Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status.

Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure-function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function.

∆Np63/p40 correlates with the location and phenotype of basal/mesenchymal cancer stem-like cells in human ER+ and HER2+ breast cancers.

ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2-driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal-type triple-negative breast cancer, some receptor-positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell-like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform-specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non-triple negative breast cancers and the presence of this population associated with improved survival in patients with ER- /HER2+ tumours (p = 0.006). Furthermore, 41% of ER+ /PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+ /ALDH- . In vitro studies revealed that MCF7 and T47D (ER+ ) and BT-474 (HER2+ ) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.

Small molecule antagonists of the sigma-1 receptor cause selective release of the death program in tumor and self-reliant cells and inhibit tumor growth in vitro and in vivo.

The acquisition of resistance to apoptosis, the cell's intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the sigma-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. sigma antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic sigma-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even sigma receptor-rich neurons are resistant to the apoptotic effects of sigma antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in sigma receptor coupling rather than levels of expression. In susceptible cells only, sigma antagonists evoke a rapid rise in cytosolic calcium that is inhibited by sigma-1 agonists. In at least some tumor cells, sigma antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3'-kinase pathway signaling. Systemic administration of sigma antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a sigma receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.

Response to trastuzumab by HER2 expressing breast tumour xenografts is accompanied by decreased Hexokinase II, glut1 and [18F]-FDG incorporation and changes in 31P-NMR-detectable phosphomonoesters.

PURPOSE: Trastuzumab, effective in about 15 % of women with breast cancer, downregulates signalling through the Akt/PI3K and MAPK pathways. These pathways modulate glucose and phospholipid metabolism which can be monitored by [(18)F]FDG-PET and (31)P-NMR spectroscopy, respectively. Here, the relationship between response of HER-2 overexpressing tumours and changes in [(18)F]-FDG incorporation and (31)P-NMR-detectable phosphomonoesters were examined. EXPERIMENTAL: Xenografts derived from HER2-overexpressing MDA-MB-453 human breast tumour cells were grown in SCID mice, treated with trastuzumab for 15 days, then [(18)F]-FDG uptake determined and (31)P-NMR carried out on chemical extracts of the tumours. Western blots were carried out to determine protein expression of Hexokinase II and glut1. RESULTS: [(18)F]-FDG incorporation, Hexokinase II and glut1 protein expression and the concentration of phosphocholine and phosphoethanolamine in chemical extracts subjected to (31)P-NMR were significantly decreased in the xenografts in the trastuzumab-treated mice compared with xenografts from the PBS-injected group. CONCLUSIONS: Changes in FDG incorporation and (31)P-NMR spectral changes can accompany response of HER2-expressing breast cancer xenografts to trastuzumab. This is the first study to show parallel changes in [(18)F]FDG- and (31)P-NMR-detectable metabolites accompany response to targeted anticancer treatment.

Differential contextual responses of normal human breast epithelium to ionizing radiation in a mouse xenograft model.

Radiotherapy is a key treatment option for breast cancer, yet the molecular responses of normal human breast epithelial cells to ionizing radiation are unclear. A murine subcutaneous xenograft model was developed in which nonneoplastic human breast tissue was maintained with the preservation of normal tissue architecture, allowing us to study for the first time the radiation response of normal human breast tissue in situ. Ionizing radiation induced dose-dependent p53 stabilization and p53 phosphorylation, together with the induction of p21(CDKN1A) and apoptosis of normal breast epithelium. Although p53 was stabilized in both luminal and basal cells, induction of Ser392-phosphorylated p53 and p21 was higher in basal cells and varied along the length of the ductal system. Basal breast epithelial cells expressed ΔNp63, which was unchanged on irradiation. Although stromal responses themselves were minimal, the response of normal breast epithelium to ionizing radiation differed according to the stromal setting. We also demonstrated a dose-dependent induction of γ-H2AX foci in epithelial cells that was similarly dependent on the stromal environment and differed between basal and luminal epithelial cells. The intrinsic differences between human mammary cell types in response to in vivo irradiation are consistent with clinical observation that therapeutic ionizing radiation is associated with the development of basal-type breast carcinomas. Furthermore, there may be clinically important stromal-epithelial interactions that influence DNA damage responses in the normal breast. These findings demonstrate highly complex responses of normal human breast epithelium following ionizing radiation exposure and emphasize the importance of studying whole-tissue effects rather than single-cell systems.

p53 and gamma radiation in the normal breast.

PURPOSE: With the increasing use of radiation as adjuvant therapy in breast cancer, the effects of gamma radiation on the remaining normal breast are of increasing importance. The complexities of multiple cellular types within breast tissues and the role of the pleiotropic Tumour Protein 53 (TP53, p53) protein with its downstream transcriptional targets and cellular processes may be central to the effects on residual normal breast tissues. CONCLUSION: While a detailed understanding of p53 protein-mediated responses in normal breast tissues remains elusive, p53 appears to have a pivotal role in the effects of gamma radiation on normal breast epithelium, but not stromal cells, which may account for the differing clinical effects of gamma radiation in women treated for breast cancer.