Genomic Instability in Multiple Myeloma: Inducible Transfectants Provide a Model To Define the Role of RHAMM and Centrosome/Mitotic Spindle Stability in Myelomagenesis.

Abstract

In this study we describe stably transfected cells expressing RHAMM, the receptor for hyaluronan (HA) mediated motility, under the control of a tetracycline-inducible promoter. We created this model system to determine the potential for RHAMM to promote genomic instability in multiple myeloma (MM), a B-lineage cancer characterized by malignant plasma cells in the bone marrow. MM cells harbor extensive genetic abnormalities, and work in our lab has shown that centrosome abnormalities are present in MM, suggesting a link between the two. RHAMM is a multifunctional protein involved in HA mediated cell motility, cytoskeletal dynamics, and most recently shown by Maxwell et al. in our lab, centrosome and mitotic spindle stability (Mol Biol Cell vol. 14, 2262–2276). The latter studies have revealed that RHAMM dysregulation correlates with mitotic abnormalities that may give rise to aneuploidy, and ultimately to transformation. Furthermore, RHAMM overexpression alone, and together with increasing amounts of its -exon4 splice variant which is unable to bind microtubules, is associated with poor clinical outcome in MM. This suggests that the centrosome/mitotic spindle stabilizing function of RHAMM has real consequences for the malignant clone in MM. In previous work with transient transfection systems, RHAMM overexpression resulted in mitotic arrest, mitotic abnormalities and ultimately apoptosis; this may explain the difficulty in creating stable transfectants expressing RHAMM in an unregulated manner. Using a tetracycline-inducible system, we have identified stably transfected RHAMM-expressing clones, some exhibiting up to forty-fold increase in RHAMM transcripts on induction, as measured by real-time PCR. Induced RHAMM protein was observed as early as 48 hours post-induction by immunofluorescence, in association with the mitotic spindle, the cytoskeleton, the cell membrane, and in a fraction of the population, interphase nuclei. Centrosomal localization of RHAMM was also observed, in most cases after centrosome replication and subsequent migration during prophase. In the induced cells, phenotypes similar to the transient system were observed: a trend towards multipolar and disorganized spindles, multinucleated and apoptotic cells by immunofluorescence, and a dramatic onset of apoptosis as early as 72 hours post-induction as measured by annexin V staining and flow cytometric analysis. More detailed studies characterizing RHAMM overexpressing cells that survive aberrant mitoses are ongoing. By finely controlling the expression of RHAMM in this system, something not easily done in transiently transfected cells, we can assess the effects of incremental increases in RHAMM expression, which may more accurately reflect the events that occur during malignant transformation. We speculate that oncogenesis may involve upregulation of yet to be defined genetic mechanisms that allow tumor cells to escape the apoptotic effects of overexpressed RHAMM and thereby promoting the survival and selection of mitotically abnormal clonal variants having misseggregated chromosomes and increasingly aggressive characteristics. In this way, stably transfected cell lines currently under development will provide relevant model systems to assess the role of RHAMM and associated centrosome, mitotic spindle, and microtubule motor proteins in malignant transformation and progression of MM.