ACTN1 promotes malignant progression in Cutaneous Squamous Cell Carcinoma by reducing the p53 signaling pathway

Abstract

Abstract Background: Cutaneous Squamous Cell Carcinoma (CSCC) presents a complex pathological landscape. While extensive research has been conducted to elucidate the molecular underpinnings of CSCC tumorigenesis, the role of ACTN1 remains ambiguous. ACTN1 has been implicated in various malignancies, but its relationship with CSCC is not yet clearly defined. Objectives: This study aims to investigate the functional role and underlying molecular mechanisms of ACTN1 in the progression of CSCC. Methods: A total of 10 normal skin tissues, 30 Bowen's disease tissues and 80 CSCC tissues between January 2019 and June 2021 in the Department of Pathology and Dermatology in our hospital were enrolled. The ACTN1 expression in 120 tissues samples were conducted by immunohistochemistry staining methods. The 3 patients with CSCC were enrolled to further verified the ACTN1 protein expression level difference between CSCC tissues and paracancerous tissues. The functions and potential molecular mechanisms of ACTN1 in CSCC is still unclear. Firstly, the ACTN1 protein and mRNA expression between CSCC cell lines (Colon-16, SCL-1 and A431) and a control cell line (hacat cell) were investigated. Besides, the ability of proliferation, migration and invasion after siRNA-mediated ACTN1 knockdown in SCL-1 and A431 cell lines were performed via cell counting kit-8 (CCK-8), wound healing and transwell assays, respectively. The RNA-Seq analysis of the si-con and si-ACTN1 in SCL-1 cells was performed to further investigate the molecular mechanisms underlying ACTN1-mediated malignant biological behavior in CSCC. Finally, the apoptosis-promoting and apoptosis-inhibiting proteins, cell cycle proteins and p53 protein and mRNA levels were investigated. Results: ACTN1 expression levels was significantly elevated in CSCC compared to Bowen's disease (P<0.001). The protein was predominantly localized in the cytoplasm, manifesting as brownish-yellow and tan granules. Elevated ACTN1 levels was correlated with worsen cell differentiation and increased depth of skin invasion (P<0.05). The ACTN1 protein expression levels in CSCC tissues were significantly higher than that in paired paracancerous tissues. Besides, when compared with the control group (hacat cell), higher expression of ACTN1 protein and mRNA in A431, SCL-1 and Colon-16 line cells were also verified. After interfering with the expression of ACTN1, the ability of proliferation, migration and invasion ability in the SCL-1 and A431 cells were significantly decreased (P<0.05). A total of 294 gens were differential expressed between the si-con and si-ACTN1 in SCL-1 cells line, with 165 up-regulated and 129 down-regulated. The KEGG enrichment analysis demonstrated that the top five significantly enriched related signaling pathways were complement and coagulation cascades, p53 signaling pathway, IL-17 signaling pathway, PPAR signaling pathway and staphylococcus aureus infection. Furthermore, all differential expressed genes were enrolled to explore the hub genes according to degree algorithm via Cytoscape software. Two clusters were formed and theses two clusters mainly involved in the p53 signaling pathways. The qRT-PCR and Western blot results showed that after si-ACTN1, the mRNA and protein expression levels of p53 and pro-apoptotic factor Bax were significantly up-regulated, while the apoptosis inhibitor BCL2 and Cyclin D were down-regulated. After constructed the siRNA targeting p53 and ACTN1 in A431 and SCL-1 cell lines, the qRT-PCR and Western blot results showed that the mRNA and protein expression levels of p53, Bax, BCL2 and Cyclin D were not significantly changed. Conclusions: ACTN1 serves as a pivotal regulator in CSCC progression, primarily through its modulation of the p53 signaling pathway. Our findings offer new avenues for targeted therapeutic strategies in CSCC management.