Voriconazole exposure regulates distinct cell cycle and terminal differentiation pathways in primary human keratinocytes

Voriconazole is a second generation triazole antifungal used routinely in the care of solid organ and stem cell transplant recipients to both prevent and treat invasive fungal infections ¹. Voriconazole causes photosensitivity ² and has been associated with a dose-dependent increased risk of developing cutaneous squamous cell carcinoma (cSCC).³ Possible mechanisms suggested include direct phototoxicity by voriconazole or one of its metabolites ⁴, oxidative stress pathway activation ⁵, an indirect retinoid effect, or DNA damage repair inhibition ⁶. However, the exact role of voriconazole in cSCC carcinogenesis remains poorly understood.

In this study, we sought to identify molecular mechanisms of voriconazole-associated carcinogenesis in cSCC. We hypothesized that voriconazole or voriconazole n-oxide (VNO), it primary drug metabolite, might regulate key signaling pathways related to cSCC carcinogenesis. To broadly investigate these hypotheses, we used gene expression arrays of in vitro cultures of primary human keratinocytes (PHKs) and human cutaneous squamous cell carcinoma B12 (SCCB12) cell lines exposed to either voriconazole or VNO (135 uM) for 14 days. The institutional review board at the University of California, San Francisco, approved this study.

Principal component analysis of array data demonstrated that drug exposure primarily regulated gene expression in PHKs (rather than SCCB12 cells) (Figure 1a) with voriconazole (rather than VNO) as the primary driver of variance (Figure 1b). Differential gene expression analysis (fold change ≥1.5; FDR q-value <0.05) identified 318 voriconazole-regulated genes (Table S1) and 36 VNO-regulated genes (Table S2) in PHKs. No gene targets were significantly regulated by drug exposure in SCCB12 cells. Supervised hierarchical clustering was used to identify two primary clusters of gene targets regulated by drug exposure in PHKs that were also differentially expressed in cSCC (figure 1c). Cluster 1 consisted of 197 unique genes (Table S3) upregulated in voriconazole-exposed PHKs including cell cycle pathway regulators such as CDC2, DLGAP5, CDKN3, and NDC80. Cluster 2 consisted of 76 unique genes (Table S4) downregulated in voriconazole-exposed PHKs, including markers of keratinocyte terminal differentiation such as SPRR3, LCE3E, and IVL.

Figure 1. Principal Component Analysis, Supervised Hierarchic Clustering Analysis of Gene Expression Data and Quantitative PCR Validation of Gene Expression Results.

(a) Principal component analysis (PCA) of entire normalized data set showing PC1 (x-axis) and PC2 (y-axis). (b) PCA of restricted data set limited to primary human keratinocyte (PHK) conditions showing PC1 (x-axis) and PC2 (y-axis). (c) Supervised hierarchical clustering and heat map of differentially expressed gene between untreated (PHK-Control) and VNO (PHK-VNO) or Voriconazole (PHK-Vori) treated PHKs (FC ≥ 1.5 and FDR <0.05) compared with untreated SCCB12 cell lines (SCC-Control). Expression values were log2-adjusted and median-normalized. Clusters 1 and 2 are identified to the right of the heat map. (d) Average fold expression change of voriconazole and VNO-treated PHKs (PHK-Vori and PHK-VNO, respectively) and untreated SCCB12 (SCCB12-Control) cell lines relative to untreated PHKs (PHK-Control) for selected Cluster 1 genes (FOXM1, CEP55, PLK1, and AURKB). (e) Average fold expression change of voriconazole and VNO-treated PHKs (PHK-Vori and PHK-VNO, respectively) and untreated SCCB12 (SCCB12-Control) cell lines relative to untreated PHKs (PHK-Control) for selected Cluster 2 genes (IVL, LCE3E, S100 A12, and SPRR3). Two-sided t-tests were performed to assess statistical significance relative to untreated PHKs (PHK-Control). * p <0.05. ** p<0.01. undetectable gene expression.

To identify biological themes, we performed functional enrichment and gene set enrichment analysis. Voriconazole upregulated genes related to cell division including chromosome condensation, DNA replication, spindle organization, and cell cycle checkpoint control (Table S5), as well a variety of cancer-related pathways including oncogenic RAS signaling and its downstream target FOXM1 (Table S7 & S8). Voriconazole down-regulated genes related to terminal epithelial differentiation and protease inhibitor activity (Table S6) in addition to pathways related to tumor gene silencing, de-differentiation, metastasis, and oncogenic Ras and NFκB signaling (Tables S9 & S10). Differentially expressed genes with important functional significance were further validated using quantitative PCR, demonstrating upregulation of FOXM1 and its downstream targets CEP55, PLK1, and AURKB (figure 1d) and downregulation of IVL, LCE3E, S100A12, and SPRR3 (figure 1e).

Next, we used three-dimensional organotypic cultures (OTCs) of PHKs exposed to fluconazole or voriconazole (270 uM) to investigate whether voriconazole exposure impacted the structure and development of human skin. Fluconazole, another triazole used for antifungal prophylaxis (Ethier et al. 2012), was utilized to assess for drug class effect. Voriconazole exposure inhibited terminal differentiation resulting in poor formation of the granular and cornified epithelial layers, but there were no significant differences between fluconazole and untreated samples (Figure 2a). We found a similar but reduced effect at the lower voriconazole dose (135 uM) indicating a dose-dependent relationship (figure S1a). We also found reduced protein expression of terminal epithelial markers in voriconazole-treated PHKs (figure 2b). FOXM1 expression was low across all conditions (positive control; figure S1b), though we found basal layer cell proliferation across all conditions indicated by positive Ki67 staining (figure 2b). Gene expression of key markers were further validated with quantitative PCR (figure 2c and 2d).

Figure 2. Hematoxylin and Eosin (H&E) and Immunhistochemical Staining of Untreated and Drug-Treated Primary Human Keratinocytes in Organotypic Cultures with Quantitative PCR Validation of Gene Expression.

(a) Hematoxylin and Eosin (H&E) stained sections of untreated (PHK-Control), fluconazole-treated (PHK-Fluc), and voriconazole-treated (PHK-Vori) primary human keratinocytes grown as OTCs isolated at both Day 7 and Day 14. (b) Immunohistochemical stained sections of untreated (PHK-Control), fluconazole-treated (PHK-Fluc), and voriconazole-treated (PHK-Vori) primary human keratinocytes grown as OTCs isolated at Day 14. Samples were stained with antibodies against involucrin (IVL), loricrin (LOR), fillagrin (FLG), and forkhead box protein M1 (FOXM1) and visualized using an avidin-biotin complex peroxidase system. (c) Average fold expression change of fluconazole and voriconazole-treated PHKs in OTC (PHK-Fluc and PHK-Vori, respectively) relative to untreated PHKs in OTC (PHK-Control) for selected Cluster 1 genes (FOXM1, CEP55, PLK1, and AURKB). (d) Average fold expression change of fluconazole and voriconazole-treated PHKs in OTC (PHK-Fluc and PHK-Vori, respectively) relative to untreated PHKs in OTC (PHK-Control) for selected Cluster 2 genes (IVL, LCE3E, S100 A12, and SPRR3). Two-sided t-tests were performed to assess statistical significance relative to untreated PHKs (PHK-Control). * p <0.05. ** p<0.01. undetectable gene expression.

These data provide evidence that voriconazole regulates distinct cell cycle and terminal differentiation pathways in PHKs, offering novel insights into the poorly understood mechanism of voriconazole-associated carcinogenesis. Voriconazole exposure inhibited expression of terminal epithelial differentiation markers, most comprising the epithelial differentiation complex (EDC), a set of genes co-expressed in outer epithelial layers and clustered on chromosomal region 1q21 ⁸. EDC genes are typically overexpressed in early cSCC lesions likely indicating hyperplasia, but are eventually downregulated following squamous cell dedifferentiation after malignant transformation (Haider et al. 2006). In OTCs, voriconazole inhibited terminal differentiation resulting in poor formation of the granular and corneal epithelial layers. Functional stratum corneum is important for photoprotection due to its physical barrier effect and its production of urocanic acid which absorbs UV light ¹⁰; it is thus possible that voriconazole's impact on terminal differentiation might be synergistic with its UVA-sensitizing properties ⁴ and contribute to chronic photosensitivity. Though, further studies are needed to assess the impact of voriconazole exposure on terminal differentiation in the context of UV exposure.

Voriconazole exposure also regulated cell cycle pathways including the FOXM1 tumorigenesis pathway. FOXM1 is a key transcription factor expressed in proliferating cells and regulates distinct cell cycle checkpoints largely through its effect on numerous downstream targets such as Cdc25B, cyclin B1, AURKB, CENPF family genes and survivin ¹¹. FOXM1 overexpression has been linked to the majority of human malignancies¹² including epithelial cancers such as cSCC ¹³. In keratinocytes, FOXM1 overexpression has been shown to result in genomic instability potentiated by UVB exposure, likely representing a first hit in skin carcinogenesis. ¹⁴

In conclusion, this study identifies that voriconazole regulates distinct cell cycle and terminal differentiation pathways in normal keratinocytes. These effects appear to be unique to voriconazole exposure, rather than a general azole class-effect, likely due to unique structural differences between individual azole anti-fungals.¹⁵. Future in vitro and in vivo studies are needed to further explore the relationship between voriconazole, terminal differentiation, and cell cycle regulation, particularly in the context of UV exposure. Pathway effects of other azoles used routinely in the care of lung transplant recipients, such as itraconazole or posaconazole, should also be investigated. Further characterization of voriconazole-associated mechanisms of carcinogenesis utilizing these findings may lead to a better understanding of the key signaling pathways controlling cSCC and offer novel insights into preventing skin cancer among patients receiving voriconazole.

What’s already known about this topic?

• -Voriconazole is a triazole antifungal used routinely in the care of solid organ and stem cell transplant recipients to both prevent and treat invasive fungal infections.
• -Voriconazole exposure is associated with photosensitivity and an increased risk of cutaneous squamous cell carcinoma, though the exact mechanisms underlying voriconazole-associated carcinogenesis are not well understood.

What does this study add?

• -Voriconazole exposure regulates distinct cell cycle and terminal differentiation pathways in keratinocytes, which offers new insights into mechanisms of voriconazole-associated carcinogenesis.

Abbreviations

cSCC

cutaneous squamous cell carcinoma

UV

ultraviolet

VNO

voriconazole N-oxide

PHK

primary human keratinocyte

PCA

principal component analysis

GO

gene ontology

qPCR

quantitative polymerase-chain reaction