A Role for p53 and Splicing Factor SAP145 in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Lan Truong, B.S. -
Award Cycle: 2005 (Cycle 11) Grant #: 11GB-0104 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
The tumor suppressor protein p53 maintains cellular homeostasis and prevents tumorigenesis. Disruption of its pathway leads to breast cancers, and p53 mutations correlate with poor prognosis. Overexpression of the cell cycle regulator cyclin E is observed in aggressive human breast cancers. Cyclin E overexpression in breast cancers has been correlated with p53 mutations, implying a connection between p53 and cyclin E. Our data suggest that both p53 and cyclin E converge at the spliceosome through their interactions with the splicing protein SAP145. Splicing of precursor messenger RNA is carefully regulated by the spliceosome, a large RNA/protein macromolecular complex.

The primary focus of this proposal is to address the question of how p53 and cyclin E intersect at the spliceosome through SAP145 to affect pre-mRNA splicing of genes involved in the initiation and progression of breast cancer. We hypothesize that activated p53 interacts with SAP145 to affect SAP145 protein stability and consequently pre-mRNA splicing. We further hypothesize that splicing events mediated by the interaction of p53 and SAP145 are further affected by cyclin E. We will pursue the following specific aims: 1. Characterize the interactions between p53, SAP145, and cyclin E. 2. Determine the importance of SAP145 for p53 cellular outcomes. 3. Determine the importance of SAP145 for p53-mediated splicing of breast cancer genes.

To accomplish these aims, we will perform co-immunoprecipitation experiments to examine the interaction of p53 and SAP145 in breast cancer cell lines having an excess amount of cyclin E. To determine the roles of SAP145 in p53 and cyclin E activities, we will assess cell cycle arrest and apoptosis in breast cell lines before and after DNA damage by fluorescence-activated cell sorter (FACS) analysis. Pre-mRNA splicing will be examined by reverse transcription polymerase chain reaction (RT-PCR).

This project will lead to a better and novel understanding of how p53, SAP145, and cyclin E are involved in breast tumor development. The interaction of p53 and SAP145 represents a novel role for p53 in splicing. It is likely to have direct implications for breast cancer research, as p53 and cyclin E are both prognostic markers for breast cancer, and both proteins converge their pathways at the spliceosome. The understanding of their interactions and resulting functional consequences will lead to new approaches for preventing and treating breast cancer.


Final Report (2007)
To elucidate the various molecular mechanisms that lead to breast tumorigenesis, we examine two important players in breast cancer biology: (1) the tumor suppressor protein p53, and (2) the cell cycle regulator cyclin E. p53 functions to prevent tumor formation and proliferation by (1) causing growth arrest of cells and (2) inducing apoptosis, or programmed cell death. In contrast, cyclin E functions to promote progression through the cell cycle for normal cellular growth and proliferation. Abnormalities in the pathways and regulation of both proteins have been shown to contribute to breast and other human cancers.

We proposed that p53 and cyclin E intersect their pathways at the “spliceosome”, the complex responsible for generating functional gene products as well as creating diverse gene products. We hypothesize that p53 and cyclin E utilize the spliceosome through the splicing factor SAP145 in opposing ways to regulate progression of cells through the cell cycle. Our findings from year 1 suggested that another cell cycle regulatory protein, p21, may also be involved in this process. The objectives of year 2 were to examine the interactions of SAP145, p53, p21 and cyclin E and how their interplay affect cellular outcomes of cell cycle arrest and apoptosis.

We have determined that SAP145 interacts with p53 and p21, and an interaction between SAP145 and cyclin E has previously been shown. The interactions of SAP145 with p53 and p21 occur under conditions of no or low cellular stress, but are abolished following high stress coinciding with apoptosis. Furthermore, following high stress or damage, activated p53 decreases SAP145 protein levels, an effect that cannot be rescued by cyclin E. We demonstrate that the loss of SAP145 is due to cleavage of SAP145 at amino acid D276 by caspase-8 in a p53-dependent manner following conditions of high cellular stress.

The findings of this project indicate a novel mechanism of action for the p53 that is likely to have implications for breast cancer. p53-dependent, caspase-8-mediated cleavage of SAP145 correlates with conditions of apoptosis. Cleavage of various proteins is a biochemical hallmark of apoptosis, an important event that kills cells in a regulated fashion as a means to inhibit uncontrolled cell growth in tumors. The effects of activated p53 on SAP145 represent a new means by which p53 may, in part, mediate apoptosis as a function of its tumor suppressor roles in breast and other cancers that could be exploited for new therapeutic approaches for treating cancers.


Symposium Abstract (2005)
The tumor suppressor protein p53 maintains cellular homeostasis and prevents tumorigenesis. Disruption of its pathway leads to breast cancers, and p53 mutations correlate with poor prognosis. Overexpression of the cell cycle regulator cyclin E is observed in aggressive human breast cancers. Cyclin E overexpression in breast cancers has been correlated with p53 mutations, implying a connection between p53 and cyclin E. Our data suggest that both p53 and cyclin E converge at the spliceosome through their interactions with the splicing protein SAP145. Splicing of precursor messenger RNA is carefully regulated by the spliceosome, a large RNA/protein macromolecular complex.

The primary focus of this project is to address the question of how p53 and cyclin E intersect at the spliceosome through SAP145 to affect pre-mRNA splicing of genes involved in the initiation and progression of breast cancer. We hypothesize that activated p53 interacts with SAP145 to affect SAP145 protein stability and consequently pre-mRNA splicing. We further hypothesize that splicing events mediated by the interaction of p53 and SAP145 are further affected by cyclin E.

We have established that p53 interacts with SAP145 by coimmunoprecipitation of over-expressed p53 and FLAG-tagged SAP145. Furthermore, activated p53 can affect the stability of SAP145 by decreasing protein levels of both over-expressed and endogenous SAP145, as shown by Western Blotting. Over-expressed cyclin E can also affect the stability of SAP145 by increasing endogenous SAP145 protein levels in non-damaged cells. These results suggest that p53 and cyclin E affect SAP145 protein stability in opposing ways, and thus, may consequently affect pre-mRNA processing.

To study the functional roles of p53 in pre-mRNA splicing, we performed RT-PCR analysis to examine the alternative splicing patterns of two different minigenes: survival motor neuron 2 (SMN2) and alpha-globin fibronectin (a-glob FN). p53 was shown to affect the alternative splicing pattern of exon 7 of SMN2 and the EDI exon of a-glob FN transcripts. Activated p53 also affects alternative splicing of endogenous p53-inducible gene 3 (PIG3) transcripts. We will next examine whether p53 contributes to alternative splicing of the breast cancer genes BRCA1 and estrogen receptor. These RT-PCR analyses will focus on how the interplay between p53, SAP145, and cyclin E (and its consequences on SAP145 stability) affect p53-mediated alternative splicing of these gene products.

This project will lead to a better and novel understanding of how p53, SAP145, and cyclin E are involved in breast tumor development. The interaction of p53 and SAP145 represents a novel role for p53 in splicing. It is likely to have direct implications for breast cancer research, as p53 and cyclin E are both prognostic markers for breast cancer, and both proteins converge their pathways at the spliceosome. The understanding of their interactions and resulting functional consequences will lead to new approaches for preventing and treating breast cancer.