Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1^−/− mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1^WT or cyclin D1^KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1^KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1^WT and cyclin D1^KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.     

Evaluation of the newborn mouse model for chemical tumorigenesis

A total of 45 chemicals, including two aromatic hydrocarbons, five aromatic amines, three azo dyes, ten nitroso compounds, three steroids, four tryptophan metabolites and their related compounds, four naturally occurring substances, four pyrolysates of amino acids and ten miscellaneous compounds, were tested for newborn mouse tumorigenesis assay (NMTA). The results of the NMTA were compared with data from 'Survey of Compounds Which Have Been Tested for Carcinogenic Activity', NIH, NCI, USA (SCWHBTCA), and also with data from the IARC Monographs (Vols 1-41), Lyon, France (IARC). Of the 45 chemicals tested by the NMTA, 28 chemicals showed positive results in the NMTA, and the remaining 17 chemicals were found to be negative for tumor development. The correlation of the results between the NMTA and the mouse and/or rat carcinogenesis test starting at young adult age reported in the SCWHBTCA and in the IARC were compared with 37 chemicals tested; the remaining eight chemicals were found only in our NMTA results. It can be concluded that 31 out of 37 chemicals (83.8%) tested by the NMTA showed similar carcinogenic or non-carcinogenic results obtained in either adult mouse and/or rat carcinogenesis tests. The remaining six chemicals showed contradictory results between the NMTA and either adult mouse and/or rat carcinogenesis tests. Those six chemicals were N-hydroxy-4-acetylaminobiphenyl, estradiol, 3-hydroxyanthranilic acid, 3-hydroxy-L-kynurenine, isonicotinic acid hydrazide and phenobarbital. Among the 37 chemicals, 34 were comparable with the results of the adult mouse carcinogenesis test and those of the NMTA. Twenty-nine out of 34 chemicals (85.3%) showed similar results to the adult mouse carcinogenesis test. Contradictory results were obtained with the following five chemicals: N-hydroxy-acetylaminobiphenyl, 3-hydroxy-anthranilic acid, 3-hydroxy-L-kynurenine, isonicotinic acid hydrazide and phenobarbital. There were 35 chemicals which were comparable with the results of the adult rat carcinogenesis test, and 32 chemicals showed the same results as the NMTA (91.4%). Dissimilar results were obtained with the following three chemicals: estradiol, 3-hydroxy-anthranilic acid and phenobarbital. Based on the results presented in this report, it is reasonable to conclude that the NMTA is one of the most useful and reliable methods for detecting tumorigenic or non-tumorigenic chemicals, when a small amount of chemical is available for rodent carcinogenesis test and the duration of the study is limited to 1 year.     

Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis

Chromosomal instability is believed to be a common feature of most human tumors, but the stage at which such instability originates has not been defined. At the molecular level, chromosomal instability is characterized by allelic imbalance (AI), representing losses or gains of defined chromosomal regions. We have assessed AI in early colorectal tumors using newly developed methods for assessing AI in complex cell populations. A total of 32 adenomas of average size (2 mm; range, 1-3 mm) were studied. AI of chromosome 5q markers occurred in 55% of tumors analyzed, consistent with a gatekeeping role of the adenomatous polyposis coli tumor suppressor gene located at chromosomal position 5q21. AI was also detected in each of the other four chromosomes tested. The fractions of adenomas with AI of chromosomes 1p, 8p, 15q, and 18q were 10,19, 28, and 28%, respectively. Over 90% of the tumors exhibited AI of at least one chromosome, and 67% had allelic imbalance of a chromosome other than 5q. These findings demonstrate that AI is a common event, even in very small tumors, and suggest that chromosomal instability occurs very early during colorectal neoplasia.     

Uncoupling of genomic instability and tumorigenesis in a mouse model of Burkitt's lymphoma expressing a conditional box II-deleted Myc protein

Burkitt's lymphomas (BL) are characterized by the constitutive expression of c-Myc protein. In total, 50-60% of all BL cells carry mutant c-Myc proteins. Using a mouse model of spontaneously immortalized pro-B-lymphocytes (Ba/F3), we have investigated genomic instability mediated by the conditional expression of either wild-type (WT) or deletion box II Delta106-Myc proteins. We found that both proteins mediate common as well as differing types of chromosomal rearrangements as documented by spectral karyotyping (SKY). A higher level of genomic instability is induced by the Delta106-Myc protein. To examine the tumorigenic potential of WT or Delta106-driven Ba/F3 cells, in vivo tumorigenesis studies were performed in SCID mice. Under the experimental conditions of this study, WT but not Delta106-Myc expressing Ba/F3 cells triggered tumorigenesis in SCID mice. Therefore, the genomic instability phenotype induced by Delta106-Myc can be genetically uncoupled from its tumorigenic potential.     

Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland

Deregulation of cyclin E expression and/or high levels have been reported in a variety of tumors and have been used as indicators of poor prognosis. Although the role that cyclin E plays in tumorigenesis remains unclear, there is evidence that it confers genomic instability when deregulated in cultured cells. Here we show that deregulated expression of a hyperstable allele of cyclin E in mice heterozygous for p53 synergistically increases mammary tumorigenesis more than that in mice carrying either of these markers individually. Most tumors and tumor-derived cell lines demonstrated loss of p53 heterozygosity. Furthermore, this tumor susceptibility is related to the number of times the transgene is induced indicating that it is directly attributable to the expression of the cyclin E transgene. An indirect assay indicates that loss of p53 function is an early event occurring in the mammary epithelia of midlactation mammary glands in which cyclin E is deregulated long before evidence of malignancy. These data support the hypothesis that deregulated expression of cyclin E stimulates p53 loss of heterozygosity by promoting genomic instability and provides specific evidence for this in vivo. Cyclin E deregulation and p53 loss are characteristics often observed in human breast carcinoma.     

Overexpression of cyclin D1 and cyclin E in N-nitrosomethylbezylamine-induced rat esophageal tumorigenesis

Dysregulation of Gl cyclins has been implicated in several humanmalignancies. To further investigate the role of Gl cyclinsin chemical carcinogenesis, the expression of cyclin Dl andcyclin E was analyzed by RT-PCR and immunohistochemical studiesin N-nitrosomethylbenzylamine (NMBA)-induced rat esophagealtumongenesis. Cyclin Dl mRNA levels were increased 2.8-foldin 25 week (P < 0.05) and 6.8-fold in 45 week (P < 0.01)papillomas induced by NMBA, when compared with normal rat esophagealepithelium. Cyclin E mRNA levels were increased 6.2-fold in25 week (P < 0.01) and 6.9-fold in 45 week (P < 0.01)papillomas. Immunohistochemical staining revealed exclusivenuclear staining of both cyclin Dl and cyclin E. Furthermore,there was a sequential increase in cyclin Dl and cyclin E-positivecells from normal epithelium, to preneoplastic lesions, to papillomas.These findings suggest that overexpression of cyclin Dl andcyclin E occur relatively early in rat esophageal tumongenesisand participate in tumor progression in this model.     

A mouse model system to genetically dissect the molecular mechanisms regulating tumorigenesis.

The vast majority of human tumors are of epithelial origin and result from the accumulation of mutations that alter the function of pathways that control critical cellular processes, including proliferation, checkpoint regulation, and apoptosis. Authentically replicating these events in animal models is critical to understanding the biology of cancer and for testing the feasibility of novel therapies. We developed a mouse model that recapitulates the steps of epithelial tumor progression of multiple tissue types (kidney, breast, ovarian surface, and prostate epithelia), which takes advantage of the power of mouse genetics, and that allows for biochemical analysis, genetic selection, and screening. Moreover, this model enables functional interrogation of far more complex tumor genotypes, both of the tumor cells themselves, and of the cells in the tumor microenvironment. This is a crucial advantage, as human tumors result from multiple compound mutations, most of which are difficult to achieve through standard mutant mouse technology. We have applied this model to establish the role of apoptosis in epithelial solid tumor progression and in treatment response, which has provided novel opportunities for cancer therapies in humans.     

A basal‐enriched microRNA is required for prostate tumorigenesis in a Pten knockout mouse model

MicroRNAs (miRNAs) play important roles in prostate cancer development. However, it remains unclear how individual miRNAs contribute to the initiation and progression of prostate cancer. Here we show that a basal layer‐enriched miRNA is required for prostate tumorigenesis. We identify miR‐205 as the most highly expressed miRNA and enriched in the basal cells of the prostate. Although miR‐205 is not required for normal prostate development and homeostasis, genetic deletion of miR‐205 in a Pten null tumor model significantly compromises tumor progression and does not promote metastasis. In Pten null basal cells, loss of miR‐205 attenuates pAkt levels and promotes cellular senescence. Furthermore, although overexpression of miR‐205 in prostate cancer cells with luminal phenotypes inhibits cell growth in both human and mouse, miR‐205 has a minimal effect on the growth of a normal human prostate cell line. Taken together, we have provided genetic evidence for a requirement of miR‐205 in the progression of Pten null‐induced prostate cancer.     

Distal Chr 4 harbors a genetic locus (Gct1) fundamental for spontaneous ovarian granulosa cell tumorigenesis in a mouse model

The spontaneous development of juvenile-onset ovarian granulosa cell tumors in mice of the SWXJ-9 recombinant inbred strain is a model for juvenile-type granulosa cell tumors that appear in very young girls. To expedite gene discovery in this mouse model of childhood cancer, we did a gene mapping study with the SWXJ-9 recombinant inbred strain and the evolutionarily divergent Mus musculus castaneus (CAST/Ei) strain as a mapping partner. Our mapping strategy focused on autosomal determinants of susceptibility with a backcross scheme that exploited a paternal, parent-of-origin effect for a X-linked gene (Gct4) that strongly supports granulosa cell tumor development. Of 1,968 backcross females examined, we detected 81 granulosa cell tumor-bearing animals and compared their allelic inheritance patterns to non-tumor-bearing siblings in a case-control analysis. The results of our study have confirmed an important locus on mouse chromosome (Chr) 4 (Gct1) and have revealed new loci for granulosa cell tumor susceptibility (Gct7-Gct9) on Chrs 1, 2, and 13 with susceptibility alleles contributed by the SWXJ-9 progenitor. Two novel gene-gene interactions supportive for granulosa cell tumor development were also observed between loci on Chrs 17 and 18 and loci on Chrs 2 and 10. Our data substantiate the evidence that Gct1 on Chr 4 is a fundamental oncogene for granulosa cell tumorigenesis in mice and has identified additional interacting autosomal loci that support tumor development.     

A genetic mouse model for metastatic lung cancer with gender differences in survival

Lung cancer is a devastating disease with poor prognosis. The design of better therapies for lung cancer patients would be greatly aided by good mouse models that closely resemble the human disease. Unfortunately, current models for lung adenocarcinoma are inadequate due to the absence of metastases. In this study, we incorporated both K-ras and p53 missense mutations into the mouse genome and established a more faithful genetic model for human lung adenocarcinoma, the most common type of lung cancer. Mice with both mutations developed advanced lung adenocarcinomas that were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that of human lung cancer. These mice also showed a gender difference in cancer-related death. Additionally, the presence of both mutations induced pleural mesotheliomas in 23% of these mice. This mouse model recapitulates the metastatic nature of human lung cancer and will be invaluable to further probe the molecular basis of metastatic lung cancer and for translational studies.     

Deregulated CDC25A expression promotes mammary tumorigenesis with genomic instability

Checkpoint pathways help cells maintain genomic integrity, delaying cell cycle progression in response to various risks of fidelity, such as genotoxic stresses, compromised DNA replication, and impaired spindle control. Cancer cells frequently exhibit genomic instability, and recent studies showed that checkpoint pathways are likely to serve as a tumor-suppressive barrier in vivo. The cell cycle-promoting phosphatase CDC25A is an activator of cyclin-dependent kinases and one of the downstream targets for the CHK1-mediated checkpoint pathway. Whereas CDC25A overexpression is observed in various human cancer tissues, it has not been determined whether deregulated CDC25A expression triggers or promotes tumorigenesis in vivo. Here, we show that transgenic expression of CDC25A cooperates markedly with oncogenic ras or neu in murine mammary tumorigenesis. MMTV-CDC25A transgenic mice exhibit alveolar hyperplasia in the mammary tissue but do not develop spontaneous mammary tumors. The MMTV-CDC25A transgene markedly shortens latency of tumorigenesis in MMTV-ras mice. The MMTV-CDC25A transgene also accelerates tumor growth in MMTV-neu mice with apparent cell cycle miscoordination. CDC25A-overexpressing tumors, which invade more aggressively, exhibit various chromosomal aberrations on fragile regions, including the mouse counterpart of human 1p31-36, according to array-based comparative genomic hybridization and karyotyping. The chromosomal aberrations account for substantial changes in gene expression profile rendered by transgenic expression of CDC25A, including down-regulation of Trp73. These data indicate that deregulated control of cellular CDC25A levels leads to in vivo genomic instability, which cooperates with the neu-ras oncogenic pathway in mammary tumorigenesis.     

Balancing instability: dual roles for telomerase and telomere dysfunction in tumorigenesis

Telomere shortening and telomerase activation both occur in human tumors. Telomere shortening has been proposed to have two conflicting roles in tumorigenesis: tumor suppression and initiation of chromosomal instability. Similarly, while telomerase activation is suggested to be necessary for tumor growth, telomerase may help to stabilize genomic instability. Here we review what is known about these conflicting roles and propose a framework to understand the role of telomerase in cancer progression.     

Knockout mouse models and mammary tumorigenesis

The generation of transgenic mice overexpressing activated forms of oncogenes has greatly advanced our understanding into their roles in mammary tumor initiation, promotion and progression. However, targeted disruption of tumor suppressor genes often results in lethality at stages prior to mammary tumor formation. This obstacle can now be overcome using several approaches including conditional knockouts that delete genes of interest in a spatial and temporal manner. This review summarizes recent studies on tumor suppressor genes, including APC, ATM, BRCA1, BRCA2, PTEN and p53, in knockout mouse models and our understanding of the possible mechanisms underlying mammary tumorigenesis.     

Deregulated DNA polymerase beta induces chromosome instability and tumorigenesis

To reach the biological alterations that characterize cancer, the genome of tumor cells must acquire increased mutability resulting from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of DNA synthesis during DNA replication. Numeric chromosomal imbalance, referred to as aneuploidy, is the most prevalent genetic changes recorded among many types of solid tumors. We report here that ectopic expression in cells of DNA polymerase beta, an error-prone enzyme frequently over-regulated in human tumors, induces aneuploidy, an abnormal localization of the centrosome-associated gamma-tubulin protein during mitosis, a deficient mitotic checkpoint, and promotes tumorigenesis in nude immunodeficient mice. Thus, we find that alteration of polymerase beta expression appears to induce major genetic changes associated with a malignant phenotype.     

Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation

Aurora-A/STK15/BTAK, which encodes a centrosome-associated kinase, is amplified and overexpressed in multiple types of human tumors, including breast cancer. However, the causal relationship between overexpression of Aurora-A and tumorigenesis has not been fully established due to contradictory data obtained from different experimental systems. To investigate this, we generated a mouse strain that carries an MMTV-Aurora-A transgene. We showed that all the MMTV-Aurora-A mice displayed enhanced branch morphogenesis in the mammary gland and about 40% developed mammary tumors at 20 months of age. The tumor incidence was significantly increased in a p53(+/-) mutation background with about 70% MMTV-Aurora-A;p53(+/-) animals developed tumors at 18 months of age. Of note, overexpression of Aurora-A led to genetic instability, characterized by centrosome amplification, chromosome tetraploidization and premature sister chromatid segregation, at stages prior to tumor formation. Most notably, the severe chromosomal abnormality did not cause cell death owing to the activation of AKT pathway, including elevated levels of phosphorylated AKT and mammalian target of rapamycin, and nuclear accumulation of cyclin D1, which enabled continuous proliferation of the tetraploid cells. These data establish Aurora-A as an oncogene that causes malignant transformation through inducing genetic instability and activating oncogenic pathways such as AKT and its downstream signaling.     

A Tcf4‐GFP reporter mouse model for monitoring effects of Apc mutations during intestinal tumorigenesis

Apc mutations cause intestinal tumorigenesis through Tcf4 activation. However, direct techniques for studying Tcf4 activation in vivo are limited. Here, we describe the development of a Tcf4‐GFP reporter mouse model for directly studying Tcf4 activation. We first developed a GFP reporter construct (Tcf4‐GFP) and transfected it into SW480 cells that have constitutively activated Tcf4. Reporter activity increased 47‐fold. Next, we created transgenic (Tg) mice by transducing the construct into C57BL/6J mice. Fluorescence microscopy did not detect GFP in intestinal sections, but flow cytometry showed 5% of crypt cells to be GFP⁺. We then established cross‐bred mice (Tg × Apc^Min/+), which have a germline Apc mutation and sustained Tcf4 activation. Here, fluorescence microscopy showed GFP⁺ cells at or near the base of normal‐appearing crypts. In adenomas, in which Apc is inactivated, GFP⁺ signal was even greater. Immunostaining for the Tcf4 target genes survivin (BIRC5) and cyclin D1 (CCND1) showed that their expression also paralleled GFP positivity. We conclude that GFP directly reports Tcf4 activation in vivo and tracks the predicted increases in Tcf4 activation that result from Apc inactivation, and that Apc mutation contributes to survivin and cyclin D1 overexpression through Tcf4 activation. Our Tcf4 mouse should be useful in studying the effects of chemopreventive agents on Wnt signaling and changes in proliferative crypt cell populations—including stem cells—during intestinal tumorigenesis. © 2009 Wiley‐Liss, Inc.     

Circadian rhythm of tumor promotion in the two-stage model of mouse tumorigenesis

The question of whether cancer risk is influenced by time-of-day exposure to potentially carcinogenic agents was approached in this study by exposing mouse skin to a single initiating dose of 7,12-dimethylbenz [-]anthracene, followed by a 12 week regime of bi-weekly skin treatments with the tumor promoter, 12-0-tetradecanoyl-phorbol acetate (TPA), given at four different circadian clock times (CCTs). Tumor incidence, average number of tumors per mouse and tumor size showed a dominant circadian component with an acrophase occurring at 23:00 h CCT. Pre-treatment with all trans-retinoic acid, prior to bi-weekly TPA promotion, reduced tumor incidence, average number and size of tumors per animal by greater than 80%, but did not suppress the underlying circadian rhythm of sensitivity to TPA-induced tumor formation.     

ICRC mouse with congenital mega-esophagus as a model to study esophageal tumorigenesis

ICRC mouse, an inbred strain, developed at the Cancer ResearchInstitute, Bombay, exhibits mega-esophagus with markedly hyperplasticmucosa. Diethylnitrosamine (DEN) when given in drinking waterat the dose of 4 mg/kg body weight/day, induced esophageal papillomasconsistently in 100% of the animals, in a short period of 12weeks. Further, tumors were produced, even at a very low cumulativedose of 28 mg/kg body weight. Development of the esophagealpapillomas was dose dependent. DEN even induces esophageal tumorstransplacentally in the ICRC F₁ progeny. Tobacco acts predominantlyas a promoter in this system. ICRC mouse thus provides a muchneeded animal model to study esophageal tumorigenesis, includingthe two-stage carcino genesis. An interesting feature of thestudy is that initiation could be induced by exposure to lowdoses of DEN in the intra-uterine life. Tumors develop in suchF₁ animals only if they are fed tobacco, a predominant promoter,post-natally.