Dissecting the Role of Twist in Breast Cancer Metastasis

Institution: University of California, San Diego
Investigator(s): Janine Low-Marchelli, B.S. -
Award Cycle: 2008 (Cycle 14) Grant #: 14GB-0144 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2008)

During metastasis, carcinoma cells detach from their surroundings, migrate into the circulation, invade tissues, and finally establish new colonies in distant organs. Several changes that occur in metastatic cancer cells resemble an evolutionarily conserved process in embryonic development called Epithelial-Mesenchymal Transition (EMT). During EMT, epithelial cells lose cell-to-cell contact and gain motility, characteristics of mesenchymal (loosely associated connective tissue) cells. Recently, a gene-regulatory transcription factor called “Twist” was shown to have a prominent role in promoting EMT in mammalian breast cancer cells. Activation of Twist causes breast carcinoma cells to disseminate and metastasize from the mammary gland to the lung in mouse tumor models.

While recent literature points toward a significant role for Twist in EMT, the molecular mechanisms for how Twist initiates EMT are unknown. The goal of this proposal is to study breast cancer metastasis from an integrated molecular, cellular, and whole organism standpoint, so that novel biomarkers of cancer prognosis may be identified and eventually become targets for therapy. We hypothesize that Twist binds to specific DNA gene promoters and can directly control the transcription of genes that are important in initiating EMT that leads to breast cancer metastasis.

We have two main aims in the proposed research. First, we will define transcriptional target genes under the direct control of the basic-helix-loop-helix transcription factor, Twist. For this, we will employ a new technology called ChIP-Sequencing to identify gene promoters that bind Twist. Secondly, we will study the function and biological significance of Twist-induced genes in the context of EMT and breast cancer metastasis. We will use RNA interference to specifically inhibit direct targets of Twist and measure changes in EMT and metastasis by light and fluorescence microscopy, migration assays, biochemical techniques, and using mouse xenograft (human breast tumor) models.

Currently the strongest predictors for metastasis, including histological grade and lymph node status, fail to classify accurately early stage breast tumors. While chemotherapy has been shown to reduce the risk of metastasis by one-third, 70-80% of patients receiving this treatment would have survived well without it. Thus, after surgery and radiation therapy to treat their primary tumors, many patients still must decide whether to undergo adjuvant chemotherapy with little information as to whether this trying and expensive process is necessary. It is our belief then, that defining molecular targets of Twist would greatly benefit the development of prognostic tools and drugs to more accurately predict and treat breast cancer metastasis.

Final Report (2010)

The Yang lab is interested in understanding how a gene regulatory protein called Twist may promote breast cancer metastasis. The goals for this project were first, to identify the genes under the direct control of Twist using a new technology called ChIP-Sequencing; and secondly to begin to understand how some of these genes function to make the cells aggressive and metastatic.

We are continuing to compare our Chip-Sequencing results with our gene regulatory data derived from a microarray experiment. We would like to pinpoint the locations that Twist binds human DNA and how this correlates to positive, negative or no regulation of the nearby genes. Currently we believe that Twist is a strong activator of gene transcription, but as we have learned in this study, Twist has a very complex binding pattern, which may prove to be difficult to interpret. For example, we found over 14,000 statistically significant DNA binding sites for Twist on the human genome.

The majority of our work has been to try to understand the biological function of specific genes we believe are directly controlled by Twist in the angiogenesis (induced tumor cell blood vessel growth) setting. Currently we are investigating how one of these genes, called semaphorin, may modulate blood vessel growth using a chicken embryo assay which allows us to count the number of new blood vessels produced in response to the application of test cells. When we used our breast cells with high expression of Twist on the chicken embryo assay, we measured a robust and repeatable increase in blood vessel number compared to using normal breast cells. Interestingly, when we lowered semaphorin expression from these same high Twist-expressing cells, we found a small but significant reduction in angiogenesis. However, breast cells with no Twist, but with high levels of semaphoring, were unable to produce angiogenesis. Based on these results, we believe that semaphorin may be required for angiogenesis in our system but is unable to promote angiogenesis on its own. In future studies, we will test whether semaphorin is required for angiogenesis in a mouse model.

Symposium Abstract (2010)

Janine M. Low-Marchelli (PI)1,2, Veronica C. Ardi3,  Etienne Danis1, Andrew T. Chang1,2, James P. Quigley2,3, Jing Yang (mentor)1,2
University of California, San Diego1 Biomedical Sciences Program2, The Scripps Research Institute3

Most breast cancer mortalities arise from aggressive metastases. A gene-regulatory protein called Twist has been shown to promote metastasis in breast cancer and is associated with disease progression. The goals of this research project are to understand the biological and pathological functions of Twist and how breast cancer metastasis may be caused. To do this, we proposed two major goals: first, to identify what genes Twist controls directly, and second, to define what these genes do in the context of cancer.

We first identified genes under the direct control of Twist using a new technology called ChIP-Sequencing. This technology enabled us to successfully identify 14,000 places in the human genome to which Twist was bound. After matching the binding sites to specific genes using a ranking system based on a computer algorithm, we were able to create a list of genes that might be directly controlled by Twist. We compared this list of genes to another list of genes we previously had generated in our lab from a technique called microarray analysis, which tells us whether the gene is either turned "on" or "off" by Twist. Using these two methods we can begin to understand how Twist controls expression of genes in the human genome and how Twist might cause cancer cells to become aggressive.

After identifying direct targets of Twist, we next sought to understand their roles in metastasis. Other groups have identified an association between Twist expression in tumors and increased blood vessel growth, or angiogenesis, an important process that allows the tumor to survive and for metastatic cells to spread. Interestingly, genes that are classically thought to promote tumor angiogenesis such as VEGF or FGF were not found in our list of genes controlled by Twist. However, some of the genes we identified by ChIP-Sequencing and microarray analysis belong to a family of genes that direct nerve fiber growth. This family of genes also has roles in blood vessel branching or giving directional cues to blood vessels during embryonic development. It is possible that the gene pathways participating during blood vessel development may become pathologically re-activated by Twist in breast carcinogenesis. To test whether these nerve fiber genes are necessary for our cells to stimulate angiogenesis, we removed them from the Twist expressing cells and tested the ability of these cells to promote the formation of new blood vessels in an animal model. Removing one of the genes from our Twist cells caused a significant decrease in angiogenesis. In another experiment, removing a different gene from our Twist cells caused the blood vessels to form improperly, leading to hemorrhage.

These experiments suggest that Twist controls genes that are important for directing blood vessel growth to the tumor. By identifying new targets to disrupt angiogenesis, our research may lead to the development of new breast cancer metastasis therapies.