Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy
Dong Han, Wei Wang, Julie Heejin Jeon, Tao Shen, Xiangsheng Huang, Ping Yi, Bingning Dong, Feng Yang
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.
Introduction
The protein A, G, and C (AGC) family kinases consist of more than 60 evolutionarily related serine/threonine protein kinases. Many AGC kinases, such as phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (AKT), serum and glucocorticoid-inducible kinases (SGK), protein kinase C (PKC), p70 ribosomal protein S6 kinase (S6K), and p90 ribosomal protein S6 kinase (RSK) play important roles in regulating cell proliferation, apoptosis, and metabolism [1]. Besides its activity in phosphorylating/activating AKT in the phosphatidylinositol 3-kinase (PI3K) pathway, PDK1 also exhibits constitutive activity in phosphorylating many other AGC kinases, such as S6K, SGK, PKC, and RSK for tumor-promoting activities [1,2]. In the latter case, PDK1 protein expression levels determine its activities. Therefore, it is important to understand the molecular mechanism regulating PDK1 protein levels, which remains elusive.
Materials and methods
Ethics statement
All animal studies were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine under protocol AN-5220.
Reagents and antibodies
The antibodies against p-AKT T308 (Catalog 13038), p-AKT S473 (Catalog 4060), AKT (Catalog 9272), PDK1 (Catalog 3062S), eIF4E (catalog 2067), p-eIF4E (Catalog 9741), p-PKCζ/λ (Thr410/403) (Catalog 9378), GSK3β (Catalog 9315), p-GSK3β S9 (Catalog 9336), MCL-1 (Catalog 5453), and Cyclin D1 (Catalog 2978) were from Cell Signaling Technology. Other antibodies used include anti-MAPK4 (Abgent, Catalog AP7298b), anti-MAPK4 (Origene, Catalog TA505872), anti-PDK1 (Santa Cruz, Catalog sc-17765), anti-HA (Santa Cruz, Catalog sc-805), anti-FLAG (Millipore Sigma, catalog F3165), and anti-β-ACTIN (Abclonal, Catalog AC026).
Results
MAPK4 up-regulates PDK1 protein expression in cancer cells
We have shown that MAPK4 can activate AKT independent of the PI3K pathway [3]. However, it stays unknown why inhibiting MAPK4 greatly represses AKT phosphorylation/activation that the canonical PI3K signaling can also drive. We have previously documented human TNBC cell lines with high, medium, or low MAPK4 expressions [4]. Overexpression of MAPK4 in the MAPK4-medium SUM159 and MAPK4-low MDA-MB-436, MDA-MB-468, HCC1395, and HCC1806 cells all enhanced PDK1 protein expression, suggesting an unexpected MAPK4-PDK1 signaling axis (Fig 1A). Accordingly, knockdown/knockout of MAPK4 in the MAPK4-medium SUM159 and MAPK4-high HS578T, HCC1937, and MDA-MB-231 cells reduced PDK1 protein expression (Fig 1B and 1C) and ectopic expression of MAPK4 in the MAPK4-KO MDA-MB-231 and SUM159 cells rescued PDK1 expression (Fig 1D). Neither overexpression nor knockdown of MAPK4 affected PDK1 mRNA expression in the examined cell lines (S1A Fig), suggesting that MAPK4 promotes PDK1 protein expression through a posttranscriptional process, such as regulation of protein synthesis and stability.
Citation: Han D, Wang W, Jeon JH, Shen T, Huang X, Yi P, et al. (2023) Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy. PLoS Biol 21(8): e3002227. https://doi.org/10.1371/journal.pbio.3002227
Academic Editor: Albana Gattelli, Consejo Nacional de Investigaciones Científicas y Técnicas: Consejo Nacional de Investigaciones Cientificas y Tecnicas, ARGENTINA
Received: October 4, 2022; Accepted: June 30, 2023; Published: August 2, 2023
Copyright: © 2023 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This research was supported by grants from the Department of Defense Congressionally Directed Medical Research Programs https://cdmrp.health.mil/ (W81XWH-17-1-0043 to FY), the Cancer Prevention and Research Institute of Texas https://www.cprit.state.tx.us/ (RP130651 and RP200439 to FY), and the National Institute of Health https://www.nih.gov/ (1R01CA276341 to FY). JHJ was supported by the National Institute of Health https://www.nih.gov/ T32CA203690. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: 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.
Abbreviations: BCA, bicinchoninic acid; CHX, cycloheximide; MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; SGK, serum and glucocorticoid-inducible kinase; TNBC, triple-negative breast cancer; WT, wild type
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002227#abstract0
