HOX Transcriptional Regulation of Angiogenesis

Institution: University of California, San Francisco
Investigator(s): Aubri  Charboneau , Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8FB-0013 Award: $39,029
Award Type: Postdoctoral Fellowship
Research Priorities
Pathogenesis>Outbreak -- how cancer spreads: angiogenesis, invasion, and metastasis



Initial Award Abstract (2002)
The growth of new blood vessels (angiogenesis) is needed for breast tumors to grow beyond a few millimeters and to act as routes for breast tumor cells to spread (metastasize). The critical cells for angiogenesis are normal endothelial cells (EC), which become influenced by the tumor microenvironment and growth factors to migrate, divide and form the new tumor microvasculature as the first stage in the process. Our laboratory has been studying at the molecular level how a group of master homeobox (HOX) genes control the growth of endothelial cells. HOX genes are "master" genes, since they control the expression of other genes (e.g., MMP's, growth factors, or integrins) that are involved with cell processes necessary for angiogenesis. Our research points to two key HOX genes, HOX D3 and D10, as the most relevant for breast cancer angiogenesis. Interestingly these two HOX genes appear to act in opposite ways. HOX D3 is associated with positive regulation of angiogenesis, and HOX D10 inhibits angiogenesis.

We will use a mouse model to study angiogenesis of human EC in response to breast tumor cells. The human microvascular EC are first modified to express high levels of HOX D10 or to be deficient in HOX D3. We will then directly observe the angiogenic response by microscopy. To better identify the EC angiogenic genes, which these HOX genes regulate, we will use a protocol to link these HOX proteins to the DNA. To examine the potential contribution of the co-factor PBX1a to HOX regulation of angiogenesis, we will study EC in which we either eliminate its expression or introduce mutant forms of PBX 1a both in tissue culture and in the mouse model of angiogenesis.

One of the major limitations of many current mouse tumor angiogenesis models is that the angiogenic responses being studied are those which arise from vessels of the host animal (i.e. mouse, chick embryo) and not vessels of human origin. However, in these proposed studies we will be able to study the ability of genetically manipulated human endothelial cells to form new blood vessels in response to human breast tumor cells in mice. In addition, this project is unique in that it will characterize naturally occurring master HOX genes as an alternative method to limit new blood vessel growth in response to breast tumor cells in order to ultimately inhibit breast tumor growth and metastasis.


Final Report (2003)
Note: this fellowship grant was resigned after one year, because the PI relocated to the University of North Carolina.

The growth of new blood vessels (angiogenesis) is needed for breast tumors to grow beyond a few millimeters and to act as routes for breast tumor cells to spread. The critical cells for angiogenesis are endothelial cells (EC), which are stimulated by the tumor microenvironment to divide, migrate and form new tumor microvasculature. Our laboratory has been studying at the molecular level how a group of master homeobox (Hox) genes control EC during this process. Hox genes encode "master" transcription factors that control the expression of other genes (e.g., proteases and protease inhibitors) necessary for angiogenesis. Our research points to two key Hox genes, Hox D3 and D10, as the most relevant for breast cancer angiogenesis. Interestingly, these two Hox factors act in opposite ways, whereby Hox D10 suppresses expression of many of the same genes which Hox 3 factors up-regulate during angiogenesis. The purpose of this project was to determine whether sustaining high levels of Hox D10, which is associated with quiescent EC in breast tissue, or suppressing the expression or activity of a positive regulator of angiogenesis, Hox D3, blocks angiogenesis in response to breast tumor cells. We also wanted to investigate which genes involved in the angiogenic process are direct DNA targets of these Hox transcription factors as well as the contribution of the Hox co-factor, Pbx1 in modulating the expression of the angiogenic phenotype.

In these studies we confirmed that Hox D10 up-regulates gene expression by binding DNA using modified human EC which express high levels of either wild type or a mutant Hox D10 incapable of binding DNA. We examined the mRNA levels of plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator receptor (uPAR). Hox D10 needs to bind DNA in order to up-regulate PAI-1, a protease inhibitor, but not to suppress expression of a protease receptor, uPAR. This suggests DNA binding is important for induction, but not for suppression of gene expression.

Studies have shown that the pro-angiogenic Hox 3 factors depend on interactions with a co-factor, Pbx1, to bind and activate target DNA. We observed that “active” Pbx1 is present at higher levels in angiogenic as compared to quiescent EC. By “knocking down” (i.e., reducing expression) this gene in angiogenic EC, we were able to prevent Hox binding to target DNA and endothelial cell migration and angiogenesis. Hox D10 is expressed at lower levels in angiogenic EC and we observed that introduction of Hox D10 in angiogenic EC also prevents the nuclear localization of Pbx1, where transcription occurs. Furthermore, we found that a Hox D10 mutant incapable of interacting with Pbx1 and altering its localization and activity can no longer block the expression of uPAR. This suggests that Hox D10 indirectly suppresses gene expression by preventing the formation of transcriptionally active complexes of Pbx1 and pro-angiogenic Hox factors.

These findings suggest a mechanism to explain how HOX D10 can act dominantly over pro-angiogenic HOX to maintain a quiescent phenotype in an angiogenic environment. Hox factors coordinately control the angiogenic process and perhaps targeting these factors would provide a more comprehensive alternative method to limit new blood vessel growth in response to breast tumor cells to ultimately inhibit breast tumor growth and metastasis.


Symposium Abstract (2003)
The growth of new blood vessels (angiogenesis) is needed for breast tumors to grow beyond a few millimeters, and these vessels act as routes for breast tumor cells to spread. The critical cells for angiogenesis are normal endothelial cells (EC), which are influenced by the tumor microenviron- ment to migrate, divide and form a new tumor microvasculature. Our laboratory has been studying, at the molecular level how a group of master homeobox (HOX) genes control EC during this process. HOX genes encode ?master? transcription factors that control the expression of other genes (e.g., proteases which degrade fibrin and protease inhibitors) necessary for angiogenesis. Our research points to two key HOX genes, HOX D3 and D10, as the most relevant for breast cancer angiogenesis. Interestingly, these two HOX genes act in opposite ways. HOX D3 positively regulates angiogenesis, while Hox D10 expression is associated with quies- cent EC in breast tissue and blocks angiogenesis in response to breast tumor cells. HOX D10?inversely regulates expression of many of the same genes which HOX 3 factors up-regulate during angiogen- esis.

In these studies we wanted to confirm that HOX D10 regulates gene expression by binding DNA. We modified human EC to express high levels of either wild type or mutant HOX D10 incapable of binding DNA. We then examined the mRNA levels of uPAR and PAI-1. HOX D10 needs to bind DNA in order to ?up-regulate? PAI-1, a protease inhibitor, but not to suppress expression of a protease receptor, uPAR. This suggests DNA binding is important for induc- tion, but not for suppression of gene expression.

Other studies have shown that the pro-angiogenic HOX 3 factors must interact with a co-factor, PBX, to bind and activate target DNA. We observed in EC that HOX D10 prevents nuclear localization of PBX, where transcription occurs. This suggests that HOX D10 indirectly suppresses gene expression by preventing the formation of transcriptionally active complexes of PBX1 and pro-angiogenic Hox factors. We also confirmed that PBX is required for angio- genesis by knocking down this gene in EC.

These findings suggest a mechanism to explain how HOX D10 can act dominantly over pro-angiogenic HOX to maintain a quiescent phenotype in an angiogenic environment. This project aims to characterize the naturally occurring master HOX genes as a method to limit breast tumor blood vessel growth, which potentially may inhibit both breast tumor size and metastasis.