Library of 1,000-plus Expressible Breast Cancer Proteins Made Public

cDNA Collection Demonstrated with Model System of Human Breast

In research that could significantly advance the pace of drug discovery for breast cancer, HMS investigators announce that they have created the first public library of reliably expressible proteins of a human disease, in this case, breast cancer. Their report in the Journal of Proteome Research appeared online Feb. 8 and will be published in the March 3 issue.

Photos by Graham Ramsay (Brugge) and Marc Raila

As leaders of the Breast Cancer 1000 initiative, Joan Brugge and Joshua LaBaer have made available a library of 1,300 protein-expressing complementary DNAs from genes related to breast cancer. They have also demonstrated the collection’s utility by using it to establish a model system mirroring human breast cells.

The researchers also expressed a subset of the 1,300 protein-producing complementary DNAs in the library into a model system mimicking cells of a human breast, enabling the investigators to study on a wide scale how these proteins might contribute to the development of breast cancer. Through this comprehensive approach, they identified potentially novel functional activities for both well-known and lesser-known breast cancer–related proteins.

“The process of carcinogenesis is complex and involves the activation of many different cellular programs,” said Joan Brugge, chair of the Department of Cell Biology, HMS professor of cell biology, and co–principal investigator of this initiative, called Breast Cancer 1000. “A significant limitation for breast cancer research has been the inability to distinguish whether certain proteins that are altered in breast tumor cells are the cause or the effect of conversion of normal breast cells to malignancy. The systematic approach that we’ve enabled and demonstrated will allow researchers to track cancer-causing proteins in simulated environments, with the goal of learning how to impede them.”

“The availability of this collection will enable pilot experimentation and accelerate the development of faster techniques for studying breast cancer in a mammalian setting,” said Joshua LaBaer, director of the Harvard Institute of Proteomics, HMS lecturer on biological chemistry and molecular pharmacology, and co–principal investigator. “To advance breast cancer research quickly, we are making the BC1000 library publicly available.” The library can be viewed from the Harvard Institute of Proteomics website (www.hip.harvard.edu).

Defining Disease Roles
To assess the range and functionality of the cDNAs in the library, the investigators introduced the first 265 constructed cDNAs into a line of immortalized breast epithelial cells and subjected these cells to a single screen to examine their relationships to cell migration, proliferation, and morphogenesis. From this screen, the researchers identified cDNAs already known to play roles in each, validating this approach as a means to identify relevant cDNAs. They also received hits from less-studied breast cancer genes, demonstrating the capability of using unbiased functional proteomics approaches to identify novel genes related to various aspects of disease biology.

“The migration findings are particularly important, as historically the roles of genes in the process of invasion and metastasis—the most devastating aspects of cancer—have been very difficult to test,” said LaBaer, “but tools such as BC1000 make this research much more accessible.”

Several unexpected cDNAs were found capable of inducing migration cooperatively when a known cancer-associated cell-signaling pathway was also activated. For example, proteins SGK (serum and glucocorticoid-regulated kinase-1) and TNFRSF10B (tumor necrosis factor receptor, 10B) were both identified as promigratory; however, they were previously recognized for their involvement in cell survival. The finding that cDNAs involved in other cellular processes may also play a role in migration suggests that this approach may help uncover unanticipated activities for previously identified proteins.

Validating Proteomics
“Drug design teams in the pharmaceutical industry traditionally have not used proteomics approaches to screen for potential targets, primarily because systematic proteomic tools are in their infancy,” said Steven Carr, who was not part of this research team and who leads the proteomics group at the Broad Institute. “While this work is highly in vitro and needs further validation, the tools and approaches demonstrated in this study show a potentially valuable screening tool for drug companies, primarily as a means to triage for novel targets to design drugs around.”

Prior to joining the Broad, Carr was director of Computational and Structural Sciences at SmithKline Pharmaceuticals (now GlaxoSmithKline) and led protein science and proteomics groups at Millennium Pharmaceuticals. “This study helps lay the groundwork for new and refined proteomics tools for cancer and other diseases,” he said.

Researchers in BC1000 created a sequence-validated cDNA collection of roughly 1,300 breast cancer–related cDNAs from genes that ranged from well-studied oncogenes to less conspicuous breast cancer–associated genes.

Selection of the cDNAs for inclusion in the BC1000 library was a multipart effort. The first 200 genes were suggested by Boston-area experts in breast cancer research. Another 50 were shown to be overexpressed in ductal carcinoma. The remainder were identified by MedGene, a literature-mining software application developed by the Harvard Institute of Proteomics that searches all titles and abstracts in the Medline database to identify cDNAs cocited with a particular disease and utilizes statistical methods to rank the relative strengths of these gene–disease relationships based on the frequency of total citation and cocitation.

“The work to isolate, sequence, and validate the BC1000 cDNAs was an immense undertaking, with multiple parties involved,” said LaBaer. “While the library covers a broad spectrum of breast cancer–related genes, it is not all-inclusive. The addition of new genes to this collection, including genes more recently linked to breast cancer and genes more difficult to clone, is an ongoing effort.”

—John Lacey

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