Letrozole treatment alters hippocampal gene expression in common marmosets (Callithrix jacchus)

Abstract

Aromatase inhibitors (AIs) are a class of drugs commonly given to patients with estrogen receptor (ER)-dependent breast cancers to reduce estrogenic stimulation. However, AIs like Letrozole are associated with negative side effects such as cognitive deficits, sleep disturbances and hot flashes. We have previously shown that these negative effects can be recapitulated in common marmosets (Callithrix jacchus) treated with Letrozole (20 μg daily) for 4 weeks and that marmosets treated with Letrozole show increased levels of estradiol in the hippocampus (Gervais et al., 2019). In order to better understand the mechanisms through which AIs affect cognitive function and increase steroid levels in the hippocampus, we used bulk, paired-end RNA-sequencing to examine differentially expressed genes among Letrozole-treated (LET; n = 8) and vehicle-treated (VEH; n = 8) male and female animals. Gene ontology results show significant reduction across hundreds of categories, some of the most significant being inflammatory response, stress response, MHC Class II protein complex binding, T-cell activation, carbohydrate binding and signaling receptor binding in LET animals. GSEA results indicate that LET females, but not LET males, show enrichment for hormonal gene sets. Based on the transcriptional changes observed, we conclude that AIs may differentially affect the sexes in part due to processes mediated by the CYP-450 superfamily. Ongoing studies will further investigate the longitudinal effects of AIs on behavior and whether AIs increase the risk of stress-induced neurodegeneration.

Keywords: Aromatase inhibitor; Estradiol; Hippocampus; Letrozole; RNA-sequencing.

Figure 1.. Experimental Design and Differentially Expressed Genes among LET and VEH animals.

(A) Common marmosets were assigned to vehicle (VEH; n = 3 females, n = 5 males) or Letrozole (LET; n = 4 females, n = 4 males). The right hippocampus was dissected from each sample and processed for bulk, paired-end RNA-sequencing. (B) The data processing workflow involved downloading forward and reverse FASTQ files and mapping them to a reference genome. Reads were counted before assessing differentially expressed genes (DEGs) via gene set enrichment analyses (GSEAs) or functional enrichment analyses. (C) Post-data normalization and filtering for lowly expressed genes, 15,149 genes were assessed for differential expression between Letrozole- and Vehicle-treated groups. Horizontal dotted lines indicate the threshold at which individual genes’ p-value < 0.05, whereas vertical lines represent a log2 fold change greater than 0.5 (a fold change of 1). The triangle shapes for individual genes represent genes which have a significant fold change but non-significant p-value.

Figure 2.. Brain Matrix Design.

(A) 3D-printed brain matrix and accompanying blade holders were designed using SOLIDWORKS^® software and a matrix template provided by the Silva lab (Guy et al., 2016). An MRI of a marmoset brain was superimposed in a sagittal position on the matrix. (B) The brain matrix and blade holders were printed in onyx and dissected over dry ice to preserve the brain while obtaining ~ 2mm sections before tissue punch and homogenization.

Figure 3.. Functional profiling of upregulated and downregulated genes (FDR<0.05) in LET animals.

The open source version of gProfiler (g:GOSt) (Raudvere et al., 2019) enables functional profiling of genes significantly downregulated in LET animals. Each circle dot reflects a specific gene ontology (GO) term within eleven distinct categories: molecular function (MF), cellular component (CC), biological processes (BP), KEGG pathways (KEGG), reactome (REAC), transcription binding sites (TF), genes targeted by miRNAs (MIRNA), and their respective adjusted p-values. The BP category returned the most categories with 1,174 GO terms.

Figure 4.. Top GO terms for downregulated genes in three GO categories: biological processes (BP); cellular component (CC); molecular function (MF).

GOplot (Walter et al., 2015) was used in R to reduced redundant or significantly overlapping GO IDs in each category. Size of the GO term bubble indicates number of significantly downregulated genes within GO term ID.

Figure 5.. Visualization of interactions between significant GO terms and overlapping genes with a logFC ≤ −1.

A circular ribbon chat depicts interaction between top significant GO terms and genes with a logFC < −1. Ribbons connect individual genes on the left to their appropriate, color-coded GO category on the right. These gene names reflect some of the most significant transcripts that were affected within each GO category.

Figure 6.. Venn Diagram of downregulated transcripts.

(A) Downregulated transcripts are shown for LET (Letrozole-treated animals relative to vehicle), Male (Letrozole-treated males relative to vehicle males), and Female (Letrozole-treated females relative to vehicle females) (FDR < 0.05). Only 8 transcripts were found to be shared between all contrasted groups while females had a five-fold difference in uniquely downregulated transcripts (45) than males (9). (B) Significantly fewer genes were upregulated across all groups.

Figure 7.. Functional profiling of uniquely downregulated genes in LET Females.

Using gOSt functionality as part of the gProfiler2 package (Raudvere et al., 2019), 45 downregulated genes unique to the LET females were submitted for functional profiling.

Figure 8.. Gene set enrichment analyses reveal sex differences.

LET females show enrichment for genes involved in the regulation of hormone levels (M11178), steroid hormone biosynthesis (M14933) and response to estradiol (M10509) compared to vehicle-treated females. LET males do not show enrichment for genes involved in the regulation of hormone levels compared to control males (FDR = 0.67) and only vehicle-treated males show enrichment for genes involved in steroid hormone biosynthesis and response to estradiol.

Figure 9.. Gene set testing results reveal sex differences not observed in aggregated group.

Gene set for Alzheimer’s disease (M12921) shows no effect when LET animals are contrasted against control animals. (FDR = 0.88). Gene set testing for Alzheimer’s disease (AD) and drug metabolism show no effect on LET animals relative to VEH animals (FDR > 0.25). Interestingly, an enrichment for Alzheimer’s disease was found for LET females, while genes involved in drug metabolism (M9257) were enriched in LET males (FDR = 0.06; FDR = 0.09). Finally, there was no effect when comparing VEH males and VEH females for genes enriched in Alzheimer’s disease, however the drug metabolism gene set was also enriched in VEH males (FDR = 0.05).

Figure 10.. Estrogen metabolism pathway for LET animals.

Using Cytoscape, the enrichment of genes involved in the estrogen metabolism pathway are shown with up- and downregulated transcripts. Metabolic byproducts (e.g. estradiol) are shown in the light tan, rectangular boxes. Black arrows denote the metabolic byproducts while the enzymes responsible for these products are shown in octagonal shapes colored in red (upregulated), blue (downregulated) or gray (no effect).