Abstract PS16-19: Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer
Authors: El-Dana Fouad , Yuan Bin , Ly Stanley , Anand Vivek , Battula Venkata Lokesh 
Published: February 2021

p53 mutations occur in 80% of triple-negative breast cancer (TNBC) cases. Several types of p53 mutations have been reported, among which gain of function mutations have been associated with increased cancer stem cells, chemotherapy resistance, and disease relapse in several malignancies. Particularly, hotspot p53 mutations (mutations at amino acid location 157, 175, 248, 249, 273 predict worse overall survival in some cancer types. We have previously reported that Ganglioside GD2, a cell surface glycosphingolipid, identifies breast cancer stem cells and promotes tumorigenesis. We have also reported that GD3 synthase, a key enzyme which catalyzes the rate-limiting step of the GD2 biosynthesis pathway, is significantly upregulated in breast tumors with p53 mutations. Here, we hypothesize that mutant p53 promotes stemness in TNBC cells by regulating GD3 synthase and GD2 expression. To identify specific p53 mutations that contribute to TNBC stemness, we stratified TNBC cell lines (N=18) based on p53 mutation status including mutation type, location, and domain affected. We measured GD2 and GD3 synthase expression in the available TNBC cell lines by flow cytometry and RT-PCR, respectively. We found that GD3 synthase expression is significantly upregulated in TNBC cell lines with p53 hotspot mutations compared to cell lines with other p53 mutations (Median relative expression 0.0114 compared to 0.0003, p=0.005). Similarly, we found that GD2 expression is significantly higher in TNBC cell lines with hotspot mutations compared to non-hotspot counterparts (Median GD2+ cells 15.2% compared to 0.97%, p= 0.013) Additionally, GD2 and GD3 synthase are upregulated in basal-type TNBC cell lines and TCGA patient samples compared to other TNBC molecular subtypes. Interestingly, we also found that GD2 expression is not always directly correlated with GD3S expression, suggesting that GD2 synthase expression is also a key factor in the regulation of GD2 expression. To examine the effect of mutant p53 protein levels on TNBC stemness, we measured p53 expression in all the available TNBC cell lines by western blot, and found that p53 protein levels do not correlate with GD2 or GD3S expression. To validate these findings, we analyzed RPPA data from the MD Anderson Cancer Cell Lines Project and found similar results. These data suggest that it is the type of p53 mutation, but not the amount of p53 protein in the cells that determines GD2 and GD3 synthase expression in TNBC cells. For instance, p53 hotspot mutations involving amino acid position 248 have been shown to be strongly associated with increased cancer stemness. We found that cell lines with p53 mutations at this specific location have significantly higher GD3 synthase expression compared to other mutant p53 forms. We are currently examining the direct role of mutant p53 in the regulation of GD3S expression by stabilizing mutant p53 using MDM2 inhibitors as well as stable knockdown of p53 gene in multiple TNBC cell lines to establish a clear link between mutant p53 and stem cell marker expression in TNBC cell lines.

In conclusion, stem cell markers are highly expressed in p53 mutant TNBC cell lines compared to wild type p53 counterparts. Specifically, cell lines with hotspot p53 mutations significantly correlate with increased TNBC stemness. The type of p53 mutation, rather than level of its expression correlates with stem cell marker expression in TNBC cells.

Citation Format: Fouad El-Dana, Bin Yuan, Stanley Ly, Vivek Anand, Venkata Lokesh Battula. Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-19.