Dysregulation, But Not Mutation Of p53 Signaling Pathway In Breast Implant-Associated Anaplastic Large Cell Lymphoma Cell Lines
Authors: Wang Hai , Xie Chao , Li Shiwu , George Eva V. , Reeves Westley H. , Epstein Alan L. , Yang Li-Jun 
Published: October 2019

Abstract
A consistent feature of over 100 reported cases of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is their complex cytogenetic abnormalities, suggesting that genomic instability may drive lymphomagenesis and/or tumor progression. Loss of heterozygosity(LOH) of the TP53 tumor suppressor gene locus on the short arm of chromosome 17 (17p13.1) is a frequent finding. Human p53 plays an important role in cell cycle arrest, DNA repair, and apoptosis and it maintains genome stability by preventing mutations. Recently, three T cell breast lymphoma (TLBR) cell lines were derived from patients’ BIA-ALCL primary tumor biopsy specimens. These cell lines are IL-2 dependent, ALK-negative, CD30+activated cytotoxic T cells closely resembling the original tumor cells. Thus, the cell lines may serve as an important tool for studying this newly recognized disease entity. Because of its rarity, the clinical pathologic features, tumor cell biology, and genetics of BIA-ALCL have yet to be fully defined.
Here we tested the hypothesis that the p53 signaling pathway is defective in TLBR cells. We initially examined TP53 transcript expression among the cell lines. By qRT-PCR, p53 transcripts were detected in all three lines, with the highest level in TLBR-2. Next we examined p53 protein expression and p53 activation in response to ultraviolet (UV) or gamma irradiation. By Western blotting, all TLBR cell lines expressed much lower levels of p53 protein following UV irradiation (400 J/m2) than Karpas (ALK+ ALCL) cells and failed to show ATM/ATR-induced phosphorylation of p53 on serine 15, an early indicator of p53 activation. Genetic defects (deletion, mutation) of the p53 coding sequence were not found by Sanger sequencing. Interestingly, a polymorphism at p53 codon 72 (Arg72Pro), a normal variant associated with increased susceptibility to breast cancer, was detected in TLBR-1 and -3 (derived from indolent BIA-ALCL), but not in the aggressive BIA-ALCL line TLBR-2. Thus, TLBR cells exhibit defective regulation of the p53 pathway in response to DNA damage, suggesting that their ability to sense DNA damage or the regulation of p53 stability may be impaired.
We next examined the DNA damage sensing pathway upstream of p53 in the presence and absence of the DNA demethylating agent 5-aza-2'-deoxycytidine (AZA, 10µM for 48hrs). In all TLBR lines, ATM and ATR transcripts were expressed at much lower levels (qRT-PCR) than normal, and their expression was not significantly affected by AZA. However, compared with human T cells, CHK2 (phosphorylate P53 at Ser20) transcripts were very low in TLBR-1 and -2, but not in TLBR-3 cells. CHK2 and p21 (the main p53 target gene) transcripts after AZA were greatly increased in TLBR-2, mildly elevated in TLBR-3, and unchanged in TLBR-1 cells, suggesting that DNA methylation of the CHK2 and p21 genes may partly explain the defective p53 signaling in TLBR-2 cells. This was confirmed by detecting of CHK2 phorphrylation only in TLBR-3 cells. Mdm2, a major negative regulator of p53 protein stability, was either normal or low (qRT-PCR), and was unaffected by AZA. However, immunobloting with Mdm2 antibodies revealed increased levels of two isoforms following UV of TLBR-1 and -2, but only the small isoform was expressed in TLBR-3 cells and there was little response to UV treatment. Treatment of TLBR cells with 5 µM Nutlin-3 (Mdm2 antagonist, p53 activator, and apoptosis inducer) inhibited cell growth by 40% at day 5 (MTT assay). We conclude that these three BIA-ALCL derived cell lines share dysregulation of the p53 signaling pathway, which may contribute to the genomic instability characteristic of these BIA-ALCL cases.
First two authors have equally contributed to this abstract.

Disclosures:
No relevant conflicts of interest to declare.