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A Functional Genomic Approach To Identify Potential Breast Cancer Therapeutic Targets
A major obstacle for breast cancer drug discovery is target identification. Despite the wealth of information on copy number alterations and mutations recently available from sequencing efforts such as The Cancer Genome Atlas, identifying new targets for breast cancer therapeutics has proved challenging because causative perturbations cannot be determined from benign changes without functional studies. Genetic shRNA screens are powerful tools for identifying loss-of-function phenotypes in mammalian cells and can be used to complement sequencing efforts in the identification of new drug targets. To perform such screens, we have developed bar-coded shRNA libraries in retroviral and lentiviral vectors that target the entire human genome. The hypothesis of this proposal is that genetic loss-of-function screens in combination with bioinformatic and genomic analyses can identify new targets for breast cancer therapeutics. Towards this end, we aim to 1) perform genome-wide shRNA lethality screens to identify genes that are selectively required for the proliferation and survival of breast cancer cells but not normal mammary epithelial cells, which we term here as Breast Cancer Lethal (BCAL) genes; 2) investigate the contribution of these candidates to proliferative and pro-survival signaling networks which support tumorigenesis; 3) examine the status of these candidates in human breast tumor samples; and 4) determine the effect of candidate loss-of-function in mouse models of human breast cancer.
The loss-of-function breast cancer lethality screens proposed here measure changes in the relative abundance of tens of thousands of pooled shRNAs using microarrays to track each shRNA?s barcode. shRNAs targeting genes that are essential for proliferation and survival can be identified because their abundance will be reduced following cell passaging and will thus ?drop-out? of the shRNA population. We describe in this proposal a pilot screen comparing HCC1954 breast cancer cells and telomerase-immortalized normal human mammary epithelial cells (HMECs) to demonstrate the feasibility of this approach. In this pilot study, we identified and validated a group of genes commonly required for cell proliferation and survival of both cell types as well as a group of BCAL genes selectively required for HCC1954 viability. Such BCAL genes are of great interest for further study as they represent potential targets for new breast cancer therapies.
As a result of this successful pilot study, we will perform genome-wide shRNA lethality screens on multiple breast cancer and normal mammary epithelial cell lines to identify BCAL genes required for the viability of all breast cancer cell lines tested or of a subset of breast cancer cell lines with common malignant perturbations. Candidates for further study will be prioritized by validation studies using alternative methods to assess cell viability and exclude shRNA off-target effects and by bioinformatic analysis to employ a breast cancer subtype-specific approach to target discovery. To complement this analysis, databases of genomic, transcriptional, functional, and protein-protein interaction information will be intersected with our dataset to determine the potential relevance of each candidate. Furthermore, we will investigate the mechanisms and signaling pathways through which candidate genes contribute to oncogenesis using biochemical and molecular biological approaches. Finally, the in vivo role of candidate BCAL genes in breast cancer progression will be investigated by examining candidate protein expression in clinical samples of human breast cancer and by inhibiting candidate gene function in mouse xenograft models of human breast cancer using genetic or pharmacological means.
This study proposes the first genome-wide, loss-of-function screen to identify and characterize genes that are selectively required for breast cancer cell viability. This effort will provide a genome-scale view of the genetic dependencies and vulnerabilities specific to breast cancer cells. Importantly, this strategy should uncover many previously unrecognized targets for breast cancer therapeutic intervention and has the potential to provide additional, more effective treatment options for breast cancer patients.
Target identification is a major obstacle to the development of new breast cancer therapies. An ideal target for breast cancer therapeutic intervention is one that upon inhibition leads to preferential killing of breast cancer cells rather than normal breast cells. Screens that measure an outcome after inhibiting individual genes on a large-scale basis are powerful experiments that could identify such targets, but until recently these screens could not be performed in human cells. To perform these screens, our laboratory has developed a new technology to individually inactivate every gene in the human genome, approximately 23,000 genes. This proposal hypothesizes that we can identify new targets for potential breast cancer therapies using this technology to determine which genes when inactivated lead to cell death of breast cancer cells but not normal breast cells. To demonstrate the feasibility of this approach, we performed a pilot screen and individually inactivated ~10% of the genes of the human genome in a breast cancer cell line derived from a patient with breast cancer and in a normal breast cell line. In this pilot study, we identified and validated a group of genes commonly required for survival of both the breast cancer and normal cells examined as well as a group of genes that were selectively required for survival of these breast cancer cells. This latter group of genes is of great interest for further study as they represent potential targets for new breast cancer therapies.
In this proposal, we aim to perform similar screens inactivating every gene in the entire human genome of multiple breast cancer and normal cell lines, representing many different subtypes of breast cancer, to identify genes selectively required for breast cancer cell viability. In addition to identifying such genes, we also propose to characterize the mechanisms by which breast cancer cells are dependent on these genes. Furthermore, we will examine whether these candidates are increased in clinical samples of human breast tumors and whether inhibiting these genes reduce tumor size in mouse models of breast cancer. Since many target-based therapies currently exist to treat a wide range of human conditions, it is likely that therapeutics exist which inhibit some of the candidates identified by this research. However, the potential efficacy of these therapies at treating breast cancer may have been unrealized until this study. We will prioritize our research to first examine the effects of these existing therapeutic agents in mouse models of breast cancer and will collaborate with clinical researchers on pre-clinical and clinical trials using these existing therapies. For potential breast cancer therapeutic targets against which no current therapies exist, we will seek collaborations aimed at identifying therapeutic inhibitors against these targets which could lead to pre-clinical and clinical trials of these therapies.
This study proposes the first screen of its kind to identify and characterize genes that are selectively required for breast cancer cell viability. This effort will provide a genome-scale view of the dependencies and vulnerabilities specific to breast cancer cells. Importantly, this strategy should uncover many previously unrecognized targets for breast cancer therapeutic intervention and has the potential to provide additional, more effective treatment options for breast cancer patients.