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Proteomic Analysis of Arylamine N-Acetyltransferase 1 Knockout Breast Cancer Cells

ABSTRACT: A phase II metabolic enzyme, NAT1 is frequently upregulated in breast cancers. Previous studies have shown that inhibition or depletion of NAT1 in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. A 2DLC-MS approach using TMT 10-plex labeling performed comparing three three groups (n=3 per group) plus a pooled internal standard/normalization channel. These groups include a parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines differing by proliferation rates. Relative protein abundances were compared by Scaffold Q+S v4.4.5 at the 99.9% CI at the peptide and protein levels to estimate protein group identification and differential abundances by Kruskal-Wallis and log fold change differences. MetaCore was used to explore pathway analyses and to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Michael L. Merchant, PhD

PROVIDER: MSV000089446 | MassIVE | Mon May 09 09:23:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Extracellular, cancer-specific methylated DNA has been shown to be a prognostic marker when detected in serum or plasma. In this study we investigated the effect of treating cancer cells with differentially methylated CpG DNA. When breast cancer cell lines were treated with methylated CpG DNA, a consistent upregulation of CHAC1 mRNA expression was observed. CHAC1 was recently described to be a novel component of the unfolded protein response pathway. To elucidate the role of CHAC1 mRNA expression in cancer in more detail, we analyzed expression of this gene in breast (n=107) and ovarian cancer (n=107) and found a strong correlation with tumor differentiation. Poorly differentiated tumors exhibited higher CHAC1 expression levels (p=0.004 for breast and p=0.031 for ovarian cancer). Additionally, hormone receptor (HR)-negative breast cancers (p<0.001) and advanced stage disease ovarian cancers (p=0.026) also demonstrated high CHAC1 mRNA levels. mRNA expression analysis of the two known CHAC1 isoforms showed a strong association of expression above the median with poor outcome in breast cancer patients in a multivariate analysis (isoform a: relative risk (RR) of death 3.2 (95% CI 1.6-6.5; p<0.01); RR of relapse 3.9 (95% CI 1.6-9.8; p<0.01); isoform b: relative risk (RR) of death 3.5 (95% CI 1.6-7.3; p<0.01); RR of relapse 6.6 (95% CI 2.4-18.5; p<0.01)). Univariate analysis in ovarian cancer showed that CHAC1 mRNA expression above the median was associated with a poor relapse free survival (p=0.03). In younger ovarian cancer patients (age < median age), a high CHAC1 mRNA expression was associated with overall survival (p=0.007) and relapse free survival (p=0.015). Finally, we show that downregulation of CHAC1 by small interfering RNA suppressed breast cancer cell migration and proliferation, whereas overexpression resulted in an observed increase in these cellular behaviours. This is the first report demonstrating that a gene (CHAC1) whose expression is triggered by methylated, but not unmethylated DNA, is involved in tumour biology. Human breast cancer cell lines (BT-20, Hs578T) were treated with methylated and unmethylated DNA oligos, and differentially expressed genes were identified between treatments with methylated and un-methylated oligos.

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Proteomic Analysis of Arylamine N-Acetyltransferase 1 Knockout Breast Cancer Cells

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