Establishment of a novel experimental system using single cell-derived pleomorphic rhabdomyosarcoma cell lines expressing K-RasG12V and deficient in p53.

Pleomorphic rhabdomyosarcoma (PRMS) predominantly arises in adult skeletal musculature and is usually associated with poor prognosis. Thus, effective treatments must be developed. PRMS is a rare tumor; therefore, it is critical to develop an experimental system to understand the cellular and molecular mechanisms of PRMS. We previously demonstrated that PRMS develops after p53 gene deletion and oncogenic K-Ras expression in the skeletal muscle tissue. In that study, oncogenic K-Ras-expressing cells were diverse and the period until disease onset was difficult to control. In this study, we developed an experimental system to address this problem. Single cell-derived murine cell lines, designated as RMS310 and RMSg2, were established by limiting the dilution of cells from a lung metastatic tumor colony that were positive for various cancer stem cells and activated skeletal muscle-resident stem/progenitor cell marker genes by RT-PCR. All cell lines stably recapitulated the histological characteristics of human PRMS as bizarre giant cells, desmin-positive cells, and lung metastases in C57BL/6 mice. All subclones of the RMSg2 cells by the limiting dilution in vitro could seed PRMS subcutaneously, and as few as 500 RMSg2 cells were sufficient to form tumors. These results suggest that the RMSg2 cells are multipotent cancer cells that partially combine the properties of skeletal muscle-resident stem/progenitor cells and high tumorigenicity. Thus, our model system's capacity to regenerate tumor tissue in vivo and maintain stable cells in vitro makes it useful for developing therapeutics to treat PRMS.

CTLA4 blockade elicits paraneoplastic neurological disease in a mouse model.

CTLA4 is an inhibitory regulator of immune responses. Therapeutic CTLA4 blockade enhances T cell responses against cancer and provides striking clinical results against advanced melanoma. However, this therapy is associated with immune-related adverse events. Paraneoplastic neurologic disorders are immune-mediated neurological diseases that develop in the setting of malignancy. The target onconeural antigens are expressed physiologically by neurons, and aberrantly by certain tumour cells. These tumour-associated antigens can be presented to T cells, generating an antigen-specific immune response that leads to autoimmunity within the nervous system. To investigate the risk to develop paraneoplastic neurologic disorder after CTLA4 blockade, we generated a mouse model of paraneoplastic neurologic disorder that expresses a neo -self antigen both in Purkinje neurons and in implanted breast tumour cells. Immune checkpoint therapy with anti-CTLA4 monoclonal antibody in this mouse model elicited antigen-specific T cell migration into the cerebellum, and significant neuroinflammation and paraneoplastic neurologic disorder developed only after anti-CTLA4 monoclonal antibody treatment. Moreover, our data strongly suggest that CD8 + T cells play a final effector role by killing the Purkinje neurons. Taken together, we recommend heightened caution when using CTLA4 blockade in patients with gynaecological cancers, or malignancies of neuroectodermal origin, such as small cell lung cancer, as such treatment may promote paraneoplastic neurologic disorders.

K-rasG12V mediated lung tumor models identified three new quantitative trait loci modifying events post-K-ras mutation.

A high incidence of oncogenic K-ras mutations is observed in lung adenocarcinoma of human cases and carcinogen-induced animal models. The process of oncogenic K-ras-mediated lung adenocarcinogenesis can be dissected into two parts: pre- and post-K-ras mutation. Adoption of transgenic lines containing a flox-K-rasG12V transgene eliminates the use of chemical carcinogens and enables us to study directly crucial events post-K-ras mutation without considering the cellular events involved with oncogenic K-ras mutation, e.g., distribution and metabolism of chemical carcinogens, DNA repair, and somatic recombination by host factors. We generated two mouse strains C57BL/6J-Ryr2(tm1Nobs) and A/J-Ryr2(tm1Nobs) in which K-rasG12V can be transcribed from the cytomegalovirus early enhancer/chicken beta actin promoter in virtually any tissue. Upon K-rasG12V induction in lung epithelial cells by an adenovirus expressing the Cre recombinase, the number of tumors in the C57BL/6J-Ryr2(tm1Nobs/+) mouse line was 12.5 times that in the A/J-Ryr2(tm1Nobs/+) mouse line. Quantitative trait locus (QTL) analysis revealed that new three modifier loci, D3Mit19, D3Mit45 and D11Mit20, were involved in the differential susceptibility between the two lines. In addition, we found that differential expression of the wild-type K-ras gene, which was genetically turn out to be anti-oncogenic activity on K-rasG12V, could not account for the different susceptibility in our two K-rasG12V-mediated lung tumor models. Thus, we provide a genetic system that enables us to explore new downstream modifiers post-K-ras mutation.

Persistent release of IL-1s from skin is associated with systemic cardio-vascular disease, emaciation and systemic amyloidosis: the potential of anti-IL-1 therapy for systemic inflammatory diseases.

The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1ß antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.

Reduced bone morphogenetic protein receptor type 1A signaling in neural-crest-derived cells causes facial dysmorphism.

Bone morphogenetic protein (BMP) receptor type 1A (BMPR1A) mutations are associated with facial dysmorphism, which is one of the main clinical signs in both juvenile polyposis and chromosome 10q23 deletion syndromes. Craniofacial development requires reciprocal epithelial/neural crest (NC)-derived mesenchymal interactions mediated by signaling factors, such as BMP, in both cell populations. To address the role of mesenchymal BMP signaling in craniofacial development, we generated a conditional knockdown mouse by expressing the dominant-negative Bmpr1a in NC-derived cells expressing the myelin protein zero(Mpz)-Cre transgene. At birth, 100% of the conditional mutant mice had wide-open anterior fontanelles, and 80% of them died because of cleft face and cleft palate soon after birth. The other 20% survived and developed short faces, hypertelorism and calvarial foramina. Analysis of the NC-derived craniofacial mesenchyme of mutant embryos revealed an activation of the P53 apoptosis pathway, downregulation of both c-Myc and Bcl-XL, a normal growth rate but an incomplete expansion of mesenchymal cells. These findings provide genetic evidence indicating that optimal Bmpr1a-mediated signaling is essential for NC-derived mesenchymal cell survival in both normal nasal and frontal bone development and suggest that our model is useful for studying some aspects of the molecular etiology of human craniofacial dysmorphism.

Development and preclinical efficacy of novel transforming growth factor-ß1 short interfering RNAs for pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of transforming growth factor (TGF)-ß1 as the major player in the pathogenesis of the disease. However, attempts to control its expression and to improve the outcome of pulmonary fibrosis have been disappointing. We tested the hypothesis that TGF-ß1 is the dominant factor in the acute and chronic phases of pulmonary fibrosis and developed short interfering (si)RNAs directed toward molecules implicated in the disease. This study developed novel sequences of siRNAs targeting the TGF-ß1 gene and evaluated their therapeutic efficacy in two models of pulmonary fibrosis: a model induced by bleomycin and a novel model of the disease developed spontaneously in mice overexpressing the full length of human TGF-ß1 in the lungs. Intrapulmonary delivery of aerosolized siRNAs of TGF-ß1 with sequences common to humans and rodents significantly inhibited bleomycin-induced pulmonary fibrosis in the acute and chronic phases of the disease and in a dose-dependent manner. Aerosolized human-specific siRNA also efficiently inhibited pulmonary fibrosis, improved lung function, and prolonged survival in human TGF-ß1 transgenic mice. Mice showed no off-target effects after intratracheal administration of siRNA. These results suggest the applicability of these novel siRNAs as tools for treating pulmonary fibrosis in humans.

Nitrative and oxidative DNA damage caused by K-ras mutation in mice.

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

Overexpression of phospholipase Cε in keratinocytes upregulates cytokine expression and causes dermatitis with acanthosis and T-cell infiltration.

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε-deficient mice that PLCε plays a critical role in activation of cytokine production in non-immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL-23, in keratinocytes, and with the accumulation of CD4(+) T cells producing IL-22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL-23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL-22-producing T-cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL-23 from keratinocytes, leading to the activation of IL-22-producing T cells.

Functional roles of Otx2 transcription factor in postnatal mouse retinal development.

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/-; Crx-/- retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx-/- retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/-; Crx-/- retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon.

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

Crucial role of phospholipase Cepsilon in chemical carcinogen-induced skin tumor development.

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) epsilon as an effector of Ras. Here we have examined the role of PLCepsilon in de novo skin chemical carcinogenesis by using mice whose PLCepsilon is genetically inactivated. PLCepsilon(-/-) mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCepsilon(-/-) mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCepsilon(+/+) mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCepsilon background. The skin of PLCepsilon(-/-) mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCepsilon in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCepsilon as a candidate molecular target for the development of anticancer drugs.