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Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy [1]

['Dong Han', 'Department Of Molecular', 'Cellular Biology', 'Baylor College Of Medicine', 'Houston', 'Texas', 'United States Of America', 'Wei Wang', 'Julie Heejin Jeon', 'Tao Shen']

Date: 2023-08

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.

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.

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.

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.

Here, using TNBC cell models, we report that besides directly phosphorylating/activating AKT (an MAPK4-AKT axis [ 3 , 4 ]), MAPK4 also greatly promotes PDK1 protein expression, representing an MAPK4-PDK1 axis to enhance PDK1 expression/activation. Blocking MAPK4 both inhibits the PI3K-independent MAPK4-AKT signaling axis [ 3 , 4 ] and represses PDK1 protein expression to block the canonical PI3K-PDK1-AKT pathway. Together, these lead to greatly repressed AKT phosphorylation/activation. We further demonstrate that by enhancing PDK1 protein expression/activity, MAPK4 also enhances PDK1-dependent but AKT-independent signaling. Accordingly, co-blockade of AKT and PDK1 largely blocks MAPK4 tumor-promoting activity. Our studies collectively identify a novel mechanism promoting PDK1 protein expression and further advance our knowledge of the molecular mechanism underlying the tumor-promoting activity of MAPK4, an emerging novel therapeutic target for human cancers.

Triple-negative breast cancer (TNBC) is a devastating disease accounting for 15% to 20% of all breast cancer but with limited therapeutic options. We previously reported that MAPK4 is highly expressed in a large fraction of TNBC, and repressing MAPK4 is effective in inhibiting TNBC cell and xenograft growth [ 4 ]. Currently, there is no MAPK4-specific inhibitor(s) for potential clinical testing to treat TNBC. Further dissecting MAPK4 downstream signaling nodes, especially those with clinical inhibitor(s) being used in the clinic or tested in clinical trials, may provide an alternative route to treat MAPK4-high TNBC.

Mitogen-activated protein kinase 4 (MAPK4) is an atypical MAPK not well studied. We recently reported that MAPK4 can promote cancer by noncanonically activating AKT independent of the PI3K/PDK1 signaling axis [ 3 ]. The parallel actions of MAPK4 and PI3K-PDK1 in activating AKT predict MAPK4 activities in regulating cell response to PI3K blockade. Indeed, inhibiting MAPK4 sensitizes cancer cells to PI3K blockade [ 4 ]. However, it remains unknown why inhibiting MAPK4 (knockdown/knockout) greatly represses AKT phosphorylation/activation that the canonical PI3K pathway can also drive.

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.

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. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002227.g001 The molecular mechanism regulating PDK1 protein stability remains unknown until the recent identification of SPOP as an E3 ubiquitin ligase for PDK1 protein ubiquitination and degradation [5]. To examine whether MAPK4 plays a major role in regulating PDK1 protein stability, we first examined PDK1 protein stability in 7 TNBC cancer cell lines, including the MAPK4-low HCC1806, MDA-MB-468, HCC1395 cells, and the MAPK4-medium/high SUM159, MDA-MB-231, HCC1937, and HS578T cell lines. We observed that PDK1 protein is very stable in MDA-MB-231, HCC1937, HS578T, HCC1806, and MDA-MB-468 cells, while notable PDK1 protein degradation was only evident in SUM159 and HCC1395 cells after prolonged protein synthesis inhibitor cycloheximide (CHX) treatment (S1B Fig). As an additional control, we also confirmed a rapid degradation of MCL-1 and Cyclin D1 in the CHX-treated MDA-MB-231, HCC1937, HS578T, and SUM159 cells. Therefore, there appears a lack of correlation between MAPK4 protein expression levels and PDK1 protein stability. Besides, proteasome inhibitor MG132 treatment did not greatly affect PDK1 protein levels in either control or MAPK4-knockdown SUM159 and HS578T cells (S1C Fig). Together, our data suggest that MAPK4 neither regulates PDK1 mRNA expression nor notably affects PDK1 protein stability. Accordingly, we next examined whether MAPK4 enhances PDK1 protein translation.

eIF4E mediates MAPK4 regulation of PDK1 protein expression Most eukaryotic mRNAs carry a methyl-7-guanosine (m7G) cap at the 5′ end. The eukaryotic translation initiation complex eIF4F plays a critical role in protein synthesis from these m7G capped mRNAs. eIF4F translation initiation complex consists of eukaryotic translation initiation factor eIF4E, eIF4G, and eIF4A [6]. Among these factors, eIF4E is the least abundant initiation factor providing the rate-limiting step to bind to the m7G cap for translation initiation. Although m7G capped mRNA relies on eIF4E for translation, not all mRNAs are equally sensitive to altered eIF4E activity. There is a specific subset of “eIF4E-sensitive mRNAs” that often encode proteins involved in cell growth and survival [7]. Therefore, we investigated whether MAPK4-enhanced PDK1 protein expression is sensitive to altered eIF4E levels. Knockdown of eIF4E greatly repressed PDK1 protein expression in both the control and MAPK4-overexpressing HCC1806 and SUM159 cells (S2A Fig), indicating a critical role of eIF4E in PDK1 protein synthesis, including MAPK4-induced PDK1 protein expression. eIF4E phosphorylation at Serine 209 (S209) may prime eIF4E activation to enhance the translation of certain mRNAs to promote cancer [8]. Hence, we next examined whether MAPK4 regulates eIF4E phosphorylation at S209. Dox-induced MAPK4 overexpression in SUM159, MDA-MB-468, HCC1395, and HCC1806 cells all greatly enhanced eIF4E S209 phosphorylation (S2B Fig). Conversely, knockdown of MAPK4 in HS578T, SUM159, and HCC1937 cells reduced such phosphorylation (S2C Fig). Furthermore, MAPK4 appeared to bind to eIF4E in the co-IP assays (S2D Fig) and GST-pulldown assays (S2E Fig). Together, these data suggest that MAPK4 can bind eIF4E and enhance its phosphorylation at S209.

MNK1/2 inhibition blocks MAPK4-induced eIF4E S209 phosphorylation but does not affect MAPK4 enhancing PDK1 expression or cancer cell growth MNK1 and MNK2 are the only known protein kinases catalyzing eIF4E phosphorylation at S209 [9–11]. MNK1/2-mediated eIF4E S209 phosphorylation is believed to be a key event promoting cancer, and MNK1/2-specific inhibitors such as eFT508 are being examined in cancer clinical trials [12]. Since we showed that MAPK4 overexpression enhances eIF4E phosphorylation at S209 and MAPK4 knockdown represses this phosphorylation (S2B and S2C Fig), a candidate working model would be that MAPK4 enhances PDK1 protein synthesis by promoting MNK1/2-mediated eIF4E S209 phosphorylation and activation. To test this hypothesis, we treated the cells with 2 different MNK1/2 inhibitors SLV-2436 (SEL201) and eFT508 [13,14]. Treatments using either inhibitor robustly blocked the basal and MAPK4-induced eIF4E S209 phosphorylation in HCC1806 and SUM159 cells, indicating that MNK1/2 are essential kinases for eIF4E S209 phosphorylation as reported and that MNK1/2 mediate MAPK4 activities in enhancing eIF4E S209 phosphorylation (S3A Fig). However, none of these inhibitor treatments affected basal or MAPK4-induced PDK1 protein levels in HCC1806 and SUM159 cells (S3A Fig). SLV-2436 and eFT508 treatments similarly repressed eIF4E S209 phosphorylation but exhibited little effect on PDK1 protein levels in wild type (WT) or MAPK4-KO SUM159 and MDA-MB-231 cells (S3B Fig). Finally, SLV-2436 and eFT508 treatments did not affect the basal or MAPK4-induced HCC1806 and SUM159 cell growth, nor the growth of WT or MAPK4-KO MDA-MB-231 cells (S3C and S3D Fig). Collectively, these data suggest that MAPK4 promotes MNK1/2-mediated eIF4E S209 phosphorylation; however, unexpectedly, eIF4E S209 phosphorylation does not play a notable role in MAPK4 promotion of PDK1 protein expression or tumor growth.

PDK1 partially mediates MAPK4 tumor-promoting activity We have previously documented that MAPK4 can directly activate AKT independent of the canonical PI3K/PDK1 pathway to promote cancer growth [3]. Besides phosphorylating and activating AKT in a PI3K-dependent manner, PDK1 also exhibits constitutive activity on most substrates beyond AKT. Therefore, our newly discovered MAPK4-PDK1 signaling axis may exhibit PI3K-independent and/or AKT-independent tumor-promoting activities. Besides, by enhancing PDK1 protein expression, MAPK4 may further enhance the canonical PI3K-PDK1-AKT pathway to promote cancer growth. To investigate PDK1 function in mediating MAPK4 biology, we performed knockdown of PDK1 in control and MAPK4-overexpressing SUM159 and HCC1806 cells using 2 independent shRNAs. Both shRNAs produced robust knockdown of PDK1, which greatly inhibited the phosphorylation of PDK1 substrates PKCζ/λ in all these cells (Fig 2A). In contrast, knockdown of PDK1 only greatly reduced AKT phosphorylation in the control SUM159 and HCC1806 cells but had little effect in MAPK4-overexpressing cells, further supporting our previously reported MAPK4-AKT signaling axis independent of PI3K/PDK1 [3]. In accord with this, while knockdown of PDK1 profoundly repressed control SUM159 and HCC1806 cell growth, it only partially inhibited MAPK4-induced cell growth (Fig 2B–2F). We conclude that by blocking both the PI3K/PDK1-induced AKT phosphorylation/activation and the PI3K-independent and/or AKT-independent PDK1 signaling cascade, knockdown of PDK1 is very effective in inhibiting control cell growth. In contrast, knockdown of PDK1 leaves the MAPK4-AKT signaling axis largely intact in the MAPK4-overexpressing cells, which may account for the partially maintained tumor cell growth. Finally, these data also support that PDK1 partially mediates MAPK4 tumor-promoting activity. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002227.g002

MAPK4-induced PDK1 protein expression alone lacks robust activity but cooperates with AKT to promote tumor cell growth In our rescue studies (Figs 3 and 4), we overexpressed PDK1 in MAPK4-KO SUM159 and MDA-MB-231 cells to levels considerably higher than those of the WT cells. Yet, we can only partially rescue the MAPK4-mediated biology, such as promoting cancer cell/xenograft growth and their resistance to PI3K blockade. To critically define whether MAPK4-induced PDK1 at physiologically relevant levels regulates cancer cell growth and their response to PI3K blockade, we also used our previously described MAPK4D254A mutant lacking affinity to AKT to address this question [3]. When ectopically expressed in SUM159 and HCC1806 cells, MAPK4D254A exhibited similar activities as WT MAPK4 in promoting PDK1 protein expression and activation (phosphorylation of PKCζ/λ, Fig 5A). However, consistent with our previous observations, MAPK4D254A largely lost its activities in enhancing AKT phosphorylation and promoting SUM159 and HCC1806 cell growth, including anchorage-independent growth (Fig 5A–5D). These data suggest that without MAPK4 directly engaging/activating AKT (MAPK4-AKT axis), MAPK4-induced PDK1 protein expression/activation (MAPK4-PDK1 axis) is not sufficient to promote robust cancer cell growth. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002227.g005 To further define whether MAPK4-induced PDK1 (MAPK4-PDK1 axis) plays any remarkable roles in promoting cancer cell growth, especially when combined with AKT activation, we investigated how co-expression of MAPK4D254A (with activation of MAPK4-PDK1 axis) and a constitutively activated AKT1 (AKT1T308D/S473D, AKT1-DD, to simulate MAPK4-AKT axis) affects SUM159 and HCC1806 cell growth. While ectopic expression of AKT1-DD alone showed some activity, co-expression of MAPK4D254A further enhanced cell growth, and the activity of co-expressed MAPK4D254A and AKT1-DD largely recapitulated WT MAPK4 tumor-promoting activity in the proliferation assays (Fig 5E and 5F). Together, these data suggest that while MAPK4-induced PDK1 protein expression (MAPK4-PDK1 axis) alone is not sufficient to robustly promote AKT phosphorylation/activation and tumor cell growth, it does provide a route to enhance the tumor-promoting activity of activated AKT. Lastly, co-expression of MAPK4D254A and AKT1-DD only partially recapitulated the tumor-promoting activity of WT MAPK4 in the soft-agar assays, suggesting that MAPK4 activation of additional signaling cascade beyond PDK1 and AKT may be important to promote tumor cell anchorage-independent growth (Fig 5G).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002227

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