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.