Evguenia M. Alexandrova

p63 is a prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/− glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/− glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/− mice again exhibit normal epithelial architecture by conventional histology. However, using RosaLSL-LacZ;WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/− glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/− ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.

Role of p53 family members p73 and p63 in human hematological malignancies

Abstract p53, mutated in over half of human cancers and about 13% of all hematological malignancies, maintains genomic integrity and triggers cellular senescence and apoptosis of damaged cells. In contrast to p53, the homologs p73 and p63 play critical roles in development of the central nervous system and skin/limbs, respectively. Moreover, dependent on the context they can exert tumor suppressor activities that cooperate with p53. Unlike p53, p73 and p63 are rarely mutated in cancers. Instead, up-regulation of the anti-apoptotic dominant-negative ΔNp73 and ΔNp63 isoforms is the most frequent abnormality in solid cancers. In hematological malignancies the most frequent p73 defect is promoter methylation and loss of expression, associated with unfavorable clinical outcomes. This suggests an essential tumor suppressor role of p73 in blood cells, also supported by genetic mouse models. Many therapeutic approaches aiming to restore p73 activity are currently being investigated. In contrast, the most frequent p63 abnormality is protein overexpression, associated with higher disease grade and poorer prognosis. Surprisingly, although available data are still scarce, the emerging picture is up-regulation of transactivation-competent TAp63 isoforms, suggesting a tumor-promoting role in this context.

p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo

Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/− tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data – while cautioning that not all genetic mouse models faithfully represent the human situation – demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

Mutant p53 – Heat Shock Response Oncogenic Cooperation: A New Mechanism of Cancer Cell Survival

The main tumor suppressor function of p53 as a “guardian of the genome” is to respond to cellular stress by transcriptional activation of apoptosis, growth arrest, or senescence in damaged cells. Not surprisingly, mutations in the p53 gene are the most frequent genetic alteration in human cancers. Importantly, mutant p53 (mutp53) proteins not only lose their wild-type tumor suppressor activity but also can actively promote tumor development. Two main mechanisms accounting for mutp53 proto-oncogenic activity are inhibition of the wild-type p53 in a dominant-negative fashion and gain of additional oncogenic activities known as gain-of-function (GOF). Here, we discuss a novel mechanism of mutp53 GOF, which relies on its oncogenic cooperation with the heat shock machinery. This coordinated adaptive mechanism renders cancer cells more resistant to proteotoxic stress and provides both, a strong survival advantage to cancer cells and a promising means for therapeutic intervention.

p73 is dispensable for commitment to neural stem cell fate, but is essential for neural stem cell maintenance and for blocking premature differentiation

p73 is dispensable for commitment to neural stem cell fate, but is essential for neural stem cell maintenance and for blocking premature differentiation

Depleting stabilized GOF mutant p53 proteins by inhibiting molecular folding chaperones: a new promise in cancer therapy

Depleting stabilized GOF mutant p53 proteins by inhibiting molecular folding chaperones: a new promise in cancer therapy

Generation of p53-Deficient Induced Pluripotent Stem Cells from Mouse Embryo Fibroblasts

Here we describe a method for generating induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). Recombinant retroviruses carrying human transcription factors for Klf4, Oct3/4, Sox2, with or without c-Myc, are used to transduce early passage MEFs several times. Based on morphologic criteria, the resulting iPS colonies are picked manually at first, and then propagated and expanded by standard methods. iPS cells can then be differentiated into virtually any cell type or lineage, thus allowing for discoveries of new functions of p53 and mutant p53.

Ganetespib synergizes with cyclophosphamide to improve survival of mice with autochthonous tumors in a mutant p53-dependent manner

The DNA-alkylating cytotoxic agent cyclophosphamide (CTX) is commonly used in the clinic to treat hematological malignancies like lymphomas and leukemias as well as solid tumors, but shows dose-dependent potentially life-threatening toxicities and can induce secondary malignancies. Thus, the clinical utility of CTX would be improved if a companion drug could be identified that allows lowering the CTX dose, while maintaining or even increasing its antineoplastic therapeutic efficacy. In mouse models, high-dose CTX (300 mg/kg) is effective in treating T-lymphomas, while low dose (defined here as 100 mg/kg) is ineffective. We previously showed that the HSP90 inhibitor ganetespib potently suppresses T-lymphoma initiation and progression and extends overall survival (OS) in hotspot knockin mice expressing the p53 gain-of-function mutants R175H and R248Q (mutp53) by 30–59%. Here we asked whether ganetespib could potentiate the effect of low-dose CTX (100 mg/kg) in the autochthonous T-lymphoma-bearing mutp53 R248Q mouse model. Indeed, combinatorial CTX/ganetespib synergistically suppresses growth of autochthonous T-lymphomas in R248Q (p53Q/−) but not p53−/− control mice by reducing mutp53 levels and triggering apoptosis. Combinatorial treatment extends progression-free (PFS) and OS in p53Q/− mice significantly longer than in p53−/− mice. Specifically, PFS of p53Q/− mice improves 8.9-fold over CTX alone versus 3.6-fold in p53−/− mice. Likewise, OS of R248Q/− mice improves 3.6-fold, but worsens in p53−/− mice (0.85-fold) over CTX alone. Moreover, half of the p53Q/− mice on combinatorial treatment lived over 60 days, and one animal reached 121 days. In contrast, p53Q/− mice on single-drug treatment and p53−/− mice on any treatment lived less than 24 days. In sum, ganetespib synergizes with a sub-effective dose of CTX in mutp53 T-lymphomas by suppressing tumor growth and extending survival. Our results provide a potential strategy to reduce the effective clinical dose of CTX in mutant p53-bearing malignancies and attenuate CTX toxicity.

Heat shock factor 1 confers resistance to lapatinib in ERBB2-positive breast cancer cells

Despite success of ERBB2-targeted therapies such as lapatinib, resistance remains a major clinical concern. Multiple compensatory receptor tyrosine kinase (RTK) pathways are known to contribute to lapatinib resistance. The heterogeneity of these adaptive responses is a significant hurdle for finding most effective combinatorial treatments. The goal of this study was to identify a unifying molecular mechanism whose targeting could help prevent and/or overcome lapatinib resistance. Using the MMTV-ERBB2;mutant p53 (R175H) in vivo mouse model of ERBB2-positive breast cancer, together with mouse and human cell lines, we compared lapatinib-resistant vs. lapatinib-sensitive tumor cells biochemically and by kinome arrays and evaluated their viability in response to a variety of compounds affecting heat shock response. We found that multiple adaptive RTKs are activated in lapatinib-resistant cells in vivo, some of which have been previously described (Axl, MET) and some were novel (PDGFRα, PDGFRβ, VEGFR1, MUSK, NFGR). Strikingly, all lapatinib-resistant cells show chronically activated HSF1 and its transcriptional targets, heat shock proteins (HSPs), and, as a result, superior tolerance to proteotoxic stress. Importantly, lapatinib-resistant tumors and cells retained sensitivity to Hsp90 and HSF1 inhibitors, both in vitro and in vivo, thus providing a unifying and actionable therapeutic node. Indeed, HSF1 inhibition simultaneously downregulated ERBB2, adaptive RTKs and mutant p53, and its combination with lapatinib prevented development of lapatinib resistance in vitro. Thus, the kinome adaptation in lapatinib-resistant ERBB2-positive breast cancer cells is governed, at least in part, by HSF1-mediated heat shock pathway, providing a novel potential intervention strategy to combat resistance.