RFP2 Modulates 20S Proteasome Activity, NF Kappa B Activation, and Tumor Cell Growth in Multiple Myeloma.

Multiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells. Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO- GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting.

At day six post transduction, 75-95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2a and Bip, and the induction of Caspase-8, 9 and 3 cleavage. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. The above described presence of ER stress, combined with the previous reports that RFP2 has E3 ubiquitin ligase activity prompted us to assess total protein ubiquitination. Concordant with its effects on ER stress, RFP2 downregulation was associated with significantly higher levels of poly-ubiquitinated proteins. Subsequently, we were able to document a significant reduction (60% inhibition) in 20S proteasome activity in RFP2 down regulated cells. Proteasome inhibition by RFP2 down regulation was confirmed in other MM cell lines and was partially abrogated by restoring RFP2 levels by overexpression. Importantly, RFP2 down regulated cells were more sensitive to bortezomib; indeed proteasome inhibition was synergistic with RFP2 downregulation in MM cells. The above results prompted us to study the mechanism whereby RFP2 impacts survival and proliferation of MM cells. Inhibition of the NF-kappa-B (NFκB) pathway is a hallmark of proteasome-related growth retardation and apoptosis and is a key pathway in MM. We show that NFkB luciferase reporter assay was associated with significant activity reduction with RFP2 downregulation. To define the mechanism of this process, we examined the level of NFkB related proteins in nuclear and cytoplasmic fractions. Interestingly, the most prominent effect observed in RFP2 down regulated cells was increased levels of IkBá in the nucleus. Altogether, these results support our supposition that the effects of RFP2 downregulation are mediated through an inhibition of the NFkB pathway that is associated with increased nuclear IkBa as well as a decrease in 20S proteasome activity.

RFP2 is a gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest, mediated by proteasome inhibition and reduced NFkB activity. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, targeting RFP2 may represent a novel therapeutic approach in MM and other lymphoid malignancies.

No relevant conflicts of interest to declare.