TRIM44 mediated p62 deubiquitination enhances DNA damage repair by increasing nuclear FLNA and 53BP1 expression

Abstract

Cancer cells show increases in protein degradation pathways, including autophagy, during progression to meet the increased protein degradation demand and support cell survival. On the other hand, reduced autophagy activity during aging is associated with a reduced DNA damage response and increased genomic instability. Therefore, it is a puzzling how DNA repair can be increased in cancer cells that are resistant to chemotherapies or during progression when autophagy activity is intact or increased. We discovered that tripartite motif containing 44 (TRIM44) is a pivotal element regulating the DNA damage response in cancer cells with intact autophagy. TRIM44 deubiquitinates p62, an autophagy substrate, which leads to its oligomerization. This prevents p62 localization to the nucleus upon irradiation. Increased cytoplasmic retention of p62 by TRIM44 prevents the degradation of FLNA and 53BP1, which increases DNA damage repair. Together, our data support TRIM44 a potential therapeutic target for therapy-resistant tumor cells with intact autophagy.

Figure 1.. TRIM44 promotes ionizing radiation (IR) resistance and protects MM cells from IR-induced cell death.

(A) TRIM44^OE MM cells show decreased IR-induced apoptosis, which was measured using flow cytometry based on annexin V/7-AAD staining. TRIM44^OE-CON and TRIM44^OE MM cells were treated with 2 Gy IR. (B) TRIM44^KD MM cells show increased IR-induced apoptosis, which was measured using flow cytometry based on annexin V/7-AAD staining. TRIM44^KD-CON and TRIM4^KD MM cells were treated with 2 Gy IR. (C) TRIM44^OE MM cells are resistant to the IR-mediated inhibition of colony formation. TRIM44^OE-CON and TRIM44^OE RPMI MM cells (10³) were irradiated with 2 Gy IR and seeded in semisolid methylcellulose medium. Colonies containing >40 cells were counted after 10 – 14 days. Colony formation was calculated as the IR-treated colony number/untreated colony number × 100%. Scale bars, 100 μm. (D) Colony formation after IR was greatly decreased in TRIM44^KD MM cells. TRIM44^KD-CON and TRIM44^KD RPMI MM cells (10³) were irradiated with 2 Gy IR and seeded in semisolid methylcellulose medium. Colony formation was calculated as described in (C). (E) TRIM44^OE MM cells display fewer IR-induced γH2AX foci after irradiation (2 Gy). After 1 hour, TRIM44^OE-CON and TRIM44^OE MM cells were immunostained with anti-γH2AX (red) and DRAQ5 (blue) and analyzed using confocal microscopy. Scale bars, 20 μm. (F) Graphic presentation of the percentage of γH2AX+ TRIM44^OE-CON and TRIM44^OE MM cells with or without irradiation. The cells containing > 5 γH2AX foci were recorded as γH2AX+ cells, whose percentage was averaged from at least 100 cells. The data are displayed as mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) TRIM44^KD MM cells show enhanced IR-induced γH2AX foci in MM cells, which indicates more susceptibility to irradiation. γH2AX+ cells were determined as described in (E). (H) Graphic presentation of the percentage of γH2AX+ TRIM44^KD-CON and TRIM44^KD MM cells with or without irradiation. The percentage of γH2AX+ cells were calculated as described in (F).

Figure 2.. TRIM44 enhances DNA damage repair in MM cells.

(A) TRIM44^OE MM cells show a decrease in IR-induced γH2AX foci with faster kinetics compared to control cells (OE-CON). At 1, 8 and 24 hours after irradiation (2 Gy), cells were immunostained with anti-γH2AX (red) and DRAQ5 (blue) and analyzed using confocal microscopy. Scale bars, 20 μm. (B) Graphic presentation of the relative percentage of γH2AX+ TRIM44^OE-CON and TRIM44^OE MM cells. The cells containing > 5 γH2AX foci were recorded as γH2AX+ cells, whose percentage was averaged from at least 100 cells. The number of γH2AX foci at 1 hour were normalized to 100%. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) TRIM44^KD MM cells show delayed DNA damage repair after irradiation (2 Gy). γH2AX+ cells were determined as described in (A). (D) Graphic presentation of the relative percentage of γH2AX+ TRIM44^KD-CON and TRIM44^KD MM cells. The relative percentage of γH2AX+ cells were calculated as described in (B). NS, non-significant. (E) The COMET assay confirmed that TRIM44 enhances DNA damage repair in MM cells. Cells were treated with 2 Gy IR, and after 8 hours, COMET images were captured using fluorescence microscopy. Scale bars, 100 μm. (F) Graphic presentation of the tail moment of TRIM44^OE-CON and TRIM44^OE MM cells. The tail moment was calculated as the tail length multiplied by the tail DNA percentage in at least 100 cells. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) The COMET assay confirmed that TRIM44^KD MM cells exhibit delayed DNA repair after irradiation. The tail moment was calculated as described in (E). (H) Graphic presentation of the tail moment of TRIM44^KD-CON and TRIM44^KD MM cells. The tail moment were calculated as described in (F).

Figure 3.. TRIM44 promotes NHEJ-mediated DNA repair and genome instability in MM.

(A, B) TRIM44 increases NHEJ activity. The BamHI-digested mCherry2-N1 and pDsRed2 plasmids, which was used as internal control (Fig. S4O), were co-transfected into TRIM44^OE-CON, TRIM44^OE, TRIM44^KD-CON and TRIM44^KD HEK 293T cells. After 48 h, the cells were harvested, and the intensity of mCherry fluorescence was measured using flow cytometry. (C, D) XRCC6, XRCC5, and PRKDC expression was increased in TRIM44^OE MM cells (C) but decreased in TRIM44^KD MM cells (D), as measured using qRT-PCR. (E-G) TRIM44^OE MM cells showed an increase in IR-induced 53BP1 focus expression. Cells were collected at 1 h post-IR and immunostained with 53BP1 (green) and DRAQ5 (blue) (E). Scale bars, 20 μm. The cells containing > 5 53BP1 foci were recorded as 53BP1+ cells under confocal microscopy. The percentages of 53BP1+ cells were displayed as the mean plus SD of three counts, and statistical significance was performed (F). Quantification of fluorescence intensity was performed from randomized districts on slides from each treatment group. Results were expressed as average fluorescence intensity of nuclear 53BP1 per cell and statistical analysis was performed. Results are shown as means ± SD of 100 randomized cells obtained from three independent experiments (G). Statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H-J) TRIM44^KD MM cells showed fewer IR-induced 53BP1 foci compared to the control. 53BP1+ cells were determined as described in (F). Quantification of fluorescence intensity was calculated as described in (G).

Figure 4.. TRIM44 upregulates HR-mediated DNA repair mechanisms and genome instability in MM.

(A, B) TRIM44^OE cells exhibit increased HR-mediated DNA repair activity, but TRIM44^KD cells exhibit decreased HR-mediated DNA repair activity. The I-SceI, pDRGFP, and pDsRed2 plasmids, which was used as internal control (Fig. S4O), were cotransfected into TRIM44^OE-CON, TRIM44^OE, TRIM44^KD-CON and TRIM44^KD HEK 293T cells. After 48 h, the cells were harvested, and the intensity of GFP fluorescence was measured using flow cytometry. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C-E) TRIM44 expression in MM cells greatly increased the expression of RAD51 after irradiation (2 Gy). Cells were collected at 8 h post-IR and immunostained with RAD51 (green), γH2AX (red) and DRAQ5 (blue) (C). Scale bars, 20 μm. The cells containing > 5 RAD51 foci were recorded as RAD51⁺ cells under confocal microscopy. The percentages of RAD51+ cells were displayed as the mean plus SD of three counts, and statistical significance was performed (D). Quantification of fluorescence intensity was performed from randomized districts on slides from each treatment group. Results were expressed as average fluorescence intensity of nuclear RAD51 colocalized with γH2AX per cell and statistical analysis was performed. Results are shown as means ± SD of 100 randomized cells obtained from three independent experiments (E). Statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F-H) TRIM44^OE MM cells show an increase in RAD51 foci after irradiation (2 Gy). After 1, 8 and 24 hours, cells were immunostained with anti-RAD51 (red, Alexa-594) and DRAQ5 (blue) and analyzed under confocal microscopy (F). Scale bars, 20 μm. The cells containing > 5 RAD51 foci were recorded as RAD51+ cells. The percentage was averaged from at least 200 cells. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented was performed (G). Quantification of fluorescence intensity was performed from randomized districts on slides from each treatment group. Results were expressed as average fluorescence intensity of nuclear RAD51 per cell and statistical analysis was performed. Results are shown as means ± SD of 100 randomized cells obtained from three independent experiments (H). Statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (I-K) TRIM44^KD MM cells show fewer RAD51 foci after irradiation (2 Gy) compared to controls (KD-CON). The percentage was averaged from at least 200 cells. RAD51+ cells were determined as described in (G). Quantification of fluorescence intensity was calculated as described in (H)

Figure 5.. TRIM44 interacts with p62.

(A) TRIM44^OE MM cell lysates were immunoprecipitated with anti-TRIM44 and immunoblotted using TRIM44 or p62 antibodies. (B) Confocal analyses show colocalization between TRIM44 and p62. U266 cells were treated with MG132 (5 μM) for 1 hour, and the results were visualized with antibodies against p62 and TRIM44. Scale bars, 10 μm. (C) Illustrations of different deletional TRIM44 constructs. GFP-tagged wild-type (WT) TRIM44; full-length (FL) TRIM44 (aa 1–344); ZF domain (aa 13–48); BB domain (aa 174–215); CC domain (aa 290 to 325); dCC domain (aa 1 to 290), a construct lacking the CC domain; and dZF domain (aa 48 to 344), a construct lacking the ZF domain. (D) Different deletional GFP-TRIM44 mutants were transfected into HeLa cells with full-length HA-p62 to identify the TRIM44 domains responsible for p62 binding. Overlapping immunofluorescence in the images is shown in yellow. All experiments were replicated three independent times with similar results. Scale bars, 10 μm.

Figure 6.. TRIM44 promotes p62 deubiquitination.

(A) GFP-TRIM44 and HA-p62 plasmids were transfected into HeLa cells and immunofluorescence imaging were analyzed using confocal microscopy (Left). Graphic presentation of the average volume of p62 oligomerization 48h after transfection. The volume of p62 oligomerization in 50 cells was counted for each data point shown (each bar) (Right). Student’s t test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 10 μm. (B) HA-p62 plasmids were transfected into HEK 293T cells expressing different levels of TRIM44 (TRIM44^KD-CON, TRIM44^KD, TRIM44^OE-CON, or TRIM44^OE). The cells were treated with or without DSP and lysed in RIPA buffer 2 hours after irradiation. The lysates were subjected to immunoblotting. (C) TRIM44 interacts with monomer as well as oligomerized p62. 293T TRIM44^OE cells are transfect with mCherry-p62 and then treated with MG132 (5 μM, 6 hours) and cross-linked using DSP. For reduced condition, we used 0.1%SDS. For non-Reduced condition, normal lysis buffer was used. Immunoprecipitation were performed using anti-TRIM44 followed by immunoblots using anti-p62. (D) ZF domain of TRIM44 is responsible for p62 oligomerization. HeLa cells were co-transfected with HA-p62 and full-length or different GFP-TRIM44 truncations. Shown the increased percentage of cells with p62 oligomerization compared to the cells transfected with GFP and HA-p62 (means + SD, n=3). The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, non-significant. (E) Deubiquitination assay using HEK293T cells transfected with HA empty vector or HA-p62, His-Ubiquitin and GFP-TRIM44 plasmids and treated with proteasome inhibitor MG132 (5 μM, 6 hours). Cells were collected and IP against HA antibody. Ubiquitinated HA-p62 was immunoblotted with anti-Ub antibody. (F) HA-Ub (K48R, and K63R) were expressed with or without GFP-TRIM44 in HEK293T cells. Cells were subjected to HA IP and IB. (G) Identification of the TRIM44 functional domain for p62 deubiquitination. HEK293T cells were transfected with a combination of HA-p62, His-Ub, and various truncated forms of GFP-TRIM44 (ZF UBP domain (ZF), B-box domain (BB), or coiled-coil domain (CC)). The cells were treated with MG132 (5 μM) for 6 hrs before collection. Ubiquitinated p62 was measures by immunoblotting.

Figure 7.. TRIM44 increases FLNA, RAD51 and 53BP1 expression by inhibiting p62 nuclear translocation after irradiation.

(A) TRIM44 expression inhibits p62 nuclear translocation after irradiation. TRIM44^OE-CON and TRIM44^OE, TRIM44^KD-CON and TRIM44^KD HeLa cells were transfected with mCherry-p62. Images were captured at the indicated times. (B) TRIM44 increases nuclear FLNA expression. TRIM44^OE-CON and TRIM44^OE, TRIM44^KD-CON and TRIM44^KD U266 cells were treated with or without IR. Cells were collected 2 hours after irradiation. (C) TRIM44 increases nuclear RAD51 expression in MM cells, which decreases in TRIM44^KD MM cells. Cells were treated with or without IR and collected 2 hours after irradiation. (D) TRIM44 increases nuclear 53BP1 expression in U266 cells, which decreases in TRIM44^KD MM cells. (E) TRIM44 increases the protein half-life of FLNA, 53BP1 and RAD51. TRIM44^OE-CON and TRIM44^OE MM cells were treated with IR, and cells were collected at the indicated times after CHX treatment. The protein levels of FLNA, 53BP1 and RAD51 were analyzed using immunoblots. (F) Down-regulation of TRIM44 decreases the protein half-life of FLNA, 53BP1 and RAD51. TRIM44^KD-CON and TRIM44^KD MM cells were treated with or without IR, and cells were collected at the indicated times after CHX treatment. The protein levels of FLNA, 53BP1 and RAD51 were analyzed using immunoblots.

Figure 8.. Knockdown of p62 rescues TRIM44^KD-mediated IR sensitivity.

(A) Downregulation of p62 decreases IR-induced apoptosis in TRIM44^KD MM cells. TRIM44^KD MM cells were transfected with NC (sc-37007, Santa Cruz, CA, USA) or p62 siRNA (sc-29679, Santa Cruz, CA, USA), and the cells were then treated with 2 Gy IR. Apoptosis was evaluated by using annexin V/7-AAD staining and flow cytometry. (B) Downregulation of p62 decreases IR-induced γH2AX foci in irradiated TRIM44^KD MM cells. TRIM44^KD U266 cells were transfected with NC or p62 siRNA, and the cells were then treated with 2 Gy IR. After 1 hour, the cells were immunostained with anti-γH2AX (red) and DRAQ5 (blue) and observed by confocal microscopy. Scale bars, 20 μm. (C) Graphic presentation of the percentage of γH2AX+ MM cells with or without irradiation. The cells containing > 5 γH2AX foci were recorded as γH2AX+ cells, whose percentage was averaged from at least 100 cells. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) p62 knockdown accelerated the decrease in IR-induced γH2AX foci in TRIM44^KD MM cells. TRIM44^KD U266 cells were transfected with NC or p62 siRNA, and the cells were then treated with 2 Gy IR. The cells were collected at indicated time after IR, then the cells were immunostained with anti-γH2AX (red) and DRAQ5 (blue) and observed by confocal microscopy. Scale bars, 20 μm. (E) Graphic presentation of the relative percentage of γH2AX+ MM cells after irradiation. The cells containing > 5 γH2AX foci were recorded as γH2AX+ cells, whose percentage was averaged from at least 100 cells. The data are displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) p62 knockdown preserves IR-induced 53BP1 foci in TRIM44^KD MM cells. TRIM44^KD U266 cells were transfected with NC or p62 siRNA, and the cells were then treated with 2 Gy IR. Cells were collected at 2 h post-IR and immunostained with 53BP1 (green) and DRAQ5 (blue) and observed by confocal microscopy. Scale bars, 20 μm. (G) Graphic presentation of the relative percentage of 53BP1+ MM cells after irradiation. The cells containing > 5 53BP1 foci were recorded as 53BP1+ cells under confocal microscopy, whose percentage was averaged from at least 100 cells. The data are displayed were displayed as the mean plus SD of three counts, and statistical significance was calculated and represented as the P value. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Downregulation of p62 stabilizes nuclear FLNA, 53BP1 and RAD51 in TRIM44 knockdown cells. TRIM44^KD U266 cells were transfected with NC or p62 siRNA, and the cells were collected 2 hours after irradiation. The protein levels of FLNA, 53BP1 and RAD51 were analyzed using immunoblots. LaminB1-Nucleus, β-Actin-Total.