AKT as Locus of Hydrogen Bond Network in Cancer

Generation and maintenance of a cancer complexity and robustness are impossible without hydrogen element. It is essential element for the cancer signaling through the AKT locus. Hyperactivated AKT locus by a positive feedback loops from the cancer hypoxic microenvironment generates a hydrogen bond network. Such network initiates protein–protein interaction at the AKT active site and at the same time stabilizes signal propagation. A hydrogen bond network conforms an entropy/enthalpy energetic process used for the interconversion of the AKT protein in metastasis formation and maintenance. Targeting the AKT locus by the redox balance change or hydrogen balance change or proton beam radiation disrupts a hydrogen bond network leading to the disappearance of a cancer complexity and robustness causing failure of the complex energy system in solid cancers and hematological malignancy. J. Cell. Biochem. 119: 130–133, 2018. © 2017 Wiley Periodicals, Inc.     

Lysosome activates AKT inducing cancer and metastasis

Hyperactivated lysosome causes cancer and induces metastasis or cancer relapse. Such activation occurs during excessive, intense, and protracted oxidative burst in the lysosome. The burst induces the formation of the constitutively active (permanently active) AKT locus generating cancer complexity and robustness. Such condition has the tendency to persist by stabilized intense signaling inducing upregulation of cell function and metabolic setup at the higher level. Most intense activator of the lysosome is the fungus Aspergillus fumigatus, which activates the AKT, a critical element in lysosome control, inducing cancer development, metastatic progression, or cancer relapse. Targeting the AKT active site of hydrogen network, by redox balance change or hydrogen balance change or muon-catalyzed fusion or laser-induced fusion with anti- A. fumigatus medication converts active AKT locus into inactive element, inducing disappearance of malignant phenotype.     

AKT as locus of fragility in robust cancer system

Metastatic cancer is a complex positive feedback loop system. Such as system has a tendency to acquire extreme robustness. Signaling pathways controlling that robustness can fail completely if an essential element from the signaling is removed. That element is a locus of fragility. Targeting that locus represents the best way to target the cancer robustness. This prospect presents another locus of fragility in signaling complex system network, controlling the cell cycle progression through the PI3K/AKT/mTOR/RAN pathway and cell migration and angiogenesis through the VEGF/PI3K/AKT/NO/ICAM-1 pathway. The locus of fragility of these pathways is AKT, which is regulated by a balance of catalase/H2O2 or by AKT inhibitor. Tiny and trivial perturbations such as change in redox state in the cells by antioxidant enzyme catalase, scavenging H2O2 signaling molecule, regulates robust signaling molecule AKT, abolishing its phosporilation and inducing cascading failure of robust signaling pathways for cell growth, proliferation, migration, and angiogenesis. An anticancer effect of the antioxidant is achieved through the AKT locus, by abolishing signals from growth factors VEGF, HGF, HIF-1alpha and H2O2. Previously reported locus of fragility nitric oxide (NO) and locus AKT are close in the complex signaling interactome network, but they regulate distinct signaling modules. Simultaneously targeted loci represents new principles in cancer robustness chemotherapy by blocking cell proliferation, migration, angiogenesis and inducing rather slow then fast apoptosis leading to slow eradication of cancer.     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.     

AKT as locus of cancer angiogenic robustness and fragility

Angiogenesis get full robustness in metastatic cancer, relapsed leukemia or lymphoma when complex positive feedback loop signaling systems become integrative. A cancer hypoxic microenvironment generates positive loops inducing formation of the vascular functional shunts. AKT is an upstream angiogenic locus of integrative robustness and fragility activated by the positive loops. AKT controls two downstream nodes the mTOR and NOS in nodal organization of the signaling genes. AKT phosphorylation is regulated by a balance of an oxidant/antioxidant. Targeting AKT locus represents new principle to control integrative angiogenic robustness by the locus chemotherapy. J. Cell. Physiol. 228: 21–24, 2013. © 2012 Wiley Periodicals, Inc.     

Insights into a highly conserved network of hydrogen bonds in the agonist binding site of nicotinic acetylcholine receptors: A structural and theoretical study

Structural and theoretical studies on the geometrical features of a hydrogen‐bond network occurring in the binding site of nicotinic acetylcholine receptors (nAChRs) and composed of interconnected WxPD (Trp‐x‐Pro‐Asp) and SWyz (Ser‐Trp‐yz) sequences from loops A and B, respectively, have been carried out. Multiple sequence alignments using as template the sequence of the apoform of Aplysia californica acetylcholine binding protein (Ac‐AChBP) show the strict conservation of serine and tryptophan residues of the loop B SWyz sequence. Considering a sample of 19 high resolution AChBP structures, the strong conformational preferences of the key tryptophan residue has been pointing out, whatever the form, free or bounded, of AChBP. The geometry of the motif hydrogen‐bond network has been characterized through the analyses of seven distances. The robustness of the various hydrogen‐bond interactions is pointed out, the one involving the aspartate carboxylate group and the serine residue being the shortest of the network. The role of a cooperative effect involving a NH(His145)…OH (Ser142) hydrogen bond is highlighted. Density functional theory calculations on several simplified models based on the motif hydrogen‐bond network allow probing the importance of the various hydrogen‐bond interactions. The removal of the Ser142 hydroxyl group induces strong structural rearrangements, in agreement with the structural observations. Molecular electrostatic potential calculations on model systems highlight the importance of a cooperative effect in the whole hydrogen‐bond network. More precisely, the key role of the Ser142 hydroxyl group, involved in several hydrogen bonds, is underlined. Proteins 2014; 82:2303–2317. © 2014 Wiley Periodicals, Inc.     

Singularity analysis of the AKT signaling pathway reveals connections between cancer and metabolic diseases

Connections between cancer and metabolic diseases may consist in the complex network of interactions among a common set of biomolecules. By applying singularity and bifurcation analysis, the phenotypes constrained by the AKT signaling pathway are identified and mapped onto the parameter space, which include cancer and certain metabolic diseases. By considering physiologic properties (sensitivity, robustness and adaptivity) the AKT pathway must possess in order to efficiently sense growth factors and nutrients, the region of normal responses is located. To optimize these properties, the intracellular concentration of the AKT protein must be sufficiently high to saturate its enzymes; the strength of the positive feedback must be stronger than that of the negative feedback. The analysis illuminates the parameter space and reveals system-level mechanisms in regulating biological functions (cell growth, survival, proliferation and metabolism) and how their deregulation may lead to the development of diseases. The analytical expressions summarize the synergistic interactions among many molecules, which provides valuable insights into therapeutic interventions. In particular, a strategy for overcoming the limitations of mTOR inhibition is proposed for cancer therapy.     

AKT as locus of cancer positive feedback loops and extreme robustness

A positive feedback loops induce extreme robustness in metastatic cancer, relapsed leukemia, myeloma or lymphoma. The loops are generated by the signaling interactome networks of autocrine and paracrine elements from cancer hypoxic microenvironment. The elements of the networks are signaling proteins synthesized in hypoxic microenvironment such as the vascular endothelial growth factor, HIF‐1α, hepatocyte growth factor, and molecules nitric oxide and H₂O₂. The signals from upstream or rebound downstream pathways are amplified by the short or wide positive feedback loops, hyperstimulating AKT‐inducing cancer extreme robustness. Targeting the phosphorylated AKT locus by an oxidant/antioxidant modulation induces collapse of positive feedback loops and establishment of negative feedback loops leading to stability of the system and disappearance of cancer extreme robustness. This is a new principle for the conversion of cancer positive loops into negative feedback loops by the locus chemotherapy. J. Cell. Physiol. 228: 522–524, 2013. © 2012 Wiley Periodicals, Inc.     

Thermodynamic consequences of disrupting a water-mediated hydrogen bond network in a protein:pheromone complex

The mouse pheromones (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT) and 6-hydroxy-6-methyl-3-heptanone (HMH) bind into an occluded hydrophobic cavity in the mouse major urinary protein (MUP-1). Although the ligands are structurally unrelated, in both cases binding is accompanied by formation of a similar buried, water-mediated hydrogen bond network between the ligand and several backbone and side chain groups on the protein. To investigate the energetic contribution of this hydrogen bond network to ligand binding, we have applied isothermal titration calorimetry to measure the binding thermodynamics using several MUP mutants and ligand analogs. Mutation of Tyr-120 to Phe, which disrupts a hydrogen bond from the phenolic hydroxyl group of Tyr-120 to one of the bound water molecules, results in a substantial loss of favorable binding enthalpy, which is partially compensated by a favorable change in binding entropy. A similar thermodynamic effect was observed when the hydrogen bonded nitrogen atom of the heterocyclic ligand was replaced by a methyne group. Several other modifications of the protein or ligand had smaller effects on the binding thermodynamics. The data provide supporting evidence for the role of the hydrogen bond network in stabilizing the complex.     

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1‐dependent inhibition of EGFR. In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context‐dependent manner. EGFR inhibition can only sensitize EGFR‐high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR‐low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR‐low cancer as a promising therapeutic approach.     

Hydrogen bonds and the catalytic mechanism of human carbonic anhydrase II

A new model for catalysis of human carbonic anhydrase II is suggested. The model is based on the X-ray structure of the hydrogen bond network in the catalytic site. The outer part of the network is proposed to adjust the p K(a) of the catalytic site to the experimentally observed value of about 7. The inner part of the network is proposed to become a low-barrier hydrogen bond network in the transition state. The energy released in forming the low-barrier hydrogen bond network is used to catalyse the interconversion of CO(2) and HCO(3)(-). The suggested molecular mechanism is consistent with the generally accepted kinetic scheme for human carbonic anhydrase II.     

Locus of fragility in robust breast cancer system

Functional heterogeneous redundancy of breast cancer makes this tumor to be robust. Signaling mechanisms which control cancer responses are crucial for controlling robustness. Identification of locus of fragility in cancer represents basic mechanism to target robustness. The goal of this prospect is to present locus of fragility in breast cancer robust system, and how disruption of this locus induces failure of robustness. My recent research show, that locus of fragility in breast cancer cells is suppression of nitric oxide (NO). When it was targeted, dynamics of cancer to generate robustness failed that it blocked cancer cell proliferation dependent on the NO/Rb pathway, blocked cell migration and angiogenesis dependent on the VEGF/PI3K/AKT/NO/ICAM-1 pathway, and induced breast cancer cell apoptosis through the NO/ROCK/FOXO3a signaling pathway. This tiny and trivial perturbation in breast cancer cells such as suppression of NO represents locus of fragility (weakness) and new approach for breast cancer chemotherapy.     

Vacuolar-type H(+)-ATPase inhibitory activity of synthetic analogues of the concanamycins: is the hydrogen bond network involving the lactone carbonyl,the hemiacetal hydroxy group,and the C-19 hydroxy group essential for the biological activity of the concanamycins?

Synthetic analogue of the concanamycins, which lacks the hydrogen bond network existing in the concanamycin structure, retains vacuolar-type H⁺-ATPase (V-ATPase) inhibitory activity and induces apoptosis to cancer cells that overexpressing epidermal growth factor receptors (EGFR).     

ALDOLASE A regulates invasion of bladder cancer cells via E-cadherin-EGFR signaling

Glycolysis and glycogenesis are known to be tightly associated with cancer cell migration. However, their roles in bladder cancer have not been reported. In this study, ALDOLASE A (ALDOA) was identified in a coexpression network generated using glycolysis- and glycogenesis-related genes in Kyoto Encyclopedia of Genes and Genomes. ALDOA was located in the central region in the network, and the cancer genome atlas (TCGA) data suggest that ALDOA expression levels are associated with viability in patients with cancer at the middle and late stages. Bladder cancer cell lines, T24 and RT4, were used to knockdown (sh) or overexpress (OE) ALODA to analyze its role. The sh-ALDOA reduced cell viability, colony formation rate, and invasion cell number; while OE had an opposite effect compared with sh-ALDOA. Further, the sh-ALDOA expression induced E-cadherin level while reduced N-cadherin and vimentin levels. The OE cells reduced E-cadherin and induced N-cadherin and vimentin levels. In addition, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and AKT serine/threonine kinase (AKT) phosphorylation levels are all reduced in sh-ALODA while activated in OE cells compared with the control group. But either sh-ALODA or OE did not change total protein levels of EGFR, MAPK, and AKT. To further analyze E-cadherin function in ALDOA regulation on bladder cancer cells, sh-ALDOA and sh-E-cadherin were cotransfected in T24 and RT4 cells. The results indicated that sh-ALDOA and sh-E-cadherin expressions eliminated sh-ALDOA function, resulting similar cell viability, colony formation rate, and invasion cell number with control group. Also, sh-ALDOA and shE-cadherin expressions increased EGFR, MAPK, and AKT phosphorylation levels; and the levels were similar to the control group. But, sh-ALDOA and sh-E-cadherin expressions did not change N-cadherin and vimentin levels, which maintain similar levels with sh-ALDOA-expressing cells. Taken together, these results suggest that ALDOA might play an important function in bladder cancer and its action may be though E-cadherin-EGFR signaling.     

EGF-ERBB signalling: towards the systems level

Signalling through the ERBB/HER receptors is intricately involved in human cancer and already serves as a target for several cancer drugs. Because of its inherent complexity, it is useful to envision ERBB signalling as a bow-tie-configured, evolvable network, which shares modularity, redundancy and control circuits with robust biological and engineered systems. Because network fragility is an inevitable trade-off of robustness, systems-level understanding is expected to generate therapeutic opportunities to intercept aberrant network activation.     

Dual inhibition of autophagy and the AKT pathway in prostate cancer

Genetic inactivation of PTEN through either gene deletion or mutation is common in metastatic prostate cancer, leading to activation of the phosphoinositide 3-kinase (PI3K-AKT) pathway, which is associated with poor clinical outcomes. The PI3K-AKT pathway plays a central role in various cellular processes supporting cell growth and survival of tumor cells. To date, therapeutic approaches to develop inhibitors targeting the PI3K-AKT pathway have failed in both pre-clinical and clinical trials. We showed that a novel AKT inhibitor, AZD5363, inhibits the AKT downstream pathway by reducing p-MTOR and p-RPS6KB/p70S6K. We specifically reported that AZD5363 monotherapy induces G2 growth arrest and autophagy, but fails to induce significant apoptosis in PC-3 and DU145 prostate cancer cell lines. Blocking autophagy using pharmacological inhibitors (3-methyladenine, chloroquine and bafilomycin A₁) or genetic inhibitors (siRNA targeting ATG3 and ATG7) enhances cell death induced by AZD5363 in these prostate cancer cells. Importantly, the combination of AZD5363 with chloroquine significantly reduces tumor volume compared with the control group, and compared with either drug alone in prostate tumor xenograft models. Taken together, these data demonstrate that AKT inhibitor AZD5363, synergizes with the lysosomotropic inhibitor of autophagy, chloroquine, to induce apoptosis and delay tumor progression in prostate cancer models that are resistant to monotherapy, with AZD5363 providing a new therapeutic approach potentially translatable to patients.     

Prediction of a highly deleterious mutation E17K in AKT-1 gene: An in silico approach

The AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a member of most frequently activated proliferation and survival signaling pathway in cancer. Recently, hyperactivation of AKT1, due to functional point mutation in the pleckstrin homology (PH) domain of AKT1 gene, has been found to be associated with human colorectal, breast and ovarian cancer. Thus, considering its crucial role in cellular signaling pathway, a functional analysis of missense mutations of AKT1 gene was undertaken in this study. Twenty nine nsSNPs (non-synonymous single nucleotide polymorphism) within coding region of AKT1 gene were selected for our investigation and six SNPs were found to be deleterious by combinatorial predictions of various computational tools. RMSD values were calculated for the mutant models which predicted four substitutions (E17K, E319G, D32E and A255T) to be highly deleterious. The insight of the structural attribute was gained through analysis of, secondary structures, solvent accessibility and intermolecular hydrogen bond analysis which confirmed one missense mutation (E17K) to be highly deleterious nsSNPs. In conclusion, the investigated gene AKT1 has twenty nine SNPs in the coding region and through progressive analysis using different bioinformatics tools one highly deleterious SNP with rs121434592 was profiled. Thus, results of this study can pave a new platform to sort nsSNPs for several important regulatory genes that can be undertaken for the confirmation of their phenotype and their correlation with diseased status in case control studies.     

Solution Structure of the Oncogenic MIEN1 Protein Reveals a Thioredoxin-Like Fold with a Redox-Active Motif

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as −225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.