, SERPINB3, S100A8, RAB31, STAT1, SPRR1B, S100A9) between CSCC and healthy skin tissues in GSE32628 and our proteomics results were found. Besides, the proteins of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in CSCC were significantly up-regulated, while COL3A1, COL1A1 and CD36 were significantly down-regulated relative to Bowen disease in proteomics results. These proteins were mainly involved in multiple pathways, including Focal adhesion, ECM-receptor interaction, Human papillomavirus infection, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway and others. These eight proteins were selected for further validation. According to the Western blotting analysis, when compared with the Bowen disease and healthy skin tissues, we found that the relative expression levels of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in the CSCC were significantly increased, while COL1A1 and CD36 were significantly decreased, and the differences were statistically significant (P < 0.05). Furthermore, the relative expression levels of TNC, FSCN1, SERPINB1 in the Bowen disease were also significantly increased, while the COL3A1 were also significantly decreased relative to the healthy control. SERPINB1 siRNA inhibited the expression of SERPINB1 at mRNA and protein levels in the A431 cells. After interfering with the expression of SERPINB1, the migration and invasion ability in the A431 cells were significantly decreased (P < 0.05).

Conclusions
This study highlights that eight proteins, TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1, CD36, were significantly associated with the mechanisms of invasion and metastasis in Bowen’s disease.