Role of Integrins in Lymphangiogenesis During Breast Cancer

Institution: University of California, San Diego
Investigator(s): Barbara Susini, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0006 Award: $134,530
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
Recent studies suggest that lymphangiogenesis, the growth of lymphatic vessels, plays a crucial role in the formation of local and distant tumor metastases, including those to the bone marrow. As breast cancer spreads predominantly via the lymphatic system, an understanding of the role of lymphangiogenesis in breast cancer could help develop new therapies to prevent the spread of breast cancer. Little is currently known about the roles of integrins in lymphangiogenesis. The altered expression and function of integrins contribute significantly to invasion and migration of cancer cells. Integrins are cell surface receptor molecules and consist of two associated proteins, termed α and β chains. They bind extracellular matrix proteins such as fibronectin, laminin and collagen. Their interior, cytoplasmic domains bind other proteins that connect them to the cytoskeleton or have signaling functions. Therefore, integrins are viewed as cell surface receptors that influence cell behavior including adhesion and migration, including metastatic spread of cancer cells.

We propose to investigate the roles of key integrins in the regulation of lymphangiogenesis in breast cancer, especially for their role in promoting the migration and survival of lymphatic endothelial cells. The aims of the project include: 1) The expression and function of integrins during lymphangiogenesis in whole animals will be examined using antagonists of integrins in animal models of lymphangiogenesis. 2) Determine the expression and function of integrins on lymphatic endothelial cells in culture. The expression of vascular integrins α2β1, α4β1, αvβ3, α5β1 and αvβ5 on purified human and mouse lymphatic endothelial cells. We will explore the functional roles of lymphatic integrins. 3) Examine the role of integrins in regulating metastasis via the lymphatic vessels using antagonists of integrins in mouse models of spontaneous and xenograft breast tumor growth and metastasis.

The expression of integrins on lymphatic endothelial cells will determined by flow cytometry and immunohistochemistry (i.e., antibody staining) on purified human and mouse lymphatic endothelial cells and tissue sections from normal and tumor tissues. The roles of integrins in the promotion of lymphatic endothelial cell migration and tube formation will be examined by evaluating the effects of integrin antagonists or inhibitory, siRNA using purified lymphatic endothelial cells in culture. The roles of integrins in the promotion of lymphangiogenesis and breast tumor metastasis will be evaluated in animal models using integrin antagonists and siRNA approaches.

Regional lymph node metastasis is common even in patients with small breast tumors and there is a strong correlation between the number of involved nodes and a poor prognosis. As tumor metastasis has been directly associated with a high level of lymphatic vessels in breast cancer, an understanding of the mechanisms that regulate lymphangiogenesis lead to new methods to inhibit breast tumor metastasis. If our hypothesis is correct, then integrin antagonists may be used to suppress breast tumor metastasis by suppressing lymph angiogenesis. Thus my studies may lead to new therapies to suppress the formation of metastases, as several anti-integrin drugs are currently in the clinic for other applications.


Final Report (2008)
Invasive, metastatic breast cancer is responsible for most of the deaths that are caused by breast cancer. Breast cancer spreads predominantly through lymphatic vessels and lymph nodes. Thus, lymph nodes are routinely removed during breast tumor surgery for diagnosis and determination of treatment options. The lymphatic vessels surround breast tissue and consist of a single layer of cells, named lymphatic endothelium. When At the start of this fellowship, I had found that the number of lymphatic vessels in breast tissue increases dramatically during breast tumor development (lymphangiogenesis). However, little was known about the mechanisms driving this increase in lymphatic endothelial cells or the mechanisms that promote breast tumor cell invasion of the lymphatic vessels.

We made significant progress in this research. My first aim was to determine which cell adhesion proteins were expressed by and played a role in promoting the growth of lymphatic vessels in animals and in human tissues, while my second aim was to perform similar studies on lymphatic endothelial cells. We found that only one cell adhesion protein, called integrin alpha4/beta1, is expressed by growing lymphatic vessels and cells. This integrin controls the migration of lymphatic endothelial cells and the growth of lymphatic vessels in animals. Other cell adhesion proteins, including integrins alpha5/beta1 and alphav/beta3 are not expressed by lymphatic endothelial cells, while integrin alphav/beta5 is expressed but plays no functional role in the growth or migration of these cells. These findings will be submitted for publication in a high impact journal. Most importantly, antibody and peptide antagonists of integrin alpha4/beta1 could block the growth of lymphatic vessels in experimental models of lymphatic vessel growth and in experimental tumors. VCAM (vascular endothelial adhesion molecule), the alpha4/beta1 ligand, is expressed by breast cancer cells. Moreover, in vitro studies have shown that carcinoma cell adhesion to lymphatic endothelial cells can be inhibited by both alpha4/beta1 and VCAM antagonists. VCAM expression on metastatic breast cancer cells in the lymph nodes could promote the adhesion to the neo-lymphatic vessels. Antagonists to either molecule blocked tumor metastasis. These findings demonstrate that lymph node lymphangiogenesis is necessary and sufficient to promotes metastases by providing “docking sites” for metastatic tumor cells.

We found that other factors in addition to alpha 4/beta 1 & VCAM were involved in the metastatic process. The CCL21 cytokine and its receptor CCR7 participate to tumor cells recruitment to the lymph nodes by enhancing the attachment of tumor cells to lymphatic endothelium.

We were also able to identify which molecules interact with alpha 4/beta 1 to induce lymphatic endothelial cells migration. Paxillin, an alpha 4/beta 1 cytoskeletal adaptor that participates in the integrin's ability to anchor to the cytoskeleton, is necessary to lymphangiogenesis. Lymphatic endothelial cells in which alpha 4 can't bind paxillin are not able to migrate. Moreover, transgenic mice that lost alpha 4’s ability to bind Paxillin develop smaller tumors compare to wild type mice, and this is associated with an inhibition of tumor metastasis to the lymph nodes. Also, bone marrow transplants between those mice and wild type mice shown that both lymphangiogenesis and metastasis are not only restricted to an endothelial effect, but both need the participation of medullar cells.

Importantly, a key discovery in the course of this funded research was that tumors caused dramatic growth of lymphatic vessels not only in tumors, but also in lymph nodes. Studies of mice with spontaneous breast tumors showed that tumors caused the growth of lymphatic vessels in lymph nodes that were nearby as well as quite distant from tumors. The growth of lymphatic vessels in lymph nodes always preceded tumor metastasis to lymph nodes. A stimulation of lymph node lymphangiogenesis can accelerate tumor metastasis to that lymph node. As these growing lymphatic vessels expressed integrin alpha4/beta1, drugs that block this adhesion molecule could stop metastasis to lymph nodes. Indeed, inhibitors of alpha4/beta1 blocked lymphangiogenesis as well as tumor metastasis to the lymph nodes in mice with tumors that were grown under the skin.

These studies may have great impact on the future of breast cancer therapy due to a better appreciation of the mechanism by which breast cancer metastasis requires lymph node lymphangiogenesis. At least two treatment targets (integrin alpha 4/beta 1 and VCAM antagonists) can prevent cancer metastasis through lymph nodes. These drugs need further evaluation in clinical trials to test their effects in breast cancer therapy.


Symposium Abstract (2005)
Recent studies suggest that lymphangiogenesis, the growth of lymphatic vessels, plays a crucial role in the formation of local and distant tumor metastases, including those to the bone marrow. As breast cancer spreads predominantly via the lymphatic system, an understanding of the role of lymphangiogenesis in breast cancer could help develop new therapies to prevent the spread of breast cancer. Little is currently known about the roles of integrins in lymphangiogenesis. The altered expression and function of integrins contribute significantly to invasion and migration of cancer cells. Integrins are cell surface receptor molecules and consist of two associated proteins, termed α and β chains. They bind extracellular matrix proteins such as fibronectin, laminin and collagen. Their interior, cytoplasmic domains bind other proteins that connect them to the cytoskeleton or have signaling functions. Therefore, integrins are viewed as cell surface receptors that influence cell behavior including adhesion and migration, including metastatic spread of cancer cells.

We propose to investigate the roles of key integrins in the regulation of lymphangiogenesis in breast cancer, especially for their role in promoting the migration and survival of lymphatic endothelial cells. The aims of the project include: 1) The expression and function of integrins during lymphangiogenesis in whole animals will be examined using antagonists of integrins in animal models of lymphangiogenesis. 2) Determine the expression and function of integrins on lymphatic endothelial cells in culture. The expression of vascular integrins α2β1, α4β1, αvβ3, α5β1 and αvβ5 on purified human and mouse lymphatic endothelial cells. We will explore the functional roles of lymphatic integrins. 3) Examine the role of integrins in regulating metastasis via the lymphatic vessels using antagonists of integrins in mouse models of spontaneous and xenograft breast tumor growth and metastasis.

The expression of integrins on lymphatic endothelial cells will determined by flow cytometry and immunohistochemistry (i.e., antibody staining) on purified human and mouse lymphatic endothelial cells and tissue sections from normal and tumor tissues. The roles of integrins in the promotion of lymphatic endothelial cell migration and tube formation will be examined by evaluating the effects of integrin antagonists or inhibitory, siRNA using purified lymphatic endothelial cells in culture. The roles of integrins in the promotion of lymphangiogenesis and breast tumor metastasis will be evaluated in animal models using integrin antagonists and siRNA approaches.

Regional lymph node metastasis is common even in patients with small breast tumors and there is a strong correlation between the number of involved nodes and a poor prognosis. As tumor metastasis has been directly associated with a high level of lymphatic vessels in breast cancer, an understanding of the mechanisms that regulate lymphangiogenesis lead to new methods to inhibit breast tumor metastasis. If our hypothesis is correct, then integrin antagonists may be used to suppress breast tumor metastasis by suppressing lymph angiogenesis. Thus my studies may lead to new therapies to suppress the formation of metastases, as several anti-integrin drugs are currently in the clinic for other applications.


Symposium Abstract (2007)
Lymph nodes are the initial sites of metastasis for most solid tumors, including breast carcinoma and melanoma1-2. We show here that primary tumors can precondition lymph nodes for tumor metastasis by inducing lymphangiogenesis in draining and distal lymph nodes, thereby facilitating the appearance of metastatic lesions. Lymphangiogenesis in lymph nodes is required for tumor metastasis to lymph nodes and this depends on the ligation of two receptors expressed by lymph node lymphatic endothelium, integrin a4ß1 and VEGFR3, a lymphatic endothelial cell receptor for VEGF-C3. Moreover, VEGF-C can account for this activity since localized stimulation of lymphangiogenesis in lymph nodes by intradermal injections of VEGF-C accelerates tumor metastasis selectively to the stimulated lymph node. VEGF-C stimulation of lymph node lymphangiogenesis facilitates long range tumor homing to the lymph node even after injection of tumor cells into footpads of non-tumor bearing mice. Significantly, inhibition of local lymph node lymphangiogenesis with antagonists of VEGF-R3 or integrin a4ß1 prevented metastasis to lymph nodes without affecting primary tumor lymphangiogenesis or tumor growth while systemic administration of these antagonists suppressed lymphangiogenesis and metastasis to local and distant lymph nodes. These studies demonstrate that lymph node lymphangiogenesis is both necessary and sufficient to promote lymph node metastases and that integrin a4ß1, VEGF-R3 and VEGF-C play fundamental roles in tumor invasion through the lymphatics.

Integrins in angiogenesis and lymphangiogenesis.
Periodical:Nature Reviews. Cancer
Index Medicus: Nat Rev Cancer
Authors: Avraamides CJ, Garmy-Susini B, Varner JA
Yr: 2008 Vol: 8 Nbr: 8 Abs: Pg:604-17

Fluorescent LYVE-1 Antibody to Image Dynamically Lymphatic Trafficking of Cancer Cells In Vivo.
Periodical:Journal of Surgical Research
Index Medicus: J Surg Res
Authors: McElroy M, Hayashi K, Garmy-Susini B, et al.
Yr: 2008 Vol: Nbr: Jan 2008 Abs: Pg:ePub

Methods to study lymphatic vessel integrins.
Periodical:Methods in Enzymology
Index Medicus: Methods Enzymol
Authors: Garmy-Susini B, Makale M, Fuster M, Varner JA
Yr: 2007 Vol: 426 Nbr: Abs: Pg:415-38