Discussion
PDT is FDA approved treatment for actinic keratosis, and existing clinical research indicates that PDT may be effective in treating Bowen Disease, a type of primary cSCC.11,12 Previous laboratory research demonstrated that 5-ALA PDT significantly increases cell apoptosis in cSCC and fibroblast cells treated with the same parameters used in this protocol.18,27 The goal was to determine the genes and pathways altered in the residual population of ALA PDT treated cells that did not undergo apoptosis or other forms of cell death. RNA-Seq was performed using RNA from cSCC and fibroblast cells collected at 0-, 4-, and 24 hours following PDT. Statistical analysis did not identify any genes that were differentially expressed after correcting for multiple testing (FDR < 0.05). Transcription factor and pathway analysis were compared with differentially expressed genes with a p-value < 0.05. Bioinformatic analysis identified key transcription factors and pathways related to cellular proliferation and cancer pathogenesis were downregulated.
The findings of this RNA-Seq experiment conform to previous microarray and transcriptomic studies using PDT on AKs and cSCCs. Joly et al. compared differential expression in cSCC lesional, perilesional, and unexposed skin in immunocompromised patients up to 18 weeks following MAL PDT treatment.19 MAL PDT downregulated cancer and cell proliferation genes, while extracellular matrix-associated genes were upregulated.19 MAL PDT downregulated genes associated with proliferation, such as cyclins and the Kinesin Family member (KIF) proteins, were similarly identified in our testing with a p <0.05. KIF proteins are involved with microtubule organization during mitosis and have been previously found to be associated with cancer.28-31 Knockdown of KIF22 inhibits tongue SCC proliferation and xenograft tumor growth.28 In laboratory studies, PDT treatment protocols decreased cSSC tumor proliferation and increased apoptosis and autophagy via regulation of MAPK protein, particularly AKT.20-22 Compared to the present study, autophagy was one of upregulated KEGG 2021 pathways with a p < 0.05, but FDR > 0.05.
Transcription factors associated with cancer proliferation and pathogenesis and known to be upregulated in cSCC, including E2F4 and FOXM1, were found in our study to be downregulated post ALA PDT.32-34 The E2F family of transcription factors bind to DNA and Rb and regulate the cell cycle.32 E2F4 is a transcriptional activator. Upregulation of E2F transcription factors has been linked to cancer progression in head and neck SCC.32 FOXM1 is an ultraviolet master regulator and is upregulated in cSCC.33,35,36 Inhibition of FOXM1 with thiostrepton and CRISPR depletion led to decreased cell viability and proliferation in cSCC.33,35,36
Limitations of this study include the lack of abundant commercially available cSCC cell lines at the onset of this study. Due to the inability to significantly identify differential expression of individual genes with an FDR <0.05, the pathway analysis may indicate that small changes in gene expression may result in changes in pathways. This could be addressed by increasing the number of cell lines tested, and could be performed in future experiments. For RNA-Seq, there is no consensus regarding the number of biological replicates with n ranging from 3 to 12.37 4 samples were used, but the cell types differed between normal (i.e., HDF) and cancerous (i.e., cSCC). In the future, it may be necessary to test additional biological replicates and specifically increase the number of cSCC cell lines. Historically, there were a limited number of commercially available cSCC cell lines. However, multiple cSCC from transplant/immunocompromised and immunocompetent donors have recently become available. Several other potential explanations may explain why the PDT treatment did not result in significant differential expression after multiple testing, including dose response and genetic heterogeneity among the cell types. The PCA and heatmaps in Figure 1A demonstrate clustering among the two fibroblast cell lines, AG13145 and CRL-2617, and heterogeneity between the cSCC cell lines, SCC-13 and A431. Additionally, there were greater differences between samples than between treatment and control (Figure 1B), indicating that small, but potentially meaningful changes occur in the individual cell lines post ALA PDT.
In conclusion, RNA-Seq analysis of cSCC and HDF differential expression after ALA PDT did not identify any significant genes after correcting for multiple testing. However, pathways associated with proliferation and carcinogenesis were downregulated. These findings using ALA PDT are similar to previously published studies using MAL PDT that indicate that residual cells post PDT undergo changes consistent with a less aggressive cancerous phenotype.19,20 Additional laboratory and clinical studies need to be performed to confirm the efficacy of ALA PDT for cSCC.