TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation

Abstract

Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice. This finding shows the functional significance of TSGΔ154-1054 in preventing the ubiquitin-proteasome proteolysis of TSG101, which increases tumor malignancy and hints at its potential as a therapeutic target in cancer treatment.

Keywords: TSG101; alternative splicing; nasopharyngeal carcinoma; tumorigenesis; ubiquitination.

Figure 1. TSGΔ154-1054 mRNA and protein

A. Structures of full-length (FL)-TSG101 and TSGΔ154-1054 mRNA. Numbered boxes illustrate the coding exons. B. Schematic illustration of the domain structures of TSG101 and TSGΔ154-1054. C. Alignment of the TSG101 amino acid sequence and the presumptive sequence of TSGΔ154-1054. A dashed line represents the amino acids deleted from the TSGΔ154-1054, compared to TSG101.

Figure 2. TSGΔ154-1054 perturbs the degradation of TSG101 protein

A-B. TW01 cells were transfected with control vector or TSGΔ154-1054 expression plasmids; then, the transfectants were pulsed (A) in ³⁵S-[Met]-containing medium and chased (B) for the times indicated. Following immunoprecipitation with anti-TSG101 antibody, the immunocomplexes were analyzed by autoradiography (upper panel). One representative result of three independent experiments is shown. The degradation of endogenous TSG101 is plotted in the lower panel.

Figure 3. TSGΔ154-1054 protects TSG101 from proteasomal degradation mediated by Tal but not MDM2

A. TW01 cells were co-transfected with the respective expression plasmids. MG132 (10 mM) treatment was administered at 18 h post-transfection and incubated for 6 h before harvesting. Immunoprecipitation-western blot assay was carried out to assess the amounts of Myc-Ub -polymerized TSG101. IgG specifies the heavy chain of the immunoglobulin G. Asterisks indicate non-specific cross-reactive bands. RT-PCR analysis (Bottom) was conducted to demonstrate the expression levels of transfected TSGΔ154-1054 transcripts and DAD-1 served as cDNA loading control. B. Effect of TSGΔ154-1054 on Tal-mediated TSG101 proteolysis. TW01 cells introduced with HA-Tal as indicated, Myc-TSG101 (1 μg), and TSGΔ154-1054 (0.4 μg) plasmids were administrated with MG132 as described above, and harvested for western blot analysis at 24 h post-transfection. Asterisk indicates an unidentified cross-reactive band. C. Comparison of the effects of TSGΔ154-1054 on Tal- and MDM2-mediated TSG101 degradation. Both TW01 and U2OS cells were transfected with the plasmids indicated and harvested at 24 h for western blot analysis. Densitometric analysis of Myc-TSG101 was accomplished by means of IMAGEQUANT. Similar results were obtained in three independent experiments.

Figure 4. TSGΔ154-1054 competes with TSG101 for binding Tal thereby diminishing Tal-mediated polyubiquitination of TSG101

A. Binding of TSGΔ154-1054 to Tal but not TSG101. Glutathione-Sepharose-bound GST-TSGΔ154-1054 and the in vitro translated HA-Tal or TSG101 were applied to in vitro binding assay followed by western blotting. B. Binding of Tal and its deletion mutants to TSGΔ154-1054 and TSG101. A schematic illustration of the domain structures of Tal and its deletion mutants is shown (top). Tal comprises a leucine-rich repeat (LRR), ezrin-radixin-moesin (ERM) domain, coiled-coil (CC) region, sterile alpha motif (SAM), RING finger (RF), and a double PTAP motif. These HA-Tal mutants were subjected to in vitro transcription and translation, and applied to in vitro binding assay together with bead-bound GST-TSGΔ154-1054 or GST-TSG101. The result was visualized by western blotting (bottom). C. Competitive binding between TSGΔ154-1054 and TSG101 to Tal. Glutathione-Sepharose-bound GST-TSG101 and the in vitro translated HA-Tal were subjected to an in vitro competitive binding assay in the presence of increasing amounts of in vitro translated TSGΔ154-1054, and the result was revealed by western blotting (upper panel). An in vivo competitive binding between TSGΔ154-1054 and TSG101 to Tal was accomplished on TW01 cells co-transfected with HA-Tal, TSG101 and increasing level of GFP-TSGΔ154-1054 using co-immunoprecipitation coupled with western blot (lower panel). Co-immunoprecipitation was performed using anti-TSG101 antibody, and anti-GST antibody as an irrelevant antibody control. D. Effect of TSGΔ154-1054 on Tal-mediated ubiquitination of TSG101. The bead-bound GST-TSG101 was incubated with an in vitro ubiquitination reaction containing E1, E2 (UbcH5a), Ub, immunopurified HA-Tal, and in vitro translated TSGΔ154-1054. Immunoblot was performed using anti-Ub antibody.

Figure 5. TSGΔ154-1054 augments the functional activities of TSG101

A. Proliferation of TSGΔ154-1054 stably expressed Au565 and TW01 cells. Cells were cultured in medium in the presence of [³H]-thymidine for 3 days. The incorporation of [³H]-thymidine into cells was counted. The protein amounts of TSG101 and internal control GAPDH are indicated by parallel blotting of the whole-cell lysate. The intensities of TSG101 proteins are specified at the bottom of TSG101 blot after normalizing with their corresponding GAPDH densities. B. Proliferation of TSGΔ154-1054 stable lines upon TSGΔ154-1054 depletion. siTSGΔ154-1054 and siE7 (an irrelevant siRNA control) were transfected twice into TW01 TSGΔ154-1054 lines. The transfectants were then reseeded onto 6-well plate for [³H]-thymidine incorporation assay (top), as well as western blot (middle) and northern blot (bottom) analysis. Level of 18S serves as loading control. The intensities of TSG101 proteins and TSG101/TSGΔ154-1054 transcripts are shown after standardizing with GAPDH and 18S, respectively. C. Effect of TSGΔ154-1054 on TSG101-induced cell proliferation. TW01 cells transfected with siE7 and siTSG101 for two passages were reseeded and transfected again with the respective plasmids as indicated. [³H]-thymidine incorporation assay was carried out and the expression of TSG101 was assessed by western blotting (Lower). Above each individual graph (A-C) is a representative result from three independent experiments performed in duplicate (mean ± SD, *p < 0.05 by two-tailed t test). D. Effect of TSGΔ154-1054 on anchorage-independent cell growth. Soft agar colony formation assay was achieved, and the forming foci numbers of Au565 and TW01 stable lines were counted at day 30 and day 14, respectively. Magnification is 100x. Above each individual graph is a representative result from three independent experiments performed in triplicate (mean ± SD, ^‡p < 0.005 by two-tailed t test). E. Typical tumor growth of TW01 TSGΔ154-1054 stable lines in nude mice. The photograph demonstrated a comparable size difference of control vector- and TSGΔ154-1054 stable clone-derived tumors. Asterisk denotes tumor mass is undetectable. The expression of TSGΔ154-1054 transcript in tumor mass was assessed by RT-PCR (bottom). This animal experiment was repeated twice (n=20), and the TSGΔ154-1054-expressing xenografts of these mice (60%, 12/20) developed into large tumors in 3 to 4 weeks after initial injection (p<0.005, two-tailed t-test). A representative result is shown.