DNA hunt uncovers important target gene

"We screened the DNA from breast cancer cells for amplifications that are associated with tumour development. The identification of these new potential cancer-causing genes is critical to uncovering novel pathways that drive the conversion of a normal cell to a cancerous one," said senior author Daniel Haber.

The YAP gene has been most associated with the fruit fly Drosophila melanogaster. However, previous research has suggested that YAP function in human cells did not support the notion that YAP might be a cancer causing gene because its overexpression actually promoted cell death rather than cell survival (like Yki in Drosophila).

Nevertheless, due to the amplification of YAP in a mouse breast tumour, the team from Harvard Medical School (HMS) and Massachusetts General Hospital (MGH) and colleagues decided to examine the functions of YAP in a 3D mammary culture model developed in HMS.

Through microarray analysis of a mammary tumour in a BRCA1/p53 deficient mouse model, Haber's group discovered an amplified region of DNA in the mouse breast tumour that contained only one known gene, called YAP.

"A similar region of DNA is also amplified in some human tumours, but this amplified region often contains other genes that are known to promote cell survival," said Haber, who worked with co-authors Jianmin Zhang and Gromoslaw Smolen, both research fellows at MGH.

"Thus, whether the YAP gene could play a role in these cancers had been largely ignored. The amplified region we discovered excluded these other genes, which allowed us to focus on YAP as a new candidate."

Earlier studies on YAP function in human cells did not support the notion that YAP might be a cancer causing gene because its overexpression actually promoted cell death rather than cell survival (like Yki in Drosophila). Nevertheless, due to the amplification of YAP in a mouse breast tumour, Overholtzer and colleagues decided to examine the functions of YAP in a 3D mammary culture model developed in Brugge's lab.

In this model, they grew cells in a 3-dimensional protein matrix rather than in 2-dimensions on plastic, which allows mammary cells to adopt an architecture in culture that is similar to what occurs in the human breast.

They had previously uncovered the effects of other genes using this model that would be missed in more conventional 2D models.

Using these 3D cultures, the authors were able to show that the overexpression of YAP caused a dramatic change in cell behaviour associated with invasion into the protein matrix.

This type of invasive activity is normally associated with strong acting cancer-promoting genes. The authors were further able to show, in 3D cultures and other assays that YAP over expression both activated cell growth and inhibited cell death, just as one might have predicted from the studies of Yki in Drosophila.

Moreover, YAP overexpression was able to turn their non-cancerous mammary cells into cancer-like cells in the lab, as evidenced by the ability of YAP expressing cells to grow in soft agar, an assay that measures cancerous potential.

"What we would like to understand next is how YAP is controlled by the Hpo-Sav-Wts pathway in human cells," said Overholtzer.

"Also, although we found the YAP amplification in a mouse breast tumour, in human cancers this amplicon is actually much more common in other types such as lung, pancreatic, ovarian, and others. Thus it is possible that YAP plays an important role in the development of many different types of cancer.

The work is published in the Aug. 8 online early edition of the Proceedings of the National Academy of Sciences and will appear in the Aug. 15 print edition.