Overexpression of the Receptor for Hyaluronan-Mediated Motility (RHAMM) Characterizes the Malignant Clone in Multiple Myeloma: Identification of Three Distinct RHAMM Variants

Abstract

The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM−48, and RHAMM−147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM−48 and RHAMM−147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM−48, and 4% were RHAMM−147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn’s disease. RHAMM−48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn’s disease. RHAMM−147 was undetectable in normal and Crohn’s disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1.2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM−48, and RHAMM−147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo–activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

Subcellular distribution, calmodulin interaction, and mitochondrial association of the hyaluronan‐binding protein RHAMM in rat brain

“…RHAMM forms with apparent molecular weights (Mr) less than 82 kDa may arise by alternate splicing of RHAMM mRNA (Hall et al, 1995; Assman et al, 1999; Crainie et al, 1999), although we found no evidence for RHAMM splice variants in rat brain (unpublished observations). Those forms with Mr greater than predicted from RHAMM cDNA sequence likely arise from posttranslational modifications, either alone or in concert with alternate splicing, that influence migration rates on SDS‐PAGE gels.…”

“…Overexpression of RHAMM was reported to increase cell migration, and mutant RHAMM lacking HA binding domains abrogated this motile response (Hall et al, 1995). Several RHAMM isoforms have been identified, and some of these appear to be generated by alternative splicing of RHAMM mRNA (Entwistle et al, 1995; Assmann et al, 1999; Crainie et al, 1999). Participation of RHAMM in cell signaling was suggested by its involvement in events leading to phosphorylation of erk1/2, paxillin, and focal adhesion kinase (Hall et al, 1994; Lokeshwar and Selzer, 2000).…”

Identification of sequence, protein isoforms, and distribution of the hyaluronan‐binding protein RHAMM in adult and developing rat brain

“…Alternate splicing of RHAMM has been reported in other systems, including B cells of the multiple myeloma lineage and HeLa cells (Crainie et al, 1999; Assmann et al, 1999). By using PCR primers flanking alternatively spliced sequence (primer pair 6; Table 1), we were able to confirm the presence of RHAMM alternate splicing in HeLa cells (not shown).…”

Rhamm−/− fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair

“…These results suggest that Rhamm has both extracellular and intracellular functions. However, the role of Rhamm as a cell surface HA receptor became controversial partly because cloning of the human (Wang et al, 1996;Hofmann et al, 1998;Crainie et al, 1999) and mouse genes (Hofmann et al, 1998) revealed an absence of both a signal peptide required for export through the Golgi/ER and membrane spanning domains common to most cell surface receptors. We now know that Rhamm resembles a group of intracellular proteins that also lack these signature characteristics of classical cell surface proteins but that are nevertheless found Rhamm −/− fi broblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair R hamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers.…”