Induction of Ovarian Leiomyosarcomas in Mice by Conditional Inactivation of Brca1 and p53

Background

Approximately one out of every ten cases of epithelial ovarian cancer (EOC) is inherited. The majority of inherited cases of EOC result from mutations in the breast cancer associated gene 1 (BRCA1). In addition to mutation of BRCA1, mutation of the p53 gene is often found in patients with inherited breast and ovarian cancer syndrome.

Methodology/Principal Findings

We investigated the role of loss of function of BRCA1 and p53 in ovarian cancer development using mouse models with conditionally expressed alleles of Brca1 and/or p53. Our results show that ovary-specific Cre-recombinase-mediated conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP resulted in ovarian or reproductive tract tumor formation in 54% of mice, whereas conditional inactivation of either allele alone infrequently resulted in tumors (≤5% of mice). In mice with conditionally inactivated Brca1^LoxP/LoxP and p53^LoxP/LoxP, ovarian tumors arose after long latency with the majority exhibiting histological features consistent with high grade leiomyosarcomas lacking expression of epithelial, follicular or lymphocyte markers. In addition, tumors with conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP exhibited greater genomic instability compared to an ovarian tumor with inactivation of only p53^LoxP/LoxP.

Conclusions/Significance

Although conditional inactivation of both Brca1 and p53 results in ovarian tumorigenesis, our results suggest that additional genetic alterations or alternative methods for targeting epithelial cells of the ovary or fallopian tube for conditional inactivation of Brca1 and p53 are required for the development of a mouse model of Brca1-associated inherited EOC.     

Cooperativity of Rb,Brca1, and p53 in Malignant Breast Cancer Evolution

Breast cancers that are “triple-negative” for the clinical markers ESR1, PGR, and HER2 typically belong to the Basal-like molecular subtype. Defective Rb, p53, and Brca1 pathways are each associated with triple-negative and Basal-like subtypes. Our mouse genetic studies demonstrate that the combined inactivation of Rb and p53 pathways is sufficient to suppress the physiological cell death of mammary involution. Furthermore, concomitant inactivation of all three pathways in mammary epithelium has an additive effect on tumor latency and predisposes highly penetrant, metastatic adenocarcinomas. The tumors are poorly differentiated and have histologic features that are common among human Brca1-mutated tumors, including heterogeneous morphology, metaplasia, and necrosis. Gene expression analyses demonstrate that the tumors share attributes of both Basal-like and Claudin-low signatures, two molecular subtypes encompassed by the broader, triple-negative class defined by clinical markers.     

抗癌基因──p53和Rb基因甲基化的研究进展

介绍p53和Rb基因甲基比研究的最新进展.CpG序列是DNA甲基化和基因突变的频发位点.p53和Rb基因的CpG序列易发生甲基化.其高度甲基化可能与基因功能失活有关,从而丧失抑制细胞增殖的功能,可能为肿瘤等发生的重要原因之一.     

Fgfr1 Inactivation in the Mouse Telencephalon Results in Impaired Maturation of Interneurons Expressing Parvalbumin

Fibroblast growth factors (Fgfs) and their receptors (Fgfr) are expressed in the developing and adult CNS. Previous studies demonstrated a decrease in cortical interneurons and locomotor hyperactivity in mice with a conditional Fgfr1 deletion generated in radial glial cells during midneurogenesis (Fgfr1^f/f;hGfapCre+). Here, we report earlier and more extensive inactivation of Fgfr1 in neuroepithelial cells of the CNS (Fgfr1^f/f;NesCre+). Similar to findings in Fgfr1^f/f;hGfapCre+ mice, parvalbumin positive (PV+) cortical interneurons are also decreased in the neocortex of Fgfr1f/f;NesCre+ mice when compared to control littermates (Fgfr1f/f). Fgfr1f/f;NesCre+ embryos do not differ from controls in the initial specification of GABAergic cells in the ganglionic eminence (GE) as assessed by in situ hybridization for Dlx2, Mash1 and Nkx2. Equal numbers of GABAergic neuron precursors genetically labeled with green fluorescent protein (GFP) were observed at P0 in Fgfr1^f/f;hGfapCre+;Gad1-GFP mutant mice. However, fewer GFP+ and GFP+/PV+ interneurons were observed in these mutants at adulthood, indicating that a decrease in cortical interneuron markers is occurring postnatally. Fgfr1 is expressed in cortical astrocytes in the postnatal brain. To test whether the astrocytes of mice lacking Fgfr1 are less capable of supporting interneurons, we co-cultured wild type Gad1-GFP+ interneuron precursors isolated from the medial GE (MGE) with astrocytes from Fgfr1f/f control or Fgfr1^f/f;hGfapCre+ mice. Interneurons grown on Fgfr1 deficient astrocytes had small soma size and fewer neurites per cell, but no differences in cell survival. Decreased soma size of Gad67 immunopositive interneurons was also observed in the cortex of adult Fgfr1^f/f;NesCre+ mice. Our data indicate that astrocytes from Fgfr1 mutants are impaired in supporting the maturation of cortical GABAergic neurons in the postnatal period. This model may elucidate potential mechanisms of impaired PV interneuron maturation relevant to neuropsychiatric disorders that develop in childhood and adolescence.     

Inactivation of arf-bp1 induces p53 activation and diabetic phenotypes in mice

It is well accepted that the Mdm2 ubiquitin ligase acts as a major factor in controlling p53 stability and activity in vivo. Although several E3 ligases have been reported to be involved in Mdm2-independent p53 degradation, the roles of these ligases in p53 regulation in vivo remain largely unknown. To elucidate the physiological role of the ubiquitin ligase ARF-BP1, we generated arf-bp1 mutant mice. We found that inactivation of arf-bp1 during embryonic development in mice resulted in p53 activation and embryonic lethality, but the mice with arf-bp1 deletion specifically in the pancreatic β-cells (arf-bp1(FL/Y)/RIP-cre) were viable and displayed no obvious abnormality after birth. Interestingly, these mice showed dramatic loss of β-cells as mice aged, and >50% of these mice died of severe diabetic symptoms before reaching 1 year of age. Notably, the diabetic phenotype of these mice was largely reversed by concomitant deletion of p53, and the life span of the mice was significantly extended (p53(LFL/FL)/arf-bp1(FL/Y)/RIP-cre). These findings underscore an important role of ARF-BP1 in maintaining β-cell homeostasis in aging mice and reveal that the stability of p53 is critically regulated by ARF-BP1 in vivo.     

Brca2 and Trp53 Deficiency Cooperate in the Progression of Mouse Prostate Tumourigenesis

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.     

Morphological Differentiation of the Microvasculature During Follicular Development, Ovulation and Luteinization of Mouse Ovaries

The chronological changes of the microvasculature during follicular development, ovulation and luteinization of mouse ovaries were examined by observation of serial histological sections, lectin angiographs and resin-corrosion casts. Graafian follicles possessing oocytes with germinal vesicles were surrounded by a few layers of basket-like capillary wreath adjacent to the follicular basement membrane. Just before ovulation 11–12 hr after hCG administration, some theca cells differentiated into hypertrophic cells, and the follicular basement membrane underwent fragmentation. Then the capillaries within the theca interna became dilated, and hyperpermeable and appeared to be injured. The capillary wreath extended into the follicle via the hypertrophied theca interna. After ovulation, the follicular wall became markedly edematous. Capillary branches invaded the granulosa cell layer of the ruptured follicle from the region of extravasation to form an intricate capillary network. The capillary network occupied the whole corpus luteum until 24 hr after hCG administration.     

Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats

Objective

Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity).

Methods and Results

PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 μM PFT.

Conclusions

Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis.     

Control of the annual testicular cycle of the marbled-newt by p53, p21, and Rb gene products

p53, p21, and Rb are proteins with an important role in cell-cycle control. The expression and distribution of these gene products and the apoptotic rate were studied in the marbled-newt testis along the annual cycle to know the role of these factors in the control of spermatogenesis and glandular tissue formation. The study was carried out using Western blot analysis and immunohistochemistry. The results differed from those, previously reported in mammals showing constant spermatogenesis. Greater expression of p53 and p21 was found in the quiescence period and was detected in PCGs (primordial germ cells), spermatogonia, follicular, interstitial cells, and glandular tissue. Greater expression of Rb and phosph-Rb was present in the proliferation period, in PCGs, and spermatogonia. Apoptosis was only detected in secondary spermatogonia (quiescence and spermiogenesis periods) and primary spermatocytes (proliferation and spermiogenesis periods). In the quiescence period, the increase in p53 expression activates p21 expression, which inhibits Rb phosphorylation and arrests the cell cycle in G1. In the proliferation period and, in a lesser degree, in the spermiogenesis period, the expressions of p53 and p21 decrease and phosph-Rb increases, enhancing cell proliferation. These gene products do not seem to be related to apoptosis.     

Inactivation of Myc-induced p53-dependent apoptosis in human tumors

The Myc family of oncoproteins promote cell growth and are frequently overexpressed in human tumors. However, Myc can also trigger cell death by apoptosis. This is at least in part mediated via the ARF-p53 pathway. Myc activation leads to a selection for inactivation of ARF or p53, allowing cell survival and tumor progression. Restoration of p53-dependent apoptosis by various means is an attractive approach for new cancer therapy.     

Immunohistochemical expression of the p53, mdm2, p21/Waf-1, Rb, p16, Ki67, cyclin D1, cyclin A and cyclin B1 proteins and apoptotic index in T-cell lymphomas

Fifty-seven cases of T-cell lymphomas (TCL) including 5 lymphoblastic (T-LBL) and 52 peripheral TCL (PTCL) were analyzed by immunohistochemistry for the expression of p53, mdm2, p21, Rb, cyclin D1, cyclin A, cyclin B1, and Ki67/MIB1 proteins and 39/52 PTCL were also analyzed for the expression of p16 protein and for the presence of apoptotic cells by the TUNEL method. The aim was to search for abnormal immunoprofiles of p53 and Rb growth control pathways and to determine the proliferative activity and the apoptotic index of TCL. Abnormal overexpression of p53, p21 and mdm2, in comparison to normal lymph nodes, was found in 12/57, 10/57 and 2/57 cases of TCL, respectively. Abnormal loss of Rb and p16 expression was found in 1/57 and 2/39 cases, respectively, whereas abnormal overexpression of cyclin D1 was not detected in any of the 57 cases. Our data revealed entity-related p53/p21/mdm2 phenotypes. Indeed, most nodal and cutaneous CD30+ anaplastic large cell lymphomas (ALCL) showed concomitant overexpression of p53 and p21 proteins (7/8 cases), and mdm2 was overexpressed in 2 p53-positive nodal ALCL. In contrast, overexpression of p53 was found in 3/17 cases of nodal peripheral TCL unspecified (PTCL-UC) and 2/7 non-ALCL cutaneous pleomorphic TCL. Overexpression of p21 protein was detected in 2/3 p53-positive PTCL-UC and in 1/2 p53-positive non-ALCL cutaneous pleomorphic TCL. Finally, all the remaining 25 cases of TCL did not show p53 and p21 overexpression. Overall, the p53+/p21+ phenotype in 10/57 TCL suggests wild-type p53 capable of inducing p21 expression. The highest apoptotic index (AI) was found in ALCL and a positive correlation between apoptotic index and Ki67 index (p<0.001) was detected. Ki67, cyclin A and cyclin B1 expression was found in all 57 TCL and on the basis of the combined use of these 3 variables, 3 groups of proliferative activity could be determined: a) high in ALCL and T-LBL, b) low in mycosis fungoides (MF) and gammadelta hepatosplenic TCL, and c) intermediate in the remaining TCL entities. The proliferative activity in the 12 p53 overexpressing cases was higher in comparison to the 45 p53-negative cases. Ki67 expresion in more than 25% of tumour cells showed significant correlation with p53 overexpression (p<0.001). Rb expression tended to be parallel to Ki67, cyclin A and cyclin B1 expression in all but one case of nodal PTCL-UC which displayed loss of RB expression. Interestingly, this case was p53-negative, whereas the p53-positive cases were Rb-positive. These findings suggest that different pathogenetic routes may function in some TCL, involving either the p53 or, less frequently, the Rb pathways.     

Inactivation of p53 by human T-cell lymphotropic virus type 1 Tax requires activation of the NF-kappaB pathway and is dependent on p53 phosphorylation

p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-kappaB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kappaB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IkappaB mutant. Tax-NF-kappaB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-kappaB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-kappaB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.