Altered extracellular matrix-related pathways accelerate the transition from normal to prefibroid myometrium in Black women

Abstract

Background: Black women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms. The etiology underlying this racial disparity remains elusive.

Objective: The aim of this study was to evaluate the molecular differences in normal myometrium (fibroid-free uteri) and at-risk myometrium (fibroid-containing uteri) tissues in Black and White women.

Study design: We conducted whole-genome RNA-seq on normal and at-risk myometrium tissues obtained from both self-identified Black and White women (not Hispanic or Latino) to determine global gene expression profiles and to conduct enriched pathway analyses (n=3 per group). We initially assessed the differences within the same type of tissue (normal or at-risk myometrium) between races. Subsequently, we analyzed the transcriptome of normal myometrium compared to at-risk myometrium in each race and determined the differences between them. We validated our findings through real-time PCR (sample size range=5-12), western blot (sample size range=5-6), and immunohistochemistry techniques (sample size range=9-16).

Results: The transcriptomic analysis revealed distinct profiles between Black and White women in normal and at-risk myometrium tissues. Interestingly, genes and pathways related to extracellular matrix and mechanosensing were more enriched in normal myometrium from Black than White women. Transcription factor enrichment analysis detected greater activity of the serum response transcription factor positional motif in normal myometrium from Black compared to White women. Furthermore, we observed increased expression levels of myocardin-related transcription factor-serum response factor and the serum response factor in the same comparison. In addition, we noted increased expression of both mRNA and protein levels of vinculin, a target gene of the serum response factor, in normal myometrium tissues from Black women as compared to White women. Importantly, the transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women. Specifically, we observed that extracellular matrix-related pathways are involved in the transition from normal to at-risk myometrium and that these processes are exacerbated in Black women. We found increased levels of Tenascin C, type I collagen alpha 1 chain, fibronectin, and phospho-p38 MAPK (Thr180/Tyr182, active) protein levels in at-risk over normal myometrium tissues from Black women, whereas such differences were not observed in samples from White women.

Conclusion: These findings indicate that the racial disparities in uterine fibroids may be attributed to heightened production of extracellular matrix in the myometrium in Black women, even before the tumors appear. Future research is needed to understand early life determinants of the observed racial differences.

Keywords: at-risk myometrium; health disparity; leiomyomas; mechanotransduction; uterine fibroid risk.

Figure 1.. Extracellular matrix - and mechanosensing-related pathways are enriched in MyoN from Black compared to White women.

A) Principal component analysis plot (PCA) based on RNA-seq gene expression data, B) Heatmap performed by unsupervised hierarchical clustering of differentially expressed genes, and C) Volcano plot (Log2FC>1.5) in normal (MyoN) myometrium tissues in samples from Black (n=3) and White (n=3) patients. D) Top 15 Reactome pathways overrepresented in Black MyoN over White MyoN. Note: bolded pathways are related to extracellular matrix signaling. E) mRNA levels of Integrin α5 (Itga5, top) and Integrin β1 (Itgb1, bottom) in MyoN from Black (n= 8–10) and White women (n=11–12). F) Transcription Factor Target Overrepresentation Analysis focused on Serum Response Factor (SRF) in Black versus White women normal myometrium (n=3 each). G) Myocardin-related transcription factor A (MRTF-A) and SRF gene expression (shown as Log2CPM values) in Black versus White women normal myometrium (n=3 each). H) mRNA levels of vinculin (VCL) in Black (n=6) and White (n=5) MyoN. I) Violin plot showing total intensity of positive/mm2 (means are indicated with pink lines) and representative IHC images (20X magnification, scale bar = 200 μm) of VCL (Vinculin) in Black (n=9) and White (n=12) MyoN. J) mRNA levels of ROCK1 in Black (n=10) and White (n=11) MyoN. The data are shown as the mean ± SEM. ns= not significant. * = p< 0.05, Student’s t test. MyoN: Normal myometrium tissue. ACTA1: Actin Alpha 1, Skeletal Muscle; ACTC1: Actin Alpha 1, Cardiac Muscle; ACTG2: Actin Gamma 2, Smooth Muscle; KIAA1324: endosome-lysosome associated apoptosis and autophagy regulator 1; MUC16: Mucin-16; MYH2: myosin 2; MYH3: myosin 3; PIGR: polymeric immunoglobulin receptor; TNC: Tenascin C.

Figure 2.. Transcriptomic profiles of at-risk myometrium tissues are distinct between races.

A) Principal component analysis (PCA) plot based on RNA-seq gene expression data. B) Heatmap performed by unsupervised hierarchical clustering of differentially expressed genes. C) Volcano plot (Log₂FC>1.5) in at-risk (MyoF) myometrium tissues in samples from Black (n=3) and White (n=3) patients. D) Top 15 Reactome pathways overrepresented in Black MyoF over White MyoF. Note: the bolded pathways are related to extracellular matrix signaling. ADAMTS5: A disintegrin and metalloproteinase with thrombospondin motifs 5; CLDN1: Claudin-1; CTNNA2: Catenin Alpha 2; IDO2: indoleamine 2,3-dioxygenase 2; TNN: Tenascin N.

Figure 3.. The transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women.

A) Principal component analysis (PCA) plots and B) unsupervised hierarchical heatmap plots in at-risk (MyoF; n=3 Black, n=3 White) over normal (MyoN; n=3 Black, n=3 White) myometrium tissues in Black (left) and White (right). C) Venn diagrams illustrating the overlap of differentially expressed genes in MyoF over MyoN that changed in the same (left diagram) or in the opposite (right diagram) direction between races. D) Volcano plots showing genes with fold changes (Log₂FC>1.5) that were also statistically significant in the Black MyoF over Black MyoN (right) and White MyoF over White MyoN (left) comparisons. ACTA1: Actin Alpha Skeletal Muscle 1; ACTC1: Actin Alpha Cardiac Muscle 1; ATF3: Activating Transcription Factor 3; CTNNA2: Catenin Alpha 2; FOSB: FosB proto-oncogene, AP-1 transcription factor subunit; GABRP: Gamma-Aminobutyric Acid Type A Receptor Subunit Pi; JUNB: JunB proto-oncogene, AP-1 transcription factor subunit; MUC16: Mucin 16; MYH3: Myosin Heavy Chain 3; RASD1: Dexamethasone-induced Ras-related protein 1.

Figure 4.. The role of extracellular matrix-related pathways in the transition from normal to at-risk myometrium is exacerbated in Black women.

Bubble chart of overrepresentation analysis (ORA) using the Reactome MSigDB collection in at-risk (MyoF) and normal myometrium (MyoN) in samples from A) Black (n=3 MyoN, n=3 MyoF) and B) White women (n=3 MyoN, n=3 MyoF). C) Reactome extracellular matrix (ECM)-related pathway overrepresented analysis in Black MyoF over MyoN and Black MyoF over MyoN. D) Tenascin (TCN) mRNA levels in samples from Black (MyoN n=7; MyoF n=11) and White (MyoN n=7; MyoF n=6) women. E) TCN protein levels and representative gel in myometrial tissues obtained from Black (MyoN n=5; MyoF n=6) and White (MyoN n=5; MyoF n=5) women. Bar plots showing total intensity of positive/mm2 and representative IHC images (20X magnification, scale bar = 200 μm) of F) Fibronectin and G) collagen type I alpha 1 (COL1A1) in MyoN and MyoF from samples of Black and White women. Means are indicated with pink lines. The data are shown as the mean ± SEM. ns= not significant. * = p< 0.05, Student’s t test (MyoF vs. MyoN). H) Protein levels and representative gel of phospho-p38 MAPK (Thr180/Tyr182) in in samples from Black (MyoN n=6; MyoF n=5) and White (MyoN n=5; MyoF n=6) women. The data were normalized to GAPDH protein levels.

Figure 5.. Figure depicting the racial disparities in the underlying increased risk and incidence of uterine fibroids (UFs) between Black and White women.

1) Anthropological risk factors specific to Black women. The elevated prevalence of helminths in Africa dating back thousands of years has driven the selection of genotypes that promote an augmented Th2 immune response . These genotypes confer heightened resistance against helminthic infections and play a role in a subset of fibroproliferative disorders demonstrating augmented incidence and severity among individuals of African descent. 2) Risk factors for developing UF, encompassing endocrine-disrupting chemical exposure, obesity, vitamin D deficiency, which is also more prevalent in Black women, contribute to ECM accumulation and chronic inflammation, thereby increasing the vulnerability of the myometrium to the initiation of tumorigenesis. 3) These environmental influences extend to the social sphere, including racism, which has been linked to an increased risk of UF in Black women. 4) The accumulation of ECM and disrupted mechanotransduction processes promote the induction of DNA damage and subsequent emergence of mutations. These genetic alterations trigger pathways implicated in promoting cellular proliferation, inhibiting apoptosis, and sustaining ECM remodeling. These events collectively contribute to the origin and advancement of UF. 5) Black women are disproportionately affected by UF, experiencing an earlier onset, greater burden, and more severe symptoms.