Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function.

DIRAS3 is an imprinted tumor suppressor gene encoding a GTPase that has a distinctive N-terminal extension (NTE) not found in other RAS proteins. This NTE and the prenylated C-terminus are required for DIRAS3-mediated inhibition of RAS/MAP signaling and PI3K activity at the plasma membrane. In this study, we applied biochemical, biophysical, and computational methods to characterize the structure and function of the NTE. The NTE peptide recognizes phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 with rapid kinetics and strong affinity. Lipid binding induces NTE structural change from disorder to amphipathic helix. Mass spectrometry identified N-myristoylation of DIRAS3. All-atom molecular dynamic simulations predict DIRAS3 could adhere to the membrane through both termini, suggesting the NTE is involved in targeting and stabilizing DIRAS3 on the membrane by double anchoring. Overall, our results are consistent with DIRAS3's function as a tumor suppressor, whereby the membrane-bound DIRAS3 can effectively target PI3K and KRAS at the membrane.

Development and evaluation of a java-based deep neural network method for drug response predictions.

Accurate prediction of drug response is a crucial step in personalized medicine. Recently, deep learning techniques have been witnessed with significant breakthroughs in a variety of areas including biomedical research and chemogenomic applications. This motivated us to develop a novel deep learning platform to accurately and reliably predict the response of cancer cells to different drug treatments. In the present work, we describe a Java-based implementation of deep neural network method, termed JavaDL, to predict cancer responses to drugs solely based on their chemical features. To this end, we devised a novel cost function and added a regularization term which suppresses overfitting. We also adopted an early stopping strategy to further reduce overfit and improve the accuracy and robustness of our models. To evaluate our method, we compared with several popular machine learning and deep neural network programs and observed that JavaDL either outperformed those methods in model building or obtained comparable predictions. Finally, JavaDL was employed to predict drug responses of several aggressive breast cancer cell lines, and the results showed robust and accurate predictions with r 2 as high as 0.81.

Mechanisms of MCL-1 Protein Stability Induced by MCL-1 Antagonists in B-Cell Malignancies.

PURPOSE: Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i-induced Mcl-1 protein stability. EXPERIMENTAL DESIGN: Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein-protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling. RESULTS: Using both in vivo and in vitro analysis, we demonstrate that MCL-1i-induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors. CONCLUSIONS: We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.

EZH2 engages TGFß signaling to promote breast cancer bone metastasis via integrin ß1-FAK activation.

Bone metastases occur in 50-70% of patients with late-stage breast cancers and effective therapies are needed. The expression of enhancer of zeste homolog 2 (EZH2) is correlated with breast cancer metastasis, but its function in bone metastasis hasn't been well-explored. Here we report that EZH2 promotes osteolytic metastasis of breast cancer through regulating transforming growth factor beta (TGFß) signaling. EZH2 induces cancer cell proliferation and osteoclast maturation, whereas EZH2 knockdown decreases bone metastasis incidence and outgrowth in vivo. Mechanistically, EZH2 transcriptionally increases ITGB1, which encodes for integrin ß1. Integrin ß1 activates focal adhesion kinase (FAK), which phosphorylates TGFß receptor type I (TGFßRI) at tyrosine 182 to enhance its binding to TGFß receptor type II (TGFßRII), thereby activating TGFß signaling. Clinically applicable FAK inhibitors but not EZH2 methyltransferase inhibitors effectively inhibit breast cancer bone metastasis in vivo. Overall, we find that the EZH2-integrin ß1-FAK axis cooperates with the TGFß signaling pathway to promote bone metastasis of breast cancer.

A gene signature consisting of ubiquitin ligases and deubiquitinating enzymes of SKP2 is associated with clinical outcome in breast cancer.

The ubiquitination of SKP2, an oncoprotein, is controlled by its E3 ligases, including APC/CFZR1 and deubiquitinases such as USP10. We identified a two-gene signature for the ubiquitination of SKP2, consisting of the copy number of FZR1 compared to the copy number of USP10. The signature reflects the level of SKP2 activity, stratifying BC patients into two groups with significantly different protein levels of SKP2 ubiquitination substrate p27 (t-test p < 0.01) and recapitulating the expression patterns of SKP2 between tumor and normal tissue (Spearman's ρ = 0.39.) The signature is also highly associated with clinical outcome in luminal BC but not other subtypes, characterizing patients into two groups with significantly different overall survival times (log-rank p = 0.006). In addition, it is dramatically associated with tumor grade (Chi-squared p = 6.7 × 10-3), stage (Chi-squared p = 1.6 × 10-11), and the number of positive lymph nodes (negative binomial regression coefficient p = 2.0 × 10-3). Our study provides a rationale for targeting the SKP2 ubiquitination pathway in luminal BC and for further investigation of the use of ubiquitinase/deubiquitinase genes as prognosis and treatment biomarkers.

Co-Production of Dimethyl Carbonate, Dimethoxymethane and Dimethyl Ether from Methanol: Process Design and Exergy Analysis.

Dimethyl carbonate is an important green chemical that has been widely used in the chemical industry. In the production of dimethyl carbonate, methanol oxidative carbonylation has been studied, but the conversion ratio of dimethyl carbonate using this method is too low, and the subsequent separation requires a large amount of energy due to methanol and dimethyl carbonate being azeotrope. In this paper, the strategy of "reaction instead of separation" is proposed. Based on this strategy, a novel process is developed to combine the production of DMC with that of dimethoxymethane (DMM) and dimethyl ether (DME). The co-production process was simulated using Aspen Plus software, and the product purity was up to 99.9%. The exergy analysis of the co-production process and the existing process was carried out. The exergy destruction and exergy efficiency were compared with those of the existing production processes. The results show that the exergy destruction of the co-production process is about 276% less than that of the single-production processes, and the exergy efficiencies in the developed co-production process are significantly improved. The utility loads of the co-production process are significantly lower than that of the single-production process. The developed co-production process increases the methanol conversion ratio to 95%, with a reduced energy requirement. It is proved that the developed co-production process can provide an advantageous option over the existing processes with improved energy efficiency and material savings. The strategy of "reaction instead of separation" is feasible. A new strategy is proposed for azeotrope separation.

PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer.

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P2 and PI(3,4,5)P3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

Directed evolution of cyclic peptides for inhibition of autophagy.

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

Conversion of RNA Aptamer into Modified DNA Aptamers Provides for Prolonged Stability and Enhanced Antitumor Activity.

Aptamers, synthetic single-strand oligonucleotides that are similar in function to antibodies, are promising as therapeutics because of their minimal side effects. However, the stability and bioavailability of the aptamers pose a challenge. We developed aptamers converted from RNA aptamer to modified DNA aptamers that target phospho-AXL with improved stability and bioavailability. On the basis of the comparative analysis of a library of 17 converted modified DNA aptamers, we selected aptamer candidates, GLB-G25 and GLB-A04, that exhibited the highest bioavailability, stability, and robust antitumor effect in in vitro experiments. Backbone modifications such as thiophosphate or dithiophosphate and a covalent modification of the 5'-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the stability of the aptamers in vivo by reducing nuclease hydrolysis and renal clearance, and achieved high and sustained inhibition of AXL at a very low dose. Treatment with these modified aptamers in ovarian cancer orthotopic mouse models significantly reduced tumor growth and the number of metastases. This effective silencing of the phospho-AXL target thus demonstrated that aptamer specificity and bioavailability can be improved by the chemical modification of existing aptamers for phospho-AXL. These results lay the foundation for the translation of these aptamer candidates and companion biomarkers to the clinic.

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Methylation of HSP70 Orchestrates Its Binding to and Stabilization of BCL2 mRNA and Renders Pancreatic Cancer Cells Resistant to Therapeutics.

Pancreatic cancer is a lethal disease owing to its intrinsic and acquired resistance to therapeutic modalities. The altered balance between pro- and antiapoptosis signals within cancer cells is critical to therapeutic resistance. However, the molecular mechanisms underlying increased antiapoptosis signals remain poorly understood. In this study, we report that PRMT1 expression is increased in pancreatic cancer tissues and is associated with higher tumor grade, increased aggressiveness, and worse prognosis. PRMT1 overexpression increased arginine methylation of HSPs of 70 kDa (HSP70); this methylation enhanced HSP70 binding and stabilization of BCL2 mRNA through AU-rich elements in 3'-untranslated region and consequentially increased BCL2 protein expression and protected cancer cells from apoptosis induced by cellular stresses and therapeutics. RNA binding and regulation function of HSP70 was involved in pancreatic cancer drug resistance and was dependent on protein arginine methylation. These findings not only reveal a novel PRMT1-HSP70-BCL2 signaling axis that is crucial to pancreatic cancer cell survival and therapeutic resistance, but they also provide a proof of concept that targeted inhibition of this axis may represent a new therapeutic strategy. SIGNIFICANCE: This study demonstrates that a PRMT1-mediated stabilization of BCL2 mRNA contributes to therapeutic resistance in pancreatic cancer and that targeting this pathway could overcome said resistance.

An up-to-date overview of computational polypharmacology in modern drug discovery.

INTRODUCTION: In recent years, computational polypharmacology has gained significant attention to study the promiscuous nature of drugs. Despite tremendous challenges, community-wide efforts have led to a variety of novel approaches for predicting drug polypharmacology. In particular, some rapid advances using machine learning and artificial intelligence have been reported with great success. AREAS COVERED: In this article, the authors provide a comprehensive update on the current state-of-the-art polypharmacology approaches and their applications, focusing on those reports published after our 2017 review article. The authors particularly discuss some novel, groundbreaking concepts, and methods that have been developed recently and applied to drug polypharmacology studies. EXPERT OPINION: Polypharmacology is evolving and novel concepts are being introduced to counter the current challenges in the field. However, major hurdles remain including incompleteness of high-quality experimental data, lack of in vitro and in vivo assays to characterize multi-targeting agents, shortage of robust computational methods, and challenges to identify the best target combinations and design effective multi-targeting agents. Fortunately, numerous national/international efforts including multi-omics and artificial intelligence initiatives as well as most recent collaborations on addressing the COVID-19 pandemic have shown significant promise to propel the field of polypharmacology forward.

Targeting Forward and Reverse EphB4/EFNB2 Signaling by a Peptide with Dual Functions.

The tyrosine kinase receptor EphB4 is frequently overexpressed in ovarian and other solid tumors and is involved in interactions between tumor cells and the tumor microenvironment, contributing to metastasis. Trans-interaction between EphB4 and its membrane-bound ligand ephrin B2 (EFNB2) mediates bi-directional signaling: forward EFNB2-to-EphB4 signaling suppresses tumor cell proliferation, while reverse EphB4-to-EFNB2 signaling stimulates the invasive and angiogenic properties of endothelial cells. Currently, no small molecule-based, dual-function, EphB4-binding peptides are available. Here, we report our discovery of a bi-directional ephrin agonist peptide, BIDEN-AP which, when selectively internalized via receptor-mediated endocytosis, suppressed invasion and epithelial-mesenchymal transition of ovarian cancer cells. BIDEN-AP also inhibited endothelial migration and tube formation. In vivo, BIDEN-AP and its nanoconjugate CCPM-BIDEN-AP significantly reduced growth of orthotopic ovarian tumors, with CCPM-BIDEN-AP displaying greater antitumor potency than BIDEN-AP. Both BIDEN-AP and CCPM-BIDEN-AP compromised angiogenesis by downregulating epithelial-mesenchymal transition and angiogenic pathways. Thus, we report a novel EphB4-based therapeutic approach against ovarian cancer.

Structural Characterization of the Aurora Kinase B "DFG-flip" Using Metadynamics.

Aurora kinase B (AKB), a Ser/Thr kinase that plays a crucial role in mitosis, is overexpressed in several cancers. Clinical inhibitors targeting AKB bind to the active DFG "in" conformation of the kinase. It would be beneficial, however, to understand if AKB is susceptible to type II kinase inhibitors that bind to the inactive, DFG "out" conformation, since type II inhibitors achieve higher kinome selectivity and higher potency in vivo. The DFG "out" conformation of AKB is not yet experimentally determined which makes the design of type II inhibitors exceedingly difficult. An alternate approach is to simulate the DFG "out" conformation from the experimentally determined DFG "in" conformation using atomistic molecular dynamics (MD) simulation. In this work, we employed metadynamics (MTD) approach to simulate the DFG "out" conformation of AKB by choosing the appropriate collective variables. We examined structural changes during the DFG-flip and determined the interactions crucial to stabilize the kinase in active and inactive states. Interestingly, the MTD approach also identified a unique transition state (DFG "up"), which can be targeted by small molecule inhibitors. Structural insights about these conformations is essential for structure-guided design of next-generation AKB inhibitors. This work also emphasizes the usefulness of MTD simulations in predicting macromolecular conformational changes at reduced computational costs.

Tankyrase disrupts metabolic homeostasis and promotes tumorigenesis by inhibiting LKB1-AMPK signalling.

The LKB1/AMPK pathway plays a major role in cellular homeostasis and tumor suppression. Down-regulation of LKB1/AMPK occurs in several human cancers and has been implicated in metabolic diseases. However, the precise upstream regulation of LKB1-AMPK pathway is largely unknown. Here, we report that AMPK activation by LKB1 is regulated by tankyrases. Tankyrases interact with and ribosylate LKB1, promoting its K63-linked ubiquitination by an E3 ligase RNF146, which blocks LKB1/STRAD/MO25 complex formation and LKB1 activation. LKB1 activation by tankyrase inhibitors induces AMPK activation and suppresses tumorigenesis. Similarly, the tankyrase inhibitor G007-LK effectively regulates liver metabolism and glycemic control in diabetic mice in a LKB1-dependent manner. In patients with lung cancer, tankyrase levels negatively correlate with p-AMPK levels and poor survival. Taken together, these findings suggest that tankyrase and RNF146 are major up-stream regulators of LKB1-AMPK pathway and provide another focus for cancer and metabolic disease therapies.

Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia.

The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.

pH-sensitive ZnO/carboxymethyl cellulose/chitosan bio-nanocomposite beads for colon-specific release of 5-fluorouracil.

To develop relatively green and ecofriendly smart vehicles for colon-specific drug delivery, carboxymethyl cellulose (CMC) and chitosan (CS) pH-sensitive biopolymers were used in this study. To overcome the weaknesses of CMC carriers, such as poor mechanical performance and an explosive drug release, zinc oxide (ZnO) nanoparticles were incorporated into CMC beads and then coated with a CS layer via a self-assembly technique to form core-shell polyelectrolyte complexes. An anticancer drug, 5-fluorouracil (5-FU), used as a model drug, was loaded into ZnO/CMC/CS bio-nanocomposite beads. Fourier transform infrared spectroscopy, scanning and transmission electron microscopy, and thermogravimetric analysis were used to characterize the chemical structure, morphological changes, and thermal properties of the developed drug carrier, respectively. By studying their swelling and in vitro 5-FU release profiles under simulated gastrointestinal conditions, the pH sensitivity of the developed bio-nanocomposite hydrogel beads could be investigated. The obtained beads with reduced porosity could effectively encapsulate 5-FU and showed self-sustained release behavior depending on the concentrations of CMC, CS, and ZnO nanoparticles. The developed beads also demonstrated a capacity for biodegradation. The results indicated that the ZnO/CMC/CS bio-nanocomposite beads exhibited pH-sensitivity and could be applied efficiently as biodegradable carriers for colon-specific 5-FU delivery.

Interaction between nanoparticles and charged phospholipid membranes.

Charged lipids in cell membranes and subcellular organelles are arranged in the form of a bilayer with the hydrocarbon tails sequestered away from the water and the polar head groups exposed to the aqueous environment. Most of them bear net negative charges leading to the negatively charged cell membranes. Charged lipid-lipid and lipid-protein interactions are generally dynamic and heavily depend on their local molecular concentrations. To examine the electrostatic properties of charged lipid layers in contact with an electrolyte solution, we incorporate the single chain mean field theory with Poisson-Boltzmann theory to explore the equilibrium structure of charged phospholipid membranes. Using the three bead coarse-grained model we reproduced the essential equilibrium properties of the charged phospholipid bilayer. We also investigate the influence of the mobile ions on the thickness of the layer, the area per lipid (APL), and the electrostatic potential of the membrane. Then we investigate the attraction-repulsion property of two charged nanoparticles which are stuck on the charged lipid molecules surrounded with mobile ions. After that we simulated the interaction between the Pleckstrin homology domain (PH domain) of Akt and the cytoplasmic membrane. Taking into account the electrostatic interaction, we observe the structure changes of the membrane at different concentrations of mobile ions in its equilibrium state. Also we discuss the influence of mobile ions on the size of the pore opened in the membrane by the charged protein. Such an observation may shed light on the activation of oncogenic Akt (or protein kinase B) around the membrane at the molecular level.

The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance.

PI3K/Akt signaling is activated in cancers and governs tumor initiation and progression, but how Akt is activated under diverse stresses is poorly understood. Here we identify AMPK as an essential regulator for Akt activation by various stresses. Surprisingly, AMPK is also activated by growth factor EGF through Ca2+/Calmodulin-dependent kinase and is essential for EGF-mediated Akt activation and biological functions. AMPK phosphorylates Skp2 at S256 and promotes the integrity and E3 ligase activity of Skp2 SCF complex leading to K63-linked ubiquitination and activation of Akt and subsequent oncogenic processes. Importantly, AMPK-mediated Skp2 S256 phosphorylation promotes breast cancer progression in mouse tumor models, correlates with Akt and AMPK activation in breast cancer patients, and predicts poor survival outcomes. Finally, targeting AMPK-mediated Skp2 S256 phosphorylation sensitizes cells to anti-EGF receptor targeted therapy. Our study sheds light on how stress and EGF induce Akt activation and new mechanisms for AMPK-mediated oncogenesis and drug resistance.