A novel splice variant of supervillin, SV5, promotes carcinoma cell proliferation and cell migration
Introduction
The actin cytoskeleton has important roles in cell proliferation, growth, and migration [1], [2]. Dysregulation of many actin-associated proteins is associated with tumorigenesis [3], [4]. Therefore, proteins involved in actin dynamics provide potential markers of cancer progression and targets for treatment regimens.
Alternative splicing enables multiple potential protein products to be generated from a single gene. A genome-wide study has shown widespread alteration in mRNA splicing in patients with cancer, indicating a potential involvement in tumorigenesis [5], [6]. Specific protein isoforms promote neoplastic transformation, cancer progression and therapeutic resistance [7]. Actin-associated proteins with specific splicing variants may increase cell motility or cancer metastasis [8], [9], [10], [11].
Supervillin is a member of the Gelsolin/Villin super-family of actin-binding proteins, with 48%–50% similarities to Gelsolin and Villin within the supervillin carboxyl-terminus [12], [13]. Supervillin associates with both F-actin and Myosin II at membranes and mediates myosin II activation and actin dynamics during cell contractility, cell spreading, cytokinesis and invasion of extracellular matrices [14], [15], [16], [17]. Also, supervillin plays important roles in transcriptional activation, histone modification, cell survival and signaling transduction [18], [19], [20], [21].
Four major isoforms of human supervillin have been identified, all transcribed from the same gene. The first isoform of supervillin, SV1, was isolated from detergent-resistant neutrophil plasma membranes and cloned from HeLa cells [12], [13]. SV2, the biggest splicing isoform of supervillin, also called archvillin, was discovered in skeletal muscle; SV2 contains four more coding exons (exons 3, 4, 5 and 9), as compared to SV1 [22]. The third splicing isoform (SV3), also called smooth muscle archvillin or SmAV, was cloned from ferret aorta and contains sequence encoded by coding exon 4 in addition to the SV1 sequence. SV4, which contains coding exons 3, 4 and 5 and is only 32 residues smaller than SV2, is extensively expressed in human tumor cells [20]. Although SV1 and SV4 share highly conserved domains and exhibit partial functional redundancy in nonmuscle cells, the differential expression of supervillin splicing variants across tissues and in tumor cells versus normal but immortalized cell lines suggests exclusive. For example, SV4 enhances cancer cellular survival through reducing the stability of tumor suppressor protein p53 [20].
In the present study, we have identified and characterized SV5, a novel splicing isoform of supervillin from tumor cell lines, which contains sequences from coding exons 3 and 5, but not from 4 or 9. We also report that SV5 is predominantly detectable in skeletal muscle, smooth muscle and in proliferating cells, but is present in only low amounts or at undetectable levels in most normal tissues. Finally, we demonstrate a specific requirement for SV5 during cancer cell proliferation and cell migration, a function that cannot be rescued by SV1 expression. Thus, the SV5 splice variant may differentially contribute to the function of supervillin during growth and cell motility.
Section snippets
Western blot analysis
Briefly, cells or tissues were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail (Roche; Indianapolis, USA) and phosphatase inhibitor cocktail (Roche; Indianapolis, USA). Protein concentrations were determined with Bradford assays (Beyotime Biotechnology; Beijing, China). Lysates were boiled for 10 min in a 100 °C heat block. Protein samples (40 μg) were separated on SDS-PAGE, and transferred to 0.22 μm porosity PVDF membranes (Millipore;
Identification of the novel supervillin splicing variant SV5
Previous studies in tumor cell lines revealed that supervillin isoforms including SV1 and SV4 are generated by alternative splicing [20], [22]. As expected, a PCR fragment of the SV4 N-terminus was amplified with primers complementary to 5'-UTR and the SV4-specific coding exon 4 (Fig. 1A and B/a, Lane#1 and Table 1). But, three PCR products were observed after amplifications with primers located in the 5'-UTR and coding exon 10 (Fig. 1 and B/a, Lane#2 and Table 1), suggesting the presence of
Discussions
Reorganization of the actin cytoskeleton underlay cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion [25]. The splicing pattern of specific isoforms is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, and survival properties [26].
Supervillin is tightly associated with membranes and the actin cytoskeleton [12]. Previously, two
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (No. 31571433 and No. 31501171), and Anhui Provincial Natural Science Foundation (No. 1508085SMC214 and No. 1608085MH180), and partially supported by Visiting Professorships Foundation of Hefei Institutes of Physical Science (No. Y3BZOH3058). We thank Dr. Elizabeth Luna for manuscript reading and helpful discussions.
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