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. 2023 Aug 2;21(8):e3002227.
doi: 10.1371/journal.pbio.3002227. eCollection 2023 Aug.

Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy

Dong Han  1 , Wei Wang  1 , Julie Heejin Jeon  1 , Tao Shen  1 , Xiangsheng Huang  1 , Ping Yi  2 , Bingning Dong  1   3 , Feng Yang  1   4
Affiliations

Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy

Dong Han et al. PLoS Biol. .

Abstract

Phosphoinositide-dependent kinase-1 (PDK1) is a master kinase of the protein A, G, and C (AGC) family kinases that play important roles in regulating cancer cell proliferation, survival, and metabolism. Besides phosphorylating/activating AKT at the cell membrane in a PI3K-dependent manner, PDK1 also exhibits constitutive activity on many other AGC kinases for tumor-promoting activity. In the latter case, PDK1 protein levels dominate its activity. We previously reported that MAPK4, an atypical MAPK, can PI3K-independently promote AKT activation and tumor growth. Here, using triple-negative breast cancer (TNBC) cell models, we demonstrate that MAPK4 can also enhance PDK1 protein synthesis, thus phosphorylate/activate PDK1 substrates beyond AKT. This new MAPK4-PDK1 axis alone lacks vigorous tumor-promoting activity but cooperates with our previously reported MAPK4-AKT axis to promote tumor growth. Besides enhancing resistance to PI3K blockade, MAPK4 also promotes cancer cell resistance to the more stringent PI3K and PDK1 co-blockade, a recently proposed therapeutic strategy. Currently, there is no MAPK4 inhibitor to treat MAPK4-high cancers. Based on the concerted action of MAPK4-AKT and MAPK4-PDK1 axis in promoting cancer, we predict and confirm that co-targeting AKT and PDK1 effectively represses MAPK4-induced cancer cell growth, suggesting a potential therapeutic strategy to treat MAPK4-high cancers.

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Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: FY is the co-founder and CEO of 4therapeutics Inc. He also holds an equity stake there. The other authors have declared that no conflict of interest exists.

Figures

Fig 1
Fig 1. MAPK4 up-regulates PDK1 protein expression in cancer cells.
Western blots on (A) engineered SUM159, MDA-MB-436, MDA-MB-468, HCC1395, and HCC1806 cells with 0.5 μg/ml Dox-induced ectopic expression of MAPK4 (iMAPK4) or control (iCtrl), (B) engineered HS578T, SUM159, and HCC1937 cells with 4 μg/ml Dox-induced knockdown of MAPK4 (iG2 and iG4) or control (iNT), (C) the parental vs. MAPK4-KO MDA-MB-231 cells (clone# 2, 3) and SUM159 cells (clone# 1, 2), and (D) the parental vs. MAPK4-KO MDA-MB-231 (clone# 3) and SUM159 cells (clone# 2, KO cells) vs. MAPK4-KO cells with rescued 0.5 μg/ml Dox-induced MAPK4 expression (KO+iMAPK4). All cells in Panel D were treated with 0.5 μg/ml Dox. Data are representative of at least 2–3 independent experiments. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.
Fig 2
Fig 2. PDK1 partially mediates MAPK4 tumor-promoting activity.
(A) Western blots, (B) proliferation assays, (C, D) plate clonogenic assays, and (E, F) soft-agar assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl). The cells were also engineered with stable knockdown of PDK1 (shPDK1-1, shPDK1-2) or control (NT). Quantification data as means ± SD. Scale bar: 500 μm. P values by two-way ANOVA followed by Sidak’s multiple comparisons. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.
Fig 3
Fig 3. Overexpression of PDK1 partly rescues MAPK4-KO tumor cell growth.
(A) Western blots, (B) proliferation assays, (C) plate colony formation assays, and (D) soft-agar assays on WT or engineered MAPK4-KO SUM159 and MDA-MB-231 cells with 0.5 μg/ml Dox-induced expression of PDK1 (iPDK1), MAPK4 (iMAPK4), or control (iCtrl). Scale bar: 500 μm. (E, F) 1 × 106 WT or the engineered MAPK4-KO SUM159 and MDA-MB-231 cells with Dox-inducible expression of PDK1 (iPDK1), MAPK4 (iMAPK4), or control (iCtrl) in 1:2 Matrigel were injected into the mammary fat pad of SCID mice. All mice also received 0.5 mg/ml Dox in drinking water. Tumors were measured and harvested as indicated. Shown are the tumors’ images at collection, growth curve (means ± SEM), and weights. Quantification data as means ± SD other than otherwise indicated. P values by one-way ANOVA followed by Sidak’s multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild type.
Fig 4
Fig 4. Overexpression of PDK1 partly rescues MAPK4-KO tumor cell resistance to PI3K blockade.
(A) Plate colony formation assays and (B) soft-agar assays on WT or engineered MAPK4-KO SUM159 and MDA-MB-231 cells with 0.5 μg/ml Dox-induced expression of PDK1 (iPDK1), MAPK4 (iMAPK4), or control (iCtrl). Scale bar: 500 μm. Quantification data as means ± SD. P values by two-way ANOVA followed by Sidak’s multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild type.
Fig 5
Fig 5. MAPK4-induced PDK1 works with AKT to promote tumor cell growth.
(A) Western blots, (B) proliferation assays, (C) plate colony formation assays, and (D) soft-agar assays on engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iWT), MAPK4D254A (iD254A), or control (iCtrl). Scale bar: 500 μm. (E) Western blots, (F) proliferation assays, and (G) soft-agar assays on engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iWT), MAPK4D254A (iD254A), or control (iCtrl). These cells were also infected with lentivirus expressing AKT1-DD mutant or control. Quantification data as means ± SD. P values by one-way ANOVA followed by Sidak’s multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.
Fig 6
Fig 6. Knockdown of PDK1 partially sensitizes MAPK4-overexpressing tumor cells to PI3K blockade.
(A) Plate colony formation assays and (B) soft-agar assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl), also with stable knockdown of PDK1 (shPDK1-1 and shPDK1-2) or control (NT). The cells were also treated with PI3K inhibitor Alpelisib (1 μm or 2 μm) or DMSO control. Scale bar: 500 μm. Quantification data as means ± SD. P values by two-way ANOVA followed by Sidak’s multiple comparisons. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.
Fig 7
Fig 7. MAPK4 promotes cancer cell resistance to PI3K and PDK1 co-blockade.
(A) Proliferation assays, (B) plate colony formation assays, and (C) soft-agar assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl). The cells were also treated with DMSO control, PDK1 inhibitor GSK2334470 (2 μm), PI3K inhibitor Alpelisib (2 μm), or both inhibitors. Scale bar: 500 μm. Quantification data as means ± SD. P values by two-way ANOVA followed by Sidak’s multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.
Fig 8
Fig 8. Co-targeting AKT and PDK1 is effective in repressing MAPK4-induced cancer cell growth.
(A) Western blot on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl), treated with 2 μm or 5 μm of PDK1 inhibitor GSK2334470 or DMSO control. (B) Western blots and (C) proliferation assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl). The cells were also treated with DMSO control, PDK1 inhibitor GSK2334470 (2 μm), AKT inhibitor MK2206 (2 μm), or both inhibitors. (D) Western blots and (E) proliferation assays on the WT and MAPK4-KO MDA-MB-231 cells treated with DMSO control, PDK1 inhibitor GSK2334470 (1 μm), AKT inhibitor MK2206 (1 μm), or both inhibitors. Data are means ± SD from at least 3 separate experiments. P values by two-way ANOVA followed by Sidak’s multiple comparisons. ns, not significant, ****P < 0.0001. The numerical values underlying the figures can be found in S1 Data. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild type.
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