Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice and exposed them to AAI in vitro (50 μM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.

Exposure to aristolochic acid (AA) is linked to kidney disease and urothelial cancer in humans. The major carcinogenic component of the AA plant extract is aristolochic acid I (AAI). The tumour suppressor p53 is frequently mutated in AA-induced tumours. We previously showed that p53 protects from AAI-induced renal proximal tubular injury, but the underlying mechanism(s) involved remain to be further explored. In the present study, we investigated the impact of p53 on AAI-induced gene expression by treating Trp53(+/+), Trp53(+/-), and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for six days. The Clariom™ S Assay microarray was used to elucidate gene expression profiles in mouse kidneys after AAI treatment. Analyses in Qlucore Omics Explorer showed that gene expression in AAI-exposed kidneys is treatment-dependent. However, gene expression profiles did not segregate in a clear-cut manner according to Trp53 genotype, hence further investigations were performed by pathway analysis with MetaCore™. Several pathways were significantly altered to varying degrees for AAI-exposed kidneys. Apoptotic pathways were modulated in Trp53(+/+) kidneys; whereas oncogenic and pro-survival pathways were significantly altered for Trp53(+/-) and Trp53(-/-) kidneys, respectively. Alterations of biological processes by AAI in mouse kidneys could explain the mechanisms by which p53 protects from or p53 loss drives AAI-induced renal injury in vivo.

Background: Lobular carcinoma in situ (LCIS) is typically clinically undetectable but is being increasingly diagnosed as a result of breast screening mammography and is often found associated with other breast pathologies such as invasive lobular breast cancer (ILC), invasive carcinoma of ductal /no special type (IDC) and ductal carcinoma in situ (DCIS). It is also considered a risk factor for the development of subsequent invasive breast disease. The aim of this study was to understand the genetic relationship between LCIS that presents with synchronous DCIS, IDC and/or ILC in order to ascertain whether the components have common precursors and also to understand the clonal relationship between LCIS and subsequent invasive disease. Methods: 25 cases of LCIS with synchronous ILC, 7 cases of LCIS with synchronous DCIS & IDC, and 8 pure LCIS that developed a subsequent invasive recurrence were identified from the GLACIER study (MREC 06/Q1702/64). DNA was extracted from archival paraffin embedded tissue and underwent copy number analysis using either the Oncoscan™ Array (Affymetrix) or HumanCytoSNP FFPE-12 BeadChip (Illumina). Four of 7 cases of LCIS with synchronous DCIS & IDC also underwent targeted sequencing using a custom 121 breast cancer-associated gene panel (SureSelectXT HS kit, Agilent Technologies). Clonal relatedness was assessed using a novel methodology based on the presence of shared copy number aberration breakpoints and mutations. Results: Of the 25 synchronous LCIS and ILC cases, 17 appeared related, 4 were ambiguous (sharing the typical lobular signature of 1q gain and 16q loss) and 4 demonstrated no evidence of relatedness. Of the 7 cases with synchronous LCIS, DCIS and IDC, all had copy number data available and 4 had mutation data available. In 3 cases the synchronous LCIS, DCIS and IDC were clonally related according to copy number and for two there was mutation data that supported this (one sharing PIK3CA and CDH1 mutations, the other a TP53 mutation). In two cases the LCIS was not related to the DCIS or IDC, but the DCIS and IDC were related to each other; while in one case LCIS was related to IDC but not to the DCIS by copy number but all components shared the same CHEK2 mutation. Finally in one case none of the three components were related to each other by copy number but the LCIS and IDC shared a PIK3CA mutation, albeit at much lower allele frequency in LCIS than in IDC. Of the 8 patients with pure LCIS, 4 developed an ipsilateral invasive recurrence of various combinations of morphologies: 1 ILC & LCIS, 1 ILC & DCIS, 1 IDC & DCIS, 1 ILC & IDC, and 4 a contralateral recurrence (1 tubular, 1 IDC, 2 ILC), with a median time to recurrence of 69 months (range 34-175). The primary LCIS was related to at least one component of the recurrent disease in all four ipsilateral cases; in two the primary LCIS and all components of the recurrent disease were related, and in the remainder we observed a variety of putative evolutionary patterns. Conclusions: The majority (68%) of cases of synchronous LCIS and ILC appeared to be clonally related by copy number. 50% of cases of co-existing LCIS and IDC appeared to have a common clonal origin by either copy number or targeted sequencing. As these are genomically stable tumours, copy number data may also be underestimating relatedness. In the four cases of pure primary LCIS that developed an ipsilateral recurrence, different subtypes of breast cancer were noted as the recurrence morphology, supporting the historical view that LCIS is a risk lesion rather than a true precursor. However, in all cases the preceding LCIS was found to be related to at least one component of the subsequent invasive tumour including DCIS and IDC. This data shows that clonal relatedness between LCIS and both synchronous and asynchronous invasive disease and DCIS is more complex than previously thought, with LCIS acting as a precursor lesion even in some cases of IDC. Citation Format: Elinor Sawyer, Mateja Sborchia, Anargyros Megalios, Vandna Shah, Salpie Nowinski, Cloe Vassart, Anita Grigoriadis, Alastair Thompson, Ian Tomlinson, Rebecca Roylance, Sarah Pinder. Clonal relatedness of LCIS with synchronous and asynchronous invasive disease [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 PS16-03.