The WNT receptor ROR2 drives the interaction of multiple myeloma cells with the microenvironment through AKT activation.

Multiple myeloma is the second most frequent hematological cancer after lymphoma and remains an incurable disease. The pervasive support provided by the bone marrow microenvironment to myeloma cells is crucial for their survival. Here, an unbiased assessment of receptor tyrosine kinases overexpressed in myeloma identified ROR2, a receptor for the WNT noncanonical pathway, as highly expressed in myeloma cells. Its ligand, WNT5A is the most abundant growth factor in the bone marrow of myeloma patients. ROR2 mediates myeloma cells interactions with the surrounding bone marrow and its depletion resulted in detachment of myeloma cells from their niche in an in vivo model, triggering apoptosis and thus markedly delaying disease progression. Using in vitro and ex vivo 3D-culture systems, ROR2 was shown to exert a pivotal role in the adhesion of cancer cells to the microenvironment. Genomic studies revealed that the pathways mostly deregulated by ROR2 overexpression were PI3K/AKT and mTOR. Treatment of cells with specific PI3K inhibitors already used in the clinic reduced myeloma cell adhesion to the bone marrow. Together, our findings support the view that ROR2 and its downstream targets represent a novel therapeutic strategy for the large subgroup of MM patients whose cancer cells show ROR2 overexpression.

Forkhead box O transcription factors in chondrocytes regulate endochondral bone formation.

The differentiation of embryonic mesenchymal cells into chondrocytes and the subsequent formation of a cartilaginous scaffold that enables the formation of long bones are hallmarks of endochondral ossification. During this process, chondrocytes undergo a remarkable sequence of events involving proliferation, differentiation, hypertrophy and eventually apoptosis. Forkhead Box O (FoxO) transcription factors (TFs) are well-known regulators of such cellular processes. Although FoxO3a was previously shown to be regulated by 1,25-dihydroxyvitamin D3 in osteoblasts, a possible role for this family of TFs in chondrocytes during endochondral ossification remains largely unstudied. By crossing Collagen2-Cre mice with FoxO1lox/lox;FoxO3alox/lox;FoxO4lox/lox mice, we generated mice in which the three main FoxO isoforms were deleted in growth plate chondrocytes (chondrocyte triple knock-out; CTKO). Intriguingly, CTKO neonates showed a distinct elongation of the hypertrophic zone of the growth plate. CTKO mice had increased overall body and tail length at eight weeks of age and suffered from severe skeletal deformities at older ages. CTKO chondrocytes displayed decreased expression of genes involved in redox homeostasis. These observations illustrate the importance of FoxO signaling in chondrocytes during endochondral ossification.

Somatic p16(INK4a) loss accelerates melanomagenesis.

Loss of p16(INK4a)-RB and ARF-p53 tumor suppressor pathways, as well as activation of RAS-RAF signaling, is seen in a majority of human melanomas. Although heterozygous germline mutations of p16(INK4a) are associated with familial melanoma, most melanomas result from somatic genetic events: often p16(INK4a) loss and N-RAS or B-RAF mutational activation, with a minority possessing alternative genetic alterations such as activating mutations in K-RAS and/or p53 inactivation. To generate a murine model of melanoma featuring some of these somatic genetic events, we engineered a novel conditional p16(INK4a)-null allele and combined this allele with a melanocyte-specific, inducible CRE recombinase strain, a conditional p53-null allele and a loxP-stop-loxP activatable oncogenic K-Ras allele. We found potent synergy between melanocyte-specific activation of K-Ras and loss of p16(INK4a) and/or p53 in melanomagenesis. Mice harboring melanocyte-specific activated K-Ras and loss of p16(INK4a) and/or p53 developed invasive, unpigmented and nonmetastatic melanomas with short latency and high penetrance. In addition, the capacity of these somatic genetic events to rapidly induce melanomas in adult mice suggests that melanocytes remain susceptible to transformation throughout adulthood.

Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16.

Oncolytic herpes simplex viruses (HSVs), in clinical trials for the treatment of malignant gliomas, are assumed to be selective for tumor cells because their replication is strongly attenuated in quiescent cells, but not in cycling cells. Oncolytic selectivity is thought to occur because mutations in viral ICP6 (encoding a viral ribonucleotide reductase function) and/or gamma34.5 function are respectively complemented by mammalian ribonucleotide reductase and GADD34, whose genes are expressed in cycling cells. However, it is estimated that only 5-15% of malignant glioma cells are in mitosis at any one time. Therefore, effective replication of HSV oncolytic viruses might be limited to a subpopulation of tumor cells, since at any one time the majority of tumor cells would not be cycling. However, we report that an HSV with defective ICP6 function replicates in quiescent cultured murine embryonic fibroblasts obtained from mice with homozygous p16 deletions. Furthermore, intracranial inoculation of this virus into the brains of p16-/- mice provides evidence of viral replication that does not occur when the virus is injected into the brains of wild-type mice. These approaches provide in vitro and in vivo evidence that ICP6-negative HSVs are 'molecularly targeted,' because they replicate in quiescent tumor cells carrying specific oncogene deletions, independent of cell cycle status.

Pten and p53 converge on c-Myc to control differentiation, self-renewal, and transformation of normal and neoplastic stem cells in glioblastoma.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Diminished lifespan and acute stress-induced death in DNA-PKcs-deficient mice with limiting telomeres.

An adequate and appropriate response to physiological and pathophysiological stresses is critical for long-term homeostasis and viability of the aging organism. Previous work has pointed to the immune system, telomeres and DNA repair pathways as important and distinct determinants of a normal healthy lifespan. In this study, we explored the genetic interactions of telomeres and DNA-PKcs, a protein involved in non-homologous end-joining (NHEJ) and immune responses, in the context of a key aspect of aging and lifespan--the capacity to mount an acute and appropriate immune-mediated stress response. We observed that the combination of DNA-PKcs deficiency and telomere dysfunction resulted in a shortened lifespan that was reduced further following viral infection or experimental activation of the innate immune response. Analysis of the innate immune response in the DNA-PKcs-deficient mice with short dysfunctional telomeres revealed high basal serum levels of tumor necrosis factor alpha (TNFalpha) and hyper-active cytokine responses upon challenge with polyinosinic-polycytidylic acid (poly-IC). We further show that serum cytokine levels become elevated in telomere dysfunctional mice as a function of age. These results raise speculation that these genetic factors may contribute to misdirected immune responses of the aged under conditions of acute and chronic stress.

Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy.

The three-dimensional architecture of the nascent microvascular network is a critical determinant of vascular perfusion in the setting of regenerative growth, vasculopathies and cancer. Current methods for microvessel visualization are limited by insufficient penetration and instability of endothelial immunolabels, inadequate vascular perfusion by the high-viscosity polymers used for vascular casting, and destruction of tissue stroma during the processing required for scanning electron microscopy. The aim of this study was to develop whole-mount tissue processing methods for 3D in situ visualization of the microvasculature that were also compatible with supplementary labeling for other structures of interest in the tissue microenvironment. Here, we present techniques that allow imaging of the microvasculature by confocal microscopy, to depths of up to 1500 mum below the specimen surface. Our approach includes labeling luminal surfaces of endothelial cells by i.v. injection of fluorescently conjugated lectin and filling the microvasculature with carbon or fluorescent nanoparticles/Mercox, followed by optical clearing of thick tissue sections to reduce light scatter and permit 3D visualization of microvessel morphology deep into the sample. Notably, tissue stroma is preserved, allowing simultaneous labeling of other structures by immunohistochemistry or nuclear dyes. Results are presented for various murine tissues including fat, muscle, heart and brain under conditions of normal health, as well as in the setting of a glioma model growing in the subcutaneous space or orthotopically in the brain parenchyma.

Haploinsufficiency of the mSds3 chromatin regulator promotes chromosomal instability and cancer only upon complete neutralization of p53.

The mSin3 corepressor complex has been linked to diverse cancer signaling pathways through its capacity to regulate target gene expression via chromatin modification. mSds3, a cell essential gene, is a key component of the mSin3 complex serving to maintain its inherent histone deacetylase activity. mSds3 also serves an essential role in the establishment of pericentric heterochromatin, and genetic ablation of mSds3 results in chromosome missegregation. In contrast, mSin3A nullizygous cells show normal chromosome dynamics and cytogenetic profiles. The integral role of mSds3 in controlling chromosome segregation and mSin3-regulated transcriptional networks prompted efforts to determine the neoplastic impact of loss of one copy of mSds3 or mSin3A. In particular, we assessed whether loss of one copy of mSds3, alone or in combination with p53 mutation, results in aneuploidy and promotes a cancer-prone condition in the mouse. We observe that, in a p53 null background, loss of one mSds3 allele results in accelerated tumor onset and increased tumor burden. Notably, these mSds3(+/-) p53(-/-) tumors exhibit a more complex cytogenetic profile characterized by marked aneuploidy and centromeric associations. The presence of even one copy of p53 is sufficient to suppress the accelerated tumorigenesis in mSds3(+/-) mice, consistent with a key role for p53 in monitoring mitotic fidelity. These observations with Sds3 mutant mice contrast with mSin3A(+/-) p53(-/-) mice, which do not show an accelerated or increased tumor incidence relative to mSin3A(+/+)p53(-/-) controls, correlating with the absence of aneuploidy detected upon mSin3A genetic inactivation. This genetic study establishes that the capacity of mSds3 to cooperate with p53 deficiency in cancer predisposition relates to its specific role in chromosome segregation, rather than its central role in maintaining a functional mSin3A complex.

Common and contrasting genomic profiles among the major human lung cancer subtypes.

Lung cancer is the leading cause of cancer mortality worldwide. With the recent success of molecularly targeted therapies in this disease, a detailed knowledge of the spectrum of genetic lesions in lung cancer represents a critical step in the development of additional effective agents. An integrated high-resolution survey of regional amplifications and deletions and gene expression profiling of non-small-cell lung cancers (NSCLC) identified 93 focal high-confidence copy number alterations (CNAs), with 21 spanning less than 0.5 Mb with a median of five genes. Most CNAs were novel and included high-amplitude amplification and homozygous deletion events. Pathogenic relevance of these genomic alterations was further reinforced by their recurrence and overlap with focal alterations of other tumor types. Additionally, the comparison of the genomic profiles of the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC), showed an almost complete overlap with the exception of one amplified region on chromosome 3, specific for SCC. Among the few genes overexpressed within this amplicon was p63, a known regulator of squamous cell differentiation. These findings suggest that the AC and SCC subtypes may arise from a common cell of origin and they are driven to their distinct phenotypic end points by altered expression of a limited number of key genes such as p63.

Induction of melanoma in murine macrophage inflammatory protein 2 transgenic mice heterozygous for inhibitor of kinase/alternate reading frame.

The molecular and genetic events that contribute to the genesis and progression of cutaneous malignant melanoma are poorly understood, attributable in large part to the different genetic alterations accompanying tumorigenesis. Inhibitor of kinase 4a (INK4a) is often inactivated in families with hereditary melanoma. Loss of INK4a/alternate reading frame (ARF) in mice is associated with increased incidence of other tumors such as lymphoma and fibrosarcoma. However, the incidence of melanoma in INK4a/ARF-deficient mice is very low. Our previous studies have revealed that the CXC chemokine, CXCL1, is overexpressed in human malignant melanoma cells and is linked to transformation of immortalized murine melanocytes. To study the direct role of CXCL1 on the genesis of primary melanoma lesions, transgenic mouse lines were established that express the murine homologue of CXCL1, murine macrophage inflammatory protein 2 (MIP-2), under the transcriptional control of the tyrosinase promoter/enhancer (Tyr-MIP-2) in the mice that were deficient or not deficient for INK4a/ARF. Strong MIP-2 immunoreactivity was associated with pigmented melanocytes in the hyperproliferative hair follicles in the Tyr-MIP-2 transgenic mice, and the level of MIP-2 expression was similar in both INK4a/ARF heterozygous or wild-type mice. After treatment of mice with 7,12-dimethylbenz(a)anthracene, cutaneous melanomas formed in 12% (17/145) of the Tyr-MIP-2 transgene-positive mice, whereas only 2% (3/146) of the Tyr-MIP-2 transgene-negative mice developed melanoma. When melanocytes cultured from MIP-2 transgenic mice null for INK4a/ARF were transplanted into nude mice, melanoma formation occurred in 83% (10/12) of the cases with a latency period of 3 months. However, no melanoma lesions arose in nude mice injected with INK4a/ARF -/- melanocytes, which did not express the MIP-2 transgene. Our results demonstrate that constitutive expression of MIP-2 in INK4a/ARF-deficient melanocytes facilitates formation of malignant melanoma.

Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-null MEFs exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19Arf or p53 (refs 4, 5). Furthermore, p16Ink4a deficiency was associated with an increased incidence of spontaneous and carcinogen-induced cancers. These data establish that p16Ink4a, along with p19Arf, functions as a tumour suppressor in mice.

Telomere dysfunction triggers developmentally regulated germ cell apoptosis.

Telomere dysfunction results in fertility defects in a number of organisms. Although data from fission yeast and Caenorhabditis elegans suggests that telomere dysfunction manifests itself primarily as defects in proper meiotic chromosome segregation, it is unclear how mammalian telomere dysfunction results in germ cell death. To investigate the specific effects of telomere dysfunction on mammalian germ cell development, we examined the meiotic progression and germ cell apoptosis in late generation telomerase null mice. Our results indicate that chromosome asynapsis and missegregation are not the cause of infertility in mice with shortened telomeres. Rather, telomere dysfunction is recognized at the onset of meiosis, and cells with telomeric defects are removed from the germ cell precursor pool. This germ cell telomere surveillance may be an important mechanism to protect against the transmission of dysfunctional telomeres and chromosomal abnormalities.

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.

The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.

Pancreatic cancer is among the leading causes of cancer death. Although a genetic profile for pancreatic cancer is emerging, many biological aspects of this disease are poorly understood. Indeed, fundamental questions regarding progenitor cell lineages, host stromal milieu, and the role of specific genetic alterations in tumor progression remain unresolved. A mouse model engineered with signature mutations would provide a powerful ally in the study of pancreatic cancer biology and may guide improved prognostic assessment and treatment for the human disease. In this review, we discuss the molecular basis for normal pancreatic development and the genetics of human pancreatic adenocarcinoma in the hope of charting a course for the development of a faithful mouse model for this lethal cancer.

Modeling chromosomal instability and epithelial carcinogenesis in the telomerase-deficient mouse.

Human carcinomas are intimately linked to advancing age. These cancers have complex cytogenetic profiles, including aneuploidy and chromosomal structural aberrations. While aged humans sustain a high rate of carcinomas, mice bearing common tumor suppressor gene mutations typically develop soft tissue sarcomas and lymphomas. One marked species distinction between human and mouse that bears on the predisposition to carcinogenesis lies in the radical differences in length and regulation of the telomere, nucleoprotein complexes that cap the ends of eukaryotic chromosomes. Recent cancer modeling studies in the telomerase knockout p53 mutant mice revealed that telomere dynamics might be relevant to carcinogenesis. In these mice, there is a shift in the tumor spectrum towards epithelial carcinomas, and these cancers emerge with complex cytogenetic profiles classical for human carcinomas. In this review, we suggest that the mechanism of fusion-bridge-breakage-translocation, triggered by critically short telomeres, may be one of the generators of genomic instability commonly seen in human carcinomas.

Telomere dysfunction and evolution of intestinal carcinoma in mice and humans.

Telomerase activation is a common feature of advanced human cancers and facilitates the malignant transformation of cultured human cells and in mice. These experimental observations are in accord with the presence of robust telomerase activity in more advanced stages of human colorectal carcinogenesis. However, the occurrence of colon carcinomas in telomerase RNA (Terc)-null, p53-mutant mice has revealed complex interactions between telomere dynamics, checkpoint responses and carcinogenesis. We therefore sought to determine whether telomere dysfunction exerts differential effects on cancer initiation versus progression of mouse and human intestinal neoplasia. In successive generations of ApcMin Terc-/- mice, progressive telomere dysfunction led to an increase in initiated lesions (microscopic adenomas), yet a significant decline in the multiplicity and size of macroscopic adenomas. That telomere dysfunction also contributes to human colorectal carcinogenesis is supported by the appearance of anaphase bridges (a correlate of telomere dysfunction) at the adenoma-early carcinoma transition, a transition recognized for marked chromosomal instability. Together, these data are consistent with a model in which telomere dysfunction promotes the chromosomal instability that drives early carcinogenesis, while telomerase activation restores genomic stability to a level permissive for tumor progression. We propose that early and transient telomere dysfunction is a major mechanism underlying chromosomal instability of human cancer.