DKK1 is an Immediate Early Gene in Multiple Myeloma Cells Modulated by Cell Density and IL-6

Abstract 1928

Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/b-catenin signaling, is actively transcribed in early embryogenesis but DKK1 expression is largely absent in adult tissues. Consistent with concept that cancer recapitulates early development, we discovered that multiple myeloma (MM) cells, but not plasma cells from normal healthy donors express and secrete DKK1. Elevated DKK1 also characterizes most solid tumors. An important link between DKK1 and osteolytic bone loss in MM and solid tumor bone metastases has now been established. Given its central role in MM pathogenesis, we studied the molecular basis of DKK1 activation in MM. DKK1 gene and protein expression is elevated in approximately 80% of newly diagnosed disease. The LB and MF molecular subtypes, with the lowest incidence of lytic bone disease, also have the lowest expression of DKK1. Interphase FISH and aCGH on over 100 cases revealed that DKK1 activation is not a result of copy number changes or mutations of the gene locus at chromosome 10q11.2. Reduced methylation of CpG islands in the DKK1 proximal promoter in a subset of primary MM was not linked to DKK1 mRNA expression. Although there is variation, there is also no significant difference in DKK1 expression in cells taken at diagnosis and relapse in 95 cases. Emerging evidence suggests that elevated DKK1 expression in MM cells is correlated with bone disease in MGUS and increased risk of progression of MGUS to MM. Thalidomide and lenalidomide, but not bortezomib induces DKK1 mRNA and protein expression in primary MM cells following in-vivo exposure and in MM cell lines (MMCL) in-vitro. Consistent with DKK1 gene expression being actively modulated, we discovered that DKK1 levels were significantly different in RNA isolated from the INA6 MMCL obtained from different laboratories. We used RT-PCR in a time course experiment to show that DKK1 mRNA levels spiked greater than 10-fold within minutes of adding the cells to fresh culture media. These levels were maintained for several hours and then rapidly dropped to basal levels for the remainder of the 3-day culture period. Comparable results were obtained when CD138+ selected cells from primary disease were cultured in fresh media. In contrast to the spike in DKK1 mRNA, ELISA of culture supernatants revealed that DKK1 protein levels rose steadily throughout the time course. DKK1 spikes of comparable magnitude occurred in PCR-based DKK1-positive (INA6+ and M144) and DKK1-negative (OPM1 and L363) MMCL, using the day three level as a comparator. To explore if the upregulation of DKK1 following a change in culture media might be related a change in cell density, DKK1 was measured in INA6 cells cultured at varying initial cell concentrations. Interestingly, co-culture of MMCL with as little as 1ng/ml of IL6 could significantly reduce the DKK1 spike induced by low cell density. Taken together these data suggests that DKK1 expression in MM cells, and perhaps other cancer cells, is very dynamic. DKK1 spikes induced by low MM cell density in vitro may be recapitulating what metastatic cancer cells do in vivo. We suspect that high DKK1 production by plasma cells at low cell density ensures that local MSC proliferate, at the expense of osteoblast differentiation, and produce IL6, which will, depending on the MM cell density and the concentration of IL6 (reflecting MSC pool size), in turn downregulate DKK1 expression in tumor cells.