Overview: Prostate cancers (PCa) have an astonishing ability to disseminate, invade and survive in the marrow. Once there, disseminated tumor cells (DTCs) may lie dormant for years. Previously we showed that during metastasis circulating PCa cells target the ?niche? that houses hematopoietic stem cells (HSC). The niche regulates HSC quiescence. Continuing this work, we show that DTCs recovered from the niche are highly enriched in the CD133+/CD44+ population and express genes associated with a ?stem-like? phenotype. The shift in phenotype from CD133- /CD44- to CD133+/CD44+ populations is not seen in cells recovered from other tissues. Further studies show that the HSC niche itself is central to the shift in the DTC phenotype towards a less mature, more cancer stem-like cell?a phenotype which is resistant to chemotherapy. The goal is to more fully understand the biology of the CD133+/CD44+ population. Hypothesis: The acquisition of a cancer stem cell-like state by DTCs once they engage the HSC niche represents a molecular pathway which facilitates dormancy and resistance to therapy.
Aim 1 : Characterize the biology of CD133+/CD44+ DTC cells isolated from the marrow. We will determine if CD133+/CD44+ isolated from marrow generate (i) spheres in vitro and (ii) generate s.c. tumors in limiting dilution in vivo, (iii) express higher levels of stem cell markers compared to non-niche engaged DTCs, and (iv) using double labeling we will identify which population of DTCs (CD133+/CD44+ or CD133-/CD44-) leads to bone metastases.
Aim 2 : Elucidate the mechanisms regulating the conversion of CD133-/CD44- into CD133+/CD44+ cells. The change in DTC phenotype upon entering the niche is dependent on growth arrest specific-6. To define the receptors involved shRNA will target each of the three receptor tyrosine kinases (Tyro3, Axl, Mer receptors) that bind to GAS6. (ii) The intracellular signaling pathways activated by GAS6 signaling will be defined to identify mechanisms to block the conversion of CD133-/CD44- into CD133+/CD44+ cells.
Aim 3 : Identify the extent to which DTCs in humans express CD133, CD44 and GAS6 receptors. We know that 0.5-8% of primary tumor cells express a stem-like phenotype, yet in our models in marrow, 20- 30% of the DTCs express CD133+/CD44+. Based on these data sets, we predict that 20% or more of the DTCs recovered from men will express the stem-like phenotype. To test our hypothesis, 20 sets of paired samples in each of the following patient populations, men pre-prostatectomy (primary, CTCs, DTCs), patients with hormone naive PCa after primary therapy with rising PSA values (DTCs & CTCs), and castrate resistant patients with clinically evident metastatic disease (CTC and DTCs) will be evaluated for the expression of CD133, CD44 and GAS6 receptors.
Skeletal metastases represent a critical clinical challenge for men with PCa. The central hypothesis of this proposal is that when DTCs engage the HSC niche, their phenotype is converted by the niche towards a more ?stem-like? state. We further hypothesize that this shift by DTCs into a more ?stem-like? state provides the basis for the development of metastasis and chemoresistance. These studies are therefore designed to uncover the molecular mechanisms used by DTCs to establish footholds in marrow ? as it is this early activity which ultimately leads to the production of bone metastases. This information will better position the field to develop innovative strategies to combat metastasis.
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