Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

Abstract

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of fulllength HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70 -80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of "gene lesions responsible for human inherited disease" were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2-4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA) 3 is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11-1...

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

“…Thus, the dimers are kept together by strong interactions. In a recent study it was demonstrated that HAS1 forms multimers through intermolecular disulfide bonds (36). However, the HAS2 dimers we observed were seen both in the presence and absence of reducing agents, making it unlikely that disulfide bonds are involved.…”

Chain Gangs: New Aspects of Hyaluronan Metabolism

“…Aberrant splice variants of HAS1 are able to modulate expression of the normally spliced variant [25]. Abnormalities of the HA synthase enzymes have been associated with transformation and tumor metastases [2, 26].…”

Targeting Hyaluronic Acid Family for Cancer Chemoprevention and Therapy

“…Several members of the hyaluronic acid (HA) family of molecules, HA synthases (i.e., HAS1, HAS2, HAS3), HA receptors (i.e., CD44 and RHAMM), and hyaluronidases (mainly HYAL-1), are critical determinants of tumor growth and progression (Adamia, Pilarski, Belch, & Pilarski, 2013; Ghosh, Kuppusamy, & Pilarski, 2009; Golshani et al, 2007; Karbownik & Nowak, 2013; Orian-Rousseau, 2010; Simpson & Lokeshwar, 2008; Sironen et al, 2011). HA family members promote malignant behavior of tumor cells in vitro , and tumor growth, metastasis, and angiogenesis in xenograft models (Bharadwaj et al, 2009; Chao, Muthukumar, & Herzberg, 2007; Gurski et al, 2012; Li, Li, Brown, & Heldin, 2007; Lokeshwar, Cerwinka, & Lokeshwar, 2005; Lokeshwar et al, 2006; Siiskonen, Poukka, Tyynela-Korhonen, Sironen, & Pasonen-Seppanen, 2013; Tan et al, 2011).…”

“…In experimental model systems such as breast, bladder, prostate, and colon, studies have been mainly focused on modulating the expression of individual HA family molecules and assessing their effects on tumor cell phenotypes both in vitro and in vivo . Each HA synthase or HYAL-1, either alone or coexpressed, contributes to tumor cell proliferation, motility, and invasion, and to enhanced tumor growth, metastasis, and angiogenesis in xenografts; in contrast, knockdown of these genes inhibits tumor cell functions (Adamia et al, 2013; Bharadwaj et al, 2009; Chao et al, 2007; Ghosh et al, 2009; Golshani et al, 2007; Li et al, 2007). In the case of HYAL-1, promotion of tumor cell function is dose dependent.…”