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. 2014 Aug 21;33(34):4330-9.
doi: 10.1038/onc.2013.383. Epub 2013 Sep 16.

Genetic Inactivation or Pharmacological Inhibition of Pdk1 Delays Development and Inhibits Metastasis of Braf(V600E)::Pten(-/-) Melanoma

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Free PMC article

Genetic Inactivation or Pharmacological Inhibition of Pdk1 Delays Development and Inhibits Metastasis of Braf(V600E)::Pten(-/-) Melanoma

M Scortegagna et al. Oncogene. .
Free PMC article

Abstract

Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of Braf(V600E)::Pten(-/-) or Braf(V600E)::Cdkn2a(-/-)::Pten(-/-) mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in Braf(V600E)::Pten(-/-) mice. Pdk1(-/-) melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1(-/-) melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Loss of PDK1 delays onset of melanoma development
(a) Representative pictures of pigmented animals 17 days following perinatal administration of 4-HT to Pdk1 WT (BrafV600E::Pten−/−::Cdkn2a−/−::Pdk1+/+) or Pdk1 KO (BrafV600E::Pten−/−::Cdkn2a−/−::Pdk1−/−) animals. (b) Kaplan-Meier survival curve of mice with the indicated Pdk1 WT (n = 8) and KO (n = 12) genotypes under the genetic background of BrafV600E::Pten−/−::Cdkn2a−/−, following perinatal administration of 4-HT. Log-rank (Mantel-Cox) test of survival plots reveals a statistically significant difference between the Pdk1 WT and KO genotypes (p < 0.0001). (c) Representative immunostaining for proliferation marker (BrdU; red) in skin sections from mice harboring the Pdk1 WT or KO genotypes (on the background of BrafV600E::Pten−/−::Cdkn2a−/−). Quantification shown is of >500 cells in three separate analyses (*P < 0.05 using t-test). (d and e) H&E stain (d) and Tyrp1 immunostaining (e) of skin from BrafV600E::Pten−/−::Cdkn2a−/−::Pdk1+/+ and BrafV600E::Pten−/−::Cdkn2a−/−::Pdk1−/− 17 days following 4-HT administration. (f) Kaplan-Meier survival curve of mice with the indicated Pdk1 WT or KO genotypes (n = 9 for each group) under the genetic background of BrafV600E::Pten−/−::Cdkn2a−/−, following local administration of 4-HT. Log-rank (Mantel-Cox) test of survival plots reveals a statistically significant difference (p = 0.0005) between the Pdk1 WT and KO genotypes. (g) Tumor growth curves for BrafV600E/Pten−/− and BrafV600E/Pten−/−::Cdkn2a−/− genotypes (n = 9 for each group). (h) Immunostaining for proliferation marker (Ki67 stain, panel H) and programed cell death (cleaved caspase 3, panel i) of locally induced melanomas of the indicated Pdk1 WT or KO genotypes (BrafV600E::Pten−/−::Cdkn2a−/−). Quantification of >1000 cells in four analyses is shown. Error bars represent SEM. *P < 0.05. Bar = 50 µm.
Figure 2
Figure 2. Genetic inactivation of Pdk1 inhibits metastases
(a-c) Representative pictures for &E stain of lymph node (a), lung (b), and spleen (c) 16 days after systemic administration of 4-HT to induce expression of BrafV600E and inactivation of Pten, Cdkn2a and Pdk1. Quantifications shown on the lower panels represent analyses from eight animals for each group. P < 0.0005. The quantification of tumor metastasis was performed using immunohistochemical and H&E stains in a blinded manner by an expert histopathologist (MB). Pigment deposition in lung metastases correlates well with tumor deposits in the lung, but does not in lymph nodes, where melanin pigment-laden macrophages are commonly observed in lymph node sinuses. Lung metastases were uniformly small (less than 0.5 mm) occurred as clusters of tumor cells. These clusters were counted on a full cross section of lung to quantitate metastatic burden. The lymph node deposits in Pdk1 WT (and untreated) melanomas were larger and more irregular, so the percentage of the cross sectional area of a full section of lymph node was used to determine metastatic burden to lymph nodes. (d) Representative H&E stain of lymph node after local induction of melanoma in BrafV600E::Pten−/−::Cdkn2a−/− for Pdk1 WT and Pdk1 KO genotypes. Eight lymph nodes were quantified from eight animals for each group. (e-f) Representative pictures of S100 stain of lymph node (e) and lung (f) 16 days after systemic administration of 4-HT to induce expression of BrafV600E and inactivation of Pten, Cdkn2a and Pdk1. (g) Representative S100 stain of lymph node after local induction of melanoma in BrafV600E::Pten−/−::Cdkn2a−/− for Pdk1 WT and Pdk1 KO genotypes. Quantifications shown on the right panels represent analyses from three animals for each group. Error bars represent SEM. *P < 0.005. Bars in panels a-b, e-g = 50 µm, bar in panel c = 100 µm, bar in panel d = 200 µm.
Figure 3
Figure 3. Pdk1 inactivation attenuates AKT and PKC signaling
(a and b) Western blot analysis was performed using protein lysates from melanoma tumors and their derived primary melanomas cultures under the genetic background BrafV600E::Pten−/−::Cdkn2a−/− with the indicated antibodies. (c) Heat map showing select genes and pathways that were up- or down-regulated in Pdk1–/::BrafV600E::Pten−/−::Cdkn2a−/− melanomas (n = 4). The selected genes have p-values less than or equal to 0.05 and fold changes are >2. The normalized expression signals in the heat map are shown from green (lower) to red (higher signal). Significant genes with similar expression pattern are indicated in bars at the left of the heat map (see Tables S1 to S4 for details). (d) Representative immunostaining of pAKT 308 and Foxo3a in the Pdk1 WT and KO melanomas (BrafV600E::Pten−/−::Cdkn2a−/−). Enlarged areas are shown in insets. (e) Nuclear and cytoplasmic fractionation was performed using protein lysates from primary melanoma cultures under the genetic background BrafV600E::Pten−/−::Cdkn2a−/−, western blot was performed and the membranes were probed with the indicated antibodies. (f) Primary melanoma cultures established from the indicated Pdk1 WT and KO tumors (BrafV600E::Pten−/−::Cdkn2a−/−) were analyzed for cell proliferation (ATP Lite assay) following their transfection with Foxo3a siRNA expression vector. (g) Cultures used in panel (e) were monitored for colony formation assay (representative image of colonies formed in culture is shown on the right panel) following their infection with Foxo3a shRNA expression vector. The graph represents the quantification of the number of colony-forming cells after 7 days in culture. Analysis was performed in triplicates and repeated two times. (h) Cultures used in panel (f) were monitored for changes in gene expression following transfection with two different Foxo3a siRNAs, using QPCR analysis for Bim and p21 mRNA levels. *P < 0.0005.
Figure 4
Figure 4. PDK1 inhibitor GSK2334470 delays melanoma formation and inhibits metastases
(a and b) representative H&E stain of skin (a) and lungs (b) in vehicle and GSK2334470 treated mice (BrafV600E::Pten−/−::PDK1−/−) following perinatal administration of 4-HT. Quantification shown for number of lung metastases is based on six sections 100 µm apart that were analyzed from three mice. Bar = 100 µm. (c) Representative S100 stain of lymph node in vehicle and GSK2334470 treated mice (BrafV600E::Pten−/−::PDK1−/−) following perinatal administration of 4-HT. Bar = 50 µm (d) Representative immunostaining of pAKT308 in the skin of vehicle and GSK2334470 treated mice following perinatal administration of 4-HT (BrafV600E::Pten−/−::PDK1−/−). Bar = 50 µm. (e) Representative S100 stain of lymph node in vehicle and GSK2334470 treated mice (BrafV600E::Pten−/−::PDK1−/−) following local administration of 4-HT. Bar = 50 µm. Quantifications shown on the right panels of panels c and e represent analyses from three animals for each group. Error bars represent SEM.
Figure 5
Figure 5. PDK1 inhibition attenuates AGC kinases in both Pten WT and mutant melanomas
(a) Western blot analysis was performed using protein lysates from human melanoma cells with Pten mutation (793, Lu1205, UACC903) or Pten WT (WM35, WM1346, and WM3629) with the indicated antibodies. (b) Human melanoma cell lines Lu1205 and WM35 were treated with either vehicle or GSK2334470 (2.5 µM, changing the media every other day) and monitored for colony formation assay. The graph represents the quantification of the mean relative area after 7 days (Lu1205) and 12 days (WM35) in culture (representative images of colonies grown in culture are shown on the right panel). Analysis was performed in triplicates and repeated two times. Error bars represent SEM. (c) Expression of PDK1 was studied in a large cohort of primary and metastatic melanomas and in nevi. Examples of strong and weak staining are shown. (d) Box plots demonstrate differences in expression in the three categories of samples, with the PDK1 H-score (scale 0–300) on the Y-axis. Expression was significantly lower in nevi and primary lesions than metastases (P = 0.0001).

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