Cell adhesion molecule CD166 drives malignant progression and osteolytic disease in multiple myeloma

Multiple myeloma (MM) is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in MM cell lines and primary bone marrow (BM) cells from patients. CD166+ MM cells homed more efficiently than CD166− cells to the BM of engrafted immunodeficient NSG mice. CD166 silencing in MM cells enabled longer survival, a smaller tumor burden and less osteolytic lesions, as compared to mice bearing control cells. CD166 deficiency in MM cell lines or CD138+ BM cells from MM patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Further, CD166 deficiency in MM cells also reduced formation of osteolytic disease in vivo after intra-tibial engraftment. Mechanistic investigation revealed that CD166 expression in MM cells inhibited osteoblastogenesis of BM-derived osteoblast progenitors by suppressing RUNX2 gene expression. Conversely, CD166 expression in MM cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in MM cell homing to the BM and MM progression, rationalizing its further study as a candidate therapeutic target for MM treatment

CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche

We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function

ROLE OF CD166 IN MULTIPLE MYELOMA CELL HOMING TO THE BONE MARROW MICROENVIRONMENT AND DISEASE PROGRESSION

poster abstractMultiple myeloma (MM) is a plasma cell malignancy characterized by multiple lytic lesions throughout the skeleton, suggesting that trafficking of MM cells from the bone marrow (BM) and lodgment of these cells at secondary sites is important in disease progression. CD38+CD138- MM cells were previously characterized as putative MM stem cells (MMSC, Cancer Res. 2008; 68(1):190-7.). We analyzed CD38+CD138- cells contained within the MM cell line H929 and determined that a fraction of these cells (29.9%±1.4%) expresses CD166. CD166 is a member of the immunoglobulin superfamily capable of mediating both homophilic and heterophilic (CD6) interactions and has been shown to enhance metastasis and invasion in several tumors including breast cancer and melanoma. Studies from our laboratory suggest that CD38+CD138-CD166+ MM cells possess many functional properties commonly associated with MMSC including cell cycle quiescence, maintenance and propagation of daughter cells on a stromal substrate and gene expression profile. We hypothesized that CD166 promotes MM cell trafficking to the BM and is critical for disease progression. To test this hypothesis, H929-GFP myeloma cells were injected intravenously into NSG mice and GFP cells were recovered from the BM 14hr later. While only 3.3%±1.5% of total H929-GFP cells express the CD38+CD138- phenotype, the frequency of CD38+CD138- cells contained in BM-homed H929-GFP cells was significantly higher (53.4%±3.7%, n=3, p<0.01), suggesting a preferential homing of MMSC to the marrow microenvironment. Interestingly, whereas only 29.9%±1.4% of CD38+CD138- cells expressed CD166 prior to injection, 84.1%±10.8% of BM-homed H929-GFP CD38+CD138- cells expressed CD166 (n=3, p<0.01), suggesting that CD166 plays a critical role in directing homing of MM cells to the BM. Next, CD166 expression on H929-GFP cells was knocked down (KD) with shRNA in order to examine if reduced CD166 expression inhibit the homing of MM cells to the BM. The number of BM-homed GFP cells was significantly decreased for CD166KD cells (5658±904, n=6) compared to mock control (8551±848, n=6; p<0.05). Interestingly, cells in which suppression of CD166 expression was not achieved with shRNA homed preferentially to the BM (4.3%±0.3% CD166+cells in CD166 KD H929-GFP before injection versus 29.3%±3.6% in BM-homed GFP cells). Then we compared the progression of MM in NSG mice initiated with mock control or CD166 KD H929-GFP cells. Disease progression in mice receiving control cells was more rapid compared to that in mice receiving CD166KD cells as evidenced by serum levels of human IgA (kappa) at 4 weeks posttransplantation (240.5±67.1ng/ml versus 45.1±33.0ng/ml, n=3; p<0.05). We next examined the potential role of CD166 in osteolytic lesions using a novel Ex Vivo Organ Culture Assay (EVOCA) in which MM cells are co-cultured over calvariae from 10d-old pups for 7 days creating an in vitro 3D system for the interaction of MM cells with bone microenvironment. Data from EVOCA with H929 cells showed that bone osteolytic lesions are substantially reduced when CD166 is absent on either MM (CD166- fraction) or osteoblast lineage cells (calvariae from CD166-/- mice). Furthermore, co-culturing CD166+ or CD166- H929 cells with bone marrow stromal cells (BMSC) from WT or CD166-/- mice revealed that mRNA levels of receptor activator of NF-κB ligand (RANKL) are decreased when CD166 is absent on either MM or stromal cells while mRNA levels of osteoprotegerin (OPG), an important inhibitor of osteoclastogenesis, are not altered. This resulted in decreased RANKL/OPG ratios in cultures containing a CD166- component suggesting reduced MM-induced osteoclastogenesis in the absence of CD166. Interestingly, levels of M-CSF and IL-6 were similar in all these cultures suggesting that loss of CD166 may mediate suppression of osteolytic lesions through the downregulation of RANKL. Together, these results suggest that CD166 plays an important role in homing and retention of MM cells in the BM and promotes MM disease progression as well as bone-lytic disease and that CD166 may serve as a therapeutic target in the treatment of MM

Differential Stem and Progenitor Cell Trafficking by Prostaglandin E2

SUMMARY To maintain lifelong production of blood cells, hematopoietic stem cells (HSC) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSC (LT-HSC) reside in several, perhaps overlapping, niches that produce regulatory molecules/signals necessary for homeostasis and increased output following stress/injury 1–5. Despite significant advances in specific cellular or molecular mechanisms governing HSC/niche interactions, little is understood about regulatory function within the intact mammalian hematopoietic niche. Recently, we and others described a positive regulatory role for Prostaglandin E2 (PGE2) on HSC function ex vivo 6,7. While exploring the role of endogenous PGE2 we unexpectedly observed hematopoietic egress after nonsteroidal anti-inflammatory drug (NSAID) treatment. Surprisingly, this was independent of the SDF-1/CXCR4 axis. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin (OPN). Hematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in higher species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced EP4 receptor signaling. These results not only uncover unique regulatory roles for EP4 signaling in HSC retention in the niche but also define a rapidly translatable strategy to therapeutically enhance transplantation