Molecular monitoring of short- and long-term transcriptional effects of hair growth stimulating agents

Introduction

Male-pattern hair loss (MPHL), also known as androgenic alopecia (AGA), is a progressive condition caused by circulating androgens, oxidative stress, the scalp microbiome and genetic factors. The only effective treatments available for MPHL are oral finasteride, topical minoxidil or invasive surgical transplantation. Evaluation of hair growth treatments for MPHL have mainly focussed on physical methods such as visual inspection, the hair comb test and hair pull test to assess hair thinning and hair fall severity. Previous transcriptomic studies identified androgen signalling, WNT signalling, adipogenesis, and other processes to be involved in the progression of MPHL pathophysiology. In this study, the authors evaluated hair growth promoting agents using RNA-seq and miRNA-seq to identify transcriptomic signatures that could be used to evaluate efficacy of hair growth treatments.

Main Points

• 89 men were randomly assigned and treated with placebo, serum A, Serum B or serum C after which RNA was extracted from plucked hair follicles. Serum A and B were plant-based treatments and serum C was a commercially available hair-loss preventing compound containing a pyrimidine active agent.
• Hair follicles were analysed at baseline and after treatment for 4 days and 6 weeks. 17 data sets were sequenced for miRNA and 21 data sets for mRNA.
• Serum B had no differentially expressed genes or miRNA.
• 53 genes and miRNAs were identified as differentially expressed in the other serum treated groups (FDR<0.05). Most of the differentially expressed genes were detected in the long-term 6 week treatment, 50 genes. In serum A treated follicles 12 genes were differentially expressed at day 4 and consistently differentially expressed at 6 weeks.
• The strongest differentially expressed gene in both serum A and C was NDUFB7, involved in electron transport, which has been demonstrated to be reduced in dermal papilla cells from balding humans. NDUFB7 can also be upregulated by dihydrotestosterone (DHT).
• GPX4 was upregulated with long-term treatment with serum C and is involved in hair follicle cycling.
• Other differentially expressed genes previously implicated in MPHL included DAPK3 in serums A and C and ZBTB20 in serum C.
• Differentially expressed genes involved in other types of hair loss included CALML5 and GNAS – linked to alopecia areata, ITGA2 – linked to senescent alopecia, MVD – linked to lichen planopilaris, miR-197-3p and miR92a-1-5p – linked to female-pattern hair loss and CDK2- linked to chemotherapy-induced hair loss.
• Gene set enrichment analysis was done on a less stringent gene set pV<0.05. 17 pathways across the different serums were identified including Prolactin (PRL) signaling, which has been implicated in hair cycle regulation and MPHL.
• More pathways implicated in hair loss included adipogenesis, brain-derived neurotrophic factor (BDNF) signalling, VEGFA /VEGFR2 signalling, polycomb repressive complex 2 function, WNT signalling and insulin- or insulin-like growth factor signalling.

Conclusion

To complement the physical methods used to assess hair growth promoting therapeutics, transcriptional profiling could identify mechanism of action and potentially even enable personalised treatments based on a patient’s genetic makeup. In this study the authors used a minimally invasive plucked hair model to assess the effects of different growth serums on transcription. Many of the differentially expressed genes were in pathways known to be implicated in MPHL such as WNT signalling and PRL signalling. Molecular signatures have the potential to identify treatment effects early, which could save time and money and the method could be utilised in other therapeutic areas.

EPISTEM SERVICES

Epistem have pioneered the use of gene expression in plucked hairs to monitor drug response and engagement. Using our pharmacogenomic expertise we have developed our own hair growth signature based on differential gene expression between telogen and anagen phases of the hair cycle.

In addition to using plucked hairs to look at the potential efficacy and mechanism of action of treatments for MPHL (similar to reported in this publication), plucked hair is routinely used as a pharmacodynamic biomarker tissue.

Biomarker Discovery Platform.

Our plucked hair biomarker platform is the ideal solution for minimally invasive repeat sampling in biomarker discovery and clinical evaluation.

The preclinical Ex Vivo stage of the platform has been used to identify biomarker signatures in the PI3K, RAF/MAPK, NOTCH, hair growth and DNA damage pathways. We have also confirmed and identified biomarkers from a diverse range of clinical trial samples. In addition to gene expression, several protein biomarkers have been employed at pre-clinical and clinical stages using immunohistochemistry on cross sections of plucked hair.

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Gene Expression.

We offer GCLP-accredited NGS and qPCR services for gene expression analysis and have experience extracting RNA from most tissue types. We specialise in extracting quality RNA from challenging tissues.

Bioinformatics.

All of our models are supported by bioinformatics analysis, including differential expression and pathway analysis. We also offer biomarker signature discovery and analysis using our proprietary software in clinical and preclinical samples.

Histology and IHC

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Epistem offers a fully automated IHC and RNAscope service utilising the Ventana Discovery Ultra with digital images created and labelling quantified using the Aperio platform. Protocols may be directly transferred or developed in-house which are tailored to your biomarkers of interest.