Role of the BH3-only protein PUMA in tumorsuppression

The tumor suppressor p53 is targetted during the development of >50% of all human tumors, yet the precise mechanism for the tumorsuppression is unknown. Current models argue that a combination of cell-cycle arrest and apoptosis signals downstream of p53explain it`s tumorsuppressor function. Downstream molecular targets have been identified for these two separate functions of p53: p21Waf1 has been shown to be essential for p53 induced cell cycle arrest (but not for cell death) and the BH3-only protein PUMA was demonstrated to be essential for p53 induced cell death (but not for cell cycle arrest). However, gene targetting of either p21Waf1 or PUMA in mice did not lead to a spontaneous tumorphenotype, comparable to that observed in p53 targetted mice, in either system. We speculate that the two pathways can substitute for one another in the functional context of tumor suppression. In fact in vitro data using ES cells suggest that this may be the case. The proof of this principle in a more physiologic context would be of great value for the development of cancer targetting strategies. The proposed project will address the role of the pro-apoptotic "BH3-only" protein PUMA in tumor suppression. Two questions will be asked: 1) Is it possible to reconstitute the tumor phenotype of mice with targeted p53 by producing mice, deficient for both PUMA and the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and 2) Is overexpression of PUMA under physiological regulation able to lead to a tumor suppressor phenotype, similar to super-p53 mice? Project part one will make use of p21Waf1- and PUMA targetted mice on a pure C57BL/6 background and employ a cross to obtain doubly-deficient mice. A tumour follow up will determine incidence and type of tumors, as well as allow insight into patterns of loss of heterozygosity in tumours from doubly-heterozygous mice. In addition the study will allow insight into mechanisms involved in potential residual tumor suppressor activity downstream of p53. Project part two will generate mice carrying additional genomic copies of PUMA flanked by the endogenous regulatory sequences. These mice should, therefore, express enhanced pro-apoptotic responses to p53 dependent signals. A such the may mimick the properties of the super-p53 mouse, that displays resistence towards tumor inducing conditions. This experiment can validate PUMA as a target for cancer therapy in a near-physiological context.

The tumor suppressor p53 is targetted during the development of >50% of all human tumors, yet the precise mechanism for the tumorsuppression is unknown. Current models argue that a combination of cell-cycle arrest and apoptosis signals downstream of p53explain it`s tumorsuppressor function. Downstream molecular targets have been identified for these two separate functions of p53: p21Waf1 has been shown to be essential for p53 induced cell cycle arrest (but not for cell death) and the BH3-only protein PUMA was demonstrated to be essential for p53 induced cell death (but not for cell cycle arrest). However, gene targetting of either p21Waf1 or PUMA in mice did not lead to a spontaneous tumorphenotype, comparable to that observed in p53 targetted mice, in either system. We speculate that the two pathways can substitute for one another in the functional context of tumor suppression. In fact in vitro data using ES cells suggest that this may be the case. The proof of this principle in a more physiologic context would be of great value for the development of cancer targetting strategies. The proposed project will address the role of the pro-apoptotic "BH3-only" protein PUMA in tumor suppression. Two questions will be asked: 1) Is it possible to reconstitute the tumor phenotype of mice with targeted p53 by producing mice, deficient for both PUMA and the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and 2) Is overexpression of PUMA under physiological regulation able to lead to a tumor suppressor phenotype, similar to super-p53 mice? Project part one will make use of p21Waf1- and PUMA targetted mice on a pure C57BL/6 background and employ a cross to obtain doubly-deficient mice. A tumour follow up will determine incidence and type of tumors, as well as allow insight into patterns of loss of heterozygosity in tumours from doubly- heterozygous mice. In addition the study will allow insight into mechanisms involved in potential residual tumor suppressor activity downstream of p53. Project part two will generate mice carrying additional genomic copies of PUMA flanked by the endogenous regulatory sequences. These mice should, therefore, express enhanced pro- apoptotic responses to p53 dependent signals. A such the may mimick the properties of the super-p53 mouse, that displays resistence towards tumor inducing conditions. This experiment can validate PUMA as a target for cancer therapy in a near-physiological context.