Carolyn S. Van Pelt

The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences.

Photothermal-Chemotherapy with Doxorubicin-Loaded Hollow Gold Nanospheres: A Platform for Near-Infrared Light-Trigged Drug Release

Photothermal ablation (PTA) is an emerging technique that uses near-infrared (NIR) laser light-generated heat to destroy tumor cells. However, complete eradication of tumor cells with PTA is difficult because of uneven heat distribution in the treatment volume. We hypothesized that combining PTA with chemotherapy using a single multifunctional nanoconstruct that mediates simultaneous photothermal cell killing and drug release (photothermal-chemotherapy) would result in enhanced antitumor activity and reduced toxicity compared to chemotherapy alone. Doxorubicin (DOX) was loaded to hollow gold nanospheres (HAuNS) coated with polyethylene glycol (PEG). The pharmacokinetics and biodistribution of both DOX and HAuNS in the resulting nanoconstruct, DOX@PEG-HAuNS having different DOX:PEG:HAuNS ratios, were evaluated using dual isotope labeling techniques. The antitumor activity of DOX@PEG-HAuNS with DOX:PEG:HAuNS weight ratio of 1:3:1 (NP3) in combination with NIR laser was studied in vitro and in vivo using human MDA-MB-231 breast cancer and A2780 ovarian cancer cells. In vitro, NP3 mediated PTA of both cancer cells and DOX release upon NIR laser treatment. In vivo, NP3 showed slower clearance in blood and greater accumulation in tumors than free DOX. NP3-plus-NIR laser demonstrated greater antitumor activity than free DOX, NP3, or liposomal DOX. Moreover, NP3 displayed significantly decreased systemic toxicity compared to free DOX or liposomal DOX. Enhanced antitumor effect with NP3-plus-laser can be attributed to both the cytotoxic effect of DOX released from NP3 and the photothermal effect mediated by HAuNS. Slow release of DOX from NP3 in normal tissues contributed to reduced systemic toxicity. Photothermal-chemotherapy exemplified by a single-agent nanoconstruct NP3 is a promising approach to anticancer therapy.

Impaired angiogenesis in aminopeptidase N-null mice.

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.

Haploinsufficiency of Mdm2 and Mdm4 in Tumorigenesis and Development

ABSTRACT The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Eμ-myc Mdm4+/− mice show a delayed onset of B-cell lymphomas compared to Eμ-myc mice. Additionally, Mdm2+/−Mdm4+/− double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/−Mdm4+/− mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction.

Multiple stress signals activate mutant p53 in vivo

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 murine double minute 2 (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 showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, 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 oftentimes 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.

Transitional Cell Hyperplasia and Carcinomas in Urinary Bladders of Transgenic Mice with Keratin 5 Promoter-Driven Cyclooxygenase-2 Overexpression

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in approximately 10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

Overexpression of the Low Molecular Weight Cyclin E in Transgenic Mice Induces Metastatic Mammary Carcinomas through the Disruption of the ARF-p53 Pathway

In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor-bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy.

Spontaneous tumorigenesis in mice overexpressing the p53-negative regulator Mdm4.

High levels of the critical p53 inhibitor Mdm4 is common in tumors that retain a wild-type p53 allele, suggesting that Mdm4 overexpression is an important mechanism for p53 inactivation during tumorigenesis. To test this hypothesis in vivo, we generated transgenic mice with widespread expression of Mdm4. Two independent lines of transgenic mice, Mdm4(Tg1) and Mdm4(Tg15), developed spontaneous tumors, the most prevalent of which were sarcomas. To determine whether overexpression of Mdm4 also cooperated with p53 heterozygosity to induce tumorigenesis, we generated Mdm4(Tg1) p53(+/-) mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53(+/-) or Mdm4(Tg1) mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4(Tg1) p53(+/-) tumors. Mdm4 is thus a bona fide oncogene in vivo and cooperates with p53 heterozygosity to drive tumorigenesis. These Mdm4 mice will be invaluable for in vivo drug studies of Mdm4 inhibitors.

Loss of Mdm4 Results in p53-Dependent Dilated Cardiomyopathy

Background— Although several loci for familial dilated cardiomyopathy (DCM) have been mapped, the origin of a large percentage of DCM remains unclear. Mdm2, a p53-negative regulator, protects cardiomyocytes from ischemic and reperfusion-induced cell death. Mdm4, a homolog of Mdm2, inhibits p53 activity in numerous cell types. It is unknown whether Mdm4 plays a role in the inhibition of p53 in fully differentiated tissues such as adult cardiomyocytes and whether this role is associated with DCM. Methods and Results— The conditional knockout of Mdm4 in the heart by use of cardiomyocyte-specific Cre (&agr;MyHC-Cre) allele does not result in any developmental defects. With time, however, mice with deletion of Mdm4 in the adult heart developed DCM and had a median survival of 234 days. More interestingly, the onset of DCM occurs significantly earlier in male mice than in female mice, which mimics human DCM disease. DCM in Mdm4 mutant mice was caused by loss of cardiomyocytes by apoptosis, and it was p53-dose dependent. Conclusion— Activity of p53 was inhibited by Mdm4 even in the fully differentiated cardiomyocyte. Elevated apoptosis mediated by the p53 pathway in cardiomyocytes may be a mechanism for DCM.

Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

Mouse models can be useful for increasing the understanding of lung tumorigenesis and assessing the potential of chemopreventive agents. We explored the role of inflammation in lung tumor development in mice with knockout of the tumor suppressor Gprc5a. Examination of normal lung tissue and tumors from 51 Gprc5a+/+ (adenoma incidence, 9.8%; adenocarcinoma, 0%) and 38 Gprc5a−/− mice (adenoma, 63%; adenocarcinoma, 21%) revealed macrophage infiltration into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 42% of adenomas and 88% of adenocarcinomas in the knockout mice. Gprc5a−/− mouse lungs contained higher constitutive levels of proinflammatory cytokines and chemokines and were more sensitive than lungs of Gprc5a+/+ mice to stimulation of NF-κB activation by lipopolysaccharide in vivo. Studies with epithelial cells cultured from tracheas of Gprc5a−/− and Gprc5a+/+ mice revealed that Gprc5a loss is associated with increased cell proliferation, resistance to cell death in suspension, and increased basal, tumor necrosis factor α–induced, and lipopolysaccharide-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by reexpression of Gprc5a. Compared with Gprc5a+/+ cells, the Gprc5a−/− cells produced higher levels of chemokines and cytokines and their conditioned medium induced more extensive macrophage migration. Silencing Gprc5a and the p65 subunit of NF-κB in Gprc5a+/+ and Gprc5a−/− cells, respectively, reversed these effects. Thus, Gprc5a loss enhances NF-κB activation in lung epithelial cells, leading to increased autocrine and paracrine interactions, cell autonomy, and enhanced inflammation, which may synergize in the creation of a tumor-promoting microenvironment. Cancer Prev Res; 3(4); 424–37. ©2010 AACR.