Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

MECHANISMS OF TUMORIGENESIS IN KERATINOCYTES BY HYDROCHLOROTHIAZIDE USE Benjamin Cutler, OMS-II 1 , Jordan Lehman, OMS-II 1 , Madison Altman, OMS-I 1 , Tom Lindsey, D.O. FACOS CHSE 1 , Amy Hicks, Ph.D. MPH 1 . 1. Edward Via College of Osteopathic Medicine – Carolinas Campus, Spartanburg, SC

Introduction

• Hydrochlorothiazide (HCTZ) is the most prescribed diuretic in the US for hypertension. • Squamous cell carcinoma (SCC) is associated with HCTZ use in fair skinned individuals 1 . • HCTZ may enhance DNA and protein damage in UV-irradiated human keratinocytes and may trigger inflammation and upregulate oncogenes and anti-apoptotic proteins 2 . This research aims to: (i) assess the link between HCTZ treatment and tumorigenic changes leading to skin cancer ​. (ii) identify tumorigenic pathways activated by HCTZ in UVA-treated keratinocytes.

Methods

Discussion

Target Protein Identification

References • HCTZ increases markers of DNA and protein damage in human keratinocytes following exposure to UVA irradiation. • The presence of Hsp90 was greatest in cells treated with HCTZ, suggesting that HCTZ with and without UVA can induce oxidative protein damage and upregulate tumorigenesis in human keratinocytes. • Both OGG1 and P-H2AX were increased in cells treated with HCTZ and UVA, demonstrating genomic damage as early as 1 hour following irradiation. • Protein and DNA damage indicates the pathways that are activated by HCTZ towards the development of SCC in human keratinocytes. • The implications of this research are important for clinicians to consider when prescribing HCTZ to fair skinned patients, as they may be at a higher risk of developing squamous cell carcinoma. Conclusions • HCTZ appears to sensitize keratinocytes toward increased damage and tumorigenesis following exposure to UVA irradiation. • Future research should determine the effects of physiological changes in response to long-term exposure. • Additional research is planned to investigate the protective effects of specific nutrients on the tumorigenic pathways stimulated by HCTZ and UVA. 1. Jensen AØ, Thomsen HF, Engebjerg MC, Olesen AB, Sørensen HT, Karagas MR. Use of photosensitising diuretics and risk of skin cancer: a population-based case-control study. Br J Cancer . 2008;99(9):1522-1528. doi:10.1038/sj.bjc.6604686 2. Bigagli E, Cinci L, D'Ambrosio M, et al. Hydrochlorothiazide Use and Risk of Nonmelanoma Skin Cancers: A Biological Plausibility Study. Oxid Med Cell Longev . 2021;2021:6655542. Published 2021 Aug 14. doi:10.1155/Koga F, Kihara K, Neckers L. Inhibition of cancer invasion and metastasis by targeting the molecular chaperone heat- shock protein 90. Anticancer Res . 2009 Mar;29(3):797-807. 3. 2021/6655542 4. Vlahopoulos S, Adamaki M, Khoury N, Zoumpourlis V, Boldogh I. Roles of DNA repair enzyme OGG1 in innate immunity and its significance for lung cancer. Pharmacol Ther . 2019 Feb;194:59-72. doi: 10.1016/j.pharmthera.2018.09.004. 5. HTRF Human Phospho-H2AX (Ser139) Detection Kit, 500 Assay Points | Revvity. Revvity.com. Published 2025. Accessed February 14, 2025. https://www.revvity.com/product/htrf-h2ax-p-s139-kit-500 pts-64h2xpeg

Protein Extraction

UVA Irradiation Half of control and treatment groups exposed to 10 J/cm 2 for 40 minutes

Protein Quantification Protein-specific absorbance measured by ELISA

Treatment Groups

Cell Culture Human epidermal keratinocytes (HEKa)

Specific antibodies were incubated with the raw protein extract

Cells lysed with RIPA at 1hr, 6 hr, and 24hr following UVA

Control medium HCTZ + medium

Figure 1. Experimental design. HEKa cells were incubated with or without HCTZ at a concentration of 370 ng/mL for two weeks. Cells were further divided into treatment groups for UV irradiation. All treatment groups were duplicated for each time point. ELISA visualized HRP and substrate.

Results

Oxidative DNA Damage

Oxidative Protein Damage

dsDNA Breaks

Figure 6. H2AX becomes activated through phosphorylation in response to dsDNA breaks 5 .

Figure 2. HSP90 supports cancer hallmarks by chaperoning specific client proteins 3 .

Figure 4. OGG1 repairs oxidized guanine residues and increases the expression of pro-inflammatory transcription factors such as NF- κ B 4 . OGG1 Activation in Response to DNA Damage

Phosphorylation of H2Ax in Response to DNA Damage

Hsp90 Response to Protein Damage

Figure 7. Prior exposure to HCTZ increases double-stranded DNA breaks by UVA irradiation.

Figure 5. Combined exposure to HCTZ and UVA increases oxidative damage to DNA.

Figure 3. Effect of HCTZ and/ or UVA irradiation on Hsp90 expression in HEKa cells.

2025 Research Recognition Day

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