Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression

Mortality from triple negative breast cancer (TNBC) is significantly higher in African American (AA) women compared to White American (WA) women emphasizing ethnicity as a major risk factor; however, the molecular determinants that drive aggressive progression of AA-TNBC remain elusive. Here, we demonstrate for the first time that AA-TNBC cells are inherently aggressive, exhibiting elevated growth, migration, and cancer stem-like phenotype compared to WA-TNBC cells. Meta-analysis of RNA-sequencing data of multiple AA- and WA-TNBC cell lines shows enrichment of GLI1 and Notch1 pathways in AA-TNBC cells. Enrichment of GLI1 and Notch1 pathway genes was observed in AA-TNBC. In line with this observation, analysis of TCGA dataset reveals a positive correlation between GLI1 and Notch1 in AA-TNBC and a negative correlation in WA-TNBC. Increased nuclear localization and interaction between GLI1 and Notch1 is observed in AA-TNBC cells. Of importance, inhibition of GLI1 and Notch1 synergistically improves the efficacy of chemotherapy in AA-TNBC cells. Combined treatment of AA-TNBC-derived tumors with GANT61, DAPT, and doxorubicin/carboplatin results in significant tumor regression, and tumor-dissociated cells show mitigated migration, invasion, mammosphere formation, and CD44+/CD24- population. Indeed, secondary tumors derived from triple-therapy-treated AA-TNBC tumors show diminished stem-like phenotype. Finally, we show that TNBC tumors from AA women express significantly higher level of GLI1 and Notch1 expression in comparison to TNBC tumors from WA women. This work sheds light on the racial disparity in TNBC, implicates the GLI1 and Notch1 axis as its functional mediators, and proposes a triple-combination therapy that can prove beneficial for AA-TNBC.


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All the experiments were conducted thrice (biological replicates) in triplicates (technical replicates).

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Statistical analyses were exploratory. Analyses were done using R and GraphPad Prism 5. Data was transformed when necessary to achieve normality. One-way ANOVA analysis was conducted for overall comparison across multiple groups. Pairwise comparisons were done using Student's t-test with Bonferroni correction. Kaplan-Meier survival curves were reported for survival outcomes and log-rank test was conducted to compare survival outcomes between groups. Results were considered to be significant if p-value < 0.05.
We utilized a Tissue microarray consisting of 25 AA and 46 WA TNBC specimens that was generated at Yale Developmental Histology Lab using TNBC specimens that were diagnosed at the Yale-New Haven Hospital, Connecticut, USA between 1996 and 2004. TNBC status (absence of ER, PR, and HER2) was determined by IHC at the Yale Developmental Histology Lab.