Project Funding Details

Background: It is clear from recent studies that oncogene-induced senescence (OIS) is a critical tumor suppressive barrier in vivo that is commonly dismantled in cancer cells. Nearly all recent studies implicate signaling to p53 as a key component necessary to enforce the senescent arrest in normal human cells. However, the importance of p53 for inducing OIS appears to be tissue specific. In response to RAS, RAF-1, or HER2, human mammary epithelial cells (HMECs) undergo a p53- and p16-independent growth arrest distinct from the p53- and p16-dependent arrest reported in human fibroblasts. Thus, it is critical that studies aimed at understanding the signaling pathways responsible for OIS in breast hyperplasia be performed using breast epithelial cells.

Our proposal is ambitious and in line with the Breast Cancer Research Program objective to fund high-risk/high-reward research aimed at reducing the burden of breast cancer. We show that the p53- and p16-independent OIS induced by RAS or HER2 in HMECs can be prevented by coexpressing MYC. Moreover, the loss of p53 and p16, combined with RAS or HER2 and MYC expression promotes the anchorage-independent growth of HMECs, an important hallmark of transformation. Many reports implicate MYC amplification or overexpression in human cancer; however, there are still many unanswered questions relating specific MYC target genes with its tumorigenic function in mammary cancers. We propose to use innovative Validation-Based Insertional Mutagenesis (VBIM) lentiviruses to identify the critical genes that can substitute for MYC to prevent OIS and drive HMEC transformation. The VBIM lentiviruses are novel promoter insertion viruses designed to induce dominant changes in the expression of genomic sequences neighboring the insertion sites, including high-level expression of a full-length or truncated protein. Since truncated proteins may have constitutive or dominant-negative functions, we can identify genes that facilitate or inhibit anchorage-independent growth. Confirmation that the promoter-induced mutation is responsible for the mutant phenotype is provided by reversible features of the viruses. These include loxP sites and a tetracycline-repressor binding site, which permit efficient excision or silencing of the promoter, causing phenotypic reversion only in promoter-dependent mutants.

Hypothesis: Promoter insertion into a gene responsible for preventing or facilitating RAS- or HER2-mediated senescence, similar to MYC, will result in a mutant phenotype capable of anchorage-independent growth. Promoter excision/silencing will ensure that the mutant phenotype is promoter-dependent.

Specific Aims: (1) Infect HMECs with VBIM lentiviruses and select by anchorage-independent growth clones that bypass the p53- and p16-independent growth arrest. (2) Study mutants with strong promoter-dependent phenotypes by identifying insertion sites and recreating the mutations in na?ve cells.

Study Design: HMECs expressing p53-shRNA will be mutagenized with VBIM viruses and screened for genetic alterations that prevent oncogenic RAS- or HER2-induced senescence and promote anchorage-independent growth. Selected mutants will be tested for validation by removal or silencing of the inserted promoter's activity to determine (1) whether the mutant phenotype is promoter-dependent, (2) the relative strength of the promoter-dependent phenotype relative to other mutants, and (3) the role of the gene in the transformed phenotype. MYC expression, which we show promotes anchorage-independent growth in our model, will serve as a positive control while a non-mutagenic vector incapable influencing transcription beyond the bounds of the lentiviral LTRs will serve as a negative control. Successful identification of novel factors involved in HMEC transformation is a critical starting point for understanding the OIS response in HMECs, and it will ultimately help expand our knowledge of tumor suppressor/oncogene interactions in breast hyperplasia. Novel targets identified here will provide opportunities for drug development, potentially expanding the arsenal of therapies necessary for eradicating breast cancer and improving the quality of life for patients.

Great strides have been made in the treatment of breast cancer, particularly at the early stage and when the cancers express certain receptors. Still, the most effective way of eradicating breast cancer is to prevent it from occurring at all. Work over the past several years has demonstrated that drugs such as tamoxifen, raloxifene, and exemestane can reduce the incidence of hormone-positive cancers by as much as 50%. However, these agents are not effective when the hormone receptors are absent (so-called "triple negative"), they carry the risk of serious side effects, they are currently available only to women at high risk, and they must be taken long-term. It is no small thing to ask a healthy person to undertake a long-term drug regimen -- one with serious side effects and unknown health risks. Perhaps this is why so few women have chosen to use tamoxifen or its alternatives.

One of us [Conneely] together with Dr. Bert O'Malley made a sentinel observation 5 years ago that opens the door to a completely new strategy for breast cancer prevention. She studied carcinogen-induced mammary cancer in female rats. Careful experiments showed that short-term exposure to chloroquine, an antimalarial drug that has been safely taken by millions for over 60 years, prior to carcinogen treatment reduced the development of breast cancer in these rats by an astounding 39%. This effect is akin to the life-long protection afforded by early pregnancy.

Of course, the next step would be to test this finding in humans by a prospective study; however, such a study would be cost-prohibitive and take many years. Fortunately, there is a group of people who were exposed to chloroquine years ago and can now be surveyed about their breast cancer history. From 1961 until 1990, the U.S. Peace Corps sent volunteers to malaria-endemic and malaria-free countries for 2-year stints and prescribed chloroquine to prevent malaria. This Peace Corps service provides a natural experiment comparing breast cancer in the two groups today, between 20 and 50 years later.

To accomplish this, we will capitalize on the affinity that returned Peace Corps volunteers (RPCVs) have with each other as a consequence of an intense shared experience. Using several national organizations of RPCVs, we will survey both chloroquine-exposed and non-exposed members and identify those RPCVs who have died. In the latter case, we will try to determine the cause of death.

This innovative translational epidemiology proposal takes a basic science observation and immediately tests its clinical impact. Further, it exploits the opportunity of repositioning a drug with known effects, toxicities, and extremely low cost as an agent for breast cancer prevention. The impact of this study is to open an entirely new method of population research focused on the eradication of breast cancer through prevention. Rather than merely going from the "bench to the bedside," we have the opportunity of going from the bench to a broad population with the possibility of a very significant effect, delivered in a short time.

The study design combines a large population, minimal risk, and high-quality data and controls. Thus, we believe that it provides an invaluable opportunity to definitively answer a vital question about the safe, effective prevention of breast cancer. It also may represent the first possibility for chemoprevention of estrogen-negative cancers, which make up between 15% and 20% of all breast cancers. If successful, it may be the first step to making breast cancer obsolete.