Home Biology Cooperative activation of PDK1 and AKT by MAPK4 enhances most cancers progress and resistance to remedy

Cooperative activation of PDK1 and AKT by MAPK4 enhances most cancers progress and resistance to remedy

Cooperative activation of PDK1 and AKT by MAPK4 enhances most cancers progress and resistance to remedy

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Summary

Phosphoinositide-dependent kinase-1 (PDK1) is a grasp kinase of the protein A, G, and C (AGC) household kinases that play essential roles in regulating most cancers cell proliferation, survival, and metabolism. In addition to phosphorylating/activating AKT on the cell membrane in a PI3K-dependent method, PDK1 additionally displays constitutive exercise on many different AGC kinases for tumor-promoting exercise. Within the latter case, PDK1 protein ranges dominate its exercise. We beforehand reported that MAPK4, an atypical MAPK, can PI3K-independently promote AKT activation and tumor progress. Right here, utilizing triple-negative breast most cancers (TNBC) cell fashions, we display that MAPK4 can even improve PDK1 protein synthesis, thus phosphorylate/activate PDK1 substrates past AKT. This new MAPK4-PDK1 axis alone lacks vigorous tumor-promoting exercise however cooperates with our beforehand reported MAPK4-AKT axis to advertise tumor progress. In addition to enhancing resistance to PI3K blockade, MAPK4 additionally promotes most cancers cell resistance to the extra stringent PI3K and PDK1 co-blockade, a lately proposed therapeutic technique. At present, there isn’t any MAPK4 inhibitor to deal with MAPK4-high cancers. Primarily based on the concerted motion of MAPK4-AKT and MAPK4-PDK1 axis in selling most cancers, we predict and ensure that co-targeting AKT and PDK1 successfully represses MAPK4-induced most cancers cell progress, suggesting a possible therapeutic technique to deal with MAPK4-high cancers.

Introduction

The protein A, G, and C (AGC) household kinases encompass greater than 60 evolutionarily associated serine/threonine protein kinases. Many AGC kinases, similar to 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 essential roles in regulating cell proliferation, apoptosis, and metabolism [1]. In addition to its exercise in phosphorylating/activating AKT within the phosphatidylinositol 3-kinase (PI3K) pathway, PDK1 additionally displays constitutive exercise in phosphorylating many different AGC kinases, similar to S6K, SGK, PKC, and RSK for tumor-promoting actions [1,2]. Within the latter case, PDK1 protein expression ranges decide its actions. Due to this fact, you will need to perceive the molecular mechanism regulating PDK1 protein ranges, which stays elusive.

Mitogen-activated protein kinase 4 (MAPK4) is an atypical MAPK not nicely studied. We lately reported that MAPK4 can promote most cancers by noncanonically activating AKT impartial of the PI3K/PDK1 signaling axis [3]. The parallel actions of MAPK4 and PI3K-PDK1 in activating AKT predict MAPK4 actions in regulating cell response to PI3K blockade. Certainly, inhibiting MAPK4 sensitizes most cancers cells to PI3K blockade [4]. Nevertheless, it stays unknown why inhibiting MAPK4 (knockdown/knockout) significantly represses AKT phosphorylation/activation that the canonical PI3K pathway can even drive.

Triple-negative breast most cancers (TNBC) is a devastating illness accounting for 15% to twenty% of all breast most cancers however with restricted therapeutic choices. We beforehand reported that MAPK4 is extremely expressed in a big fraction of TNBC, and repressing MAPK4 is efficient in inhibiting TNBC cell and xenograft progress [4]. At present, there isn’t any MAPK4-specific inhibitor(s) for potential scientific testing to deal with TNBC. Additional dissecting MAPK4 downstream signaling nodes, particularly these with scientific inhibitor(s) getting used within the clinic or examined in scientific trials, could present another path to deal with MAPK4-high TNBC.

Right here, utilizing TNBC cell fashions, we report that apart from instantly phosphorylating/activating AKT (an MAPK4-AKT axis [3,4]), MAPK4 additionally significantly promotes PDK1 protein expression, representing an MAPK4-PDK1 axis to boost PDK1 expression/activation. Blocking MAPK4 each inhibits the PI3K-independent MAPK4-AKT signaling axis [3,4] and represses PDK1 protein expression to dam the canonical PI3K-PDK1-AKT pathway. Collectively, these result in significantly repressed AKT phosphorylation/activation. We additional display that by enhancing PDK1 protein expression/exercise, MAPK4 additionally enhances PDK1-dependent however AKT-independent signaling. Accordingly, co-blockade of AKT and PDK1 largely blocks MAPK4 tumor-promoting exercise. Our research collectively establish a novel mechanism selling PDK1 protein expression and additional advance our information of the molecular mechanism underlying the tumor-promoting exercise of MAPK4, an rising novel therapeutic goal for human cancers.

Outcomes

MAPK4 up-regulates PDK1 protein expression in most cancers cells

Now we have proven that MAPK4 can activate AKT impartial of the PI3K pathway [3]. Nevertheless, it stays unknown why inhibiting MAPK4 significantly represses AKT phosphorylation/activation that the canonical PI3K signaling can even drive. Now we have beforehand documented human TNBC cell strains with excessive, medium, or low MAPK4 expressions [4]. Overexpression of MAPK4 within the MAPK4-medium SUM159 and MAPK4-low MDA-MB-436, MDA-MB-468, HCC1395, and HCC1806 cells all enhanced PDK1 protein expression, suggesting an surprising MAPK4-PDK1 signaling axis (Fig 1A). Accordingly, knockdown/knockout of MAPK4 within the MAPK4-medium SUM159 and MAPK4-high HS578T, HCC1937, and MDA-MB-231 cells decreased PDK1 protein expression (Fig 1B and 1C) and ectopic expression of MAPK4 within the MAPK4-KO MDA-MB-231 and SUM159 cells rescued PDK1 expression (Fig 1D). Neither overexpression nor knockdown of MAPK4 affected PDK1 mRNA expression within the examined cell strains (S1A Fig), suggesting that MAPK4 promotes PDK1 protein expression by way of a posttranscriptional course of, similar to regulation of protein synthesis and stability.

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Fig 1. MAPK4 up-regulates PDK1 protein expression in most cancers 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 management (iCtrl), (B) engineered HS578T, SUM159, and HCC1937 cells with 4 μg/ml Dox-induced knockdown of MAPK4 (iG2 and iG4) or management (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 have been handled with 0.5 μg/ml Dox. Knowledge are consultant of at the least 2–3 impartial 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 stays unknown till the latest identification of SPOP as an E3 ubiquitin ligase for PDK1 protein ubiquitination and degradation [5]. To look at whether or not MAPK4 performs a serious function in regulating PDK1 protein stability, we first examined PDK1 protein stability in 7 TNBC most cancers cell strains, together with the MAPK4-low HCC1806, MDA-MB-468, HCC1395 cells, and the MAPK4-medium/excessive SUM159, MDA-MB-231, HCC1937, and HS578T cell strains. We noticed that PDK1 protein may be very steady in MDA-MB-231, HCC1937, HS578T, HCC1806, and MDA-MB-468 cells, whereas notable PDK1 protein degradation was solely evident in SUM159 and HCC1395 cells after extended protein synthesis inhibitor cycloheximide (CHX) remedy (S1B Fig). As an extra management, we additionally confirmed a fast degradation of MCL-1 and Cyclin D1 within the CHX-treated MDA-MB-231, HCC1937, HS578T, and SUM159 cells. Due to this fact, there seems a scarcity of correlation between MAPK4 protein expression ranges and PDK1 protein stability. In addition to, proteasome inhibitor MG132 remedy didn’t significantly have an effect on PDK1 protein ranges in both management or MAPK4-knockdown SUM159 and HS578T cells (S1C Fig). Collectively, our knowledge counsel that MAPK4 neither regulates PDK1 mRNA expression nor notably impacts PDK1 protein stability. Accordingly, we subsequent examined whether or not MAPK4 enhances PDK1 protein translation.

eIF4E mediates MAPK4 regulation of PDK1 protein expression

Most eukaryotic mRNAs carry a methyl-7-guanosine (m7G) cap on the 5′ finish. The eukaryotic translation initiation advanced eIF4F performs a essential function in protein synthesis from these m7G capped mRNAs. eIF4F translation initiation advanced consists of eukaryotic translation initiation issue eIF4E, eIF4G, and eIF4A [6]. Amongst these elements, eIF4E is the least ample initiation issue offering the rate-limiting step to bind to the m7G cap for translation initiation. Though m7G capped mRNA depends on eIF4E for translation, not all mRNAs are equally delicate to altered eIF4E exercise. There’s a particular subset of “eIF4E-sensitive mRNAs” that always encode proteins concerned in cell progress and survival [7]. Due to this fact, we investigated whether or not MAPK4-enhanced PDK1 protein expression is delicate to altered eIF4E ranges. Knockdown of eIF4E significantly repressed PDK1 protein expression in each the management and MAPK4-overexpressing HCC1806 and SUM159 cells (S2A Fig), indicating a essential function of eIF4E in PDK1 protein synthesis, together with MAPK4-induced PDK1 protein expression.

eIF4E phosphorylation at Serine 209 (S209) could prime eIF4E activation to boost the interpretation of sure mRNAs to advertise most cancers [8]. Therefore, we subsequent examined whether or not MAPK4 regulates eIF4E phosphorylation at S209. Dox-induced MAPK4 overexpression in SUM159, MDA-MB-468, HCC1395, and HCC1806 cells all significantly enhanced eIF4E S209 phosphorylation (S2B Fig). Conversely, knockdown of MAPK4 in HS578T, SUM159, and HCC1937 cells decreased such phosphorylation (S2C Fig). Moreover, MAPK4 appeared to bind to eIF4E within the co-IP assays (S2D Fig) and GST-pulldown assays (S2E Fig). Collectively, these knowledge counsel that MAPK4 can bind eIF4E and improve its phosphorylation at S209.

MNK1/2 inhibition blocks MAPK4-induced eIF4E S209 phosphorylation however doesn’t have an effect on MAPK4 enhancing PDK1 expression or most cancers cell progress

MNK1 and MNK2 are the one identified protein kinases catalyzing eIF4E phosphorylation at S209 [911]. MNK1/2-mediated eIF4E S209 phosphorylation is believed to be a key occasion selling most cancers, and MNK1/2-specific inhibitors similar to eFT508 are being examined in most cancers scientific trials [12]. Since we confirmed that MAPK4 overexpression enhances eIF4E phosphorylation at S209 and MAPK4 knockdown represses this phosphorylation (S2B and S2C Fig), a candidate working mannequin can be that MAPK4 enhances PDK1 protein synthesis by selling MNK1/2-mediated eIF4E S209 phosphorylation and activation. To check this speculation, we handled the cells with 2 totally different MNK1/2 inhibitors SLV-2436 (SEL201) and eFT508 [13,14]. Remedies utilizing both inhibitor robustly blocked the basal and MAPK4-induced eIF4E S209 phosphorylation in HCC1806 and SUM159 cells, indicating that MNK1/2 are important kinases for eIF4E S209 phosphorylation as reported and that MNK1/2 mediate MAPK4 actions in enhancing eIF4E S209 phosphorylation (S3A Fig). Nevertheless, none of those inhibitor therapies affected basal or MAPK4-induced PDK1 protein ranges in HCC1806 and SUM159 cells (S3A Fig). SLV-2436 and eFT508 therapies equally repressed eIF4E S209 phosphorylation however exhibited little impact on PDK1 protein ranges in wild kind (WT) or MAPK4-KO SUM159 and MDA-MB-231 cells (S3B Fig). Lastly, SLV-2436 and eFT508 therapies didn’t have an effect on the basal or MAPK4-induced HCC1806 and SUM159 cell progress, nor the expansion of WT or MAPK4-KO MDA-MB-231 cells (S3C and S3D Fig). Collectively, these knowledge counsel that MAPK4 promotes MNK1/2-mediated eIF4E S209 phosphorylation; nevertheless, unexpectedly, eIF4E S209 phosphorylation doesn’t play a notable function in MAPK4 promotion of PDK1 protein expression or tumor progress.

PDK1 partially mediates MAPK4 tumor-promoting exercise

Now we have beforehand documented that MAPK4 can instantly activate AKT impartial of the canonical PI3K/PDK1 pathway to advertise most cancers progress [3]. In addition to phosphorylating and activating AKT in a PI3K-dependent method, PDK1 additionally displays constitutive exercise on most substrates past AKT. Due to this fact, our newly found MAPK4-PDK1 signaling axis could exhibit PI3K-independent and/or AKT-independent tumor-promoting actions. In addition to, by enhancing PDK1 protein expression, MAPK4 could additional improve the canonical PI3K-PDK1-AKT pathway to advertise most cancers progress. To analyze PDK1 perform in mediating MAPK4 biology, we carried out knockdown of PDK1 in management and MAPK4-overexpressing SUM159 and HCC1806 cells utilizing 2 impartial shRNAs. Each shRNAs produced sturdy knockdown of PDK1, which significantly inhibited the phosphorylation of PDK1 substrates PKCζ/λ in all these cells (Fig 2A). In distinction, knockdown of PDK1 solely significantly decreased AKT phosphorylation within the management SUM159 and HCC1806 cells however had little impact in MAPK4-overexpressing cells, additional supporting our beforehand reported MAPK4-AKT signaling axis impartial of PI3K/PDK1 [3]. In accord with this, whereas knockdown of PDK1 profoundly repressed management SUM159 and HCC1806 cell progress, it solely partially inhibited MAPK4-induced cell progress (Fig 2B–2F). We conclude that by blocking each the PI3K/PDK1-induced AKT phosphorylation/activation and the PI3K-independent and/or AKT-independent PDK1 signaling cascade, knockdown of PDK1 may be very efficient in inhibiting management cell progress. In distinction, knockdown of PDK1 leaves the MAPK4-AKT signaling axis largely intact within the MAPK4-overexpressing cells, which can account for the partially maintained tumor cell progress. Lastly, these knowledge additionally assist that PDK1 partially mediates MAPK4 tumor-promoting exercise.

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Fig 2. PDK1 partially mediates MAPK4 tumor-promoting exercise.

(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 management (iCtrl). The cells have been additionally engineered with steady knockdown of PDK1 (shPDK1-1, shPDK1-2) or management (NT). Quantification knowledge as means ± SD. Scale bar: 500 μm. P values by two-way ANOVA adopted by Sidak’s a number of comparisons. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.


https://doi.org/10.1371/journal.pbio.3002227.g002

Overexpression of PDK1 partly rescues MAPK4-KO tumor cell progress and reduces their sensitivity to PI3K blockade

To additional consider PDK1’s perform in mediating MAPK4 tumor-promoting exercise, we overexpressed PDK1 within the MAPK4-KO SUM159 and MDA-MB-231 cells. As a constructive management, we additionally ectopically expressed MAPK4 in these MAPK4-KO cells to ranges similar to WT cells (Fig 3A). As anticipated, ectopic MAPK4 expression largely rescued PDK1 protein expression and AKT phosphorylation within the MAPK4-KO cells (Fig 3A). In distinction, PDK1 overexpression at ranges significantly larger than WT cells may solely partially rescue AKT phosphorylation (Fig 3A). In accord with these, overexpression of PDK1 may solely partially rescue the expansion, together with anchorage-independent progress of the MAPK4-KO SUM159 and MDA-MB-231 cells in vitro and xenograft progress in vivo (Fig 3B–3F). Lastly, overexpression of PDK1 appeared much less potent than MAPK4 in rescuing the expansion of those MAPK4-KO cells/xenografts (Fig 3B–3F), that are per the notion that PDK1 solely partially mediates MAPK4 tumor-promoting exercise.

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Fig 3. Overexpression of PDK1 partly rescues MAPK4-KO tumor cell progress.

(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 management (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 management (iCtrl) in 1:2 Matrigel have been injected into the mammary fats pad of SCID mice. All mice additionally acquired 0.5 mg/ml Dox in consuming water. Tumors have been measured and harvested as indicated. Proven are the tumors’ photographs at assortment, progress curve (means ± SEM), and weights. Quantification knowledge as means ± SD apart from in any other case indicated. P values by one-way ANOVA adopted by Sidak’s a number of comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not important. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild kind.


https://doi.org/10.1371/journal.pbio.3002227.g003

Now we have beforehand demonstrated that overexpression of MAPK4 enhances most cancers cell resistance to PI3K blockade [4]. We subsequent examined whether or not overexpression of PDK1 reduces MAPK4-KO cell sensitivity to PI3K inhibition. As we beforehand reported, in comparison with parental cells, the MAPK4-KO SUM159 and MDA-MB-231 cells grew slower and have been way more delicate to PI3K inhibitor Alpelisib remedy (Fig 4A and 4B). Rescued MAPK4 expression in these MAPK4-KO cells largely restored their progress and resistance to Alpelisib. In distinction, overexpression of PDK1 solely partially restored their progress and resistance to Alpelisib (Fig 4A and 4B). Collectively, these knowledge counsel that PDK1 could solely partially mediate MAPK4 actions in selling tumor cell progress and resistance to PI3K blockade.

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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 management (iCtrl). Scale bar: 500 μm. Quantification knowledge as means ± SD. P values by two-way ANOVA adopted by Sidak’s a number of comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not important. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild kind.


https://doi.org/10.1371/journal.pbio.3002227.g004

MAPK4-induced PDK1 protein expression alone lacks sturdy exercise however cooperates with AKT to advertise tumor cell progress

In our rescue research (Figs 3 and 4), we overexpressed PDK1 in MAPK4-KO SUM159 and MDA-MB-231 cells to ranges significantly larger than these of the WT cells. But, we are able to solely partially rescue the MAPK4-mediated biology, similar to selling most cancers cell/xenograft progress and their resistance to PI3K blockade. To critically outline whether or not MAPK4-induced PDK1 at physiologically related ranges regulates most cancers cell progress and their response to PI3K blockade, we additionally used our beforehand described MAPK4D254A mutant missing affinity to AKT to deal with this query [3]. When ectopically expressed in SUM159 and HCC1806 cells, MAPK4D254A exhibited related actions as WT MAPK4 in selling PDK1 protein expression and activation (phosphorylation of PKCζ/λ, Fig 5A). Nevertheless, per our earlier observations, MAPK4D254A largely misplaced its actions in enhancing AKT phosphorylation and selling SUM159 and HCC1806 cell progress, together with anchorage-independent progress (Fig 5A–5D). These knowledge counsel that with out MAPK4 instantly participating/activating AKT (MAPK4-AKT axis), MAPK4-induced PDK1 protein expression/activation (MAPK4-PDK1 axis) shouldn’t be enough to advertise sturdy most cancers cell progress.

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Fig 5. MAPK4-induced PDK1 works with AKT to advertise tumor cell progress.

(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 management (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 management (iCtrl). These cells have been additionally contaminated with lentivirus expressing AKT1-DD mutant or management. Quantification knowledge as means ± SD. P values by one-way ANOVA adopted by Sidak’s a number of comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not important. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.


https://doi.org/10.1371/journal.pbio.3002227.g005

To additional outline whether or not MAPK4-induced PDK1 (MAPK4-PDK1 axis) performs any outstanding roles in selling most cancers cell progress, particularly when mixed 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) impacts SUM159 and HCC1806 cell progress. Whereas ectopic expression of AKT1-DD alone confirmed some exercise, co-expression of MAPK4D254A additional enhanced cell progress, and the exercise of co-expressed MAPK4D254A and AKT1-DD largely recapitulated WT MAPK4 tumor-promoting exercise within the proliferation assays (Fig 5E and 5F). Collectively, these knowledge counsel that whereas MAPK4-induced PDK1 protein expression (MAPK4-PDK1 axis) alone shouldn’t be enough to robustly promote AKT phosphorylation/activation and tumor cell progress, it does present a route to boost the tumor-promoting exercise of activated AKT. Lastly, co-expression of MAPK4D254A and AKT1-DD solely partially recapitulated the tumor-promoting exercise of WT MAPK4 within the soft-agar assays, suggesting that MAPK4 activation of extra signaling cascade past PDK1 and AKT could also be essential to advertise tumor cell anchorage-independent progress (Fig 5G).

MAPK4 promotes most cancers cell resistance to mixed PI3K and PDK1 blockade

Castel and colleagues reported that inhibition of PDK1 sensitizes most cancers cells to PI3K blockade [15]. Due to this fact, a co-blockade of PI3K and PDK1 could present a simpler therapeutic method to deal with cancers. Since MAPK4 can PI3K/PDK1-independently activate AKT, we predicted and experimentally validated that MAPK4 renders most cancers cell resistance to PI3K blockade [4]. In distinction, how MAPK4 impacts most cancers cell response to the extra stringent co-blockade of PI3K and PDK1 stays unknown.

To critically tackle this query, we first examined how PDK1 knockdown impacts management and MAPK4-overexpressing SUM159 and HCC1806 cell response to PI3K inhibitor Alpelisib. In settlement with our earlier research, overexpression of MAPK4 each significantly enhanced SUM159 and HCC1806 cell progress and rendered them proof against PI3K blockade (Fig 6A and 6B). Knockdown of PDK1 in management cells each decreased their progress and significantly sensitized them to Alpelisib therapies, as anticipated [15]. Knockdown of PDK1 additionally decreased the expansion of MAPK4-overexpressing cells, as we described in Fig 2. In distinction, the PDK1-knockdown MAPK4-overexpressing cells largely maintained their progress within the presence of 1 μm Alpelisib, thus, have been proof against 1 μm Alpelisib remedy (Fig 6A and 6B). These cells have been additionally partially proof against 2 μm Alpelisib therapies (Fig 6A and 6B). For instance, whereas each 1 and a couple of μm Alpelisib therapies basically wiped off the anchorage-independent progress of PDK1-knockdown management SUM159 and HCC1806 cells, the PDK1-knockdown MAPK4-overexpressing cells maintained sturdy progress when handled with 1 μm Alpelisib and produced quite a few clones when handled with 2 μm Alpelisib (Fig 6B). Collectively, these knowledge counsel that MAPK4 each PDK1-independently and -dependently regulates tumor cell response to PI3K blockade, and MAPK4 overexpression renders most cancers cells at the least partially proof against PI3K and PDK1 co-blockade.

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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 management (iCtrl), additionally with steady knockdown of PDK1 (shPDK1-1 and shPDK1-2) or management (NT). The cells have been additionally handled with PI3K inhibitor Alpelisib (1 μm or 2 μm) or DMSO management. Scale bar: 500 μm. Quantification knowledge as means ± SD. P values by two-way ANOVA adopted by Sidak’s a number of comparisons. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not important. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.


https://doi.org/10.1371/journal.pbio.3002227.g006

To substantiate our above observations additional, we additionally handled MAPK4-overexpressing and management SUM159 and HCC1806 cells with PI3K inhibitor Alpelisib (2 μm), PDK1 inhibitor GSK2334470 (2 μm), and together. As predicted [15], co-treatment confirmed outstanding exercise in repressing management tumor cell progress, leading to few progress within the plate clonogenic assays and soft-agar assays (Fig 7A–7C). In distinction, MAPK4 overexpression each promoted cell progress and rendered tumor cells at the least partially proof against Alpelisib and GSK2334470 co-treatment (Fig 7A–7C). These knowledge additional assist MAPK4 roles in enhancing each tumor cell progress and at the least partial resistance to mixed PI3K and PDK1 blockade. Accordingly, in contrast with WT cells, the MAPK4-KO SUM159 cells have been extra delicate to PI3K inhibitor Alpelisib (1 μm), PDK1 inhibitor GSK2334470 (2 μm), and together, and ectopic expression of MAPK4 in these MAPK4-KO cells rescued their progress to WT ranges (S4 Fig).

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Fig 7. MAPK4 promotes most cancers 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 management (iCtrl). The cells have been additionally handled with DMSO management, PDK1 inhibitor GSK2334470 (2 μm), PI3K inhibitor Alpelisib (2 μm), or each inhibitors. Scale bar: 500 μm. Quantification knowledge as means ± SD. P values by two-way ANOVA adopted by Sidak’s a number of comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ns, not important. Knowledge are consultant of at the least 3 impartial experiments. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.


https://doi.org/10.1371/journal.pbio.3002227.g007

Co-targeting AKT and PDK1 is efficient in repressing MAPK4-induced most cancers cell progress

Now we have beforehand demonstrated that AKT activation is important for supporting MAPK4 tumor-promoting exercise [3]. Though excessive focus of AKT inhibitor remedy may be very efficient in inhibiting each the management and MAPK4-overexpressing most cancers cell progress, the potential toxicity of AKT inhibitors at excessive concentrations is predicted to restrict their therapeutic potential. Since our newly recognized MAPK4-PDK1 axis can work with the MAPK4-AKT axis to advertise most cancers progress, we examined whether or not co-targeting PDK1 and AKT utilizing PDK1 inhibitor GSK2334470 and AKT inhibitor MK2206 might be a legitimate method to dam MAPK4 tumor-promoting actions. We first confirmed that PDK1 inhibitor GSK2334470 (2 μm and 5 μm) can inhibit MAPK4-induced PDK1 activation (PDK1-mediated PKCζ/λ phosphorylation) in SUM159 and HCC1806 cells (Fig 8A). We then examined the results of GSK2334470 at a suboptimal focus (2 μm) and AKT inhibitor MK2206 (2 μm), alone or together, on MAPK4-regulated signaling cascade and SUM159 and HCC1806 cell progress. The MK2206 (2 μm) remedy potently blocked each the basal and MAPK4-induced AKT phosphorylation, significantly repressed management cell progress however solely partially blocked MAPK4-induced cell progress (Fig 8B and 8C). As anticipated, the suboptimal GSK2334470 (2 μm) remedy decreased PKCζ/λ phosphorylation with minor results on cell progress. In distinction, the mixed MK2206 and GSK2334470 remedy produced sturdy inhibition of each basal and MAPK4-induced PKCζ/λ and GSK3β phosphorylation and blocked MAPK4-induced SUM159 and HCC1806 cell progress (Fig 8B and 8C). We noticed the same sturdy exercise of PDK1 inhbitor GSK2334470 (1 μm) and AKT inhibitor MK2206 (1 μm) co-treatment in blocking the MAPK4-high MDA-MB-231 cell progress. In distinction, the MAPK4-KO MDA-MB-231 cells have been already delicate to MK2206 remedy (Fig 8D and 8E).

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Fig 8. Co-targeting AKT and PDK1 is efficient in repressing MAPK4-induced most cancers cell progress.

(A) Western blot on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or management (iCtrl), handled with 2 μm or 5 μm of PDK1 inhibitor GSK2334470 or DMSO management. (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 management (iCtrl). The cells have been additionally handled with DMSO management, PDK1 inhibitor GSK2334470 (2 μm), AKT inhibitor MK2206 (2 μm), or each inhibitors. (D) Western blots and (E) proliferation assays on the WT and MAPK4-KO MDA-MB-231 cells handled with DMSO management, PDK1 inhibitor GSK2334470 (1 μm), AKT inhibitor MK2206 (1 μm), or each inhibitors. Knowledge are means ± SD from at the least 3 separate experiments. P values by two-way ANOVA adopted by Sidak’s a number of comparisons. ns, not important, ****P < 0.0001. The numerical values underlying the figures could be present in S1 Knowledge. MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1; WT, wild kind.


https://doi.org/10.1371/journal.pbio.3002227.g008

Dialogue

Though PDK1 must bind PIP3 and activate AKT on the cell membrane within the PI3K-PDK1-AKT signaling pathway, PDK1 is constitutively activated, and its protein ranges dominate its exercise on most targets past AKT. Due to this fact, it’s essential to grasp how PDK1 protein expression is regulated in human cancers. Right here, utilizing human TNBC cell line fashions, we uncovered an surprising sturdy pathway for MAPK4 selling PDK1 protein expression. We additionally made related observations on MAPK4 enhancing PDK1 protein ranges in human non-small cell lung most cancers H1299 and H157 cells, colorectal most cancers HCT116 cells, and prostate most cancers DU145 cells (S5 Fig), suggesting the MAPK4-PDK1 axis past TNBC. By enhancing PDK1 protein expression, MAPK4 each prompts PDK1 substrates past AKT and helps the PI3K-PDK1-AKT signaling cascade to control most cancers cell biology.

Now we have proven that MAPK4 can activate AKT impartial of the PI3K pathway [3]. Nevertheless, it was unknown why inhibiting MAPK4 (knockdown/knockout of MAPK4) robustly represses AKT phosphorylation/activation that the canonical PI3K pathway can even drive. Our at the moment reported MAPK4-PDK1 signaling axis could present a solution to that query. For instance, knockdown/knockout of MAPK4 can instantly repress AKT phosphorylation/activation by blocking the MAPK4-AKT axis. In addition to, by blocking the MAPK4-PDK1 axis, knockdown/knockout of MAPK4 additionally decreases PDK1 protein expression to inhibit the canonical PI3K-PDK1-AKT pathway, additional decreasing AKT phosphorylation/activation.

Earlier experiences have nicely documented the AKT-independent tumor-promoting actions of PDK1. For instance, the PDK1-PLK1-MYC, PDK1-SGK1, and PDK1-PKCα signaling axis have been proven to advertise tumor progress, remedy resistance, and/or confer oncogenic transformation [1517]. Due to this fact, theoretically, by enhancing PDK1 protein expression, the MAPK4-PDK1 signaling axis will promote most cancers by activating the above PDK1-dependent however AKT-independent tumor-promoting pathway. Nevertheless, the MAPK4D254A mutant, which might promote PDK1 protein expression however can’t instantly bind and activate AKT, exhibited restricted tumor-promoting exercise (Fig 5). These knowledge counsel that the AKT-independent MAPK4-PDK1 signaling axis alone shouldn’t be enough for sturdy tumor-promoting exercise.

MNK1 and MNK2 are the one identified kinases that may phosphorylate eIF4E at S209, which has been proven essential to advertise the synthesis of sure proteins essential in cell progress [8]. Due to this fact, a number of MNK1/2 inhibitors are being examined in scientific trials [12]. We discover that MAPK4 promotes eIF4E S209 phosphorylation, and MNK1/2 inhibitor therapies (SLV-2436 and eFT508) robustly block MAPK4-induced eIF4E phosphorylation at S209. These counsel a possible MAPK4-MNK1/2-eIF4E signaling axis essential for MAPK4 selling eIF4E S209 phosphorylation. Curiously, neither of the two MNK1/2 inhibitors examined (SLV-2436 and eFT508) exhibited notable exercise in inhibiting MAPK4-induced PDK1 protein expression or tumor cell progress (S3 Fig). These point out that MNK1/2-mediated eIF4E S209 phosphorylation shouldn’t be required for MAPK4 actions in selling PDK1 protein expression or cell progress. In distinction, knockdown of eIF4E repressed MAPK4-induced PDK1 protein expression, suggesting a purposeful MAPK4-eIF4E-PDK1 axis that continues to be to be absolutely characterised (S2A Fig). Lastly, regardless of sturdy actions in blocking eIF4E phosphorylation at S209, not one of the 2 MNK1/2 inhibitors exhibited notable tumor-inhibiting actions in any of the tumor cell strains we examined, together with the MAPK4-low and MAPK4-KO most cancers cell strains (S3C and S3D Fig). Our present knowledge at the least increase warning for scientific trials testing MNK1/2 inhibitors in treating cancers with out some kind(s) of stratification.

Our beforehand reported PI3K-independent MAPK4-AKT signaling axis gives a direct mechanism for MAPK4 driving tumor cell resistance to PI3K blockade [4]. It has been reported that mixed PI3K and PDK1 blockade is extremely efficient in blocking most cancers cell progress, which can present an improved therapeutic method to deal with cancers [15]. We predict that the MAPK4-AKT axis can also maintain AKT phosphorylation/activation and supply MAPK4-high most cancers cells resistance to mixed PI3K and PDK1 inhibitors remedy. Certainly, our knowledge affirm this, suggesting the need of contemplating MAPK4 expression standing in future scientific trials testing PI3K and PDK1 co-blockade.

PDK1 clearly partially mediates MAPK4 tumor-promoting exercise (Fig 2). Nevertheless, the MAPK4-PDK1 axis itself (expression of MAPK4D254A) lacks sturdy exercise in phosphorylating/activating AKT and driving tumor cell progress. When mixed with AKT activation (expression of a constitutively activated AKT-DD), MAPK4D254A displays robust tumor-promoting exercise (Fig 5). These knowledge counsel {that a} concerted motion of MAPK4-AKT and MAPK4-PDK1 signaling axis underlies MAPK4 total tumor-promoting exercise. We additionally observed that whereas twin pharmacological blockade of AKT and PDK1 is enough to virtually fully block the expansion of MAPK4-overexpressing cells (Fig 8C), twin genetic activation of AKT and PDK1 (overexpression of MAPK4D254A and PDK1, Fig 5F and 5G) doesn’t absolutely recapitulate MAPK4 overexpression. These counsel that MAPK4 prompts extra signaling cascade(s) past PDK1 and AKT for full tumor-promoting exercise. On this case, twin activation of AKT and PDK1 is important (Fig 8C) however not enough (Fig 5F and 5G) for the total tumor-promoting exercise of MAPK4.

Now we have beforehand reported that inhibiting MAPK4 (knockdown or knockout) may be very efficient in suppressing the expansion of assorted MAPK4-high most cancers cells, together with breast most cancers, prostate most cancers, non-small cell lung most cancers, and colon most cancers [3,4,18]. Particularly, MAPK4 is extremely expressed in a big fraction of TNBC, which accounts for 15% to twenty% of all breast most cancers however with restricted therapeutic choices [4]. At present, there isn’t any MAPK4-specific inhibitor(s), which prevents us from testing our discoveries within the clinic. In distinction, many AKT and PDK1 inhibitors are being examined in most cancers scientific trials. Primarily based on the concerted actions of MAPK4-AKT and MAPK4-PDK1 signaling axis in driving MAPK4 tumor-promoting actions, we examined and confirmed that co-blockade of AKT and PDK1 successfully represses MAPK4-induced most cancers cell progress (Fig 8). This helps an thrilling therapeutic alternative to make use of AKT and PDK1 inhibitors to deal with MAPK4-high cancers, similar to a big fraction of TNBC.

Supplies and strategies

Plasmids

The pInducer20-YF vector was constructed as beforehand described [3]. pRK5 vector was supplied by Dr. Xin-Hua Feng at Baylor Faculty of Drugs, Houston, Texas. The CDS of PDK1 or eIF4E was PCR amplified from the SUM159 cell cDNA, cloned into the pInducer20-YF vector between the MluI and SalI websites, then sequencing verified. The pGIPZ lentiviral shRNA constructs for knockdown of human PDK1 have been bought from Open Biosystem (Thermo Fisher Scientific).

Cell tradition, transfection, lentivirus an infection

The human TNBC SUM159, MDA-MB-231, HCC1937, HCC1806, HCC1395, MDA-MB-468, HS578T cells, non-small cell lung most cancers H1299, H157 cells, colorectal most cancers HCT116 cells, and prostate most cancers DU145 cells have been acquired from ATCC. MDA-MB-231, HCC1937, MDA-MB-468, HS578T, and DU145 cells have been cultured in DMEM supplemented with 10% FBS. HCC1806, HCC1395, H1299, H157, and HCT116 cells have been cultured in RPMI1640 supplemented with 10% FBS (Hyclone or Invitrogen). SUM159 cells have been cultured in F12 supplemented with 10% FBS. The MAPK4-knockout (KO) SUM159, MDA-MB-231, H1299, and H157 cell strains have been described beforehand [3,4]. The pCDH based mostly lentiviral constructs have been used for lentivirus-mediated steady overexpression of MAPK4 within the MAPK4-KO cell as described earlier than [3,4].

LipoD293 DNA transfection reagent (SignaGen Laboratories, catalog SL100668) was used for DNA transfection. For lentiviral mediated gene supply, the packaging mixture of lentiviral-based expression vectors pMD2.G and psPAX2 have been transfected into the HEK293T cells. Harvested at 48 or 96 h after the transfection, viruses within the conditioned media have been collected after which filtered (0.45 μm). Earlier than drug choice, the focused cells have been contaminated with the viral media for 3 days, then expanded for additional assays. Secure and doxycycline (Dox)-inducible overexpression of MAPK4, PDK1, or eIF4E cells have been established utilizing the pCDH and pInducer20-YF-based lentiviral gene supply programs. Lentiviral-mediated steady or Dox-inducible knockdown of MAPK4, PDK1, or eIF4E cells have been established utilizing the pGIPZ and pInducer10 vectors. The inducible cells have been handled with 4 μg/ml Dox for at the least 3 days to acquire important gene knockdown or 0.5 μg/ml Dox for at the least 3 days for gene overexpression. The sequences of shRNAs are as following: shMAPK4-G2, GGGTTGGTAACAAAGTGGT, shMAPK4-G4, CGGGAGGAAGACAAGGACG, shPDK1-1: AGGAGATTGTCATAATTGC, shPDK1-2: ATAAGATACTCGTTTCCAG, sheIF4E-1: TTGGAGATCAGCCGCAGGT, sheIF4E-2: GCAACCTCCTGATTAGATT.

Plate colony formation assay and tender agar assay

For plate colony assay, 2,000 to eight,000 cells/nicely (cell line dependent) have been seeded in 12-well plates in 1 ml media and changed with recent media each week. Roughly 10% (w/v) formaldehyde was used to repair cells for 15 min after 2 to three weeks tradition, and 0.05% (w/v) crystal violet supplemented with 10% methanol and 10% ethanol was used to stain cells for 20 min at room temperature. Then eliminated the staining resolution and washed the plate with distilled water. The plates have been then air-dried and scanned utilizing a Canon scanner. Then added 500 μl 10% acetic acid to every nicely and browse the OD absorbance at 570 nm. Comfortable agar assays have been carried out as beforehand described [3,4,18,19]. Full medium containing 0.4% low melting agarose at 37°C was used to combine the cells after which plated onto supporting gel (0.8% agarose); 4 μg/ml or 0.5 μg/ml Dox was used for inducible gene knockdown or inducible overexpression. Comfortable agar was fastened utilizing 10% acetic acid and 5% methanol, after 3 to 4 weeks of tradition. ImageJ software program was used to depend the Numbers of colonies (with an estimated minimal colony dimension of fifty to 100 μm). When relevant, PI3K and/or PDK1 inhibitors on the indicated concentrations have been added through the preliminary setup and replenished in recent media each week.

GST-pull down

The GST-MAPK4 fusion protein was expressed, purified, and subjected to pull-down assay as we beforehand reported [3]. GST-HF protein was used as management. Immunoprecipitation was carried out utilizing EZview Crimson ANTI-FLAG M2 Affinity Gel (Millipore Sigma), as we beforehand reported [3,4,1820].

Supporting data

S1 Knowledge. Numerical knowledge for Figs 2B, 2C, 2D, 2E, 2F, 3B, 3C, 3D, 3E, 3F, 4A, 4B, 5B, 5C, 5D, 5F, 5G, 6A, 6B, 7A, 7B, 7C, 8C, 8E, S1A, S3C, S3D and S4.

https://doi.org/10.1371/journal.pbio.3002227.s007

(XLSX)

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