Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma.

In this study, we show that the hyaluronan synthase 1 (HAS1) gene undergoes aberrant intronic splicing in multiple myeloma (MM). In addition to HAS1 full length (HAS1(FL)), we identify 3 novel splice variants of HAS1, HAS1Va, HAS1Vb, and HAS1Vc, detected in patients with MM or monoclonal gammopathy of undetermined significance (MGUS). HAS1Vb and HAS1Vc undergo intronic splicing with creation of a premature stop codon. MM cells expressing one or more HAS1 variants synthesize extracellular and/or intracellular hyaluronan (HA). Expression of the HAS1Vb splice variant was significantly correlated with reduced survival (P = .001). Together, alternative HAS1 gene splicing, the correlations between HAS1 splicing and HA synthesis, and the correlations between HAS1 splicing and reduced survival of MM patients support the hypothesis that the family of HAS1 protein plays a significant role in disease progression. Further, expression of HAS1Vb, in conjunction with HAS1(FL) and/or other HAS1 variants, may lead to accumulation of intracellular HA molecules and an impact on receptor for HA-mediated motility (RHAMM)-mediated mitotic abnormalities in MM. This study highlights the potential importance of HAS1 and its alternative splicing in pathophysiology of MGUS and MM.

RHAMM expression and isoform balance predict aggressive disease and poor survival in multiple myeloma.

Multiple myeloma (MM) plasma cells (PCs) express receptor for hyaluronan-mediated motility (RHAMM), a hyaluronan-binding, cytoskeleton and centrosome protein. The most abundant RHAMM isoforms in MM are full-length RHAMM (RHAMMFL) and the splice variant RHAMM-exon4. We separately examined the significance of RHAMM expression, and isoform balance, in 2 groups of MM patients. In oligonucleotide microarray experiments (n=210, Arkansas), increasing RHAMM mRNA expression in MM PCs is strongly associated with osteolytic bone lesions (P <.001), and event-free (P =.05) and overall (P =.04) survival. Semiquantitative determination of RHAMM isoform expression (Alberta, Canada) used capillary electrophoretic detection and measurement of RHAMM-exon4/RHAMMFL reverse-transcriptase-polymerase chain reaction (RT-PCR) products. RHAMM isoforms are rarely expressed concurrently in single MM PCs; the pattern of isoform expression, at the single-cell level, is approximated in larger numbers of cells by the RHAMM-exon4/RHAMMFL ratio. Absolute RHAMM expression and the RHAMM-exon4/RHAMMFL ratio are only partially correlated in MM PCs; in cell lines, absolute RHAMM expression is elevated in mitosis, while RHAMM ratios remain stable. Temporal examination of MM patients' peripheral blood reveals that the RHAMM-exon4/RHAMMFL ratio increases with disease burden. The RHAMM-exon4/RHAMMFL ratio in diagnostic bone marrow samples (n=101, Alberta) is an independent prognostic factor. Thus, expression and splicing of RHAMM are important molecular determinants of disease severity in MM.

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability.

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.

Abnormal expression of hyaluronan synthases in patients with Waldenstrom's macroglobulimenia.

Little is known about the biology or spread of Waldenstrom's macroglobulinemia (WM), a lymphoplasmo-proliferative disorder. Hyaluronan synthases (HASs), plasma membrane proteins, synthesize the extracellular matrix molecule hyaluronan (HA), which plays a role in malignant cell migration and the spread of many cancers. Three isoenzymes of HAS-HAS1, HAS2, and HAS3-are detected in humans. Aberrant expression of the HASs is coupled with different abnormalities. We have analyzed the expression pattern of HASs in WM patients. HAS3 was expressed in all patients and healthy donors tested, whereas the expression of HAS1 and HAS2 varied among the WM patients. Additionally, in WM patients, we have detected novel variants of HAS1, one of which was also detected in multiple myeloma (MM) patients. We speculate that HAS1 variants synthesize the intracellular HA ligand for RHAMM (a receptor for HA). RHAMM contributes to genetic instability in MM; therefore, we speculate that it may also contribute to genetic instability in WM. Furthermore, we suggest that overexpression of HAS1 and its variants in combination with HAS3 may form an HA matrix around WM cells, thus preventing their elimination by the immune system, and it promotes their migration and may facilitate the spread of disease.