To the Editor: Cutaneous squamous cell carcinoma (CSCC) originates from the keratinocytes of the epidermal layer and (or) its associated appendages, manifesting as a rapidly proliferating, highly destructive malignancy, with almost 20,000 cases occurring in China in 2022.[1] The pathogenesis of CSCC is complex, but recent studies have made significant progress. Our previous research identified eight proteins, including actinin alpha 1 (ACTN1), tenascin C (TNC), fascin actin-bundling protein 1 (FSCN1), serpin family B member 1 (SERPINB1), ras-related protein 31 (RAB31), collagen type III alpha 1 chain (COL3A1), collagen type I alpha 1 chain (COL1A1), and cluster of differentiation 36 (CD36), which are markedly linked to CSCC invasion and metastasis.[2] Besides, our subsequent assays also revealed the elevated serum level of ACTN1 in CSCC patients, which correlated with tumor cell differentiation, invasion depth, and primary tumor diameter.[3] This underscores the potentially pivotal role of ACTN1 in CSCC pathogenesis. This study aimed to further investigate the role of ACTN1 in the pathogenesis of CSCC. The study procedures were approved by the Medical Ethics Committee of Wannan Medical College (No. 2023-184). All individuals voluntarily agreed to participate in this study and signed informed consent. A total of 10 normal skin tissues surrounding melanocytic nevi, 30 Bowen's disease tissues, and 80 CSCC tissues were collected from patients who had presented to Yijishan Hospital between January 2019 and June 2021. The ACTN1 expression in 120 tissues samples was assessed by immunohistochemistry staining. For each samples, three regions were randomly selected and the numbers of total tumor cells and ACTN1-positive tumor cells were counted, respectively to calculate the percentage of positively stained tumor cell score (no staining, 1; <30%, 2; 31–60%, 3; >61%, 4). Besides, the intensity score was set from 0 to 3 (0, no staining; 1, weak; 2, mild; 3, strong staining). The final score equals to positively stained tumor cell score multiplied by intensity score. The total scores great than or equal to 6 were defined as high expression of ACTN1, while the scores less than 6 were defined as ACTN1 low expression. To further verify the ACTN1 protein expression level differences between CSCC and paracancerous tissues, CSCC tissues and paracancerous tissues were collected from three patiens. The expression level of ACTN1 protein in CSCC and paracancerous tissues was explored by western blot. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to explore the ACTN1 mRNA expression level. The expression level of ACTN1 messenger RNA (mRNA) and protein was analyzed and compared between the control cell line (Hacat) and CSCC cell lines (Colon-16, A431, and SCL-1). Next, the cell proliferation, migration, and invasion abilities in A431 and SCL-1 cell were also explored after being transfected with small interfering (si) RNA targeting ACTN1 (si-ACTN1). Meanwhile, an empty vector was used as a negative control group (si-con). To illustrate the downstream targets and pathways regulated by ACTN1 in CSCC, the RNA sequencing (RNA-Seq) analysis of cells with si-ACTN1 and si-con was conducted (n = 5). Gene Ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to analyze the biological processes and signaling pathways that ACTN1 involves. Furthermore, the hub genes were also explored via Protein-Protein Interaction Networks (PPI) analysis of differentially expressed genes. In addition, western blot and qRT-PCR were conducted to verify the results of RNA-Seq. Besides, si-RNAs targeting p53 and ACTN1 were also used in A431 and SCL-1 to investigate the role of ACTN1 in p53 signaling pathway. The primary antibodies and primer sequences information of multiple genes for western blot and qRT-PCR experiments are shown in Supplementary Table 1, https://links.lww.com/CM9/C36. Statistical analysis was performed using SPSS Statistics for Windows, Version 17.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. The one-way analysis of variance (ANOVA) was used for comparision among multiple groups. A two-independent-sample t test was performed to assess differences between two groups. The P-value <0.05 was considered statistically significant. In RNA-Seq analysis, the criteria for the selection of significantly differentially expressed genes were set as |log2(fold change [FC])| >1 and a P-value <0.05. No statistically significant disparities in baseline age, gender, and lesion locations were observed among the normal group, Bowen's disease group, and CSCC group [Supplementary Table 2, https://links.lww.com/CM9/C36]. The expression of ACTN1 was negative in all normal skin samples and exhibited low level or absence in Bowen's disease tissues, while among the 80 cases of CSCC, 56 exhibited high ACTN1 expression and 24 showed low expression level [Supplementary Figure 1, https://links.lww.com/CM9/C34]. Compared to Bowen's disease, the rate of high ACTN1 expression in CSCC was significantly increased (P <0.001). As shown in Supplementary Table 3, https://links.lww.com/CM9/C36, the ACTN1 expression level in CSCC tissues were significantly associated with the cell differentiation degree and invasion depth (P <0.05), suggesting that ACTN1 may promote the progression of CSCC. Furthermore, CSCC tissues had a higher expression of ACTN1 protein compared with paracancerous tissues [Supplementary Figure 2A, https://links.lww.com/CM9/C34]. Besides, compared with the control group (Hacat cell), higher expression of ACTN1 protein and mRNA in A431, SCL-1, and Colon-16 CSCC line cells were also observed [Supplementary Figure 2B, C, https://links.lww.com/CM9/C34]. However, the ACTN1 showed significantly higher expression in A431 and SCL-1 cell lines relative to Colon-16 cell lines. After silencing ACTN1 in SCL-1 and A431 cell lines, the si-ACTN1 group exhibited significantly reduced proliferation, migration, and invasion abilities compared to the si-con group (P <0.05) [Supplementary Figures 3 and 4, https://links.lww.com/CM9/C34]. To sum up, these data indicated that ACTN1 could promote the cell proliferation and metastasis in CSCC. To explore the underlying mechanism of ACTN1-mediated tumor progression and metastasis in CSCC, the RNA-Seq analysis was conducted. As RNA-Seq analysis reported, 294 genes were selected with a |log2(fold change [FC])| >1 and P-value <0.05. Among these differentially expressed genes, a total of 165 genes were up-regulated, while 129 genes were down-regulated [Supplementary Figure 5, https://links.lww.com/CM9/C35]. The GO enrichment analysis of differentially expressed genes revealed that the top three significantly enriched biological processes included regulated exocytosis, negative regulation of peptidase activity, and cornification. The top three significantly enriched molecular functions included endopeptidase inhibitor activity, peptidase inhibitor activity, and peptidase regulator activity. The top significantly enriched cell components included extracellular region [Supplementary Figure 6, https://links.lww.com/CM9/C35]. The KEGG enrichment analysis unveiled the top three significantly enriched related signaling pathways, namely complement and coagulation cascades, p53 signaling pathway, and interleukin (IL)-17 signaling pathway [Supplementary Figure 6, https://links.lww.com/CM9/C35]. The significantly differentially expressed genes between si-ACTN1 group and si-con group in SCL-1 cell (top 10 gens) were presented in Supplementary Table 4, https://links.lww.com/CM9/C36. The PPI analysis of differentially expressed genes was presented in Supplementary Figure 7A, https://links.lww.com/CM9/C35, and 25 hub genes, including APOA1, APOH, APOE, HP, AHSG, and EEF2, were found [Supplemental Table 5, https://links.lww.com/CM9/C36]. As shown in Supplementary Figure 7B,C, https://links.lww.com/CM9/C35, a total of two clusters (b and c) were detected. The differentially expressed genes in cluster b were mainly from ribosomal proteins (RPs) family and the RPs were involved in cell growth and proliferation process. Multiple RPs have extra-ribosomal functions including activation of p53-dependent or p53-independent pathways, resulting in cell cycle arrest and apoptosis in cancer. Besides, the hub genes from cluster c were mainly involved in the process of cholesterol metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathways. The reprogramming of lipid metabolism plays a key role in cancer progression. Besides, the role of p53 in regulating lipid metabolism and cancer progression also had gained increasing attention. To verify the RNA-Seq data, the proteins and mRNA expression level of two hub gens (RPL17 and RPS9, family members of RPs) were assessed. The si-ACTN1 group showed markedly lower expression level of RPL17 and RPS9 mRNA and protein than that in si-con group [Supplementary Figure 8, https://links.lww.com/CM9/C35]. Therefore, the ACTN1 may be involved in the tumorigenic mechanisms of CSCC through a variety of biological pathways, and the p53 signaling pathway may be one important pathway according to enriched terms results and clusters b and c may also involve in p53 signaling pathway. KEGG-enriched results also showed that p53 signaling pathway may play an important role in CSCC. Hence, the interaction of ACTN1 and p53 was investigated. Compared with CSCC tissue, the p53 protein was significantly over-expressed in paracancerous tissues [Supplementary Figure 9, https://links.lww.com/CM9/C35]. The p53 signaling pathway is an important anticancer signaling pathway, which can exert anticancer effects by regulating cell cycle progression and/or apoptosis and other important biological processes. Previous studies have proved that multiple genes could regulate the p53 signaling pathway in tumor progression. Nevertheless, the interactions of ACTN1 and p53 signaling pathway in CSCC were still unclear. Through our above bio-informatics analysis results, it has been suggested that ACTN1 may regulate p53 signaling pathway. The qRT-PCR and western blot results showed that the mRNA and protein expression levels of p53 and pro-apoptotic factor Bax were significantly up-regulated in si-ACTN1 cells, while the apoptosis inhibitor B cell lymphoma 2 (BCL2) and cyclin D were down-regulated [Supplementary Figures 10 and 11, https://links.lww.com/CM9/C35]. These results revealed that ACTN1 gene inhibits p53 signaling pathway in CSCC. After knockdown of p53 and ACTN1 in A431 and SCL-1 cell lines, the mRNA and protein expression levels of p53, as well as its downstream target molecules were assessed. After simultaneous knockdown of both p53 and ACTN1, the qRT-PCR and western blot results revealed no significant alterations in the mRNA and protein expression levels of Bax, BCL2, and Cyclin D when compared to p53 knockdown alone [Supplementary Figures 12 and 13, https://links.lww.com/CM9/C35]. These findings suggest that p53 acts as an important intermediate factor, which regulates ACTN1 in CSCC progression. ACTN1, an integral component of the alpha-actinin protein family, is primarily involved in cellular functions such as cytokinesis, cell adhesion, and migration. Elevated ACTN1 expression has been documented in a spectrum of cancers, such as gastric cancer, hepatocellular carcinoma, and breast cancer.[4-6] Zhang et al[4] demonstrated that ACTN1 over-expression in gastric cancer disrupts the cell cycle and attenuates apoptosis by modulating the protein kinase B/glycogen synthase kinase-3β/β-catenin (Akt/GSK3β/β-catenin) signaling axis. In hepatocellular carcinoma, ACTN1 up-regulation has been linked to the suppression of the Hippo signaling pathway through its interaction with Mps one binder 1 (MOB1).[5] And in breast cancer, the silencing of ACTN1 genes could significantly decrease the tumor progression at any stage.[6] In the present study, we found that ACTN1 gene is significantly, negatively correlated with p53 signaling pathway. The p53 gene is known as a "gene guardian" via regulating the initiation and termination of the cell cycle, as well as inducing apoptosis. p53 plays a vital role in maintaining cell homeostasis and prevents the development of cancer. The hub genes in ACTN1 silenced SCL-1 cell were identified and mainly associated with RPs family. Previous studies had confirmed that RPL17 and RPS9 up-regulation was involved in non-small cell lung cancer, colorectal cancer tumorigenesis by activating extracellular signal-regulated kinase (ERK), Nimrod A (NIMA)-related kinase 2/β-catenin (NEK2/β-catenin), and signal transducer and activator of transcription 3 (STAT3) signaling pathways.[7-8] Interestingly, the ERK and STAT3 pathways could be regulated by p53 in esophageal and other carcinomas.[9] Other studies also investigated the role of p53 in CSCC development. For example, the HSP105, one of the family members of heat shock proteins (HSPs), is mainly involved in the abnormal proliferation, invasion, and apoptosis of tumor cells. Jia et al[10] confirmed that the HSP105 acts as an inhibitor of CSCC via regulating the p53 signaling pathway. This study revealed that ACTN1 promotes the development of CSCC by downregulating the p53 pathway. These results provide a theoretical basis for the development of new clinical diagnosis and treatment approaches for CSCC. Funding This work was supported by 2021 Natural Science Research Project of Universities from Anhui Province (No. KJ2021ZD0097), 2023 Key Research Projects from Universities in Anhui Provinces (No. 2023AH051781), National Natural Science Foundation of Anhui (No. 2308085QH269), and the Key Research Project of Wannan Medical College in 2022 (Nos. WK2022ZF09 and WK2022F19). Conflicts of interest None.
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