Fig. 1: ASAH1 overexpression in TNBC is regulated via p53 and PI3K-AKT signaling pathway. | Cell Death & Disease

Fig. 1: ASAH1 overexpression in TNBC is regulated via p53 and PI3K-AKT signaling pathway.

From: ASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability

Fig. 1

A Analysis of immunohistochemical (IHC) staining data from a tissue microarray (TMA) with triple-negative breast cancer samples [21] and matched adjacent normal breast tissue samples [10]. The average intensity of ASAH1 staining in the TNBC and normal breast tissue samples is shown, which is categorized as follows: 0, no staining; +1, weak staining; +2, moderate staining; or +3, high staining. B Protein expression was measured via IHC staining in a TMA containing TNBC and matched normal adjacent breast tissue (20× magnification) samples. Representative images are shown. Scale bar, 50 μm. C Transcript levels of ASAH1 and CDKN1A (p21) were evaluated by quantitative reverse-transcriptase–polymerase chain reaction (qRT-PCR) in MDA-MB-468 cells after treatment with 25 μM of APR-246 (Prima-1Met) for 24 h. D Protein levels for ASAH1 and p21 were evaluated by immunoblotting in MDA-MB-468 cells after treatment with 25 μM of APR-246 (Prima-1Met) for 24 h. ACTINB was used as a loading control. E Schematic diagram showing the p53 binding site on the ASAH1 promoter region. F MDA-MB-468 cells after treatment with 25 μM of APR-246 (Prima-1Met) for 24 h were analyzed using CUT and RUN assay to evaluate the binding of p53 to the ASAH1 promoter. G MDA-MB-468 cells expressing either nonspecific (NS) small hairpin RNA (shRNA) or mutant p53 targeting shRNAs were analyzed by performing a quantitative reverse-transcriptase–polymerase chain reaction (RT-qPCR). Mutant p53 and ASAH1 mRNA expression level in the knockdown cells is presented. H Immunoblotting of the shown protein was performed in mutant p53 shRNA-expressing cells along with NS shRNA-expressing cells. ACTINB was used as the loading control. I ASAH1 mRNA levels were evaluated by quantitative reverse-transcriptase–polymerase chain reaction (qRT-PCR) in MDA-MB-468 cells after treatment with 100 nM of Buparlisib for 24 h. J Immunoblotting of the shown protein was performed in MDA-MB-468 cells after treatment with 100 nM of Buparlisib for 24 h. ACTINB was used as the loading control. K Schematics showing PI3K-AKT signaling activates ASAH1 expression and WT p53 inhibits ASAH1 expression in TNBC cells. Data represent the mean ± standard error for three biological replicates. **P < 0.01, ***P < 0.001, ns not significant.

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