P53 Heterozygous Mice Research Articles

Endogenous p53 inhibitor TIRR dissociates systemic metabolic health from oncogenic activity

It is unclear whether metabolic health corresponds to reduced oncogenesis or vice versa. We study Tudor-interacting repair regulator (TIRR), an inhibitor of p53 binding protein 1 (53BP1)-mediated p53 activation, and the physiological consequences of enhancing tumor suppressor activity. Deleting TIRR selectively activates p53, significantly protecting against cancer but leading to a systemic metabolic imbalance in mice. TIRR-deficient mice are overweight and insulin resistant, even under normal chow diet. Similarly, reduced TIRR expression in human adipose tissue correlates with higher BMI and insulin resistance. Despite the metabolic challenges, TIRR loss improves p53 heterozygous (p53HET) mouse survival and correlates with enhanced progression-free survival in patients with various p53HET carcinomas. Finally, TIRR's oncoprotective and metabolic effects are dependent on p53 and lost upon p53 deletion in TIRR-deficient mice, with glucose homeostasis and orexigenesis being primarily regulated by TIRR expression in the adipose tissue and the CNS, respectively, as evidenced by tissue-specific models. In summary, TIRR deletion provides a paradigm of metabolic deregulation accompanied by reduced oncogenesis.

Abstract PS07-07: The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response

Abstract Background: Women with germline BRCA1 mutations are at an increased risk for developing breast cancer in their lifetime, often at a young age with more aggressive tumors. At present, prophylactic bilateral mastectomy is the most effective strategy for reducing breast cancer risk. However, this invasive procedure is irreversible and associated with potential complications. We and others have found that PARP inhibitors can delay Brca1-mutant tumor formation in mice and could be beneficial for the prevention of breast cancer. However, currently available PARP inhibitors are associated with modest toxicities that may not be acceptable to women without cancer. Thus, there remains an urgent need for the development of safe and effective therapies for the prevention of breast cancer. Here, we present data demonstrating the activity of IRX4204, a minimally toxic and highly specific agonist of the nuclear retinoid X receptor (RXR), to delay the formation of mammary tumors in a Brca1-deficient mouse model. This inhibitory effect on tumor growth is due, in part, to the role of IRX4204 in stimulating the anti-tumor immune response. Methods: We used the established MMTV-Cre, conditional Brca1 gene knockout, p53 heterozygous loss mouse model (BRCA1co/co; MMTV-Cre+/+; p53+/-) and selected for mutant female pups using PCR genotyping. At 16 weeks of age, all mice were separated into 4 treatment groups (n=10 per group): (1) sesame oil control; (2) the novel RXR agonist, IRX-4204 (10mg/kg); (3) high-dose IRX4204 (20 mg/kg); and (4) the RXR agonist, 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage five days per week, and mice were observed daily for tumor formation and toxicity. At the study endpoint, tumors and normal mammary glands were collected for additional analyses. Immunohistochemical staining was used to quantify CD8a, Ki-67 and cleaved caspase 3 expression in Brca1-deficient tumors. Oil Red O staining was used to measure changes in lipid accumulation in Brca1-deficient cell lines treated with IRX4204. qPCR was used to quantify the changes in gene expression of lipid metabolism-associated genes upon treatment with IRX4204 in vitro. Results: Vehicle-treated Brca1-deficient mice had a median time-to-tumor formation (TTF) of 211 days, with 100% developing tumors by 330 days. Mice treated with UAB 5 mg/kg had an improved median TTF of 261 days, whereas mice treated with IRX4204 10mg/kg or 20mg/kg had a median TTF of 347 and 304 days, respectively (p < 0.01). In addition, 60% of mice treated with IRX4204 10 mg/kg remained tumor-free at 330 days. IRX4204-treated tumors showed an increased infiltration of CD8-positive T-cells over vehicle-treated tumors (p < 0.05). Treatment of Brca1-deficient cell lines with IRX4204 in vitro resulted in a significant increase in lipid accumulation accompanied by a 2-fold increase of Srebf1 expression (a key transcription factor that regulates lipid homeostasis) within 24 hours of treatment (p < 0.05). Conclusion: These data demonstrate a novel use of the RXR agonist, IRX4204, to delay the formation of Brca1-deficient mammary tumors. We have found that IRX4204 treatment modulates the tumor immune response through increased infiltration of cytotoxic CD8-positive T-cells in Brca1-deficient mammary tumors in vivo. We have also determined that IRX4204 modulates lipid metabolism in breast cancer cell lines in vitro. It is known that lipid-derived antigens can stimulate T-cell activity. Our findings suggest that RXR agonists may alter lipid antigen production to activate an anti-tumor response. Additional immune and lipidomic studies are on-going. This work was supported by NCI-PREVENT grant (to PB and AM HHSN26100008) and CFP Foundation (Odyssey Fellowship to CM). Citation Format: Cassandra Moyer, Darian Coleman, Jamal Hill, Lana A. Vornik, Michelle Savage, Martin Sanders, Sei Shizuko, Altaf Mohammed, Powel Brown, Abhijit Mazumdar. The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-07.

Loss of the aryl hydrocarbon receptor increases tumorigenesis in p53-deficient mice

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.

Low-dose but not high-dose γ-irradiation elicits the dominant-negative effect of mutant p53 in vivo

Contrary to high doses irradiation (HDR), the biological consequences of dose irradiation (LDR) in breast cancer remain unclear due to the complexity of human epidemiological studies. LDR induces DNA damage that activates p53-mediated tumor-suppressing pathways promoting DNA repair, cell death, and growth arrest. Monoallelic p53 mutations are one of the earliest and the most frequent genetic events in many subtypes of cancer including ErbB2 breast cancer. Using MMTV/ErbB2 mutant p53 (R172H) heterozygous mouse model we found differential p53 genotype-specific effect of LDR vs. HDR on mammary tumorigenesis. Following LDR, mutant p53 heterozygous tumor cells exhibit aberrant ATM/DNA-PK signaling with defects in sensing of double-strand DNA brakes and deficient DNA repair. In contrast, HDR-induced genotoxic stress is sufficient to reach the threshold of DNA damage that is necessary for wtp53 induced DNA repair and cell cycle arrest. As a result, mutant p53 endows dominant-negative effect promoting mammary tumorigenesis after low-impact DNA damage leading to the selection of a genetically unstable proliferative population, with negligible mutagenic effect on tumors carrying wtp53 allele.

Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/−) mice has not been investigated. We hypothesized that p53+/− mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/− mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/− mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/− and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/− mice compared to p53+/+ mice. Importantly, p53+/− mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

Dysregulation and restoration of homeostatic network plasticity in fragile X syndrome mice

Chronic activity perturbations in neurons induce homeostatic plasticity through modulation of synaptic strength or other intrinsic properties to maintain the correct physiological range of excitability. Although similar plasticity can also occur at the population level, what molecular mechanisms are involved remain unclear. In the current study, we utilized a multielectrode array (MEA) recording system to evaluate homeostatic neural network activity of primary mouse cortical neuron cultures. We demonstrated that chronic elevation of neuronal activity through the inhibition of GABA(A) receptors elicits synchronization of neural network activity and homeostatic reduction of the amplitude of spontaneous neural network spikes. We subsequently showed that this phenomenon is mediated by the ubiquitination of tumor suppressor p53, which is triggered by murine double minute-2 (Mdm2). Using a mouse model of fragile X syndrome, in which fragile X mental retardation protein (FMRP) is absent (Fmr1 knockout), we found that Mdm2-p53 signaling, network synchronization, and the reduction of network spike amplitude upon chronic activity stimulation were all impaired. Pharmacologically inhibiting p53 with Pifithrin-α or genetically employing p53 heterozygous mice to enforce the inactivation of p53 in Fmr1 knockout cultures restored the synchronization of neural network activity after chronic activity stimulation and partially corrects the homeostatic reduction of neural network spike amplitude. Together, our findings reveal the roles of both Fmr1 and Mdm2-p53 signaling in the homeostatic regulation of neural network activity and provide insight into the deficits of excitability homeostasis seen when Fmr1 is compromised, such as occurs with fragile X syndrome.

Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth.

Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N‐Methyl‐N‐nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 ( FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121‐fold higher than YTN2. FGFR4‐deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.

Does p53 Inhibition Suppress Myocardial Ischemia-Reperfusion Injury?

p53 is well known as a regulator of apoptosis and autophagy. In addition, a recent study showed that p53 is a modulator of the opening of the mitochondrial permeability transition pore (mPTP), a trigger event of necrosis, but the role of p53 in necrosis induced by myocardial ischemia-reperfusion (I/R) remains unclear. The aim of this study was to determine the role of p53 in acute myocardial I/R injury in perfused mouse hearts. In male C57BL6 mice between 12 and 15 weeks of age, 2 types of p53 inhibitors were used to suppress p53 function during I/R: pifithrin-α, an inhibitor of transcriptional functions of p53, and pifithrin-μ, an inhibitor of p53 translocation from the cytosol to mitochondria. Neither infusion of these inhibitors before ischemia nor infusion for the first 30-minute period of reperfusion reduced infarct size after 20-minute ischemia/120-minute reperfusion. Infarct sizes were similar in p53 heterozygous knockout mice (p53+/-) and wild-type mice (WT), but recovery of rate pressure product (RRP) 120 minutes after reperfusion was higher in p53+/- than in WT. The protein expression of p53 in WT was negligible under baseline conditions, during ischemia, and at 10 minutes after the start of reperfusion, but it became detectable at 120 minutes after reperfusion. In conclusion, upregulation of p53 during the late phase of reperfusion plays a significant role in contractile dysfunction after reperfusion, although p53 is not involved in cardiomyocyte necrosis during ischemia or in the early phase of reperfusion.

Immune deficiency augments the prevalence of p53 loss of heterozygosity in spontaneous tumors but not bi-directional loss of heterozygosity in bone marrow progenitors.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.

Role of p53 in silibinin-mediated inhibition of ultraviolet B radiation-induced DNA damage, inflammation and skin carcinogenesis.

Non-melanoma skin cancers (NMSC) are a growing problem given that solar ultraviolet B (UVB) radiation exposure is increasing most likely due to depletion of the atmospheric ozone layer and lack of adequate sun protection. Better preventive methods are urgently required to reduce UV-caused photodamage and NMSC incidence. Earlier, we have reported that silibinin treatment activates p53 and reduces photodamage and NMSC, both in vitro and in vivo; but whether silibinin exerts its protective effects primarily through p53 remains unknown. To address this question, we generated p53 heterozygous (p53+/-) and p53 knockout (p53-/-) mice on SKH-1 hairless mouse background, and assessed silibinin efficacy in both short- and long-term UVB exposure experiments. In the chronic UVB-exposed skin tumorigenesis study, compared to p53+/+ mice, p53+/- mice developed skin tumors earlier and had higher tumor number, multiplicity and volume. Silibinin topical treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but silibinin' protective efficacy was significantly compromised in p53+/- mice. Additionally, silibinin treatment failed to inhibit precursor skin cancer lesions in p53-/- mice but improved the survival of the mice. In short-term studies, silibinin application accelerated the removal of UVB-induced DNA damage in p53+/+ mice while its efficacy was partially compromised in p53-/- mice. Interestingly, silibinin treatment also inhibited the UVB-induced inflammatory markers in skin tissue. These results further confirmed that absence of the p53 allele predisposes mice to photodamage and photocarcinogenesis, and established that silibinin mediates its protection against UVB-induced photodamage, inflammation and photocarcinogenesis partly through p53 activation.

Effects of chronic deoxynivalenol exposure on p53 heterozygous and p53 homozygous mice

Deoxynivalenol (DON) is a secondary metabolite associated with Fusarium species pathogenic to important food crops. A two-year feeding study reported that DON was non-carcinogenic in B6C3F1 mice. The present study was conducted to further characterize the chronic effects of DON by exposing cancer-prone transgenic p53 heterozygous (p53+/−) male mice and p53 homozygous (p53+/+) male mice to 0, 1, 5, or 10 mg DON/kg in diet for 26 weeks. Gross and microscopic organ-specific neoplastic and non-neoplastic changes and expression profiles of key hepatic and renal genes were assessed. Few toxicologic differences between p53+/+ and p53+/− mice were observed, and no tumours were observed due to DON. The results indicated that DON was non-carcinogenic and that reduced expression of the p53 gene did not play a key role in responses to DON toxicity. The lack of inflammatory and proliferative lesions in mice may be attributed to the anorectic effects of DON, which resulted in dose-dependent reductions in body weight in p53+/+ and p53+/− mice. Hepatic and renal gene expression analyses confirmed that chronic exposure to DON was noninflammatory. The effects of 26-week DON exposure on p53+/+ and p53+/−mice were consistent with those previously seen in B6C3F1 mice exposed to DON for two years.

Effects of Chronic Ochratoxin A Exposure on p53 Heterozygous and p53 Homozygous Mice.

Exposure to the mycotoxin ochratoxin A (OTA) causes nephropathy in domestic animals and rodents and renal tumors in rodents and poultry. Humans are exposed to OTA by consuming foods made with contaminated cereal grains and other commodities. Management of human health risks due to OTA exposure depends, in part, on establishing a mode of action (MOA) for OTA carcinogenesis. To further investigate OTA's MOA, p53 heterozygous (p53+/-) and p53 homozygous (p53+/+) mice were exposed to OTA in diet for 26 weeks. The former are susceptible to tumorigenesis upon chronic exposure to genotoxic carcinogens. OTA-induced renal damage but no tumors were observed in either strain, indicating that p53 heterozygosity conferred little additional sensitivity to OTA. Renal changes included dose-dependent increases in cellular proliferation, apoptosis, karyomegaly, and tubular degeneration in proximal tubules, which were consistent with ochratoxicosis. The lowest observed effect level for renal changes in p53+/- and p53+/+ mice was 200 μg OTA/kg bw/day. Based on the lack of tumors and the severity of renal and body weight changes at a maximum tolerated dose, the results were interpreted as suggestive of a primarily nongenotoxic (epigenetic) MOA for OTA carcinogenesis in this mouse model.

Abstract A06: Obesity induces a metabolic switch toward aerobic glycolysis in breast cancer cells in vitro and in vivo

Abstract Background: The prevalence of obesity is steadily increasing worldwide, impacting the incidence of a variety of diseases, including breast cancer (BCa). Although epidemiological studies show a strong association between elevated body mass index (BMI) and a heightened risk of postmenopausal BCa, the underlying etiology remains relatively unknown. One potential mechanism by which obesity contributes to BCa progression and recurrence is through induction of obesity-induced stress (OBIS). Reactive oxygen species (ROS) are byproducts of metabolism, and excessive production or accumulation of ROS is known to contribute to early tumor initiating events through redox modulation of p53 transcriptional activity. Objectives: Recent studies indicate that p53 is a key regulator of glycolysis. As the obese state is known to promote aerobic glycolysis, the preferred mode of energy production by most transformed cells, and because approximately 40% of human breast cancers harbor p53 mutations, the aim of this study was to determine the impact of obesity on rates of glucose consumption and genotoxic stress in the context of p53 status. Results: Using the MMTV-Wnt1 transgenic mouse model of spontaneous breast cancer harboring a heterozygous deletion mutation for p53, we determined the impact of diet-induced obesity (DIO) on markers of aerobic glycolysis and oxidative stress. Interestingly, thioredoxin interacting protein (TXNIP), a regulator of glucose homeostasis, was found to be induced in the normal mammary fat pad (MFP) of obese p53-heterozygous (p53-het) mice versus p53-wild type (p53-wt) MFPs. Conversely, MCF-7 breast cancer cells grown in vitro in serum from these obese mice showed a 50% decrease in TXNIP expression in comparison to cells cultured in serum from the non-obese mice. These findings are consistent with TXNIP's putative role as a tumor suppressor gene. Further, glucose transporter 1 (GLUT-1), a basal glucose transporter required to sustain cellular respiration, was upregulated 5-fold in the MFP of obese p53-het mice compared to MFP taken from non-obese p53-het mice, confirming that obesity regulates glucose uptake in the MFP independently of p53 status. Additionally, the obese p53-het mice displayed elevated serum levels of 8-isoprostane, a biomarker of oxidative stress. Additional in vitro studies demonstrate that MCF-7 cells cultured in serum derived from obese versus non-obese C57BL/6 mice (Ob-serum and non-Ob serum) displayed heightened levels of ROS and significant modulation of HIF-1α, a ROS-inducible transcription factor known to regulate glycolysis. Ob-serum also enhanced lactate dehydrogenase activity and inhibited expression of pyruvate dehydrogenase (PDH), concurrently promoting glycolysis and bypassing oxidative phosphorylation (OXPHOS). This metabolic shift was associated with the accumulation of γ-H2AX foci, a known indicator of DNA double-strand breaks (DSBs). Finally, we observed that a combined shRNA-mediated knockdown of TXNIP and p53 in MCF-7 cells resulted in a dramatic induction of DSBs, and this was exacerbated by exposure to Ob-serum. Potential Impact: Based on these findings, we conclude that obesity may enhance the rate of metabolic reprogramming in breast cancer cells, resulting in OBIS and accumulation of ROS; thereby promoting disease progression by enhancing genotoxic stress in cells harboring a p53 mutation. Citation Format: David Antonio Cavazos, Karrie Wheatly, Stephen Hursting. Obesity induces a metabolic switch toward aerobic glycolysis in breast cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A06.

Gene Expression Profiles Resulting from Stable Loss of p53 Mirrors Its Role in Tissue Differentiation

The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53’s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

The genotype-dependent influence of functionalized multiwalled carbon nanotubes on fetal development

In many cases cancer is caused by gene deficiency that is being passed along from generation to generation. Soluble carbon nanotubes (CNTs) have shown promising applications in the diagnosis and therapy of cancer, however, the potential relationship between cancer-prone individuals and response to CNT exposure as a prerequisite for development of personalized nanomedicine, is still poorly understood. Here we report that intravenous injections of multi-walled carbon nanotubes into p53 (a well-known cancer-susceptible gene) heterozygous pregnant mice can induce p53- dependent responses in fetal development. Larger sized multi-walled carbon nanotubes moved across the blood-placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas single-walled and smaller sized multi-walled carbon nanotubes showed no or less fetotoxicity. A molecular mechanism study found that multi-walled carbon nanotubes directly triggered p53-dependent apoptosis and cell cycle arrest in response to DNA damage. Based on the molecular mechanism, we also incorporated N-acetylcysteine (NAC), an FDA approved antioxidant, to prevent CNTs induced nuclear DNA damage and reduce brain development abnormalities. Our findings suggest that CNTs might have genetic background-dependent toxic effect on the normal development of the embryo, and provide new insights into protection against nanoparticle-induced toxicity in potential clinical applications.

Abstract 5438: Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation.

Abstract The tumor repressor gene, p53, is involved in a variety of cellular activities ranging from stress, differentiation and cell cycle regulation. In our previous studies we have shown p53’s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression while the same is absent in p53 null mice. This deficiency of p53 has also been shown to produce more osteosarcomas than a complete loss of p53. This suggests that the presence of p53 is able to modify the environment within pre osteoblasts based on its ability to regulate key bone specific genes. In the present study we compared changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels in C2C12 cells capable of both myoblast and osteoblast differentiation transiently, and compared the changes in gene expression of candidate genes to cells with stable p53 knockdown. Using a PCR array to assay p53 target genes, we have found differential expression profiles when comparing stable versus transient knockdown. As expected several of genes that were profoundly affected after transient p53 loss were related to apoptosis and cell cycle regulation. Stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles suggesting that long term effect of p53 loss affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were also validated by measuring promoter activities of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression profoundly affecting normal tissue physiology than its acute loss. Citation Format: Oliver Couture, Eric Lombardi, Kendra Davis, Emily Hays, Nalini Chandar. Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5438. doi:10.1158/1538-7445.AM2013-5438

Abstract 326: Overexpression of the carboxyl-terminus of MUC16 can accelerate the occurrence of phenotypes in p53 heterozygous mouse model.

Abstract Ovarian cancer is the fifth leading cause of cancer death in women. Cancer antigen 125 (CA125), also known as MUC16, is a high molecular weight glycoprotein overexpressed in sera of women with epithelial ovarian cancer, but not in normal epithelial ovarian tissue. As a biomarker, CA125 is widely used to screen, diagnose and monitor the epithelial ovarian cancer. To further understand the roles of MUC16 in tumorigenesis, recently, some mouse models related Muc16 and MUC16 were created. There is no phenotype for the Muc16 knockout mice (Cheon et al., PLoS ONE, 2009). We built a MUC16 carboxyl-terminus transgenic mouse (MUC16ΔC354) and found out that the transgenic mice appear to be viable, fertile, and develop normally through the more than two years of life. In order to investigate the relationship of p53 mutation and MUC16 overexpression in tumorigensis, we generated mice in which MUC16ΔC354 can be overxepressed with p53 heterozygote. Like the MUC16ΔC354 transgenic mice, the MUC16ΔC354; p53+/- mice develop normally. Both mouse strains are susceptible to spontaneous tumors, including sarcoma and lymphoma. However, comparing p53+/- mice, overexpression of MUC16ΔC354 significantly accelerates the occurrence of phenotypes in MUC16ΔC354; p53+/- mice. Out of a study population of 94 MUC16ΔC354; p53+/- mice, 22 (23.4%) developed obvious neoplasms by 18 months of age, 13 (11.9%) out of 109 for p53+/- mice (ρ <0.014). Tumor tissues and tumor cells carrying MUC16ΔC354 and p53+/- are transplantable. MAPK/ERK signaling pathway is upregulated in these tumors. Our findings demonstrate our hypothesis that MUC16 may play a role in tumorigensis. Citation Format: Huasong Tian, Qin Zhou, Alexia Iasonos, Spriggs David. Overexpression of the carboxyl-terminus of MUC16 can accelerate the occurrence of phenotypes in p53 heterozygous mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2013-326

Abstract A85: Silibinin exerts its protective effects against UVB-induced photodamage and skin carcinogenesis through p53

Abstract Ultraviolet (UV)-caused photodamage is the primary etiological factor for non-melanoma skin cancers (NMSCs), the most common malignancy in the United States. Better preventive strategies are urgently required to reduce UV-caused photodamage as well as the incidence of NMSCs. The tumor suppressor gene p53 is considered the most important cellular transcriptional factor for preserving genomic stability, and plays a major protective role against UVB-induced photodamage in epidermal cells. Mutated p53 patches are an early molecular biomarker and considered one of the most important characteristics of UVB-induced skin carcinogenesis. Earlier, in vitro and in vivo studies have shown that the natural flavonolignan silibinin activates p53 as well as reduces photodamage and NMSCs; but whether silibinin channels its protective effects primarily through p53 remains unknown. Herein, we generated p53 heterozygous (p53+/−) and p53 null (p53−/−) mice on SKH-1 hairless mouse background to investigate this issue. In wild-type SKH-1 mice, topical application of silibinin (9 mg/200 μl acetone) pre- or post- UVB (180 mJ/cm2) irradiation significantly reduced the formation of CPDs (cyclobutane pyrimidine dimers) after 12 h, while CPD repair by silibinin was significantly compromised in p53−/− mice. In the skin carcinogenesis study, wild type (p53+/+) and heterozygous (p53+/-) SKH-1 hairless mice were exposed to UVB (90 mJ/cm2, 5 times per week), while a subset was administered silibinin topically (9 mg/200 μl acetone) pre- or post-UVB irradiation. Mice were sacrificed at various time-points (4, 8, and 25 weeks) and skin samples were collected and analyzed. Analyses for proliferation marker (PCNA) in the early time-points (4 and 8 weeks) showed that the proliferation rate was higher in p53+/- mice compared to p53+/+ mice; furthermore, the strong inhibitory effect of silibinin on PCNA was evident in p53+/+ mice but compromised in p53+/- mice. At the end of the 25 weeks, p53+/- mice developed skin tumors earlier than p53+/+ mice; and p53+/- mice showed higher tumor number, multiplicity, and volume as compared to p53+/+ mice. Results also showed that silibinin treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but its protective efficacy was significantly compromised in p53+/- mice. In the case of p53+/+ mice, silibinin treatment (pre- or post-UVB) significantly reduced tumor volume in irradiated animals (5.3 and 18.6 mm3/per mouse compared to 109.5 mm3/per mouse in UVB-alone irradiated group; P<0.001) accounting for 95% and 83% reduction in tumor volume respectively. While in p53+/- mice, UVB irradiation induced significantly larger tumors (503.7 mm3), in the case of silibinin pre- and post-treatment the tumor volume was reduced to 293 mm3 (P<0.05 versus UVB-alone) and 425 mm3 accounting for only 42% and 15% reduction respectively. These results not only reestablished previous observations that absence of p53 allele significantly predisposes animals to photodamage and photocarcinogenesis, but also showed for the first time that silibinin mediates its protection against UVB-induced photodamage and photocarcinogenesis in a p53-dependent manner. These mechanistic studies support the use of silibinin as chemopreventive agent in NMSCs. Citation Format: Cynthia Tilley, Srirupa Roy, Gagan Deep, Chapla Agarwal, Rajesh Agarwal. Silibinin exerts its protective effects against UVB-induced photodamage and skin carcinogenesis through p53. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A85.

Dose‐dependent mesothelioma induction by intraperitoneal administration of multi‐wall carbon nanotubes in p53 heterozygous mice. Histology of a small nodule of moderately to poorly differentiated epithelioid mesothelioma

Dose‐dependent mesothelioma induction by intraperitoneal administration of multi‐wall carbon nanotubes in p53 heterozygous mice. Histology of a small nodule of moderately to poorly differentiated epithelioid mesothelioma

Effects of long term exposure to the mycotoxin fumonisin B1 in p53 heterozygous and p53 homozygous transgenic mice

The fungal toxin fumonisin B1 (FB1) is a potential human carcinogen based on evidence of renal carcinogenicity in rats and hepatocarcinogenicity in mice. The toxicity and carcinogenicity of FB1 is linked to ceramide synthase inhibition. Based on this mechanism of action and on lack of evidence of genotoxicity, FB1 is considered a non-genotoxic carcinogen. The p53 heterozygous (p53+/−) mouse is a cancer-prone model used for carcinogenesis. The effects of chronic dietary FB1 exposure were characterized in p53+/− mice to confirm non-genotoxicity using a model which is more sensitive to genotoxic than non-genotoxic carcinogens and to clarify the relationship between p53 expression, altered sphingolipid metabolism, and FB1-induced carcinogenesis. Responses to FB1 were similar in p53+/− and p53+/+ mice after 26weeks exposure to 0, 5, 50 or 150mg FB1/kg diet, supporting a non-genotoxic mechanism of action. Hepatic adenomas and cholangiomas were observed in mice exposed to 150mg/kg FB1. For a 10% increase in hepatic megalocytosis, the estimated 95% lower confidence limit of the benchmark dose (BMDL10) ranged from 0.15 and 1.11mg FB1/kg bw/day. Based on similar responses in p53+/− and p53+/+ mice, p53 and related pathways play a secondary role in responses to FB1 toxicity and carcinogenesis.