Loss of p53 Tumor Suppressor Function in Breast Cancer

Institution: City of Hope National Medical Center
Investigator(s): Jamil Momand, Ph.D. -
Award Cycle: 1995 (Cycle I) Grant #: 1KB-0102 Award: $313,848
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1995)
The p53 tumor suppressor protein is largely responsible for protecting cells from cancer-causing DNA-damaging agents. In breast cancer, the p53 protein can be inactivated by four different mechanisms. First, a mutation within the gene can result in a p53 protein that is not functional. Second, a mutation within the gene can result in no expression of the p53 protein. Third, the p53 protein can be expressed in a normal fashion, but a second protein binds to the p53 protein and inactivates it. Fourth, the p53 protein can be expressed in a normal fashion but is located in the wrong place within the cell. This proposal sets out to identify proteins that bind and inactivate p53 and to uncover the mechanism responsible for p53 mislocation in breast cancers. One protein previously demonstrated to be a natural inhibitor of p53 is mdm-2. Mdm-2 binds normal p53 and inactivates the tumor suppressor activity of p53. While recent evidence indicates that some breast cancer cells express significant levels of mdm-2 it is not known if mdm-2 actually binds p53 and inhibits p53 activity. We propose to measure the relative levels of free p53 and p53 bound to mdm-2 in breast cancer cells and determine if p53 is functional. This will test the hypothesis that cells expressing a high proportion of p53 bound to mdm-2 exhibit the lowest p53 tumor suppressor activity. p53 may be inactivated by its inability to travel to the nucleus of the cell. In order for p53 to protect cells from DNA damage it must bind DNA in the nucleus. Studies have shown that several breast cancers contain normal p53, but that it is located outside the nucleus. It is unclear what prevents p53 from entering the nucleus but preliminary work has shown that a short-lived protein is required. We propose to purify this short-lived protein and identify it by protein sequence analysis. Identification of these alternate mechanisms of p53 inactivation has immediate relevance to increasing our understanding of breast cancer pathogenesis for the following four reasons: 1) Since the p53 gene can be the first gene mutated in breast cancer, identification of the proteins responsible for p53 inactivation will directly contribute to our understanding of breast cancer initiation at the molecular level; 2) If mutations in the genes coding these putative proteins are inherited, genetic screening of breast cancer families may provide a useful risk factor; 3) These proteins may be suitable targets for the design of more effective or less invasive cancer therapies; 4) The clinical outcome of breast cancer patients may correlate with the abnormal regulation of these proteins and, therefore, their identification may be used as a guideline for future therapy modalities.

Final Report (1999)
Goal: To determine whether the majority of p53 tumor suppressor protein must accumulate into the nucleus in order to mediate transcription in breast cancer cells.

Description: The p53 tumor suppressor gene is inactivated by mutation in a wide variety of cancers. Epidemiology studies indicate that the p53 tumor suppressor gene is mutated in 25-40% of breast cancer cases. Some reports have shown that, in some breast cancer cases where p53 is not mutated, p53 protein can reside in the cytoplasm and not in the nucleus. Since it is likely that p53 must reside in the nucleus to perform its tumor suppressor function this observation has led to the suggestion non-mutant p53 may be inactivated by the tumor cell by excluding p53 from the nucleus. We asked whether p53 in such breast cancers could be activated after treatment with DNA damaging agents.

Using the MCF7 breast cancer cell line as a model, we showed that p53 does indeed reside in the cytoplasm of these cells by indirect immunofluoresence analysis. Ionizing radiation (IR) and actinomycin D (Act D) were used as p53 activators to ask whether: 1) p53 accumulates into the nucleus; 2) the p53 protein level increase; 3) the p53 effector genes WAF1/CIP1/SDI1 and MDM2 are upregulated; and 4) cells undergo growth arrest. These activities commonly correlate with p53 activation. We report that both Act D and IR increase the steady state p53 protein level, upregulate p21WAF1 and MDM2, and lead to cell growth inhibition. In comparison to 7.2 nM Act D, IR treatment (5-40 Gy) led to a lower level of p53 protein induction, MDM2 protein upregulation and p21WAF1 protein upregulation. Indirect immunofluoresence analysis indicated that the majority of p53 accumulates in the nucleus after Act D treatment but fails to accumulate in the nucleus after IR treatment. We also tested if p53 in finite lifespan human mammary epithelial cells (HMEC) could accumulate into the nucleus after DNA damage. We observed that the majority of p53 resides in the cytoplasm of these HMEC before and after IR treatment. Act D treatment, on the other hand, led to transient p53 nuclear accumulation in these HMEC within 1 h after treatment but failed to lead to significant p53 nuclear accumulation at 24 after treatment.

Conclusions: 1) The majority of immunologically detectable wild-type p53 resides in the cytoplasm of MCF7 breast cancer cells and some finite lifespan HMEC 2) IR and Act D can differ in their ability to promote p53 nuclear accumulation. 3) The data suggests that the majority of p53 need not accumulate into the nucleus of MCF7 cells to upregulate p21WAF1 and MDM2 and inhibit cell growth.

Potential impact: In breast cancer cells where p53 is shown to reside in the cytoplasm, treatment by certain agents can activate the p53 tumor suppressor gene.

Pyrrolidine dithiocarbamate prevents P53 activation and promotes p53 cysteine residue oxidation
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Wu H-H, and Momand J
Yr: 1998 Vol: 273 Nbr: 30 Abs: Pg:18898-18905

The MDM2 gene amplification database
Periodical:Nucleic Acids Research
Index Medicus: Nucleic Acids Res
Authors: Momand J, Jung D, Wilczynski S, and Niland J
Yr: 1998 Vol: 26 Nbr: 15 Abs: Pg:3453-3459

Geldanamycin prevents nuclear translocation of mutant p53
Periodical:Experimental Cell Research
Index Medicus: Exp Cell Res
Authors: Dasgupta G, and Momand J
Yr: 1997 Vol: 237 Nbr: 1 Abs: Pg:29-37

In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation
Index Medicus: Oncogene
Authors: Chin PL, Momand J, and Pfeifer GP
Yr: 1997 Vol: 15 Nbr: 1 Abs: Pg:87-99

Mdm-2: "big brother" of p53
Periodical:Journal of Cellular Biochemistry
Index Medicus: J Cell Biochem.
Authors: Momand J and Zambetti GP
Yr: 1997 Vol: 64 Nbr: 3 Abs: Pg:343-52

Solution structure of an essential region of the p53 transactivation domain.
Periodical:Folding & Design
Index Medicus:
Authors: Botuyan MV, Momand J
Yr: 1997 Vol: 2 Nbr: 6 Abs: Pg:331-342

TP53 tumor suppressor protein in normal human fibroblasts does not respond to 837 MHz microwave exposure.
Periodical:Radiation Research
Index Medicus: Radiat Res
Authors: Li JR, Chou CK, McDougall JA, Dasgupta G, Wu HH, Ren RL, Lee A, Han J, Momand J
Yr: 1999 Vol: 151 Nbr: 6 Abs: Pg:710-716