A New Mouse Model of PI3-Kinase Induced Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Jun Zhang, Ph.D., M.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13FB-0104 Award: $89,600
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2007)
PI 3-kinases have been linked to a diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. PI 3-kinases are heterodimeric (two different protein subunits) molecules composed of a regulatory and a catalytic subunit. Mutations of the PI 3-kinase gene, termed PIK3CA, which encodes the p110a catalytic subunit, are frequently detected in various human cancers. In breast cancer, PIK3CA is mutated in 8-40% patients. To date, PIK3CA mutations have been shown to not only exert direct oncogenic effects through enhanced activity of PI 3-kinase, but also play a role in drug resistance and overall prognosis. Due to the association between p110a and cancer, it is believed to be a promising drug target.

Despite the availability of various cell-based systems to dissect oncogenic transformation and human tissue samples for descriptive analysis of PIK3CA mutations, there is no mouse model system to explore its role in breast cancer initiation and progression. Such a model, if well designed, will facilitate not only the understanding of the mechanisms of the activating mutations for PIK3CA as an oncogene in breast cancer, but also provide a chance to study the interaction of this oncogene with tumor suppressors (e.g. p53 and INK4A/ARF) by crossing the mice accordingly to produce desired genetic background.

Our aims are to: (1) generate genetically manipulated mice with PIK3CA mutation(s) capable of being conditionally activated in mammary epithelial cells and cause tumorigenesis; (2) investigate the effect of loss of tumor suppressor p53 in the context of activation of PIK3CA mutation(s); and (3) test the therapeutic effects of inhibitors of PI 3-kinase and compare them with conventional chemotherapy. Standard subcloning and/or genetic recombination techniques will be exploited for the construction of a targeting PIK3CA mutation(s). The mice will be generated by introducing the mutant genes into mouse embryonic stem (ES) cells. Mammary tumorigenesis will be achieved by crossing our mice with MMTV-Cre or WAP-Cre mice to allow mammary-specific expression. We will also cross the mice with mice harboring tumor suppressor p53 that can be conditionally inactivated. Inhibitors of PI 3-kinase and its downstream effectors and/or chemotherapeutic agents will be administered appropriately after mammary tumors are formed.

We anticipate that this study will demonstrate the oncogenic effect of activating PIK3CA mutation(s) in breast cancer in a causal-effect manner. By investigating the relationship with certain tumor suppressors (e.g., p53) and comparing the effect of PI 3-kinase inhibitors with conventional chemotherapy, we will evaluate the possibility of targeting PIK3CA as an independent therapy for breast cancer.

Final Report (2009)
Note: the PI left the project early to take a staff physician position at Kingsbrook Jewish Medical Center in Brooklyn, NY.

Breast cancer can be driven by mutations of certain genes, among which, PIK3CA is frequently mutated in about 30% of breast cancer patients. Although previous studies linked PIK3CA mutation to breast cancer, there is not so far a published delicate mouse model to test oncogenic effect of PIK3CA mutation and therefore the therapeutic effects of its inhibitors in vivo. Thus, I proposed to generate a genetically manipulated mouse model harboring this mutation that can be conditionally activated in breast tissue to model breast tumorigenesis in humans. By using this model, we planned to dissect the oncogenic effect of PIK3CA mutation in vivo, compare the therapeutic effects of inhibitors targeting PIK3CA with conventional chemotherapy, and, investigate the synergistic effects of PIK3CA mutation and loss of certain tumor suppressor genes.

By using molecular biology techniques, a vector (contains all necessary genetic information) targeting PIK3CA gene has been constructed. The H1047R mutation, which has the highest frequency in breast cancer was introduced into the vector, and has been shown in vitro that can be conditionally activated. The vector was then introduced into mouse embryonic stem cells. Stem cells which contained correct incorporation of genetic information from the vector were selected and confirmed by various methods. These stem cells were then used to generate the desired genetically manipulated mice. So far, chimeric mice with all desired genetic manipulation have been successfully generated.

So far, the major accomplishments of this research project include: (1) construction of the targeting vector; (2) targeting and selection of mouse embryonic stem cells; (3) generation of chimeras. The failure of germline passage, according to UCSF Transgenic Core Facility, was due to the poor quality of ES cells. Thus, we were offered a free second ES cell targeting. But quite unfortunately, from the 400 ES clones of the 2nd batch, only two clones seemed to be promising but not perfect to pass the stringent Southern screening. Since Dr. Wayne Phillips’ lab in Australia has successfully generated a very similar mouse model targeting the exact same mutation (H1047R) and agreed to ship us their mice, we decided to stop generating our own and use their mice instead to continue this project.