The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway

Abstract

Eyes absent 1 (Eya1) is a conserved critical regulator of organ-specific stem cells. Ectopic Eya1 activities, however, promote transformation of mammary epithelial cells. Signals that instigate Eya1 oncogenic activities remain to be determined. Here, we show that Akt1 kinase physically interacts with Eya1 and phosphorylates a conserved consensus site of the Akt kinase. PI3K/Akt signaling enhances Eya1 transcription activity, which largely attributes to the phosphorylation-induced reduction of Eya1 SUMOylation. Indeed, SUMOylation inhibits Eya1 transcription activity; and pharmacologic and genetic activation of PI3K/Akt robustly reduces Eya1 SUMOylation. Wild-type but not Akt phosphorylation site mutant Eya1 variant rescues the cell migratory phenotype of EYA1-silenced breast cancer cells, highlighting the importance of Eya1 phosphorylation. Furthermore, knockdown EYA1 sensitizes breast cancer cells to the PI3K/Akt1 inhibitor and irradiation treatments. Thus, the PI3K/Akt signal pathway activates Eya1. These findings further suggest that regulation of SUMOylation by PI3K/Akt signaling is likely an important aspect of tumorigenesis.

Figure 1. Akt1 interacts with and phosphorylates Eya1

(A) A conserved substrate consensus site of Akt kinase identified in Eya1. (B and C) Immunoprecipitation (IP) using antibodies against either a pan-AKT (AKT) (B), EYA1 (C) or control IgG from BT549 cell lysates, and resultants were immunoblotted (IB) using indicated antibodies. (D and E) Co-IP of full-length mouse Akt1 and Flag-tagged Eya1 (Flag/Eya1) fragments from transfected HEK293 cells. Results are summarized in E. *, inconclusive. (F) Immunoprecipitates (Flag/Eya1 or Flag/S298A) were incubated with recombinant AKT1 (r-AKT1) in an in vitro kinase assay with γ-[³²P]-ATP. Top panel, autoradiography; bottom panel, protein immunoblot using a Flag-specific antibody. (G) Immunoprecipitates from the phospho-specific Akt substrate antibody (p-sub) were probed using indicated antibodies. Flag/S298A, an Eya1 serine 298 to alanine mutation; myr-HA/Akt1, myristoylated HA-tagged Akt1, GAPDH was used as loading control.

Figure 2. Akt1 induces Eya1 transcription activity

(A) HEK293 cells were transiently transfected with the Flag/Eya1 and/or HA/Akt1 expression constructs and analyzed by indirect immunocytochemistry using a Flag-antibody (green, 1), HA- antibody (Red, 2) and counter-stained with DAPI (blue). 3, a merged image of 1 and 2. (B–C) Combinations of Akt1, Flag/Eya1 and Myc/Six1 were co-expressed in HEK293 cells. co-IP and IB were done using indicated antibodies. (D) A transient transcription reporter assay using a Six1/Eya1-dependent reporter (SE1/luc), which was co-expressed with combinations of indicated expression constructs. Relative fold changes were calculated using basal and Renilla controls (mean ± SEM, n=5). (E) Schematic diagram of regulation of Eya1 activities. Resting state Eya1 has low transcription activity (low, in a dash box). Akt1 increases Eya1 transcription (HIGH) in part through the conserved S298 phosphorylation (p) site.

Figure 3. SUMOylation represses Eya1 transcription activity

(A–B) Immunoprecipitation (IP) of endogenous EYA1 (A) or SUMO1 (B) from HEK293 cells and immunobloted (IB) using indicated antibodies. IgG, negative control. (C) Flag/Eya1 and HA/Sumo1 were transiently expressed in HEK293 cells with SenP2 or a control (con). IP and IB were performed using indicated antibodies. (D) Combinations of lysine (K) 43, 146 and 459 to arginine (R) Eya1 mutatn variants were used to map Eya1 SUMOylation sites in an IP and IB assay. Number 1 and 2 indicate single and double SUMOylated Eya1, respectively. (E–G) Reporter assays indicated that SUMOylation represses Eya1 transcription activity. The Six1/Eya1-dependent reporter (SE1/luc) was co-transfected with indicated constructs and the luciferase reporter activity was measure at 48 hours (n=3). S1-Eya1, Eya1-S1: fusion of SUMO1 to Eya1 at N- and C-terminus, respectively.

Figure 4. Akt1 inhibits Eya1 SUMOylation

(A–C) Inhibition of PI3K/Akt signaling with an inhibitor LY294002 (25 µM, 2 h, A and B) or AKT siRNA oligo sets (C, targeting all three AKT1/2/3 isoforms, Dharmacon) enhanced endogenous EYA1 (A) and Flag/Eya1 (B and C) SUMOylation levels. IP and IB were performed using indicated antibodies. (D) S298A mutant had enhanced SUMOylation level. (E and F) Constitutively active myr-HA/Akt1 suppressed SUMOylation of wild type Eya1 (E) but not S298A mutant (F). Flag/Eya1, Flag/S298A and HA/Sumo1 were transiently expressed in HEK293 cells with gradient amount of myr-HA/Akt1. (G) Akt1 induced Eya1 transcription activity via inhibition of Eya1 SUMOylation. The Six1/Eya1-dependent reporter (SE1/luc) was co-transfected with indicated constructs and the luciferase reporter activity was measure at 48 hours. Relative fold changes were calculated using basal and Renilla controls (mean ± SEM, n=3). ns, not significant; KS/RA, K43R/K164R/S298A triple mutation.

Figure 5. Genotoxic stress decreases Eya1 SUMOylation via the PI3K/Akt pathway

(A–C) HEK293 cells, transfected with Flag/Eya1 and HA/Sumo1, were γ-irradiated with the indicated doses and allowed to recover for indicated time prior to IP and IB assays. Densitometry for quantification were presented in B and C. LY, LY294002. (D–E) Flag/Eya1- and HA/Sumo1-expressing HEK293 cells were treated with camptothecin (CPT, 10 µM, 30 min) or DMSO (D) in the presence or absence of siAKT (E) prior to the IP and IB analysis using indicated antibodies.

Figure 6. EYA1 and PI3K/Akt synergistically promote breast cancer cell survival

(A–B) Growth rate of stable clones of BT549 breast cancer cells infected with EYA1-specific shRNAs (shEYA1, 1–4) or control (shCon) lentiviral particles. Cell number was quantified after a 3-day culture (A, n=3), or over a 6-day time course (B, n=6). Values are expressed as mean ± SEM. *, p<0.01. (C) EYA1 knockdown attenuates growth of all four lines of breast cancer cells. The indicated cells were infected with either control (shCon) or EYA1-specific (shEYA1-2) shRNA lentiviral particles, and cell number was quantified after 5 days (BT549, MDA157, SUM149) or 6 days (MDA157). (D–E) Increased sensitivity of EYA1-silenced BT549 breast cancer cells to genotoxic agent camptothecin (CPT) or PI3K/Akt inhibition (LY294002). Equivalent numbers of control (shCon, solid line) and EYA1-silenced (shEya1-1, 2, 4, dotted lines) cells were treated with either LY294002 or CPT for 24 hours. Relative survival rates were compared with mock transfected cells (DMSO) (mean ± SEM, n=6). *, p<0.01. (F) Rescue of EYA1-silenced BT549 cells by wild type mouse Eya1. Equal number of cells was treated with CPT as indicated. Surviving cells were counted 24 h later (n=6). *, EYA1 vs shCon, p<0.01; **, shEYA1 vs shEYA1+Eya1, p< 0.001.

Figure 7. Eya1-mediated cell motility depends on the Akt phosphorylation site

(A–B) Migration of control BT549 cells (shCon, 1), EYA1-silenced (shEYA1-2), and the corresponding cells rescued with wild type mouse Flag/Eya1, were assayed over a 2h period. Quantified results were shown in B (mean ± SEM, n=3). (C) Several murine Eya1 variants were stably expressed in EYA1-silenced BT549 cells. The upper bar graph, real-time quantitative PCR assay; the bottom blots, immunoblots of total cell lysate using antibodies specific to Flag (for murine Eya1 variants) and GAPDH (internal loading control). Con, scrambled shRNA; vec, empty expression construct; Eya1, wild type mouse Eya1; D327A, phosphatase-dead mutation; S298A, a phosphorylation-defective mutation. All rescue constructs were Flag tagged. (D) Migration rate of stable BT549-derived cell lines was examined over a 4 h period. Number of cells was counted and quantitatively analyzed (mean ± SEM, n=3).