MOLECULAR AND CELLULAR MECHANISMS RESPONSIBLE FOR SKELETAL METASTASIS BY PROSTATE ADENOCARCINOMA.

Approximately 90% of patients with advanced prostate cancer develop skeletal metastasis. This occurrence results in a substantial reduction of the quality of life and the drastic worsening of the prognosis for a tumor that otherwise would generally progress slowly when confined to the prostate gland.

Cancer cells escaping the primitive prostate tumor will migrate throughout the body using blood and lymphatic circulation. According to a widely accepted paradigm, specific mechanisms are responsible for directing them to the skeleton. Cancer cells first adhere to the bone marrow blood vessel wall – by recognizing specific adhesion molecules – and successively respond to components of the bone tissue by migrating towards them. However, this small population of cancer cells will eventually disappear or remain indefinitely a micro-metastasis, unless bone factors support their growth and survival in the new microenvironment.

We aim to identify the molecular and cellular mechanisms conferring metastatic potential to cancer cells and the intrinsic factors which render the bone tissue such a unique and congenial environment for metastatic prostate cells.

Adhesion and extravasation of human prostate cancer cells

The mechanisms involved in this phenomenon might be very similar to those utilized by immune cells to reach inflammatory sites within different tissues. Chemokines are a family of chemotactic cytokines which play a determinant role in the adhesion of monocytes and lymphocytes to the endothelium and their extravasation into the target organs.

We have recently obtained the first evidence that human prostate epithelial cells express CX3CR1, the plasma membrane receptor for the chemokine fractalkine. This receptor is expressed in both metastatic and non-metastatic cancer cells as well as non-malignant prostate epithelial cells.

The endothelial cells of the bone marrow (BMEC) are thougth to express selected adhesion molecules that could capture prostate cancer cells from the blood stream. We found that human BMEC costitutively express fractalkine on their plasma membrane. Fractalkine has cell adhesive properties and is expressed as a trans-membrane protein, thus being well suited to resist the shear forces of the blood flow.

Possible roles of fractalkine and its receptor CX3CR1 in the adhesion of circulating prostate cancer cells to the bone marrow endothelium and their extravasation. The cell-bound fractalkine expressed on the surface of HBME cells directly functions as an adhesion molecule and allows CX3CR1-bearing cells to adhere to the vascular wall of bone marrow sinusoids (A). Adherent cancer cells migrate into the marrow stroma following the concentration gradient of soluble fractalkine, produced and released by cells of the bone microenvironment (B).

Click here to view film [.AVI] Prostate cancer cells in suspension (loaded with a red fluorescent tracer) adhere to human endothelial cells (loaded with a green fluorescent tracer), when perfused in vitro using shear forces similar to those found in human bone marrow sinusoids.

To characterize the role of the CX3CR1-fractalkine pair in the adhesion of prostate cancer cells to the bone marrow endothelial cells we perform dynamic-flow adhesion experiments.
In a typical experiment, endothelial and prostate cancer cells are labeled with green and red fluorescent probes, respectively. Prostate cancer cells in suspension are then perfused on a monolayer of endothelial cells and their interactions analyzed by real-time imaging.

Fractallkine can also be released from the plasma membrane and chemoattract different cell types. We found that human bone-resident cells, osteoblasts, express this chemokine and can release it from their surface.

Osteoblast-conditioned medium or soluble fractalkine can both attract prostate cancer cells in vitro. We hypothesize that this mechanim could occur also in vivo, thus driving prostate cancer cells adhering to the endothelial wall into the bone tissue.

We have recently found that Dihydrotestosterone (DHT) dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide, an antagonist of the androgen receptor. However, DHT is unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells upon a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. 

Survival and Growth of Metastatic Prostate Cells

Cancer cells migrated to a distant organ will succeed in producing clinically evident metastases only if they are able to survive and proliferate. Thus, the presence of specific trophic factors in the new microenvironment is a crucial requirement for the establishment of secondary tumors.

We investigate the activation of specific pro-survival signaling pathways by growth factors normally present in the bone tissue, to identify differences in the responsiveness of metastatic compared to non-metastatic prostate cancer cells.

To this end, we use two human cancer sub-lines derived from the widely used parental cells PC3. The PC3-ML cells are highly bone-metastatic whereas PC3-N cells lack metastatic potential.  We compare these two prostate cell phenotypes to DU-145 cells – derived from a brain metastasis of prostate adenocarcinoma– that also lack bone-metastatic potential.

We recently found that only bone-metastatic PC3-ML cells express the alpha receptor for Platelet Derived Growth Factor (PDGFR-α) whereas normal prostate cells as well as PC3-N and DU-145 malignant cells are negative for this receptor. Interestingly, PDGFR-α in PC3-ML is conventionally activated by its own ligand (the growth factor PDGF) but can also be atypically trans-activated by soluble molecules present in the bone marrow, binding to cell surface receptors different from PDGFR-α and recruiting intracellular mediators that we are currently attempting to identify.

We have also found that the activation of PDGFR-α by the soluble fraction of the bone marrow – which is the microenvironment in which cancer cells grow when metastasize to the skeleton – recruits the Akt signaling pathway, a crucial player in cellular survival.  It is plausible that the expression of PDGFR-α provides selected prostate phenotypes – such as the PC3-ML cells – with a survival advantage in the bone and allows them to grow into clinically evident metastases

We are currently applying both in vitro and in vivo experimental approaches described above also to the study of bone and brain metastatic potential of breast cancer cells.