Résumé
Multiple Myeloma (MM) is a fatal plasma cell malignancy characterized by deregulation of both proliferation and cell-death.
Using a collagen based (serum-free) semi-solid assay, we have evaluated the ability of 40 HMCL to form colonies either spontaneously or in the presence of exogenous growth factors (IL6 or IGF-1).
We have shown that 28 Human Myeloma Cell Lines (HMCL) formed colonies in the presence of exogenous IL6, 14 in the presence of exogenous IGF-1 and 12 have the capacity to clone spontaneously without any growth factor.
In the present study, we have addressed:
1) Autocrine growth factors implicated in the spontaneous clonogenicity
2) Gene expression profile (GEP) associated with the spontaneous clonogenicity
On the one hand, we showed that IGF-1 or SCF (CD117) autocrine loop supports the growth of 5 and 1 HMCL, respectively.
On the other hand, GEP analysis of 40 HMCLs revealed that jagged2 gene expression is statistically associated with the spontaneous clonogenicity. Fourteen cell lines express jagged2, among them 11 formed colonies spontaneously.
To confirm this correlation, we next determined JAGGED2 expression by Western-blot and showed that only spontaneous clonogenic HMCL highly expressed this protein.
JAGGED2 is a ligand of NOTCH1 and NOTCH2 receptors that also appear to be highly expressed on MM cells (Western-blot and Flow-cytometry).
Overexpression of JAGGED2 in MM was reported either to be induced by hypomethylation of jagged2 promoter or by a lack of expression of the repressor SMRT/NCoR2. However, these mechanisms are not associated with JAGGED2 expression in our 14 JAGGED2+ HMCLs, suggesting another mechanism(s) currently under investigation.
Of note, jagged2 is expressed by 40% of MM patients (as documented by RT-PCR) and thus is not restricted to HMCL.
To address the involvement of Notch pathway in the spontaneous clonogenicity of myeloma cells, we generated sh-JAGGED2 HMCL using lentiviral systems. We observed that this stable down-regulation fully abrogates spontaneous clonogenic capacity.
To further analyze the implied mechanism(s), we used a low-cellular-density liquid culture (serum-free, cytokine-free) to mimic our collagen-based assay.
In this model, stable down-regulation of JAGGED2 induces inhibition of proliferation followed by delayed cell death. The molecular mechanism implicated in this process is currently under investigation.
Our results highlight a subgroup of JAGGED2 positive HMCLs that display spontaneous clonogenic capability. Furthermore, we described a new implication of JAGGED2/NOTCH pathway in survival and proliferation of myeloma cells under stringent conditions such as collagen-based clonogenic assay.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 124.