Marcus W. Bosenberg

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.

Failure to prolyl hydroxylate hypoxia-inducible factor α phenocopies VHL inactivation in vivo

Many functions have been assigned to the von Hippel‐Lindau tumor suppressor gene product (pVHL), including targeting the alpha subunits of the heterodimeric transcription factor HIF (hypoxia‐inducible factor) for destruction. The binding of pVHL to HIFα requires that HIFα be hydroxylated on one of two prolyl residues. We introduced HIF1α and HIF2α variants that cannot be hydroxylated on these sites into the ubiquitously expressed ROSA26 locus along with a Lox‐stop‐Lox cassette that renders their expression Cre‐dependent. Expression of the HIF2α variant in the skin and liver induced changes that were highly similar to those seen when pVHL is lost in these organs. Dual expression of the HIF1α and HIF2α variants in liver, however, more closely phenocopied the changes seen after pVHL inactivation than did the HIF2α variant alone. Moreover, gene expression profiling confirmed that the genes regulated by HIF1α and HIF2α in the liver are overlapping but non‐identical. Therefore, the pathological changes caused by pVHL inactivation in skin and liver are due largely to dysregulation of HIF target genes.

Genetic analysis of Pten and Ink4a/Arf interactions in the suppression of tumorigenesis in mice

Dual inactivation of PTEN and INK4a/ARF tumor suppressor genes is a common feature observed in a broad spectrum of human cancer types. To validate functional collaboration between these genes in tumor suppression, we examined the biological consequences of Pten and/or Ink4a/Arf deficiency in cells and mice. Relative to single mutant controls, Ink4a/Arf−/−Pten+/− mouse embryonic fibroblast cultures exhibited faster rates of growth in reduced serum, grew to higher saturation densities, produced more colonies upon low density seeding, and showed increased susceptibility to transformation by oncogenic H-Ras. Ink4a/Arf deficiency reduced tumor-free survival and shortened the latency of neoplasias associated with Pten heterozygosity, specifically pheochromocytoma, prostatic intraepithelial neoplasia, and endometrial hyperplasia. Compound mutant mice also exhibited an expanded spectrum of tumor types including melanoma and squamous cell carcinoma. Functional synergy between Ink4a/Arf and Pten manifested most prominently in the development of pheochromocytoma, prompting an analysis of genes and loci implicated in this rare human neoplasm. The classical pheochromocytoma genes Ret, Vhl, and Nf-1 remained intact, a finding consistent with the intersection of these genes with pathways engaged by Pten and Ink4a/Arf. Notably, conventional and array-comparative genomic hybridization revealed frequent loss of distal mouse chromosome 4 in a region syntenic to human chromosome 1p that is implicated in human pheochromocytoma. This study provides genetic evidence of collaboration between Pten and Ink4a/Arf in constraining the growth and oncogenic transformation of cultured cells and in suppressing a wide spectrum of tumors in vivo.

Amplification of CDK4 and MDM2 in malignant melanoma

Amplification of the 12q13–15 region is a common event in several human tumors including liposarcomas, gliomas, and osteosarcomas. We have demonstrated high‐level amplification of 12q14 in a subset of uncultured malignant melanomas (3 of 53). High‐resolution mapping of the amplicon using quantitative PCR revealed a bipartite amplicon consisting of a primary 50‐kb amplicon centered on CDK4 and a secondary amplicon centered on MDM2, without amplification of the intervening 11 Mb of genomic DNA. Analysis of mRNA and protein levels in melanomas with 12q14 amplification demonstrated overexpression of target genes CDK4 and MDM2 without loss of CDKN2A‐P16 (P16INK4A) or CDKN2A‐P14ARF (P14ARF) expression, important regulators of the RB1 and TP53 pathways, which are commonly lost or mutated in melanoma. These results suggest that coamplification of CDK4 and MDM2 may substitute for loss of P16INK4A and P14ARF function in a subset of melanomas.

Components of the Rb pathway are critical targets of UV mutagenesis in a murine melanoma model

Epidemiological studies support a link between melanoma risk and UV exposure early in life, yet the molecular targets of UVs mutagenic actions are not known. By using well characterized murine models of melanoma, we provide genetic and molecular evidence that identifies components of the Rb pathway as the principal targets of UV mutagenesis in murine melanoma development. In a melanoma model driven by H-RAS activation and loss of p19ARF function, UV exposure resulted in a marked acceleration in melanoma genesis, with nearly half of these tumors harboring amplification of cyclin-dependent kinase (cdk) 6, whereas none of the melanomas arising in the absence of UV treatment possessed cdk6 amplification. Moreover, UV-induced melanomas showed a strict reciprocal relationship between cdk6 amplification and p16INK4a loss, which is consistent with the actions of UV along the Rb pathway. Most significantly, UV exposure had no impact on the kinetics of melanoma driven by H-RAS activation and p16INK4a deficiency. Together, these molecular and genetic data identify components of the Rb pathway as critical biological targets of UV-induced mutagenesis in the development of murine melanoma in vivo.

Both products of the mouse INK4a/ARF locus suppress melanoma formation in vivo

Deletion of the INK4a/ARF locus at 9p21 is detected with high frequency in human melanoma. Within a short genomic distance, this locus encodes several proteins with established tumor-suppressor roles in a broad spectrum of cancer types. Several lines of evidence support the view that p16INK4a and p19ARF exert the tumor-suppressor activities of this locus, although their relative importance in specific cancer types such as melanoma has been less rigorously documented on the genetic level. Here, we exploit a well-defined mouse model of RAS-induced melanomas to examine the impact of germline p16INK4a or p19ARF nullizygosity on melanoma formation. We demonstrate that loss of either Ink4a/Arf product can cooperate with RAS activation to produce clinically indistinguishable melanomas. In line with the common phenotypic end point, we further show that RAS+ p16INK4a−/− melanomas sustain somatic inactivation of p19ARF-p53 and, correspondingly, that RAS+ p19ARF−/− melanomas experience high-frequency loss of p16INK4a. These genetic studies provide definitive proof that p16INK4a and p19ARF cooperate to suppress the development of melanoma in vivo.

Role of Epidermal Growth Factor Receptor Signaling in RAS-Driven Melanoma

ABSTRACT The identification of essential genetic elements in pathways governing the maintenance of fully established tumors is critical to the development of effective antioncologic agents. Previous studies revealed an essential role for H-RASV12G in melanoma maintenance in an inducible transgenic model. Here, we sought to define the molecular basis for RAS-dependent tumor maintenance through determination of the H-RASV12G-directed transcriptional program and subsequent functional validation of potential signaling surrogates. The extinction of H-RASV12G expression in established tumors was associated with alterations in the expression of proliferative, antiapoptotic, and angiogenic genes, a profile consistent with the observed phenotype of tumor cell proliferative arrest and death and endothelial cell apoptosis during tumor regression. In particular, these melanomas displayed a prominent RAS-dependent regulation of the epidermal growth factor (EGF) family, leading to establishment of an EGF receptor signaling loop. Genetic complementation and interference studies demonstrated that this signaling loop is essential to H-RASV12G-directed tumorigenesis. Thus, this inducible tumor model system permits the identification and validation of alternative points of therapeutic intervention without neutralization of the primary genetic lesion.

Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse

The Rb/p16Ink4a and p53/p19Arf tumor suppressor pathways have been linked to diverse cancer-relevant processes, including those governing the cellular responses to telomere dysfunction. In this study, we sought to provide direct genetic evidence of a role for the Ink4a/Arf tumor suppressor gene, encoding both p16Ink4a and p19Arf, in modulating the cellular and tissue phenotypes associated with telomere dysfunction by using the mTerc Ink4a/Arf mouse model. In contrast to the rescue associated with p53 deficiency, Ink4a/Arf deficiency did not attenuate the degenerative phenotypes elicited by telomere dysfunction in the late-generation mTerc−/− mice. Furthermore, in contrast to accelerated cancer onset and increased epithelial cancers of late-generation mTerc−/− p53 mutant mice, late-generation mTerc−/− Ink4a/Arf mutant mice experienced a delayed tumor onset and maintained the lymphoma and sarcoma spectrum. Consistent with the negligible role of Ink4a/Arf in the telomere checkpoint response in vivo, late-generation mTerc−/− Ink4a/Arf−/− tissues show activated p53, and derivative tumor cell lines sustain frequent loss of p53 function, whereas all early generation mTerc Ink4a/Arf−/− tumor cell lines remain intact for p53. In addition, the late-generation mTerc−/− Ink4a/Arf−/− tumors showed activation of the alternative lengthening of telomere mechanism, underscoring the need for adaptation to the presence of telomere dysfunction in the absence of p16Ink4a and p19Arf. These observations highlight the importance of genetic context in dictating whether telomere dysfunction promotes or suppresses age-related degenerative conditions as well as the rate of initiation and type of spontaneous cancers.

Precancer in Mice: Animal Models Used to Understand, Prevent, and Treat Human Precancers

We present a status report from the NCI Mouse Models of Human Cancers Consortium (MMHCC) Precancers Workshop held November 8 and 9, 2004. An expert panel, the Mouse Models Group (MMG) evaluated the status of mouse models of precancer emphasizing genetically engineered mouse models, especially of lining epithelium and their utilitarian value to human carcinogenesis. An outline of the background for the panel’s considerations is provided with examples of past and current precancerous lesions in mice. The experimental use of oncogenic viruses and chemical carcinogens in mice led to operational definitions of initiation, promotion, and preneoplasia Preneoplastic and precancerous lesions are found in these models. In this precancer concept, most preneoplastic lesions are considered as potentially precancerous or at least an earlier stage in cancer development than typical pre-invasive epithelial lesions, which are often seen in these mouse models. Genetically engineered mice, used to test the oncogenicity of individual genes, develop precancers that are initiated by defined molecular and histopathologic changes. The mouse can be used to isolate and study precancers in detail, thereby providing a level of biological understanding not readily available in clinical disease. These studies suggest that genetically engineered mice are very useful preclinical models for chemoprevention and therapy.

Activated cAMP Response Element Binding Protein Is Overexpressed in Human Mesotheliomas and Inhibits Apoptosis

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy.