Invasiveness of a Growth-Migration System in a Two-dimensional Percolation cluster: A Stochastic Mathematical Approach.

We developed a framework based on the software Unstructured Reaction-Diffusion Master Equation (URDME) to address tumor cells' proliferation and migration in a heterogeneous space, herein a 2D percolation cluster. A mitogenic paracrine signaling pathway is utilized phenomenologically to reveal how cells cooperate with one another. We modeled the emerging Allee effect using low seeding density culture (LSDC) assays to fit the model parameters. A Finite time scaling (FTS) function has been formulated to quantitatively analyze invasiveness of a virtual Growth-Migration (GM) system in mimicking the cancer cell growth. Through such simulation, we analyzed the GM dynamics of virtual model in mimicking the growth of BT-474 cancer cell populations in vitro in a 2D percolation cluster and calculated the successful penetration rate (SPR). By analyzing the temporal trajectories of the SPR, we could determine the critical exponents of the critical SPR scaling relation. The SPR transition point ([Formula: see text]), which is a fundamentally different from a conventional percolation transition point, is found to be negatively correlated with the invasiveness of this cancer cell. The [Formula: see text] of the three variations of the virtual GM system distinctly designated by varying paracrine-regulated Allee (PAllee) model phenotypes is 0.3408, 0.3675, and 0.4454, respectively. FTS algorithm thereon may serve as an approach to quantify invasiveness of tumor cells. Through a phenomenological paracrine model, inter-cell cooperation and mutual mitogenic boosting are enabled to elicit the Allee effect in the GM systems. The rationale behind such computationally tunable virtual mechanism can be applied to other circumstances concerning emerging processes.

Playing cancer at its own game: activating mitogenic signaling as a paradoxical intervention.

In psychotherapy, paradoxical interventions are characterized by a deliberate reinforcement of the pathological behavior to improve the clinical condition. Such a counter-intuitive approach can be considered when more conventional interventions fail. The development of targeted cancer therapies has enabled the selective inhibition of activated oncogenic signaling pathways. However, in advanced cancers, such therapies, on average, deliver modest benefits due to the development of resistance. Here, we review the perspective of a 'paradoxical intervention' in cancer therapy: rather than attempting to inhibit oncogenic signaling, the proposed therapy would further activate mitogenic signaling to disrupt the labile homeostasis of cancer cells and overload stress response pathways. Such overactivation can potentially be combined with stress-targeted drugs to kill overstressed cancer cells. Although counter-intuitive, such an approach exploits intrinsic and ubiquitous differences between normal and cancer cells. We discuss the background underlying this unconventional approach and how such intervention might address some current challenges in cancer therapy.

Disentangling Pro-mitotic Signaling during Cell Cycle Progression using Time-Resolved Single-Cell Imaging.

Cells rely on input from extracellular growth factors to control their proliferation during development and adult homeostasis. Such mitogenic inputs are transmitted through multiple signaling pathways that synergize to precisely regulate cell cycle entry and progression. Although the architecture of these signaling networks has been characterized in molecular detail, their relative contribution, especially at later cell cycle stages, remains largely unexplored. By combining quantitative time-resolved measurements of fluorescent reporters in untransformed human cells with targeted pharmacological inhibitors and statistical analysis, we quantify epidermal growth factor (EGF)-induced signal processing in individual cells over time and dissect the dynamic contribution of downstream pathways. We define signaling features that encode information about extracellular ligand concentrations and critical time windows for inducing cell cycle transitions. We show that both extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) activity are necessary for initial cell cycle entry, whereas only PI3K affects the duration of S phase at later stages of mitogenic signaling.

EGFR signaling promotes resistance to CHK1 inhibitor prexasertib in triple negative breast cancer.

Aim: Innate resistance to the CHK1 inhibitor prexasertib has been described, but resistance mechanisms are not understood. We aimed to determine the role epidermal growth factor receptor (EGFR) plays in innate resistance to prexasertib in triple negative breast cancer (TNBC). Methods: Using a panel of pre-clinical TNBC cell lines, we measured the sensitivity to prexasertib. We examined the effect activation of EGFR had on prexasertib sensitivity. We measured the synergy of dual blockade of EGFR with erlotinib and CHK1 with prexasertib in TNBC cell lines and xenografts. Results: EGFR overexpression and activation increased resistance to CHK1 inhibition by prexasertib. EGFR promoted the phosphorylation of BCL2-associated agonist of cell death (BAD), inactivating its pro-apoptotic functions. Inhibition of EGFR reversed BAD phosphorylation, increasing sensitivity to prexasertib. Conclusion: The use of prexasertib as a monotherapy in TNBC has been limited due to modest clinical responses. We demonstrated that EGFR activation contributes to innate resistance to prexasertib in TNBC and potentially other cancers. EGFR expression status should be considered in clinical trials examining prexasertib's use as a monotherapy or combination therapy.

Fibroblast Growth Factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors.

In malignant transformation, cellular stress-response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress-targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K-Ras-driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response-checkpoint inhibitors triggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK-ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress-targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies.

Fibroblast growth factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors

In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

Potential therapeutic targets for growth arrest of colorectal cancer cells exposed to PTHrP.

Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment.

Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

Hydrogen peroxide (H2O2) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H2O2-reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion models of H2O2 signaling. The results show that at H2O2 supply rates commensurate with mitogenic signaling a H2O2 concentration gradient with a length scale of a few tenths of µm is established. Even near the supply sites H2O2 concentrations are far too low to oxidize typical targets in an early mitogenic signaling time frame. Furthermore, any inhibition of the peroxiredoxin or increase in H2O2 supply able to drastically increase the local H2O2 concentration would collapse the concentration gradient and/or cause an extensive oxidation of the peroxiredoxins I and II, inconsistent with experimental observations. In turn, the local concentrations of peroxiredoxin sulfenate and disulfide forms exceed those of H2O2 by several orders of magnitude. Redox targets reacting with these forms at rate constants much lower than that for, say, thioredoxin could be oxidized within seconds. Moreover, the spatial distribution of the concentrations of these peroxiredoxin forms allows them to reach targets within 1 µm from the H2O2 sites while maintaining signaling localized. The recruitment of peroxiredoxins to specific sites such as caveolae can dramatically increase the local concentrations of the sulfenic and disulfide forms, thus further helping these species to outcompete H2O2 for the oxidation of redox targets. Altogether, these results suggest that H2O2 signaling is mediated by localized redox relays whereby peroxiredoxins are oxidized to sulfenate and disulfide forms at H2O2 supply sites and these forms in turn oxidize the redox targets near these sites.

Structural Basis for Selective Interaction between the ESCRT Regulator HD-PTP and UBAP1.

Endosomal sorting complexes required for transport (ESCRTs) are essential for ubiquitin-dependent degradation of mitogenic receptors, a process often compromised in cancer pathologies. Sorting of ubiquinated receptors via ESCRTs is controlled by the tumor suppressor phosphatase HD-PTP. The specific interaction between HD-PTP and the ESCRT-I subunit UBAP1 is critical for degradation of growth factor receptors and integrins. Here, we present the structural characterization by X-ray crystallography and double electron-electron resonance spectroscopy of the coiled-coil domain of HD-PTP and its complex with UBAP1. The coiled-coil domain adopts an unexpected open and rigid conformation that contrasts with the closed and flexible coiled-coil domain of the related ESCRT regulator Alix. The HD-PTP:UBAP1 structure identifies the molecular determinants of the interaction and provides a molecular basis for the specific functional cooperation between HD-PTP and UBAP1. Our findings provide insights into the molecular mechanisms of regulation of ESCRT pathways that could be relevant to anticancer therapies.

Silibinin and its 2,3-dehydro-derivative inhibit basal cell carcinoma growth via suppression of mitogenic signaling and transcription factors activation.

Basal cell carcinoma (BCC) is the most common cancer worldwide, and its current treatment options are insufficient and toxic. Surprisingly, unlike several other malignancies, chemopreventive efforts against BCC are almost lacking. Silibinin, a natural agent from milk thistle seeds, has shown strong efficacy against several cancers including ultraviolet radiation-induced skin (squamous) cancer; however, its potential activity against BCC is not yet examined. Herein, for the first time, we report the efficacy of silibinin and its oxidation product 2,3-dehydrosilibinin (DHS) against BCC both in vitro and in vivo using ASZ (p53 mutated) and BSZ (p53 deleted) cell lines derived from murine BCC tumors. Both silibinin and DHS significantly inhibited cell growth and clonogenicity while inducing apoptosis in a dose- and time-dependent manner, with DHS showing higher activity at lower concentrations. Both agents also inhibited the mitogenic signaling by reducing EGFR, ERK1/2, Akt, and STAT3 phosphorylation and suppressed the activation of transcription factors NF-κB and AP-1. More importantly, in an ectopic allograft model, oral administration of silibinin and DHS (200 mg/kg body weight) strongly inhibited the ASZ tumor growth by 44% and 71% (P < 0.05), respectively, and decreased the expression of proliferation biomarkers (PCNA and cyclin D1) as well as NF-κB p50 and c-Fos in the tumor tissues. Taken together, these results provide the first evidence for the efficacy and usefulness of silibinin and its derivative DHS against BCC, and suggest the need for additional studies with these agents in pre-clinical and clinical BCC chemoprevention and therapy models.

TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases.

NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.

An Overview of Ultraviolet B Radiation-Induced Skin Cancer Chemoprevention by Silibinin.

Skin cancer incidences are rising worldwide, and one of the major causative factors is excessive exposure to solar ultraviolet radiation (UVR). Annually, ~5 million skin cancer patients are treated in United States, mostly with nonmelanoma skin cancer (NMSC), which is also frequent in other Western countries. As sunscreens do not provide adequate protection against deleterious effects of UVR, additional and alternative chemoprevention strategies are urgently needed to reduce skin cancer burden. Over the last couple of decades, extensive research has been conducted to understand the molecular basis of skin carcinogenesis, and to identifying novel agents which could be useful in the chemoprevention of skin cancer. In this regard, several natural non-toxic compounds have shown promising efficacy in preventing skin carcinogenesis at initiation, promotion and progression stages, and are considered important in better management of skin cancer. Consistent with this, we and others have studied and established the notable efficacy of natural flavonolignan silibinin against UVB-induced skin carcinogenesis. Extensive pre-clinical animal and cell culture studies report strong anti-inflammatory, anti-oxidant, DNA damage repair, immune-modulatory and anti-proliferative properties of silibinin. Molecular studies have identified that silibinin targets pleotropic signaling pathways including mitogenic, cell cycle, apoptosis, autophagy, p53, NF-κB, etc. Overall, the skin cancer chemopreventive potential of silibinin is well supported by comprehensive mechanistic studies, suggesting its greater use against UV-induced cellular damages and photocarcinogenesis.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells.

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca²âº-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation".This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo.Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.

Advanced glycation end products are mitogenic signals and trigger cell cycle reentry of neurons in Alzheimer's disease brain.

Neurons that reenter the cell cycle die rather than divide, a phenomenon that is associated with neurodegeneration in Alzheimer's disease (AD). Reexpression of cell-cycle related genes in differentiated neurons in AD might be rooted in aberrant mitogenic signaling. Because microglia and astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque components or factors secreted from plaque-activated glia induce neuronal mitogenic signaling. Advanced glycation end products (AGEs), protein-bound oxidation products of sugar, might be one of those mitogenic compounds. Cyclin D1 positive neurons are colocalized with AGEs or directly surrounded by extracellular AGE deposits in AD brain. However, a direct proof of DNA replication in these cells has been missing. Here, we report by using fluorescent in situ hybridization that consistent with the expression of cell cycle proteins, hyperploid neuronal cells are in colocalization with AGE staining in AD brains but not in nondemented controls. To complement human data, we used apolipoprotein E-deficient mice as model of neurodegeneration and showed that increased oxidative stress caused an intensified neuronal deposition of AGEs, being accompanied by an activation of the MAPK cascade via RAGE. This cascade, in turn, induced the expression of cyclin D1 and DNA replication. In addition, reduction of oxidative stress by application of α-lipoic acid decreased AGE accumulations, and this decrease was accompanied by a reduction in cell cycle reentry and a more euploid neuronal genome.

Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells.

Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p<0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro-angiogenic (VEGF, iNOS), migratory (MMP-2) and EMT promoting proteins (uPA, vimentin, N-cadherin) were up-regulated by IR in PCa cells. Interestingly, all of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR-activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3, by silibinin.

The paired basic amino acid-cleaving enzyme 4 (PACE4) is involved in the maturation of insulin receptor isoform B: an opportunity to reduce the specific insulin receptor-dependent effects of insulin-like growth factor 2 (IGF2).

Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects.

Oncofinder, a new method for the analysis of intracellular signaling pathway activation using transcriptomic data.

We propose a new biomathematical method, OncoFinder, for both quantitative and qualitative analysis of the intracellular signaling pathway activation (SPA). This method is universal and may be used for the analysis of any physiological, stress, malignancy and other perturbed conditions at the molecular level. In contrast to the other existing techniques for aggregation and generalization of the gene expression data for individual samples, we suggest to distinguish the positive/activator and negative/repressor role of every gene product in each pathway. We show that the relative importance of each gene product in a pathway can be assessed using kinetic models for "low-level" protein interactions. Although the importance factors for the pathway members cannot be so far established for most of the signaling pathways due to the lack of the required experimental data, we showed that ignoring these factors can be sometimes acceptable and that the simplified formula for SPA evaluation may be applied for many cases. We hope that due to its universal applicability, the method OncoFinder will be widely used by the researcher community.

Precocious cerebellum development and improved motor functions in mice lacking the astrocyte cilium-, patched 1-associated Gpr37l1 receptor.

In the developing cerebellum, the proliferation and differentiation of glial and neuronal cell types depend on the modulation of the sonic hedgehog (Shh) signaling pathway. The vertebrate G-protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a putative G-protein-coupled receptor that is expressed in newborn and adult cerebellar Bergmann glia astrocytes. This study shows that the ablation of the murine Gpr37l1 gene results in premature down-regulation of proliferation of granule neuron precursors and precocious maturation of Bergmann glia and Purkinje neurons. These alterations are accompanied by improved adult motor learning and coordination. Gpr37l1(-/-) mice also exhibit specific modifications of the Shh signaling cascade. Specific assays show that in Bergmann glia cells Gpr37l1 is associated with primary cilium membranes and it specifically interacts and colocalizes with the Shh primary receptor, patched 1. These findings indicate that the patched 1-associated Gpr37l1 receptor participates in the regulation of postnatal cerebellum development by modulating the Shh pathway.