Genetic prevention of lymphoma in p53 knockout mice allows the early development of p53-related sarcomas

Homozygous knockout of p53 in mice leads to early mortality from lymphoma, with almost complete penetrance, thus hampering studies of other tumor histotypes related to p53 alterations. To avoid lymphoma development, we crossed p53 knockout mice (BALB-p53 mice) with alymphocytic BALB/c Rag2^−/−;Il2rg^−/− (RGKO) mice. We compared the tumor spectrum of homozygous (BALB-p53^−/−) and heterozygous (BALB-p53^+/−) mice with alymphocytic mice (RGKO-p53^−/− and RGKO-p53^+/−). Lymphoma incidence in BALB-p53^−/− mice exceeded 80%, whereas in RGKO-p53^−/− it was strongly reduced. The prevalent tumor of RGKO-p53^−/− mice was hemangiosarcoma (incidence over 65% in both sexes, mean latency 18 weeks), other tumors included soft tissue sarcomas (incidence ~10%), lung and mammary carcinomas. Tumor spectrum changes occurred also in p53 heterozygotes, in which lymphomas are relatively rare (~20%). RGKO-p53^+/− had an increased incidence of hemangiosarcomas, reaching ~30%, and females had an increased incidence of osteosarcomas, reaching ~20%. Osteosarcomas shared with the corresponding human tumors the involvement of limbs and a high metastatic ability, mainly to the lungs. Specific alterations in the expression of p53-related genes (p16Ink4a, p19Arf, p15Ink4b, p21Cip1) were observed. Genetic prevention of lymphoma in p53 knockout mice led to new models of sarcoma development, available for studies on hemangiosarcoma and osteosarcoma onset and metastatization.     

Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase delta gene

DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired. Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis. Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2(-/-) tumours and Msh2(-/-) normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner. Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis. Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase delta (DNA pol delta), we hypothesized that the acquisition of mutations in DNA pol delta could contribute to the hypermutator phenotype and tumorigenesis in Msh2(-/-) thymic tissue. Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR. Therefore, we sequenced all 26 exons of the DNA pol delta catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2(-/-) thymic lymphomas and two wild-type controls. No DNA pol delta pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol delta sequences were observed. We conclude, therefore, that inactivating mutations in DNA pol delta are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas.     

Increased incidence of endometrioid tumors caused by aberrations in E-cadherin promoter of mismatch repair-deficient mice

Loss of E-cadherin expression is a critical step in the development and progression of gynecological tumors. Study of the precise role of E-cadherin has been hampered by the lack of satisfactory mouse model for E-cadherin deficiency. Likewise, DNA mismatch repair (MMR) is implicated in gynecological tumorigenesis, but knockout of MMR in mice predominantly causes hematologic neoplasms. Here, we show that combined disruption of E-cadherin and DNA MMR pathways increases incidence of endometrioid tumors in mice. Twenty percent of mice knockout for Msh2 enzyme and hemizygous for E-cadherin [Msh2(-/-)/Cdh1(+/-)] developed endometrioid-like tumors in the ovary, uterus and genital area. Characteristic of these tumors was a complete loss of E-cadherin expression. Sequence analysis of E-cadherin promoter region demonstrated that the loss of E-cadherin expression is caused by inactivating mutations, implying that E-cadherin is a mutational target in Msh2-deficient mice. In addition, Msh2(-/-)/Cdh1(+/-) mice showed a reduction in overall survival as compared with their Msh2(-/-) counterparts due to the development of more aggressive lymphomas, suggesting a specific role of E-cadherin in lymphomagenesis. In conclusion, Msh2(-/-)/Cdh1(+/-) mice provide a good model of gynecological tumorigenesis and may be useful for testing molecular target-specific therapies.     

Crosstalk between Desmoglein 2 and Patched 1 accelerates chemical-induced skin tumorigenesis

Aberrant activation of Hedgehog (Hh) signaling is causative of BCCs and has been associated with a fraction of SCCs. Desmoglein 2 (Dsg2) is an adhesion protein that is upregulated in many cancers and overexpression of Dsg2 in the epidermis renders mice more susceptible to squamous-derived neoplasia. Here we examined a potential crosstalk between Dsg2 and Hh signaling in skin tumorigenesis. Our findings show that Dsg2 modulates Gli1 expression, in vitro and in vivo. Ectopic expression of Dsg2 on Ptc1^+/lacZ background enhanced epidermal proliferation and interfollicular activation of the Hh pathway. Furthermore, in response to DMBA/TPA, the Dsg2/Ptc1^+/lacZ mice developed squamous lessons earlier than the WT, Ptc1^+/lacZ, and Inv-Dsg2 littermates. Additionally, DMBA/TPA induced BCC formation in all mice harboring the Ptc1^+/lacZ gene and the presence of Dsg2 in Dsg2/Ptc1^+/lacZ mice doubled the BCC tumor burden. Reporter analysis revealed activation of the Hh pathway in the BCC tumors. However, in the SCCs we observed Hh activity only in the underlying dermis of the tumors. Furthermore, Dsg2/Ptc1^+/lacZ mice demonstrated enhanced MEK/Erk1/2 activation within the tumors and expression of Shh in the dermis. In summary, our results demonstrate that Dsg2 modulates Hh signaling, and this synergy may accelerate skin tumor development by different mechanisms.     

Immunohistochemical expression pattern of MMR protein can specifically identify patients with colorectal cancer microsatellite instability

The microsatellite instability (MSI) pathway is found in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in 12 % of sporadic colorectal cancer (CRC). It involves inactivation of deoxyribonucleic acid mismatch repair (MMR) genes MLH1, MSH2, PMS2, and MSH6. MMR germline mutation detections are an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC. In this study, we analyzed for the first time in Tunisia the potential value of immunohistochemical assessment of MMR protein to identify microsatellite instability in CRC. We evaluate by immunohistochemistry MMR protein expression loss in tumoral tissue compared to positive expression in normal mucosa. Immunohistochemistry revealed loss of expression for MLH1, MSH2, MSH6, and PMS2 in 15, 21, 13, and 15 % of cases, respectively. Here, we report a more elevated frequency of MSI compared to data of the literature. In fact, by immunohistochemistry, 70 % of cases were shown to be MSS phenotype, whereas 30 % of cases, in our set, were instable. Moreover, according to molecular investigation, 71 % of cases were instable (MSI-H) and remaining cases were stable (29 %). Thus, we found a perfect association between MMR immunohistochemical analyses and MSI molecular investigation. Immunohistochemical analysis of MMR gene product expression may allow one to specifically identify MSI phenotype of patients with colorectal carcinomas.     

Msh2 deficiency increases susceptibility to benzo[a]pyrene-induced lymphomagenesis

DNA mismatch repair (MMR) is essential for repair of single-base mismatches and insertion/deletion loops. MMR proteins also participate in cellular response to DNA damaging agents such as various alkylating agents. Mice deficient in the MMR gene Msh2 develop tumors earlier after exposure to alkylating agents when compared to unexposed mice. The interaction between the MMR system and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has not been investigated in vivo. Here, we show that treatment of Msh2-deficient mice with B[a]P enhances susceptibility to lymphomagenesis. Carrying at least one intact copy of the Msh2 gene had a protective effect. B[a]P treatment only induced lymphomas in 3 of the 40 (7.5%) mice with at least one intact copy of the Msh2 gene as compared to 13 of the 17 (76.5%) Msh2-deficient mice and occurs only after a much longer time period. The B[a]P-DNA adduct levels measured in lung, liver, spleen and forestomach of B[a]P-treated Msh2-/- mice were not significantly different from B[a]P-treated Msh2+/+ mice. In summary, the results suggest that B[a]P accelerates lymphomagenesis in Msh2-deficient mice. Furthermore, Msh2 deficiency does not have any significant effect on B[a]P-DNA adduct levels.     

The mechanisms underlying MMR deficiency in immunodeficiency‐related non‐Hodgkin lymphomas are different from those in other sporadic microsatellite instable neoplasms

The spectrum of tumors showing microsatellite instability (MSI) has recently been enlarged to sporadic neoplasms whose incidence is favored in the context of chronic immunosuppression. We investigated the biological, therapeutic and clinical features associated with MSI in immunodeficiency‐related non‐Hodgkin lymphomas (ID‐RL). MSI screening was performed in 275 ID‐RL. MSI ID‐RL were further analyzed for MMR gene expression and for BRAF/KRAS mutations since these genes are frequently altered in MSI cancers. We also assessed the expression of O⁶‐methylguanine‐DNA methyltransferase (MGMT), an enzyme whose inactivation has been reported in lymphomas and may help in the selection of MMR deficient clones. Unlike other sporadic MSI neoplasms, MSI ID‐RL (N = 17) presented with heterogeneous MMR defects and no MLH1 promoter methylation. About one third of these tumors presented with normal expression of MLH1, MSH2, MSH6 and PMS2. They accumulated BRAF activating mutations (33%). Unlike other ID‐RL, MSI ID‐RL were primarily EBV‐negative NHL of T‐cell origin, and arose after long‐term immunosuppression in patients who received azathioprine as part of their immunosuppressive regimen (p = 0.05) and/or who exhibited methylation‐induced loss of expression of MGMT in tumor cells (p= 0.02). Overall, these results highlight that, in the context of deficient immune status, some MSI neoplasms arise through alternative mechanism when compared to other sporadic MSI neoplasms. They give the exact way how to make the diagnosis of MSI in these tumors and may help to define biological and clinicalrisk factors associated with their emergence in such a clinicalcontext. © 2009 UICC     

Involvement of MBD4 inactivation in mismatch repair-deficient tumorigenesis

The DNA glycosylase gene MBD4 safeguards genomic stability at CpG sites and is frequently mutated at coding poly-A tracks in mismatch repair (MMR)-defective colorectal tumors (CRC). Mbd4 biallelic inactivation in mice provided conflicting results as to its role in tumorigenesis. Thus, it is unclear whether MBD4 alterations are only secondary to MMR defects without functional consequences or can contribute to the mutator phenotype. We investigated MBD4 variants in a large series of hereditary/familial and sporadic CRC cases. Whereas MBD4 frameshifts were only detected in tumors, missense variants were found in both normal and tumor DNA. In CRC with double-MBD4/MMR and single-MBD4 variants, transition mutation frequency was increased, indicating that MBD4 defects may affect the mutational landscape independently of MMR defect. Mbd4-deficient mice showed reduced survival when combined with Mlh1^−/− genotype. Taken together, these data suggest that MBD4 inactivation may contribute to tumorigenesis, acting as a modifier of MMR-deficient cancer phenotype.     

Myeloid heme oxygenase‐1 promotes metastatic tumor colonization in mice

Heme oxygenase-1 (HO-1) is a heme degradation enzyme with antioxidant and immune-modulatory functions. HO-1 promotes tumorigenesis by enhancing tumor cell proliferation and invasion. Whether HO-1 has an effect on cancer progression through stromal compartments is less clear. Here we show that the growth of tumor engrafted subcutaneously in syngeneic mice was not affected by host HO-1 expression. However, lung metastasis arisen from subcutaneous tumor or circulating tumor cells was significantly reduced in HO-1^+/− mice comparing to wild type (WT) mice. The reduced lung metastasis was also observed in B6 mice bearing HO-1^+/− bone marrow as comparing to WT chimeras, indicating that HO-1 expression in hematopoietic cells impacts tumor colonization at the metastatic site. Further experiments demonstrated that the numbers of myeloid cells recruited to pulmonary premetastatic niches and metastatic loci were significantly lower in HO-1^+/− mice than in WT mice. Likewise, the extents of tumor cell extravasation and colonization at the metastatic loci in the early phase of metastasis were significantly lower in HO-1^+/− mice. Mechanistic studies revealed that HO-1 impacted chemoattractant-induced myeloid cell migration by modulating p38 kinase signaling. Moreover, myeloid HO-1-induced expressions of vascular endothelial growth factor and interleukin-10 promoted tumor cell transendothelial migration and STAT3 activation in vitro. These data support a pathological role of myeloid HO-1 in metastasis and suggest a possibility of targeting myeloid HO-1 for cancer treatment.     

Shh and p50/Bcl3 signaling crosstalk drives pathogenesis of BCCs in gorlin syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder that is due, in large measure, to aberrant Shh signaling driven by mutations in the tumor suppressor gene Ptch1. Here, we describe the development of Ptch1^+/−/SKH-1 mice as a novel model of this disease. These animals manifest many features of NBCCS, including developmental anomalies and are remarkably sensitive to both ultraviolet (UVB) and ionizing radiation that drive the development of multiple BCCs. Just as in patients with NBCCS, Ptch1^+/−/SKH-1 also spontaneously develops BCCs and other neoplasms such as rhabdomyomas/rhabdomyosarcomas. Administration of smoothened inhibitors (vismodegib/itraconazole/cyclopamine) or non-steroidal anti-inflammatory drug (sulindac/sulfasalazine) each result in partial resolution of BCCs in these animals. However, combined administration of these agents inhibits the growth of UVB-induced BCCs by >90%. Employing small molecule- and decoy-peptide-based approaches we further affirm that complete remission of BCCs could only be achieved by combined inhibition of p50-NFκB/Bcl3 and Shh signaling. We posit that Ptch1^+/−/SKH-1 mice are a novel and relevant animal model for NBCCS. Understanding mechanisms that govern genetic predisposition to BCCs should facilitate our ability to identify and treat NBCCS gene carriers, including those at risk for sporadic BCCs while accelerating development of novel therapeutic modalities for these patients.     

Msh2 deficiency leads to chromosomal abnormalities, centrosome amplification, and telomere capping defect

Msh2 is a key mammalian DNA mismatch repair (MMR) gene and mutations or deficiencies in mammalian Msh2 gene result in microsatellite instability (MSI+) and the development of cancer. Here, we report that primary mouse embryonic fibroblasts (MEFs) deficient in the murine MMR gene Msh2 (Msh2(-/-)) showed a significant increase in chromosome aneuploidy, centrosome amplification, and defective mitotic spindle organization and unequal chromosome segregation. Although Msh2(-/-) mouse tissues or primary MEFs had no apparent change in telomerase activity, telomere length, or recombination at telomeres, Msh2(-/-) MEFs showed an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA. These data suggest that MSH2 helps to maintain genomic stability through the regulation of the centrosome and normal telomere capping in vivo and that defects in MMR can contribute to oncogenesis through multiple pathways.     

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice

Introduction

Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm ^+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm ^+/- mice on the p53 ^+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation.

Methods

BALB/cHeA-p53 ^+/- mice were crossed with MSM/Ms-Atm ^+/- mice, and females of the F₁ progeny ([BALB/cHeA × MSM/Ms]F₁) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks.

Results

We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53 ^+/- mice of the BALB/cHeA × MSM/Ms background. The singly heterozygous p53 ^+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53 ^+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma.

Conclusion

Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53 ^+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.     

The impact of coexisting genetic mutations on murine optic glioma biology

Background

Children with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome are prone to the development of optic pathway gliomas resulting from biallelic inactivation of the NF1 gene. Recent studies have revealed the presence of other molecular alterations in a small portion of these NF1-associated brain tumors. The purpose of this study was to leverage Nf1 genetically engineered mouse strains to define the functional significance of these changes to optic glioma biology.

Methods

Nf1+/− mice were intercrossed with Nf1^flox/flox mice, which were then crossed with Nf1^flox/flox; GFAP-Cre mice, to generate Nf1^flox/mut; GFAP-Cre (FMC) mice. These mice were additionally mated with conditional KIAA1549:BRAF knock-in or Pten^flox/wt mice to generate Nf1^flox/mut; f-BRAF; GFAP-Cre (FMBC) mice or Nf1^flox/mut; Pten^flox/wt; GFAP-Cre (FMPC) mice, respectively. The resulting optic gliomas were analyzed for changes in tumor volume, proliferation, and retinal ganglion cell loss.

Results

While KIAA1549:BRAF conferred no additional biological properties on Nf1 optic glioma, FMPC mice had larger optic gliomas with greater proliferative indices and microglial infiltration. In addition, all 3 Nf1 murine optic glioma strains exhibited reduced retinal ganglion cell survival and numbers; however, FMPC mice had greater retinal nerve fiber layer thinning near the optic head relative to FMC and FMBC mice.

Conclusions

Collectively, these experiments demonstrate genetic cooperativity between Nf1 loss and Pten heterozygosity relevant to optic glioma biology and further underscore the value of employing genetically engineered mouse strains to define the contribution of discovered molecular alterations to brain tumor pathogenesis.     

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients'' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG). Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh^−/−mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh^−/− mice whereas similarly treated wild-type animals remained tumor-free.     

Spindle assembly checkpoint and p53 deficiencies cooperate for tumorigenesis in mice

The spindle assembly checkpoint (SAC) guards against chromosomal missegregation during mitosis. To investigate the role of SAC in tumor development, mice heterozygously knocked out for the mitotic arrest deficient (Mad) genes Mad1 and/or Mad2 were mated with p53^+/? mice. Increased tumor frequencies were reproducibly observed in Mad2^+/?p53^+/? (88.2%) and Mad1^+/?Mad2^+/?p53^+/? (95.0%) mice compared with p53^+/? (66.7%) mice. Moreover, 53% of Mad2^+/?p53^+/? mice developed lymphomas compared with 11% of p53^+/? mice. By examining chromosome content, increased loss in diploidy was seen in cells from Mad2^+/?p53^+/? versus p53^+/? mice, correlating loss of SAC function, in a p53^+/? context, with increased aneuploidy and tumorigenesis. The findings here provide evidence for a cooperative role of Mad1/Mad2 and p53 genes in preventing tumor development. Published 2008 Wiley‐Liss, Inc.