p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer

Abstract

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse.

Chemotherapy-induced senescent cancer cells engulf other cells to enhance their survival

“…p53 mutant tumors had a more modest enrichment (Fig. 7, C–E), consistent with other senescence-related gene signatures in this genotype (Tonnessen-Murray et al, 2018) and reflective of cell death occurring in p53 mutant cells that might have incurred enough cell damage to activate the engulfment pathway (Tonnessen-Murray et al, 2018). Cell engulfment, SA-βGAL–positive staining, and morphological changes become evident ∼3–5 d after doxorubicin treatment (Figs.…”

“…To examine interactions among cells in treated mammary tumors, we transplanted p53 wild-type MMTV- Wnt1 tumors that were transduced with lentiviruses expressing various GFP- and mCherry-conjugated markers. After tumors formed, mice were treated with doxorubicin to induce arrest and senescence as previously shown (Jackson et al, 2012; Tonnessen-Murray et al, 2018) and then harvested during the response. Using confocal microscopy of sections, we observed structures consistent with cells engulfed within other cells in the treated tumors.…”

“…To understand the molecular mechanisms altered in senescent cells, RNA sequencing (RNA-seq) and gene set enrichment analysis (GSEA) were performed on MMTV- Wnt1 tumors (Tonnessen-Murray et al, 2018) and two cell lines, MCF-7 and 4226. Strikingly, a potential program of engulfment was revealed.…”

“…Positive staining for SA-βGAL activity, caused by an expanded lysosomal compartment (Kurz et al, 2000; Lee et al, 2006), is a well-described feature of many senescent cell types, both in vivo and in vitro, in normal and cancer cells (Jackson et al, 2012; Sharpless and Sherr, 2015), including chemotherapy-treated breast cancers (te Poele et al, 2002; Jackson et al, 2012; Tonnessen-Murray et al, 2018). The function for this near-ubiquitous feature of senescent cells was previously unclear, but our observation of entire engulfed cells being processed to the lysosome suggests an obvious purpose for lysosome expansion.…”

“…All experiments were approved by the Tulane Institutional Animal Care and Use Committee, Protocol ID# 4381, and conformed to the guidelines of the United States Animal Welfare Act and the National Institutes of Health. For tumor transplants performed as previously described (Tonnessen-Murray et al, 2018), ∼4 × 10 6 cells from cryopreserved MMTV- Wnt1 tumors were resuspended in Matrigel (Corning) and injected into the #4 mammary fat pad. Tumors formed after 2–3 wk, and when they became ∼1.25 cm, they were excised, minced, trypsinized for 10 min, and filtered through a 100-µm filter.…”