Nucleocytoplasmic Translocation of UBXN2A Is Required for Apoptosis during DNA Damage Stresses in Colon Cancer Cells

Abstract

The subcellular localization, expression level, and activity of anti-cancer proteins alter in response to intrinsic and extrinsic cellular stresses to reverse tumor progression. The purpose of this study is to determine whether UBXN2A, an activator of the p53 tumor suppressor protein, has different subcellular compartmentalization in response to the stress of DNA damage. We measured trafficking of the UBXN2A protein in response to two different DNA damage stresses, UVB irradiation and the genotoxic agent Etoposide, in colon cancer cell lines. Using a cytosol-nuclear fractionation technique followed by western blot and immunofluorescence staining, we monitored and quantitated UBXN2A and p53 proteins as well as p53's downstream apoptotic pathway. We showed that the anti-cancer protein UBXN2A acts in the early phase of cell response to two different DNA damage stresses, being induced to translocate into the cytoplasm in a dose- and time-dependent manner. UVB-induced cytoplasmic UBXN2A binds to mortalin-2 (mot-2), a known oncoprotein in colon tumors. UVB-dependent upregulation of UBXN2A in the cytoplasm decreases p53 binding to mot-2 and activates apoptotic events in colon cancer cells. In contrast, the shRNA-mediated depletion of UBXN2A leads to significant reduction in apoptosis in colon cancer cells exposed to UVB and Etoposide. Leptomycin B (LMB), which was able to block UBXN2A nuclear export following Etoposide treatment, sustained p53-mot-2 interaction and had partially antagonistic effects with Etoposide on cell apoptosis. The present study shows that nucleocytoplasmic translocation of UBXN2A in response to stresses is necessary for its anti-cancer function in the cytoplasm. In addition, LMB-dependent suppression of UBXN2A's translocation to the cytoplasm upon stress allows the presence of an active mot-2 oncoprotein in the cytoplasm, resulting in p53 sequestration as well as activation of other mot-2-dependent growth promoting pathways.

Keywords: DNA damage stress; UBXN2A; colon cancer cells; mortalin-2; p53.

Figure 1

UVB induces nucleocytoplasmic translocation of UBXN2A. (A-B) HCT-116 colon cancer cells were UVB irradiated with doses of 0.05, 0.125, 0.250, 0.5, 1, 2, and 4 KJ/m², followed by 24 hours recovery time. Proteins were extracted from cytoplasmic and nuclear fractions followed by a BCA assay to measure the total level of proteins. Proteins were loaded into 4-20% gradient SDS-PAGE followed by WB analysis. (A) Results indicate that UVB irradiation upregulated UBXN2A protein in the cytoplasm and, simultaneously, the UBXN2A level decreased in the nucleus fraction in the corresponding dosages (0.5, 1, 2, and 4 KJ/m²). Upregulation of UBXN2A in the cytoplasm was accompanied by the second p53 peak in the cytoplasm fraction and accumulation of p53 in the nucleus. P47 (UBXN2C), another member of the UBXD family showed no significant change in response to low UVB, while it decreased in higher doses of UVB (0.5, 1, 2, and 4 KJ/m²) when UBXN2A is increased in the cytoplasm. UBXN2A's protein partner, p97 showed no changes in the cytoplasm. Upregulation of UBXN2A in the cytoplasm and consequent accumulation of p53 in the nucleus were associated with the second peak of cleaved PARP at 1, 2, and 4 KJ/m². Anti-GAPDH and ORC-2 antibodies were used as subcellular and loading control markers in Panels A and B. (C-D) HCT-116 colon cancer cells were UVB irradiated with biologically relevant doses of 1 and 2 KJ/m². The total proteins from cytoplasmic and nuclear fractions were measured by BCA assay followed by WB analysis. The band density of UBXN2A was quantified and normalized with GAPDH and ORC-2 loading controls for the cytoplasm and nucleus, respectively. The data is shown as mean ± SEM of three different experiments where *p<0.05 using Bonferroni's t-test. UVB irradiation significantly increases UBXN2A protein in the cytoplasm, whereas in the nucleus UVB significantly decreased the level of UBXN2A protein.

Figure 2

Nucleocytoplasmic translocation of UBXN2A in response to UVB is an early event. (A-B) Cells were exposed to UVB radiation for different time intervals followed by WB analysis of cytoplasmic and nuclear fractions. (A) The nucleocytoplasmic translocation of UBXN2A in response to UVB started 6 hours after UVB exposure and reached a maximum 24 hours after 1KJ/m² UVB exposure in the cytoplasm. Meanwhile, the levels of heat shock protein HSC70 showed no alteration in response to 1KJ/m² UVB irradiation. Upregulation of p53 in the cytoplasm and its maximized accumulation in the nucleus were coincident with the cytoplasmic upregulation of UBXN2A. Anti-cleaved PARP showed a typical fluctuation of cleaved-PARP representing the level of apoptosis after UVB exposure. (C) HCT-116 cells were treated with three different doses of UVB followed by immunoprecipitation with anti-mot-2 antibody. Immunoblotting of the pulled down protein revealed UVB-induced UBXN2A binds to mot-2 in a dose-dependent manner. (D) Following UVB treatment, cell lysates were subjected to immunoprecipitation using anti-p53 antibodies followed by WB. Results showed reduction of p53-mot-2 binding at 1 and 2 KJ/m² can coincide with increased association of UBXN2A with mot-2 (Panel D versus panel C). IgG immobilized on magnetic beads was used as control.

Figure 3

Etoposide induces nucleocytoplasmic translocation of UBXN2A in a dose- and time-dependent manner in poorly and well-differentiated colon cancer cells. (A-B) HCT-116 and LoVo cells were treated with Etoposide (20 and 50μM) for 24 hours. Nuclear and cytoplasm fractions were analyzed for UBXN2A localization. Etoposide induces UBXN2A nuclear export in both HCT-116 (poorly differentiated) and LoVo (well-differentiated) colon cancer cells. (C-D) The statistical analysis of HCT-116 data using Bonferroni's t-test revealed a significant upregulation and associated downregulation of UBXN2A levels in the cytoplasm and nucleus, respectively (n=3, *p<0.05). (E-F) HCT-116 cells were incubated with a clinical dose of Etoposide (50μM) for different time periods (4-48 hours). (E) DNA damage induced by Etoposide led to UBXN2A nuclear export following 4 hours incubation, and nucleocytoplasmic translocation was completed after 24 hours incubation. An anti-p53 antibody showed that upregulation of p53 started as early as 4 hours and reached maximum level at 48 hours, and it partially coincided with constitutively increased UBXN2A levels in the cytoplasm. Downregulation of UBXN2A after 48 hours may be due to the late-apoptosis/necrosis induced by p53.

Figure 4

Silencing of UBXN2A decreases stress-induced apoptosis in colon cancer cells. (A-B) HCT-116 p53 -/- and p53 +/+ were treated with Etoposide (20μM) for 24 hours. Cytoplasmic and nuclear lysates were subjected to WB analysis using anti-cleaved PARP and anti-UBXN2A antibodies as well as anti-GAPDH and Orc-2 antibodies. (C) Signal intensities of cleaved PARP were quantified in both cytoplasmic and nuclear fractions. The sum of pixel densities of the cleaved PARP bands, normalized to the pixel densities of corresponding loading controls, was represented by bar graph. This experiment was conducted twice and generated comparable results. (D-E) Early apoptosis marker Annexin V significantly upregulated in HCT-116 p53 +/+, indicating the Annexin V marker can reliably indicate apoptosis following Etoposide and UVB stresses. (F) WB analysis verified efficient silencing of UBXN2A protein in HCT-116 cells (clones 5 and 6) stably expressing shRNA against UBXN2A. (G-H) HCT-116 expressing scrambled shRNA (control) and clones 5 and 6 were treated with Etoposide or UVB followed by flow-cytometry analysis using Annexin V marker. Statistical analysis revealed effective silencing of UBXN2A is associated with suppression of stress-induced early apoptosis in HCT-116 p53 +/+ cells (n=3, p<0.05).

Figure 5

UBXN2A nucleocytoplasmic translocation is mediated via the CRM1 pathway. (A-B) HCT-116 cells were treated with Leptomycin B (LMB, an inhibitor of the CRM1 nuclear export pathway) and Etoposide for 24 hours. Prepared cytoplasmic and nuclear fractions were subjected to WB analysis. (A) In the cytoplasm, Etoposide-induced UBXN2A nuclear export was inhibited by LMB. (B) UBXN2A protein accumulated in the nucleus of cells following treatment with LMB in the presence of Etoposide. P47 remained unchanged in the presence and the absence of LMB. Etoposide-induced cleaved PARP proteins were observed in the presence and the absence of LMB. However, LMB caused less dramatic accumulation of cleaved-PARP following Etoposide treatment. (C-D) Quantification of the protein bands detected in WB further confirmed that the cytoplasmic UBXN2A protein levels (normalized with loading controls) were dramatically increased in the absence of LMB (Panel C: columns 2 and 3 versus 1) as compared to cells treated with LMB (Panel C: columns 5 and 6 versus 4). A similar phenomenon was observed in nuclei as analysis of protein bands (Panel D) showed the presence of LMB can neutralize UBXN2A nuclear export induced by Etoposide (Panel D, columns 4, 5, and 6 versus columns 1, 2, and 3). (E-F) Immunofluorescence analysis of UBXN2A in HCT-116 and LoVo cells treated with Etoposide for 24 hours confirmed the induction of DNA damage results in a significant translocation of UBXN2A from the nucleus to the cytoplasmic compartment. DAPI-stained cells show characteristic chromatin condensation and apoptotic bodies, particularly in LoVo well-differentiated cells treated with Etoposide. (E) The proposed working model suggests that, following genotoxic stress, UBXN2A proteins translocate from nucleus to cytoplasm. This leads to the binding of UBXN2A to mot-2 oncoprotein, which has several oncogenic functions in tumors, including cytoplasmic tethering of p53 protein.