Animal Model And Model Studies For Our Knowledge

Animal models and model studies

Good animal models for prostate cancer were for a long time absent. Recently, some progress has been made and 3 examples for the dynamics in this field were provided. Probably the best such example was documented by Dr. D. Waters with spontaneous tumors of pet dogs, which share striking similarities to their human counterparts with respect to epidemiology, histologic spectrum and metastatic behavior. Canine osteosarcoma also provides a particularly useful model system to test novel therapies directed against minimal residual disease. Studies are being designed to elucidate the association between organismal aging, oxidative stress and cancer development in dogs. Priorities for future research will be to characterize the molecular biology of particular tumors and to determine their suitability to evaluate targeted therapies.

Then, Dr. N. Schreiber-Agus reminded the study group that prostate adenocarcinoma remains one of the more elusive cancer types, in part due to a lack of understanding of the normal and diseased human prostate on the molecular and cellular levels. Several complementary animal models of prostate cancer, including the genetically manipulated mouse, have emerged as a means to confront this elusiveness. Newly characterized reagents and strategies may allow us to overcome the limitations of existing mouse models and to develop models that will illuminate the pathogenetic basis of prostate cancer initiation and progression.

Finally, Dr. W. R. Sellers brought an example for a particular pathway possibly deranged in prostate tumor. The PTEN protein acts to regulate cell-cycle progression and cell survival. These functions require PTEN to act as a lipid phosphatase and in so doing, antagonize P13K signaling. Downstream targets of P13K/PTEN, such as AKH are activated in PTEN−/− tumors. Mouse models of prostate cancer based on deregulation of this pathway are in progress.

Dr. T. L. Benjamin presented the case of Polyoma virus, which perturbs multiple signaling pathways that impinge on cell growth and can induce a broad range of solid tumors in mice. Inactivation of individual pathways in the virus may alter the histological patterns, sizes and frequencies of tumors, depending on the particular pathway and target tissue. The host genetic background can also influence the patterns and overall susceptibility to polyoma-induced tumors, thus presenting another example of the relevance of the host background not only for animal models for human tumors but also for the outcome of carcinogens on animal targets in general.

Dr. A. Berns used conditional tumor suppressor gene knockout mice to produce, in a tissue-specific fashion, specific tumors in mice. This method permitted him to perform a detailed genotype-phenotype analysis making it possible to correlate distinct genetic lesions with specific tumor characteristics. In an initial series of experiments, the loss of retinoblastoma gene (Rb) in combination with p107 and p53 was studied. Inactivation was directed to various cell types such as photo receptor cells, the pineal gland, choroid plexus astrocytes and the intermediate lobe of the pituitary gland. A range of tumors were found, including choroid plexus tumors, pineal gland tumors, pituitary tumors and medullablastomas. Interestingly, some but not other tumors required loss of p53, as measured by loss of heterozygosity (LOH), indicating a cell type-specific need for specific gene mutations. Such studies illustrate the potential of these mice to dissect tumorigenic processes. It is expected that they will also be valuable for testing therapeutic intervention protocols.

Dr. N. E. Hynes discussed the question whether a single genetically modified organ could be implanted ectopically into an animal and thus used to test natural effectors or drugs. The mammary gland is unique in that a major part of its development takes place after birth. This allowed other researchers to show, in 1959, that the entire developmental program of the mammary gland could be recapitulated following transplantation of mammary tissue fragments or cells into the cleared fat pad of syngeneic hosts. Thus, an interesting possibility for developing models of breast cancer in the orthotopic site is to introduce primary mammary cells that have been genetically manipulated to express specific transforming proteins into fat pads cleared of host tissue. Overexpression of RTK c-Met (and/or its ligand HGF) has been found in many breast tumors. Primary mammary epithelial cells ectopically expressing HGF rapidly form mammary tumors after transplantation into cleared fat pads. This model will be used to test the effects of specific inhibitors.

Dr. T. A. Van Dyke brought up the point that there appear to be cell/tissue-specific tumor suppression mechanisms for p53. In normally nondividing brain epithelial cells (chorioid plexus) p53 inactivation has no effect on an otherwise wild-type background. Upon inactivation of the Rb family proteins, however, p53-dependent apoptosis suppresses tumor growth and progression. In thymocytes, p53 inactivation predisposes to tumorigenesis, an activity that is not dependent on VDJ recombination. In contrast, VDJ recombination is required for thymic lymphoma induced by an ATM deficiency. Thus p53 and ATM suppress thymic lymphoma by distinct mechanisms.

Dr. M. E. Ewen addressed the relationship between retinoblastoma protein (pRb) and Ras. Rb-deficient mice display elevated levels (up to 30-fold) of active, GTP-bound Ras, suggesting that Ras is a downstream target of the pRb. The influence of pRb on Ras is linked to the ability of pRb to regulate differentiation. Together our previous work, his data suggest that cytoplasmic-nuclear signaling between pRb and Ras is bidirectional.

P300 and CBP constitute a closely related family of nuclear, signal integrating molecules, both of which are targets of DNA tumor viral oncoproteins. Dr. D. A. Livingston's group produced mice heterozygous for a null allele of p300 and other mice heterozygous for CBP were compared. The mice heterozygous for CBP developed several forms of hematological malignancies with LOH and CBP. The mice heterozygous for a null allele of p300 remained healthy for ≥ 20 months. Therefore, in at least one strain of mice, CBP is a tumor suppressor, and p300 is not.

Drs. R. A. DePinho reported about his work with Dr. L. Chin, in which they used regulated expression of a gene to obtain information about its function, a highly successful approach getting more attention as already shown by Dr. A. Berns. Melanocyte-specific expression of oncogenic RAS coupled with INK4a deficiency generates malignant melanoma in mice, establishing a causal role in melanoma-genesis. To determine whether initiating genetic lesions, i.e., oncogenic RAS, are still required for maintenance, the tet system was exploited to turn off RAS in established tumors. Down-regulation of RAS resulted in complete regression of primary tumors, a process highlighted initially by massive endothelial cell death. This inducible melanoma model establishes that genetic lesions remain relevant to tumor maintenance and indicates roles for tumor-associated changes in directing host support mechanisms.

Dr. S. J. Korsmeyer brought attention to apoptosis, which has been investigated not only at the murine, i.e., mammalian level, but where seed discoveries were made at the invertebrate level as well. Many BCL-2 family members exist in active and inactive conformations determined by post-translational modifications in response to proximal death and survival signals. The pro-apoptotic molecule, BAD, a “BH3 domain” only molecule is inactivated by phosphorylation of serine 112 by mitochondrial tethered PKA or serine 136 by the PI-3 kinase pathway. BID as an inactive p22 molecule resides in the cytosol and is activated by a caspase−8 cleavage following tumor necrosis factor-α/Fas engagement resulting in the translocation of truncated BID to mitochondria. The three-dimensional structure of BID suggests that BCL-2 family members can be subgrouped into constitutively inactive molecules with hidden BH3 domains and active ones with an exposed hydrophobic face.

Dr. C. M. Croce provided a classical example where new observations made at the human level will require mouse experimentation (e.g.,k/o) to solidify assumptions or to obtain deeper insights into mechanisms. He has identified a gene at 3p he named FHIT, which is mutated by deletions in a very high percentage of some of the most common human cancers, including lung cancer. The gene encodes a Ap₃ A hydrolase that cleaves Ap₃ A into AMP + ADP. Transfection of the human FHIT gene into human tumor-derived cell lines results in suppression of tumorigenicity. A study of human lung cancer for expression of the FHIT protein indicates that almost 100% of small cell carcinomas and 73% of non-small cell carcinomas (HNSCC) have lost the ability to express the FHIT protein. Interestingly, 85% of bronchial dysplastic lesions are FHIT negative, suggesting that loss of FHIT function is a very early event in lung carcinogenesis. The mouse FHIT gene was knocked out in mouse embryonal stem cells and FHIT^−/+ and FHIT^{− /−} mice were obtained. FHIT^{− /−} mice develop stomach cancers at 9 months. FHIT^+/− mice can be induced to develop stomach cancers and rhabdomyosarcomas after treatment with low doses of carcinogens.