SUMMARY Studies on the role of TP53 mutation in breast cancer response to chemotherapy are conflicting. Here, we show that, contrary to dogma, MMTV-Wnt1 mammary tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin-treated p53-mutant tumors failed to arrest proliferation leading to abnormal mitoses and cell death, while p53 wild-type tumors arrested, avoiding mitotic catastrophe. Senescent tumor cells persisted, secreting senescence-associated cytokines that exhibited autocrine/paracrine activity and mitogenic potential. Wild-type p53 still mediated arrest and inhibited drug response even in the context of a heterozygous p53 point mutation or absence of p21. Thus, we show wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy.

Cellular senescence is a permanent growth arrest in cells with damage or stress that could lead to transformation. Some tumor cells also undergo senescence in response to chemotherapy. Senescent cells secrete cytokines and other factors of the senescence-associated secretory phenotype (SASP) that contribute to tumor suppression by enforcing arrest and recruiting immune cells that remove these damaged or oncogene-expressing cells from organisms. However, some cells can develop a SASP comprising factors that are immunosuppressive and protumorigenic by paracrine mechanisms. Likewise, the SASP in treated cancers can either contribute to durable responses or drive relapse. Here, we discuss the studies that have demonstrated a complex and often conflicting role for the SASP in tumorigenesis and treatment response.

Summary MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2SNP309T or the MDM2SNP309G allele to address the impact of MDM2SNP309G on tumorigenesis. Mdm2SNP309G/G cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2SNP309G/G mice succumbed to tumors before one year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2SNP309G allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2SNP309G allele.

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Summary Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53515C (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2−/− mice. Mdm2−/− p53515C/515C mice, however, die by postnatal day 13 due to hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2−/− p53515C/515C mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS, and cell cycle arrest, senescence and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized due to ROS, and its loss increased cell cycling, and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation, protect cells from apoptosis, and enhance metastasis. In this study, we examined the IGF signaling pathway in two breast cancer cell lines selected for metastatic behavior. LCC6 was selected for growth as an ascites tumor in athymic mice from parental MDA-MB-435 cells (435P). The MDA-231BO cell line was derived from osseous metastases that formed after intracardiac injection of the MDA-MB-231 cell line in athymic mice. Compared to the parental cell lines, IGF-I treatment enhanced IRS-2 phosphorylation over IRS-1 in the metastatic variants. IGF-I stimulated cell migration in the variant cells, but not in the parental cells. To determine the role for IRS-2 in IGF-mediated motility, we transfected MDA-231BO cells with an anti-sense IRS-2 construct. Transfected cells had decreased levels of IRS-2 with diminished IGFmediated motility and anchorage independent growth when compared to control cells. However, adherence to ®bronectin was enhanced in the transfected cells compared to MDA-231BO cells. Our data show that breast cancer cells selected for metastatic behavior in vivo have increased IRS-2 activation and signaling. In these cells, IGF-I enhances cell adhesion and motility suggesting that IRS-2 may mediate these aspects of the malignant phenotype. Oncogene (2001) 20, 7318 ± 7325.

p53 levels are tightly regulated in normal cells, and thus the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly Mdm2, allowing p53 to be stabilized to activate cell cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we demonstrated that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. Additionally, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which often times contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status.

Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity.