Targeting the UFL1-AKT cascade suppresses triple-negative breast cancer progression

Abstract

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease with limited therapies. While UFL1-mediated UFMylation has been implicated in various diseases, its role in TNBC remains not fully understood. Here, we demonstrate that AKT1 directly interacts with UFL1 and undergoes UFMylation at Lys189/276/297. This modification enhances AKT phosphorylation and activation, promoting tumor growth and chemoresistance in TNBC. In turn, AKT phosphorylates UFL1 at Thr426, establishing a positive feedback loop that sustains high activity of both pro-oncogenic regulators in TNBC. Disrupting the UFL1-AKT interaction using the specific peptide PDAU-TAT significantly inhibits TNBC progression both in vitro and in vivo. Clinically, elevated pT426 UFL1 correlates with high pAKT in TNBC specimens. These findings uncover a crucial UFL1-AKT positive feedback loop that drives TNBC progression and suggest that targeting this axis could offer a promising therapeutic strategy for TNBC and potentially other aggressive cancers characterized by upregulated UFL1 and AKT activation.

Fig. 1. UFL1 exerts an oncogenic role in TNBC.

a UFL1 mRNA expression level in TNBC (n = 135) and normal breast tissues (n = 291) was analyzed via the GEPIA database (P < 0.01). In the box plot, the upper and lower whiskers represent the maximum and minimum values, respectively. The upper and lower boundaries of the box correspond to the upper and lower quartiles, and the line within the box indicates the median. b Representative immunohistochemical (IHC) staining and scores of UFL1 in TNBC and adjacent normal tissues (n = 40). The left panel displayed a low-magnification field of view (scale bar, 300 μm); the right panels were magnified views of the boxed regions (scale bar, 60 μm). c MDA-MB-231 and HCC1806 cells stably expressing control or UFL1 shRNAs were analyzed by Western blotting. d Cell proliferation of cells in (c) was examined. e Cells as in (c) were treated with the indicated concentrations of cisplatin or doxorubicin, and cell survival was determined. f, g HCC1806 cells in (c) were subcutaneously implanted into nude mice. Mice were treated with saline or cisplatin (5 mg/kg weekly, n = 6) when tumor volume reached 100 mm³. Tumors were collected (f), and tumor weights were measured (g) (means ± SD). Western blotting is representative of three independent experiments (c). Data were presented as mean ± SD of three independent experiments (d, e). Data were analyzed by two-sided one-way ANOVA in (d, g), by t-test in (a, b). Source data are provided as a Source Data file.

Fig. 2. UFL1 promotes TNBC progression via AKT activation.

a List of UFL1-associated proteins identified by mass spectrometric analysis. MDA-MB-231 cells stably expressing Flag-S-UFL1 were treated with MG132 (10 μM) for 10 h. UFL1 immunoprecipitates were subjected to mass spectrometric analysis (n = 3). b Endogenous co-immunoprecipitation confirms UFL1-AKT interaction. c Purified GST, GST-AKT1/2/3 and His-UFL1 were incubated in vitro as indicated. The interaction between UFL1 and AKT1/2/3 was examined. CBS, Coomassie blue staining. d Western blotting of MDA-MB-231 and HCC1806 cells stably expressing control or UFL1 shRNAs using the indicated antibodies. e Western blotting of cells stably expressing empty vector or Flag-UFL1 using the indicated antibodies. f Cells as in (e) were treated with vehicle or capivasertib, and a cell proliferation assay was performed. g Cells as in (f) were treated with DMSO or indicated concentrations of cisplatin or doxorubicin, and cell survival was determined. Mass spectrometric analysis and western blotting are representative of three independent experiments (a, d, e). Data were presented as mean ± SD of three independent experiments (f, g). Data were analyzed by two-sided one-way ANOVA in (f). Source data are provided as a Source Data file.

Fig. 3. UFL1 ufmylates AKT1 at Lys189, Lys276, and Lys297.

a In vivo UFMylation assay of AKT1 in HEK293T cells transfected with Flag-S-AKT1 and the components of the UFMylation system. b In vitro UFMylation assay of AKT1. Samples were subjected to western blotting with an anti-UFM1 antibody. c In MDA-MB-231 and HCC1806 cells stably expressing control or UFL1 shRNA, UFMylation of endogenous AKT1 was detected by immunoprecipitation with an anti-AKT1 antibody, followed by subsequent western blotting using an anti-UFM1 antibody. d A schematic showing the generation of the UFL1 full-length (FL), 1–247 aa and 248-C aa constructs. e Cells were transfected with vector, HA-S-UFL1 full length or HA-S-UFL1 truncation mutants (1–247 aa and 248-C aa). Cell lysates were pulled down using S-agarose, and the interaction between UFL1 and AKT1 was then examined. f A schematic showing the generation of the AKT1 full-length (FL), 1–148 aa, 148–410 aa and 410–C aa constructs. g Cells were transfected with vector, Flag-S-AKT1 truncation mutants (1–148, 148–410 and 410-C). Cell lysates were pulled down using S-agarose, and the interaction between UFL1 and AKT1 was then examined. h UFMylation assay of the Flag-S-AKT1 full length and its truncation mutants (1–148 aa, 148–410 aa or 410-C aa) was examined in HEK293T cells. i, j UFMylation assay of the HA-S-AKT1 WT and 3KR was performed in MDA-MB-231 and HCC1806 cells. Source data are provided as a Source Data file.

Fig. 4. UFL1-mediated UFMylation is critical to activate AKT in TNBC.

a, b HCC1806 cells were transfected with the indicated plasmids and treated with MG132 for 10 h before harvest. Cell lysates were pulled down using S-agarose, and the interaction between AKT1 and the indicated proteins was then examined. c AKT1/2/3-depleted HCC1806 cells were transfected with the indicated plasmids, and western blotting was performed as indicated. Cell proliferation was measured. d Cells as in (c) were treated with the indicated concentrations of cisplatin or doxorubicin, and cell survival was determined. e, f Nude mice were subcutaneously inoculated with HCC1806 cells (2 × 10⁶) stably expressing either an empty vector, HA-S-AKT1 WT, or the 3KR mutant. Treatment with saline or cisplatin (5 mg/kg, once weekly) commenced when the xenograft tumors reached approximately 100 mm³ (n = 6 per group). Tumors were subsequently harvested (e) and weighed (f). Data are presented as the mean ± SD for each group of six mice. g IHC staining and quantification of Ki67 and cleaved PARP1 expression in a subcutaneous tumor model (n = 6) from (e). Scale bars, 50 μm. Data are shown as means ± SD. Western blotting is representative of three independent experiments (c). Data were presented as mean ± SD of three independent experiments (c, d). Data were analyzed by two-sided one-way ANOVA in (c, f, g). Source data are provided as a Source Data file.

Fig. 5. AKT phosphorylates UFL1 at the T426 site to enhance its UFMylation and promote TNBC progression.

a UFMylation assay of the Flag-S-AKT1 was examined in cells treated with indicated concentrations of capivasertib. b Cells were transfected with vector or Flag-S-UFL1, followed by S-agarose pull-down. Western blotting was performed using the indicated antibodies. c Cells were transfected as indicated and treated with capivasertib (10 μM) for 24 h before harvest. Then, cell lysates were pulled down using S-agarose and western blotting was performed. d AA sequences around the T426 residue in UFL1 are conserved across different species. Arrows, threonine residues that are conserved across species. e Cells were transfected with vector, Flag-UFL1 WT or T426A mutant and cell lysates were subjected to immunoprecipitation with an anti-Flag antibody. Western blotting was performed using the indicated antibodies. f HCC1806 cells were treated with vehicle or indicated concentrations of capivasertib for 24 h, and western blotting was performed. g HCC1806 cells stably expressing control or AKT1/2/3 shRNAs were generated, and western blotting was performed using the indicated antibodies. h UFMylation assay of the Flag-S-AKT1 was examined in cells co-transfected with the indicated UFMylation system components. i Cells transfected as indicated were treated with MG132 and subjected to anti-Flag immunoprecipitation to assess UFL1 interaction with UFBP1, CDK5RAP3, or UFC1. j Cells were transfected with vector or Flag-UFL1 and treated with capivasertib (10 μM) for 24 h before harvest. Cell lysates were subjected to immunoprecipitation with an anti-Flag antibody. Western blotting was performed using the indicated antibodies. k HCC1806 cells stably expressing vector, Flag-UFL1 WT, or T426A mutant were generated, and western blotting was performed with the indicated antibodies. Cell proliferation was measured. l HCC1806 cells, as in (k), were treated with the indicated concentrations of cisplatin or doxorubicin and cell survival was determined. Western blotting is representative of three independent experiments (f, g, k). Data were presented as mean ± SD of three independent experiments (k, l). Data were analyzed by two-sided one-way ANOVA in (k). Source data are provided as a Source Data file.

Fig. 6. Disrupting the UFL1-AKT1 interaction by PDAU suppresses TNBC progression in vitro.

a Cells transfected with vector, HA-S-UFL1 truncation mutants (1–247, 10–247, 20–247, 30–247, 40–247) were pulled down using S-agarose, and the interaction between UFL1 and AKT1 was then examined. b Purified recombinant GST, GST-UFL1 truncation mutants were incubated with purified His-AKT1 in vitro to assess the interaction between UFL1 and AKT1. c Two TAT-Peptides, PDAU (20-30 aa) and NP (1-10 aa), from UFL1 are shown. d Cells transfected as indicated were treated with vehicle (ddH₂O), PDAU or NP (10 μM) for 24 h before harvest and subjected to anti-Flag immunoprecipitation to assess UFL1 interaction with AKT. e UFMylation assay of Flag-S-AKT1 was performed in HEK293T cells treated with vehicle (ddH₂O), PDAU or NP (10 μM). f MDA-MB-231 and HCC1806 cells were treated with vehicle (ddH₂O), PDAU or NP (10 μM) for 24 h and western blotting was performed indicated antibodies. g Cell proliferation assay was performed from cells in (f). h Cells as in (f) were treated with the indicated concentrations of cisplatin or doxorubicin, and cell survival was determined. Western blotting is representative of three independent experiments (f). Data were presented as mean ± SD of three independent experiments (g, h). Data were analyzed by two-sided one-way ANOVA in (g). Source data are provided as a Source Data file.

Fig. 7. Disrupting the UFL1-AKT1 interaction by PDAU suppresses TNBC progression in vivo.

a, b TNBC patient-derived xenografts (PDXs) were subcutaneously implanted into nude mice. Saline, PDAU or NP (15 mg/kg) was injected intraperitoneally every 2 days when tumor volume reached 100 mm³. Mice were then treated with saline or cisplatin (5 mg/kg weekly, n = 6). Tumors were collected (a), and tumor weights were measured (b). Results represent the mean ± SD from six mice. c Cell lysates from PDXs treated with saline, PDAU or NP were subjected to immunoprecipitation with IgG, anti-UFL1 antibodies. The immunoprecipitates were blotted with the indicated antibodies. d Cell lysates from PDXs treated with saline, PDAU or NP were subjected to western blot with the indicated antibodies. e Representative images of H&E, Ki67 and cleaved PARP1 staining in xenograft models from (a). Scale bars, 50 μm. Data were analyzed by two-sided one-way ANOVA in (b). Source data are provided as a Source Data file.

Fig. 8. Correlations between pUFL1 and pAKT expression in TNBC.

a Representative IHC staining of pUFL1 and pAKT in TNBC (n = 40). Scale bars, 60 μm. b Chi-square (χ²) test analysis (two-sided) of pAKT and pUFL1 expression in TNBC (n = 40). c Pearson correlation analysis (two-sided) between pUFL1 and pAKT score in TNBC (n = 40), r = 0.8311, P < 0.0001. d Schematic representation of the experimental model of this study.