PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation

Abstract

PI3K/AKT pathway activation is thought to be a driving force in metastatic melanomas. Members of the pleckstrin homology (PH) domain leucine-rich repeat protein Ser/Thr specific phosphatase family (PHLPP1 and PHLPP2) can regulate AKT activation. By dephosphorylating specific serine residues in the hydrophobic motif, PHLPP1 and PHLPP2 restrain AKT signalings, thereby regulating cell proliferation and survival. We here show that PHLPP1 expression was significantly downregulated or lost and correlated with metastatic potential in melanoma. Forcing expression of either PHLPP1 or PHLPP2 in melanoma cells inhibited cell proliferation, migration, and colony formation in soft agar; but PHLPP1 had the most profound inhibitory effect on metastasis. Moreover, expression of PH mutant forms of PHLPP1 continued to inhibit metastasis, whereas a phosphatase-dead C-terminal mutant did not. The introduction of activated PHLPP1-specific targets AKT2 or AKT3 also promoted melanoma metastasis, while the non-PHLPP1 target AKT1 did not. AKT2 and AKT3 could even rescue the PHLPP1-mediated inhibition of metastasis. An AKT inhibitor blocked the activity of AKT2 and inhibited AKT2-mediated tumor growth and metastasis in a preclinical mouse model. Our data demonstrate that PHLPP1 functions as a metastasis suppressor through its phosphatase activity, and suggest that PHLPP1 represents a novel diagnostic and therapeutic marker for metastatic melanoma.

Figure 1.. PHLPP1 suppresses metastasis in human melanoma.

(A) The protein level of PHLPP1 and PHLPP2 was analyzed by western blot in highly and poorly metastatic human melanoma cell lines. (B, C) Expression of PHLPP1(B) and PHLPP2 (C) in primary and metastatic melanoma tumors in the GSE7553 dataset. (D) The western blot analysis of highly metastatic melanoma A375sm cells transfected with PHLPP1, PHLPP2 or empty vector control (c). (E) Gross pulmonary metastases of A375sm cells transfected with PHLPP1 or PHLPP2 expression vectors were determined using orthotopic footpad transplantation in NSG mice. (F) The western blot analysis of poorly metastatic melanoma A375p cells transfected with shRNAs for PHLPP1 or PHLPP2. (G) Gross pulmonary metastasis of A375p cells transfected with shRNAs for PHLPP1 or PHLPP2 was determined using orthotopic footpad transplantation in NSG mice. Knockdown of endogenous PHLPP1 by shRNAs enhanced metastatic potential in A375p cells (p<0.05). shc, vector control; sh1, sh1, and sh3 are different shRNA constructs. #, no statistical difference (p>0.05); *, statistical significant difference (p<0.05).

Figure 2.. PHLPP1 inhibits metastasis through its phosphatase activity.

(A) Western blot analysis of A375sm cells transfected with vector (c), PHLPP1 (WT1, WT2), PHLPP1ΔC (ΔC1, ΔC2), PHLPP1ΔPH (ΔPH1, ΔPH2) or PHLPP2 expressing constructs. (B-G) A375sm cells transfected with the various PHLPP1- and PHLPP2-expressing constructs described in (A) were analyzed with CCK8 to assess cell proliferation (B), with transwell to determine the cell motility (C), with matrigel coated transwell to determine the cell invasiveness, with soft-agar to determine anchorage-independent colony formation (E, F) and with an experimental metastatic assay by tail vein injection in SCID mice to determine the gross pulmonary metastasis (G). NS, not significant.

Figure 3.. Constitutive expression of activated AKT2, and to a lesser extent AKT3, promotes metastasis in melanoma.

(A-E) Overexpression of activated AKT2 and AKT3 promotes metastasis in human A375p cells. Pan-AKT expression was analyzed by Western blot of A375p cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 (A). A375p cell transfected with the constructs described in (A) were analyzed by ELISA to determine phosphorylation of AKT1 (B), AKT2 (C) and AKT3 (D), and by experimental metastasis assay using tail vein injection to determine the gross pulmonary metastasis (E). c, empty vector control. Compared to vector control, total of Akt1, Akt2 and Akt3 were increased by 2.1, 1.7 and 1.9 fold respectively; phosphorylated Akt1, Akt2 and Akt3 were significantly enhanced by 2.9, 1.9 and 1.8 fold respectively. (F-J) Overexpression of activated AKT2 and AKT3 promotes metastasis in mouse B16F1 cells. Pan-AKT expression was analyzed by western blot of B16 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 (F). B16 cells transfected with the constructs described in (F) were analyzed by ELISA to determine the phosphorylation of AKT1 (G), AKT2 (H) and AKT3 (I), and by experimental metastasis assay using tail vein injection to determine the gross pulmonary metastasis (J). c, empty vector control. Compared to vector control, total of Akt1, Akt2 and Akt3 were increased by 1.9, 2.5 and 2 fold respectively; phosphorylated Akt1, Akt2 and Akt3 were significantly enhanced by 1.6, 2 and 1.7 fold respectively. (K, L) Knockdown of AKT2 and AKT3 inhibits metastasis in human A375sm cells. A375sm cell transfected with shRNA for AKT1, AKT2 and AKT3 were analyzed by western blot with Akt isoform-specific anti-Akt1, Akt2 and Akt3 antibodies (K); gross pulmonary metastasis was determined by experimental metastasis assay using tail vein injection in NSG mice (L). shc, vector control; sh1 and sh2, different shRNAs of AKT1, AKT2 or AKT3. β-actin as an internal control. (M) A375sm cell cotransfected with CRISPR/cas9 and HDR plasmid for specifically knockout of AKT1, AKT2, and AKT3 were analyzed by western blot with Akt isoform-specific anti-AKT1, AKT2 and AKT3 antibodies. β-tubulin as an internal control. (N) Gross pulmonary metastasis was determined by experimental metastasis assay using tail vein injection of knockout of AKT1 (Akt1KO), AKT2 (Akt2KO) and AKT3 (Akt3KO) cells in NSG mice.

Figure 4.. Activated AKT2 and AKT3 can rescue PHLPP1-mediated inhibition of metastasis in melanoma.

(A-I) ELISA analysis of stable A375sm expressing PHLPP1, PHLPP1ΔC and PHLPP2 cells transfected with myr-Akt1, myr-AKT2 or myr-AKT3 plasmids was performed to determine protein levels of total Akt1 (A), total Akt2 (B) and total Akt3 (C), phosphorylated Akt1 (D), phosphorylated Akt2 (E), phosphorylated Akt3 (F), phosphorylated Akt-Thr-308 (G), phosphorylated Erk1/2 (H), total Erk1/2 (I); c, empty vector control; #, no statistical difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (J) Gross pulmonary metastases of A375sm cells stably expressing PHLPP1, PHLPP1ΔC or PHLPP2 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 constructs were determined using tail vein injection in SCID mice. c, empty vector control. (K) Representative histopathology with H&E staining of lung sections with metastases from mice inoculated with stable A375sm expressing PHLPP1, PHLPP1ΔC or PHLPP2 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 constructs. c, empty vector control.

Figure 5.. Inhibition of AKT2 by MK2206 blocks in vivo tumor growth and metastasis.

(A) Whole cell lysates from A375sm cells and stable A375p-myr-AKT2 (A375p Akt2) cells treated with various doses of MK2206 were analyzed by western blot. (B-E) ELISA analysis of A375sm and stable A375p myr-AKT2 cells treated with different doses of MK2206 was performed to determine protein levels of phosphorylated AKT1 (B), phosphorylated AKT2 (C), phosphorylated AKT3 (D), and phosphorylated AKT-Thr-308 (E). #, no statistical difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (F) Gross pulmonary metastases from mice treated with MK2206 or from pretreated cultured A375p Akt2 cells with either 12 nM or 65 nM MK2206 for 36 hours in cell culture (green color). Mice harboring A375p cells stably transfected with the myr-AKT2 expression vector (A375p Akt2) were treated with two different doses of MK2206 immediately (black color) or 7 days after cells injection (red color). After pretreatment with MK2206 for 36 hours, A375p Akt2 cells were injected into NSG mice by tail vein. c, mock control. (G-H) Tumor growth curve (G) and tumor weight (H) of A375sm cells in xenograft NSG mice with/without treatment of MK2206. (I) Representative histopathology (H&E staining) or immunohistochemical staining in metastases derived from mice bearing A375sm cells treated with MK2206. H&E, hematoxylin and eosin staining; t-Akt, immunohistochemical staining with the anti-AKT antibody; p-Akt, immunohistochemical staining with anti-p-AKT antibody 20X. (J) Immunoreactivity score of total AKT (T-Akt) and phosphorylated AKT (p-Akt) expression in metastases from mice treated with MK2206. Quantitative scores analyzed using ImageScope V10.0 software from Aperio Technologies, presenting the percentage of cells in strongly positive, positive, weakly positive and negative for immunohistochemical staining with an anti-AKT or phosphorylated AKT antibody. (K-L) Whole tissue lysates from tumors in mice treated with MK2206 were analyzed by ELISA to determine protein levels of phosphorylated AKT1 (p-Akt1), phosphorylated AKT2 (p-Akt2), phosphorylated AKT3 (p-Akt3), phosphorylated AKT-Thr-308 (p-AktThr308), and phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2 (T-Erk1/2), total AKT (T-Akt1), total AKT2 (T-Akt2) and total AKT3 (T-Akt3).