Transcompp: understanding phenotypic plasticity by estimating Markov transition rates for cell state transitions.

MOTIVATION: Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. RESULTS: We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. AVAILABILITY AND IMPLEMENTATION: Freely available for download at http://github.com/nsuhasj/Transcompp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Organismal Aging and Oxidants beyond Macromolecules Damage.

The relationship between oxidants and organismal aging was first articulated through the free radical theory of aging. One of the major predictions of the free radical theory of aging is that oxidative stress shortens organisms' lifespan because of an increased level of oxidants, which are damaging to macromolecules. However, challenging the role of oxidants in age-related diseases, there is now sufficient evidence that antioxidant supplements do not provide significant health benefits. Interestingly, in addition to an increase in oxidant-mediated macromolecules damage, there is convincing experimental data to support the role of senescent cells in the process of aging. Here, the current knowledge regarding the role of oxidants and cellular senescence in organismal aging is reviewed and it is proposed that, in addition to the role of oxidants as inducers of macromolecular damage, oxidants may also function as regulators of signaling pathways involved in the establishment of cellular senescence. If this role for oxidants is established, it may be necessary to modify the free radical theory of aging from "Organisms age because cells accumulate reactive oxygen species-dependent damage over time" to: "Organisms age because cells accumulate oxidants'-dependent damage and oxidants'-dependent senescent characteristics over time."

Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress.

Although oxidative stress has been shown to induce senescence and replication stress independently, no study has implicated unresolved replication stress as the driver for cellular senescence in response to oxidative stress. Using cells exposed to increasing concentrations of hydrogen peroxide, we show that sub-lethal amount of exogenous hydrogen peroxide induces two waves of DNA damage. The first wave is rapid and transient while the second wave coincides with the cells transition from the S to the G2/M phases of cell cycle. Subsequently, cells enter growth arrest accompanied by the acquisition of senescence-associated characteristics. Furthermore, a p53-dependent decrease in Rad51, which is associated with the formation of DNA segments with chromatin alterations reinforcing senescence, and Lamin B1 that is involved in chromatin remodeling, is observed during the establishment of the senescent phenotype. On the other hand, increase in senescence associated-ß-Gal activity, a classical marker of senescence and HMGA2, a marker of the senescence-associated heterochromatin foci, is shown to be independent of p53. Together, our findings implicate replication stress-induced endogenous DNA damage as the driver for the establishment of cellular senescence upon sub-lethal oxidative stress, and implicate the role of p53 in some but not all hallmarks of the senescent phenotype.

Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts, Revealed by Comparative Modeling of Pathway Dynamics.

The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., "overshoot") was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr308 (Aktp308). The model was fit to experimental measurements of Aktp308, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp308 (at 30-60 minutes). From this we conclude that the peak and decline behavior of Aktp308 is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp308 is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient experiments.

Systematic parameter estimation in data-rich environments for cell signalling dynamics.

MOTIVATION: Computational models of biological signalling networks, based on ordinary differential equations (ODEs), have generated many insights into cellular dynamics, but the model-building process typically requires estimating rate parameters based on experimentally observed concentrations. New proteomic methods can measure concentrations for all molecular species in a pathway; this creates a new opportunity to decompose the optimization of rate parameters. RESULTS: In contrast with conventional parameter estimation methods that minimize the disagreement between simulated and observed concentrations, the SPEDRE method fits spline curves through observed concentration points, estimates derivatives and then matches the derivatives to the production and consumption of each species. This reformulation of the problem permits an extreme decomposition of the high-dimensional optimization into a product of low-dimensional factors, each factor enforcing the equality of one ODE at one time slice. Coarsely discretized solutions to the factors can be computed systematically. Then the discrete solutions are combined using loopy belief propagation, and refined using local optimization. SPEDRE has unique asymptotic behaviour with runtime polynomial in the number of molecules and timepoints, but exponential in the degree of the biochemical network. SPEDRE performance is comparatively evaluated on a novel model of Akt activation dynamics including redox-mediated inactivation of PTEN (phosphatase and tensin homologue). AVAILABILITY AND IMPLEMENTATION: Web service, software and supplementary information are available at www.LtkLab.org/SPEDRE SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The small GTPase Rac1 is a novel binding partner of Bcl-2 and stabilizes its antiapoptotic activity.

The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase-fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.

A superoxide-driven redox state promotes geroconversion and resistance to senolysis in replication-stress associated senescence.

Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with replication stress-induced senescence termed senescence-associated redox state (SA-redox state). SA-redox state is characterized by its reactivity with superoxide-sensing fluorescent probes such as dihydroethidine, lucigenin and mitosox and peroxynitrite or hydroxyl radical sensing probe hydroxyphenyl fluorescein (HPF) but not the hydrogen peroxide (H2O2) reactive fluorescent probe CM-H2DCFDA. Measurement of GSH and GSSH also reveals that SA-redox state mitigates the level of total GSH rather than oxidizes GSH to GSSG. Moreover, supporting the role of superoxide (O2.-) in the SA-redox state, we show that incubation of senescent RPE1-hTERT cells with the O2.- scavenger, Tiron, decreases the reactivity of SA-redox state with the oxidants' reactive probes lucigenin and HPF while the H2O2 antioxidant N-acetyl cysteine has no effect. SA-redox state does not participate in the loss of proliferative capacity, G2/M cell cycle arrest or the increase in SA-ß-Gal activity. However, SA-redox state is associated with the activation of NF-κB, dictates the profile of the Senescence Associated Secretory Phenotype, increases TFEB protein level, promotes geroconversion evidenced by increased phosphorylation of S6K and S6 proteins, and influences senescent cells response to senolysis. Furthermore, we provide evidence for crosstalk between SA redox state, p53 and p21. While p53 mitigates the establishment of SA-redox state, p21 is critical for the sustained reinforcement of the SA-redox state involved in geroconversion and resistance to senolysis.

Therapeutic Potential of a Senolytic Approach in a Murine Model of Chronic GVHD.

Graft-versus-host disease (GVHD) is a life-threatening systemic complication of allogeneic hematopoietic stem cell transplantation (HSCT) characterized by dysregulation of T and B cell activation and function, scleroderma-like features, and multi-organ pathology. The treatment of cGVHD is limited to the management of symptoms and long-term use of immunosuppressive therapy, which underscores the need for developing novel treatment approaches. Notably, there is a striking similarity between cytokines/chemokines responsible for multi-organ damage in cGVHD and pro-inflammatory factors, immune modulators, and growth factors secreted by senescent cells upon the acquisition of senescence-associated secretory phenotype (SASP). In this pilot study, we questioned the involvement of senescent cell-derived factors in the pathogenesis of cGVHD triggered upon allogeneic transplantation in an irradiated host. Using a murine model that recapitulates sclerodermatous cGVHD, we investigated the therapeutic efficacy of a senolytic combination of dasatinib and quercetin (DQ) administered after 10 days of allogeneic transplantation and given every 7 days for 35 days. Treatment with DQ resulted in a significant improvement in several physical and tissue-specific features, such as alopecia and earlobe thickness, associated with cGVHD pathogenesis in allograft recipients. DQ also mitigated cGVHD-associated changes in the peripheral T cell pool and serum levels of SASP-like cytokines, such as IL-4, IL-6 and IL-8Rα. Our results support the involvement of senescent cells in the pathogenesis of cGVHD and provide a rationale for the use of DQ, a clinically approved senolytic approach, as a potential therapeutic strategy.

The redox-senescence axis and its therapeutic targeting.

SIGNIFICANCE: Cellular growth arrest, associated with 'senescence', helps to safeguard against the accumulation of DNA damage which is often recognized as the underlying mechanism of a wide variety of age-related pathologies including cancer. Cellular senescence has also been described as a 'double-edged sword'. In cancer, for example, the creation of an immune-suppressive milieu by senescent tumor cells through the senescence-associated secretory phenotype contributes toward carcinogenesis and cancer progression. RECENT ADVANCES: The potential for cellular senescence to confer multi-faceted effects on tissue fate has led to a rejuvenated interest in its landscape and targeting. Interestingly, redox pathways have been described as both triggers and propagators of cellular senescence, leading to intricate cross-links between both pathways. CRITICAL ISSUES: In this review, we describe the mechanisms driving cellular senescence, the interface with cellular redox metabolism as well as the role that chemotherapy-induced senescence plays in secondary carcinogenesis. Notably, the role that anti-apoptotic proteins of the Bcl-2 family play in inducing drug resistance via mechanisms that involve senescence induction. FUTURE DIRECTIONS: Though the therapeutic targeting of senescent cells as cancer therapy remains in its infancy, we summarize the current development of senotherapeutics, including recognized senotherapies, as well as the repurposing of drugs as senomorphic/senolytic candidates.

Superoxide induced inhibition of death receptor signaling is mediated via induced expression of apoptosis inhibitory protein cFLIP.

The death inhibitory proteins, cFLIP and Bcl-2, canonically act at different steps to regulate receptor-mediated apoptosis in cancer cells. Here we report that pharmacological or genetic means to effect an increase in intracellular superoxide result in cFLIP upregulation. Interestingly, Bcl-2 overexpression is associated with a concomitant increase in cFLIP, and reducing superoxide sensitizes Bcl-2 overexpressing cancer cells to receptor-mediated apoptosis via downregulation of cFLIP. Moreover, inhibiting glycolytic flux overcomes apoptosis resistance by superoxide-dependent downregulation of cFLIP. Superoxide-induced upregulation of cFLIP is a function of enhanced transcription, as evidenced by increases in cFLIP promoter activity and mRNA abundance. The positive effect of superoxide on cFLIP is mediated through its reaction with nitric oxide to generate peroxynitrite. Corroborating these findings in cell lines, subjecting primary cells derived from lymphoma patients to glucose deprivation ex vivo, as a means to decrease superoxide, not only reduced cFLIP expression but also significantly enhanced death receptor sensitivity. Based on this novel mechanistic insight into the redox regulation of cancer cell fate, modulation of intracellular superoxide could have potential therapeutic implications in cancers in which these two death inhibitory proteins present a therapeutic challenge.

Computational identification and experimental validation of PPRE motifs in NHE1 and MnSOD genes of human.

BACKGROUND: Activation of PPARs has been reported to inhibit the proliferation of malignant cells from different lineages. They are involved in transcription regulation of genes upon activation by a ligand. The binding of PPARs to the promoter sequence either represses or activates the gene. Hence, PPARs represent promising targets for cancer treatment because of their anti-proliferative and pro-apoptotic activities. Here we computationally identified PPAR binding regions in NHE1 and MnSOD. We further validated the predictions in vitro. RESULTS: Our results computationally predicted the presence of 2 PPRE motifs in NHE1 and 3 PPRE motifs in MnSOD. We experimentally confirmed the true motifs and their regulation by PPAR. CONCLUSION: Our results suggest that both NHE1 and MnSOD have PPRE binding motif in their upstream/promoter region and hence are regulated by PPAR upon ligand binding.

Metabolic reprogramming of oncogene-addicted cancer cells to OXPHOS as a mechanism of drug resistance.

The ability to selectively eradicate oncogene-addicted tumors while reducing systemic toxicity has endeared targeted therapies as a treatment strategy. Nevertheless, development of acquired resistance limits the benefits and durability of such a regime. Here we report evidence of enhanced reliance on mitochondrial oxidative phosphorylation (OXPHOS) in oncogene-addicted cancers manifesting acquired resistance to targeted therapies. To that effect, we describe a novel OXPHOS targeting activity of the small molecule compound, OPB-51602 (OPB). Of note, a priori treatment with OPB restored sensitivity to targeted therapies. Furthermore, cancer cells exhibiting stemness markers also showed selective reliance on OXPHOS and enhanced sensitivity to OPB. Importantly, in a subset of patients who developed secondary resistance to EGFR tyrosine kinase inhibitor (TKI), OPB treatment resulted in decrease in metabolic activity and reduction in tumor size. Collectively, we show here a switch to mitochondrial OXPHOS as a key driver of targeted drug resistance in oncogene-addicted cancers. This metabolic vulnerability is exploited by a novel OXPHOS inhibitor, which also shows promise in the clinical setting.

Superoxide anion: oncogenic reactive oxygen species?

Recent evidence linking intracellular reactive oxygen species to cell survival and/or proliferation signals has resulted in a paradigm shift from the age-old dogma implicating reactive oxygen species exclusively in cell damage and death. It is now accepted that reactive oxygen species play important roles in normal physiological states and that depending on the species involved the effect could be highly varied. In this regard, the effects of the two major reactive oxygen species, superoxide and hydrogen peroxide have been extensively studied. During normal cell growth a tight balance between the two species is kept under check by the cells' anti-oxidant defense systems. Deficiency or defect in this defense armory is invariably associated with neoplasia, thus rendering the intracellular redox status in a state of imbalance and generating a "pro-oxidant" milieu. A variety of model systems have underscored the relationship between a pro-oxidant state and cancer promotion and progression. In this review, we present evidence to support the hypothesis that the effect of intracellular reactive oxygen species on oncogenesis is dependent on the ratio of intracellular superoxide to hydrogen peroxide in that a predominant increase in superoxide supports cell survival and promotes oncogenesis whereas a tilt in favor of hydrogen peroxide prevents carcinogenesis by facilitating cell death signaling.

Phosphorylation of the survival kinase Akt by superoxide is dependent on an ascorbate-reversible oxidation of PTEN.

In this report, we demonstrate that in serum-deprived mouse embryonic fibroblasts an increase in intracellular level of superoxide through pharmacological inhibition of the Cu/ZnSOD protein or the down-regulation of its expression using specific siRNA mimics growth factor-induced phosphorylation of Akt. Using the PI3K inhibitor LY294002 and PTEN knockout mouse embryonic fibroblasts, we show that phosphorylation of Akt by superoxide requires the production of PIP3 and that the target for the induction of Akt phosphorylation by O2.- is the phosphatase PTEN. Interestingly, the inhibition of PTEN involves an O2.--mediated oxidation of the phosphatase rather than regulation of its phosphorylation or decreased protein expression. Moreover, using differential reduction of oxidized protein by DTT and ascorbate, O2.--dependent oxidation of PTEN is shown to be due to S-nitrosylation of the protein. Finally, exposure of serum-deprived mouse embryonic fibroblasts to fetal bovine serum leads to a rapid and strong phosphorylation of Akt that is dependent on an ascorbate-reversible O2.--mediated oxidation of PTEN. These results support O2.- as a physiologically relevant second messenger for Akt activation through S-nitrosylation of PTEN and offer a mechanistic explanation for the mitogenic and prosurvival activities of O2.-.

A decompositional approach to parameter estimation in pathway modeling: a case study of the Akt and MAPK pathways and their crosstalk.

Parameter estimation is a critical problem in modeling biological pathways. It is difficult because of the large number of parameters to be estimated and the limited experimental data available. In this paper, we propose a decompositional approach to parameter estimation. It exploits the structure of a large pathway model to break it into smaller components, whose parameters can then be estimated independently. This leads to significant improvements in computational efficiency. We present our approach in the context of Hybrid Functional Petri Net modeling and evolutionary search for parameter value estimation. However, the approach can be easily extended to other modeling frameworks and is independent of the search method used. We have tested our approach on a detailed model of the Akt and MAPK pathways with two known and one hypothesized crosstalk mechanisms. The entire model contains 84 unknown parameters. Our simulation results exhibit good correlation with experimental data, and they yield positive evidence in support of the hypothesized crosstalk between the two pathways.

Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells.

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence, this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes, Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution, the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay, which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2% vs. 9.9%, p > 0.05). However, when 100 mM Z-VAD-FMK was added to the post-thaw culture media, there was a significant enhancement in the survival rate from 9.9% to 14.4% (p < 0.05), which was further increased to 18.7% when Z-VAD-FMK was also added to the freezing solution as well (p < 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture.

Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

Here we provide evidence to link sub-lethal oxidative stress to lysosome biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB-dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosome biogenesis under conditions of sub-lethal oxidative stress.

Resveratrol inhibits drug-induced apoptosis in human leukemia cells by creating an intracellular milieu nonpermissive for death execution.

Efficient apoptotic signaling is a function of a permissive intracellular milieu created by a decrease in the ratio of superoxide to hydrogen peroxide and cytosolic acidification. Resveratrol (RSV) triggers apoptosis in some systems and inhibits the death signal in others. In this regard, the inhibitory effect on hydrogen peroxide-induced apoptosis is attributed to its antioxidant property. We provide evidence that exposure of human leukemia cells to low concentrations of RSV (4-8 micro M) inhibits caspase activation, DNA fragmentation, and translocation of cytochrome c induced by hydrogen peroxide or anticancer drugs C2, vincristine, and daunorubicin. Interestingly, at these concentrations, RSV induces an increase in intracellular superoxide and inhibits drug-induced acidification. Blocking the activation of NADPH oxidase complex neutralized RSV-induced inhibition of apoptosis. Furthermore, our results implicate intracellular hydrogen peroxide as a common effector mechanism in drug-induced apoptosis that is inhibited by preincubation with RSV. Interestingly, decreasing intracellular superoxide with the NADPH oxidase inhibitor diphenyliodonium reversed the inhibitory effect of RSV on drug-induced hydrogen peroxide production. These data show that low concentrations of RSV inhibit death signaling in human leukemia cells via NADPH oxidase-dependent elevation of intracellular superoxide that blocks mitochondrial hydrogen peroxide production, thereby resulting in an intracellular environment nonconducive for death execution.