Volume 20, Issue 4, Pages 539-550 (November 2005) Akt Blocks Breast Cancer Cell Motility and Invasion through the Transcription Factor NFAT  Merav Yoeli-Lerner, Gary K. Yiu, Isaac Rabinovitz, Peter Erhardt, Sebastien Jauliac, Alex Toker  Molecular Cell  Volume 20, Issue 4, Pages 539-550 (November 2005) DOI: 10.1016/j.molcel.2005.10.033 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Akt Inhibits Breast Carcinoma Invasion (A and B) MDA-MB-435, MDA-MB-231, and SUM-159-PT cells were transfected with the indicated Akt plasmids encoding wild-type Akt (HA.Akt), constitutively active Akt (Myr.Akt.HA), or empty vector. Twenty-four hours after transfection, cells were subjected to a Matrigel invasion assay. Cell lysates from each transfection were immunoblotted with anti-HA. In (B), MDA-MB-231 cells were transfected with increasing concentrations (1–9 μg) of Myr.Akt.HA plasmid, followed by an invasion assay. Cell lysates were immunoblotted with anti-HA to detect Myr.Akt.HA and anti-Akt to detect endogenous Akt1. (C) MDA-MB-231 cells were transiently transfected with either vector control or the indicated Akt alleles, including wild-type (HA.Akt), constitutively active (Myr.Akt.HA, HA.Akt.T308D/S473D), PH-domain-deleted mutant (Myr.ΔPH.Akt.HA) (left panel), or kinase-inactive Myr.Akt (Myr.Akt.K/M.HA) (right panel), followed by invasion assay. Anti-HA immunoblotting of total lysates is shown in the bottom panels. (D) SUM-159-PT cells were transiently transfected with two vector-based (pSUPER) siRNA plasmids specific for Akt1 (Akt1 siRNA #1 and #2) or control (vector, pSUPER), followed by an invasion assay. Akt expression levels were determined by immunoblotting with specific antibodies (anti-Akt1 and anti-Akt2) and anti-actin as loading control. (E) SUM-159-PT cells were transiently cotransfected with wild-type Myr.Akt (Myr.Akt.HA) or an allele mutated in the siRNA recognition sequence (Myr.Akt.HA∗), along with vector control (pSUPER) or Akt1-specific siRNA (Akt1 siRNA #1), followed by an invasion assay. Expression levels were determined by immunoblotting with anti-Akt1 and anti-actin. In all cases, relative invasion is presented as fold invasion relative to empty vector control. All results are representative of three independent experiments. Error bars indicate ±SD. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Akt Blocks Cell Migration (A) MDA-MB-435 cells were transiently transfected with constitutively active Akt (Myr.Akt.HA) or empty vector. Twenty-four hours after transfection, the ability of the cells to invade through Matrigel or to migrate toward 3T3-conditioned medium without Matrigel was assayed. Cell lysates were immunoblotted against anti-HA. (B) SUM-159-PT cells were transiently transfected with the GFP expression plasmid (pEGFP-N1) together with wild-type Akt (HA.Akt), activated Akt (Myr.Akt.HA), or inactive Akt (Myr.Akt.K/M.HA), or empty vector. After transfection, cell monolayers were wounded and washed twice, and images of the wounded area were collected immediately (t = 0 hr) or after 24 hr (t = 24). Quantitation of the healed area was calculated and is presented as a percentage of vector-alone-transfected cells. A fraction of the cells was analyzed by FACS to determine transfection efficiency. Cell lysates were also immunoblotted with anti-HA. (C) SUM-159-PT cells transfected with either Myr.Akt (Myr.Akt, left panels) or GFP control (right panels) were stained with anti-HA or anti-GFP as indicated, followed by FITC-conjugated rhodamine-phalloidin (upper panels). The reduction in stress fibers in the anti-HA-positive cell compared to surrounding cells is highlighted by the arrow (left panels). In contrast, the GFP-positive cell shows a similar number of stress fibers compared to neighboring cells (arrowhead, right panels). Scale bar, 10 μM. (D) SUM-159-PT (left panel) and MDA-MB-231 (right panel) cells were serum starved and then stimulated with IGF-1 (50 ng/ml) for 16 hr, followed by a Transwell invasion or migration assays, as indicated. Cell lysates were immunoblotted with anti-phospho Akt (pSer473) and total Akt (anti-Akt). (E) SUM-159-PT cells were transfected with Akt1 siRNA or vector control (pSUPER). The cells were serum starved and then stimulated with IGF-1 (50 ng/ml) for 10 hr, followed by a Transwell migration assay. All results are representative of three independent experiments. Error bars indicate ±SD. ∗p < 0.05; ∗∗p > 0.05. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Akt Regulates NFAT1 Transcriptional Activity (A) SUM-159-PT cells were transiently transfected with the IL-2-luciferase reporter plasmid and the indicated Akt plasmids, or empty vector control. Twenty-four hours after transfection, cells were lysed and luciferase assays were performed. Lysates were immunoblotted with anti-HA. (B) SUM-159-PT cells were transiently cotransfected with vector control (pSUPER) or two distinct Akt1 siRNA sequences (Akt1 siRNA #1 and #2) along with the IL-2-luciferase reporter. Luciferase activity was assayed 48 hr after transfection. Lysates were immunoblotted with anti-Akt1. (C) SUM-159-PT cells were transiently transfected with Myr.Akt (Myr.Akt) or empty vector plus the indicated NFAT1 plasmids and the IL-2-luciferase reporter. Twenty-four hours after transfection, luciferase assays were performed. (D) SUM-159-PT cells were transfected with either vector control (pSUPER) or Akt1 siRNA, either alone or in combination with HA.DN.NFAT1. Matrigel assays were then performed. A fraction of the transfected cells was used to probe lysates with anti-HA and anti-Akt. n.s., nonspecific protein immunoreactive toward anti-HA. All results are representative of three independent experiments. Error bars indicate ±SD. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Akt Promotes NFAT1 Proteasomal Degradation (A) SUM-159-PT cells were transfected with the indicated Akt plasmids, and nuclear and cytosolic extracts were immunoblotted with anti-HA to reveal Akt and NFAT expression. n.s., nonspecific protein immunoreactive toward anti-HA. (B) SUM-159-PT cells were transfected with HA.NFAT1 and either vector control (pSUPER) or Akt1 siRNA. Lysates were immunoblotted with anti-HA, anti-Akt, and anti-actin. (C) SUM-159-PT cells were transfected with Myr.Akt.HA or vector control and used for cDNA synthesis and real-time PCR using NFAT1 and GAPDH oligonucleotides. Quantitation of NFAT product is shown relative to control. A fraction of the products was resolved by agarose gel electrophoresis. (D) SUM-159-PT cells were transfected with the indicated Akt plasmids and HA.NFAT1 or empty vector control. Twenty-four hours after transfection, cells were treated with ALLN (50 μM) or DMSO control. Lysates were immunoblotted with anti-HA and anti-actin. (E) Two independent clones of SUM-159-PT cells (SUM-159-PT.N1 clones 12 and 16) harboring tetracycline-inducible HA.NFAT were induced with doxycycline (Dox., 0.5 μg/ml) for 16 hr, followed by Matrigel invasion assays. Cell lysates were immunoblotted with anti-HA to reveal NFAT expression and with actin-actin as control. (F) SUM-159-PT.N1 cells were treated with doxycycline (0.5 μg/ml) for 16 hr in serum-free media, then stimulated with IGF-1 (50 ng/ml) for the indicated times in the presence or absence of ALLN (50 μM). Lysates were immunoblotted with anti-HA, anti-phospho-Akt (pSer473), anti-Akt, and anti-p85. (G) SUM-159-PT.N1 cells were treated with doxycycline (0.5 μg/ml) and were transfected with vector control (top panel) or Myr.Akt.HA (bottom panels). Sixteen hours after transfection, cells were treated cycloheximide (100 μg/ml) over time. Total cell lysates were immunoblotted with anti-HA to reveal expression of NFAT and Akt. All results are representative of three independent experiments. Error bars indicate ±SD. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Akt Promotes Ubiquitination of NFAT (A) SUM-159-PT.N1 cells were induced with doxycycline (0.5 μg/ml) for 16 hr and in the presence of the indicated proteasome inhibitors (MG132, 10 μM; lactacystin, 10 μM; ALLN, 50 μM), calpain inhibitor ALLM (10 μM), or vehicle alone (DMSO). Total lysates were immunoblotted with anti-HA. (B) SUM-159-PT cells were transfected with HA.ubiquitin or empty vector control. Twenty-four hours after transfection, cells were treated with MG132 (10 μM) for 16 hr, then lysed and immunoprecipitated with either anti-NFAT or control IgG and immunoblotted with anti-HA. (C) SUM-159-PT.N1 cells were transfected with Myr.Akt (Myr.Akt.HA) or empty vector, treated with doxycycline (0.5 μg/ml) for 16 hr in complete media, then treated with MG132 (10 μM). HA immunoprecipitates were immunoblotted with anti-ubiquitin and anti-HA. Total lysates were immunoblotted with anti-HA to reveal expression of Akt and NFAT. (D) SUM-159-PT.N1 cells were induced with doxycycline (0.5 μg/ml) and then serum starved for 10 hr. Cells were then treated with IGF-1 (50 ng/ml) and MG132 (10 μM) for 10 hr, or vehicle control. NFAT1 was immunoprecipitated (anti-HA) and immunoblotted with anti-ubiquitin. Total lysates were immunoblotted with anti-HA to reveal expression of NFAT. Long and short exposures of the anti-HA immunoblot were required to reveal equivalent expression of NFAT in cells treated or not with MG132, due to degradation, as indicated. All results are representative of three independent experiments. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Akt Promotes NFAT1 Degradation through HDM2 (A) SUM-159-PT cells were cotransfected with HA.NFAT1, either alone or in combination with Myr.Akt.HA or HDM2, as indicated. Cell extracts were immunoblotted with either anti-HA or anti-HDM2. (B) SUM-159-PT.N1 cells were transfected with Akt1 siRNA (Akt1 siRNA #1 and #2), HDM2 siRNA (HDM2 siRNA #1 and #2), or vector control (pSUPER). After transfection, cells were treated with doxycycline (0.5 μg/ml) for 16 hr in serum-free media, then stimulated with IGF-1 (50 ng/ml) for 2 hr. Lysates were immunoblotted with anti-HA, anti-Akt, and anti-HDM2. (C) Lysates of MDA-MB-231 cells treated with MG132 or vehicle alone were immunoprecipitated with control IgG or anti-NFAT antibody, and precipitates were immunoblotted with anti-HDM2 or anti-NFAT. (D) SUM-159-PT cells were transfected with the indicated Myr.Akt and NFAT constructs, either alone or in the presence of either vector control (−) or HDM2 siRNA (+). Cells were also transfected with HA.NFAT and HDM2. Cell extracts were immunoblotted with anti-HA and anti-HDM2. (E) SUM-159-PT.N1 cells were transfected with HDM2 or control, then treated with doxycycline (0.5 μg/ml) for 16 hr and with MG132 (10 μM) for 10 hr. NFAT was immunoprecipitated with anti-HA, then immunoblotted with anti-ubiquitin. Lysates were immunoblotted with anti-HA and anti-HDM2. All results are representative of three independent experiments. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Akt Blocks Invasion through HDM2 (A) SUM-159-PT cells were transfected with Myr.Akt (Myr.Akt.HA) either in the presence of vector control (pSUPER, −) or HDM2 siRNA (+). Forty-eight hours after transfection, Matrigel assays were performed. (B) SUM-159-PT cells were transfected with either vector control or wild-type HDM2, or mutant HDM2.S166A/S186A, either alone or with Myr.Akt (Myr.Akt.HA). Forty-eight hours after transfection, Matrigel assays were performed, and a portion of the transfected cells were immunoblotted with anti-HDM2 or anti-HA. All results are representative of three independent experiments. Error bars indicate ±SD. Molecular Cell 2005 20, 539-550DOI: (10.1016/j.molcel.2005.10.033) Copyright © 2005 Elsevier Inc. Terms and Conditions