GSK-3α aggravates inflammation, metabolic derangement, and cardiac injury post-ischemia/reperfusion.

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling. KEY MESSAGES: GSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R). GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R). GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R. GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer.

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFß and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

Interactomes of Glycogen Synthase Kinase-3 Isoforms.

Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms are highly similar in structure and are largely redundant, though there is also evidence for specific roles. Identification of isoform-specific protein interactors may elucidate the differences in function and provide insight into isoform-selective regulation. We therefore sought to identify novel GSK-3 interaction partners and to examine differences in the interactomes of the two isoforms using both affinity purification and proximity-dependent biotinylation (BioID) mass spectrometry methods. While the interactomes of the two isomers are highly similar in HEK293 cells, BioID in HeLa cells yielded a variety of preys that are preferentially associated with one of the two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored GSK-3ß, were evaluated for reciprocal interactions. The differences in interactions between isoforms may help in understanding the distinct functions and regulation of the two isoforms as well as offer avenues for the development of isoform-specific strategies.

Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

Specific Role for GSK3α in Limiting Long-Term Potentiation in CA1 Pyramidal Neurons of Adult Mouse Hippocampus.

Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of several hundred proteins, and its aberrant activity is associated with an array of prevalent disorders. The two paralogs, GSK3α and GSK3ß, are expressed ubiquitously and fulfill common as well as unique tasks throughout the body. In the CNS, it is established that GSK3 is involved in synaptic plasticity. However, the relative roles of GSK3 paralogs in synaptic plasticity remains controversial. Here, we used hippocampal slices obtained from adult mice to determine the role of each paralog in CA3-CA1 long-term potentiation (LTP) of synaptic transmission, a form of plasticity critically required in learning and memory. Conditional Camk2a Cre-driven neuronal deletion of the Gsk3a gene, but not Gsk3b, resulted in enhanced LTP. There were no changes in basal synaptic function in either of the paralog-specific knockouts, including several measures of presynaptic function. Therefore, GSK3α has a specific role in serving to limit LTP in adult CA1, a postsynaptic function that is not compensated by GSK3ß.

Novel GSK-3 kinase inhibitor Pym-5 induces GSK-3ß rather than GSK-3α-dependent melanogenesis in murine melanoma cells.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) inhibitors are considered to activate Wnt/ß-Catenin, which remains a controversial topic in melanoma treatment. OBJECTIVE: Here, we have developed Pym-5, an attractive GSK-3 inhibitor. Using Pym-5 as a chemical tool to probe the GSK-3 biology, we aimed to investigate the potential of GSK-3 inhibition as a strategy of melanoma treatment and underlying mechanisms. METHODS: Using pigment B16 and B16BL6 murine melanoma model in vitro and a zebrafish pigmentation model in vivo, we investigated Pym-5-meditaed activation of Wnt/ß-Catenin, melanogenesis and antitumor response in melanoma treatment. RESULTS: We found that Pym-5 delayed the growth and promoted melanogenesis of melanoma cells. Pym-5 activated the transcription of ß-Catenin and responsive targets genes (AXIN2 and MITF), melanin biosynthesis genes (TYR, TYRP1 and TYRP2) and eventually elevated the production of melanin. Interestingly, genetic inactivation of GSK-3ß, but not its paralogue GSK-3α, compromised Pym-5-mediated melanogenesis in B16 and B16BL6 cells. CONCLUSION: These data provide insight into the potential therapeutic benefits obtained from activation of Wnt/ß-Catenin signaling pathway and how Pym-5 can regulate melanin production and the rationale for future clinical application of GSK-3 inhibitor in melanoma patients.

Cardiomyocyte-GSK-3ß deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms.

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here, we sought to identify the potential roles of glycogen synthase kinase-3ß (GSK-3ß), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3ß knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension and contractile functions were comparable at 2 weeks post-MI, cKO mice displayed significantly preserved LV chamber and contractile function versus control mice at 4 weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at 4 and 6 weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage, as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3ß profoundly suppresses the expression and secretion of growth factors, including basic-fibroblast growth factor, angiopoietin-2, erythropoietin, stem cell factor, platelet-derived growth factor-BB, granulocyte colony-stimulating factor, and vascular endothelial growth factor, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3ß promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte-GSK-3ß could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

GSK-3 mediates nuclear translocation of p62/SQSTM1 in MPTP-induced mouse model of Parkinson's disease.

p62/SQSTM1 is a multifunctional, cytoplasmic protein with fundamental roles in autophagy and antioxidant responses. Here we showed that p62 translocated from the cytoplasm to the nucleus in nigral dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrid (MPTP)-induced mouse model of Parkinson's disease (PD). We found that p62 was physically associated with glycogen synthase kinase (GSK)-3ß, a serine/threonine protein kinase implicated in dopaminergic neurodegeneration in PD, and that MPTP treatment promoted dissociation of the complex in mice. Conditional knockout of GSK-3 prevented nuclear translocation of p62, suggesting that this translocation was detrimental to dopaminergic neurons. p62 knockout mice were used to investigate the role of p62 in MPTP-induced neuronal death. Knockout of p62 aggravated neuronal injury induced by MPTP intoxication, suggesting that p62 plays an important role in dopaminergic cell survival in stress conditions. Together, our data demonstrate that GSK-3 mediates nuclear translocation of p62 during MPTP-induced parkinsonian neurodegeneration. These findings shed new light on the role of the cytoplasmic-nuclear shuttling of p62 and the mechanism underlying GSK-3-depedent neuronal death in PD pathogenesis.

Glycogen synthase kinase 3 alpha/beta deletion induces precocious growth plate remodeling in mice.

Glycogen synthase kinase (GSK) 3 acts to negatively regulate multiple signaling pathways, including canonical Wnt signaling. The two mammalian GSK3 proteins (alpha and beta) are at least partially redundant. While Gsk3a KO mice are viable and display a metabolic phenotype, abnormal neuronal development, and accelerated aging, Gsk3b KO animals die late in embryogenesis or at birth. Selective Gsk3b KO in bone delays development of some bones, whereas cartilage-specific Gsk3b KO mice are normal except for elevated levels of GSK3A protein. However, the collective role of these two GSK3 proteins in cartilage was not evaluated. To address this, we generated tamoxifen-inducible, cartilage-specific Gsk3a/Gsk3b KO (described as "cDKO") in juvenile mice and investigated their skeletal phenotypes. We found that cartilage-specific Gsk3a/Gsk3b deletion in young, skeletally immature mice causes precocious growth plate (GP) remodeling, culminating in shorter long bones and hence, growth retardation. These mice exhibit inefficient breathing patterns at later stages and fail to survive. The disrupted GP in cDKO mice showed progressive loss of cellular and proteoglycan components, and immunostaining for SOX9, while BGLAP (osteocalcin) and COL2A1 increased. In addition, we observed increased osteoclast recruitment and cell apoptosis. Surprisingly, changes in articular cartilage of cDKO mice were mild compared with the GP, signifying differential regulation of articular cartilage vs GP tissues. Taken together, these findings emphasize a crucial role of two GSK3 proteins in skeletal development, in particular in the maintenance and function of GP. KEY MESSAGES: • Both GSK3 genes, together, are crucial regulators of growth plate remodeling. • Cartilage-specific deletion of both GSK3 genes causes skeletal growth retardation. • Deletion of both GSK3 genes decreases Sox9 levels and promotes chondrocyte apoptosis. • Cartilage-specific GSK3 deletion in juvenile mice culminates in premature lethality. • GSK3 deletion exhibits mild effects on articular cartilage compared to growth plate.

Glycogen synthase kinase-3ß inhibits tubular regeneration in acute kidney injury by a FoxM1-dependent mechanism.

Acute kidney injury (AKI) is characterized by injury to the tubular epithelium that leads to the sudden loss of renal function. Proper tubular regeneration is essential to prevent progression to chronic kidney disease. In this study, we examined the role of FoxM1, a forkhead box family member transcription factor in tubular repair after AKI. Renal FoxM1 expression increased after renal ischemia/reperfusion (I/R)-induced AKI in mouse kidneys. Treatment with thiostrepton, a FoxM1 inhibitor, reduced FoxM1 regulated pro-proliferative factors and cell proliferation in vitro, and tubular regeneration in mouse kidneys after AKI. Glycogen synthase kinase-3 (GSK3) was found to be an upstream regulator of FoxM1 because GSK3 inhibition or renal tubular GSK3ß gene deletion significantly increased FoxM1 expression, and improved tubular repair and renal function. GSK3 inactivation increased ß-catenin, Cyclin D1, and c-Myc, and reduced cell cycle inhibitors p21 and p27. Importantly, thiostrepton treatment abolished the improved tubular repair in GSK3ß knockout mice following AKI. These results demonstrate that FoxM1 is important for renal tubular regeneration following AKI and that GSK3ß suppresses tubular repair by inhibiting FoxM1.

GSK-3ß Contributes to Parkinsonian Dopaminergic Neuron Death: Evidence From Conditional Knockout Mice and Tideglusib.

Glycogen synthase kinase-3 (GSK-3) dysregulation has been implicated in nigral dopaminergic neurodegeneration, one of the main pathological features of Parkinson's disease (PD). The two isoforms, GSK-3α and GSK-3ß, have both been suggested to play a detrimental role in neuronal death. To date, several studies have focused on the role of GSK-3ß on PD pathogenesis, while the role of GSK-3α has been largely overlooked. Here, we report in situ observations that both GSK-3α and GSK-3ß are dephosphorylated at a negatively acting regulatory serine, indicating kinase activation, selectively in nigral dopaminergic neurons following exposure of mice to 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP). To identify whether GSK-3α and GSK-3ß display functional redundancy in regulating parkinsonian dopaminergic cell death, we analysed dopaminergic neuron-specific Gsk3a null (Gsk3a ΔDat ) and Gsk3b null (Gsk3b ΔDat ) mice, respectively. We found that Gsk3b ΔDat , but not Gsk3a ΔDat , showed significant resistance to MPTP insult, revealing non-redundancy of GSK-3α and GSK-3ß in PD pathogenesis. In addition, we tested the neuroprotective effect of tideglusib, the most clinically advanced inhibitor of GSK-3, in the MPTP model of PD. Administration of higher doses (200 mg/kg and 500 mg/kg) of tideglusib exhibited significant neuroprotection, whereas 50 mg/kg tideglusib failed to prevent dopaminergic neurodegeneration from MPTP toxicity. Administration of 200 mg/kg tideglusib improved motor symptoms of MPTP-treated mice. Together, these data demonstrate GSK-3ß and not GSK-3α is critical for parkinsonian neurodegeneration. Our data support the view that GSK-3ß acts as a potential therapeutic target in PD and tideglusib would be a candidate drug for PD neuroprotective therapy.

Emerging roles of GSK-3α in pathophysiology: Emphasis on cardio-metabolic disorders.

Glycogen synthase kinase-3 (GSK-3) is a widely expressed serine/threonine kinase regulates a variety of cellular processes including proliferation, differentiation and death. Mammals harbor two structurally similar isoforms GSK-3α and ß that have overlapping as well as unique functions. Of the two, GSK-3ß has been studied (and reviewed) in far greater detail with analysis of GSK-3α often as an afterthought. It is now evident that systemic, chronic inhibition of either GSK-3ß or both GSK-3α/ß is not clinically feasible and if achieved would likely lead to adverse clinical conditions. Emerging evidence suggests important and specific roles for GSK-3α in fatty acid accumulation, insulin resistance, amyloid-ß-protein precursor metabolism, atherosclerosis, cardiomyopathy, fibrosis, aging, fertility, and in a variety of cancers. Selective targeting of GSK-3α may present a novel therapeutic opportunity to alleviate a number of pathological conditions. In this review, we assess the evidence for roles of GSK-3α in a variety of pathophysiological settings.

Molecular stratification within triple-negative breast cancer subtypes.

Triple-negative breast cancer (TNBC) has been subdivided into six distinct subgroups: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR). We recently identified a subgroup of TNBC with loss of the tumor suppressor PTEN and five specific microRNAs that exhibits exceedingly poor clinical outcome and contains TP53 mutation, RB1 loss and high MYC and WNT signalling. Here, show that these PTEN-low/miRNA-low lesions cluster with BL1 TNBC. These tumors exhibited high RhoA signalling and were significantly stratified on the basis of PTEN-low/RhoA-signalling-high with hazard ratios (HRs) of 8.2 (P = 0.0009) and 4.87 (P = 0.033) in training and test cohorts, respectively. For BL2 TNBC, we identified AKT1 copy gain/high mRNA expression as surrogate for poor prognosis (HR = 3.9; P = 0.02 and HR = 6.1; P = 0.0032). In IM, programmed cell death 1 (PD1) was elevated and predictive of poor prognosis (HR = 5.3; P = 0.01 and HR = 3.5; P < 0.004). Additional alterations, albeit without prognostic power, characterized each subtype including high E2F2 and TGFß signalling and CXCL8 expression in BL2, high IFNα and IFNγ signalling and CTLA4 expression in IM, and high EGFR signalling in MSL, and may be targeted for therapy. This study identified PTEN-low/RhoA-signalling-high, and high AKT1 and PD1 expression as potent prognostications for BL1, BL2 and IM subtypes with survival differences of over 14, 2.75 and 10.5 years, respectively. This intrinsic heterogeneity could be exploited to prioritize patients for precision medicine.

A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells.

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/ß-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3ß) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5-1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3ß (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2-2.5 fold, p < 0.05), a result associated with an increase in total ß-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (-86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

How to continually make the case for fundamental science: from the perspective of a protein kinase.

The strength of the scientific process is its immunity from human frailties. The built-in error correction and robustness of principles protect and nurture truth, despite both intended and unintended errors and naivety. What it doesn't secure is understanding of how the scientific sausage is made. Here, a scientific journey revolving around a single protein that spans nearly 35 years is used to illustrate the twists and turns that can accompany any scientific path. Lessons learned from such exploration speak to the need for story-telling in communicating scientific meaning - and the effectiveness of this will influence future investment and understanding of the scientific endeavor.

Cardiomyocyte-GSK-3α promotes mPTP opening and heart failure in mice with chronic pressure overload.

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.

A subgroup of microRNAs defines PTEN-deficient, triple-negative breast cancer patients with poorest prognosis and alterations in RB1, MYC, and Wnt signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) represents a heterogeneous group of ER- and HER2-negative tumors with poor clinical outcome. We recently reported that Pten-loss cooperates with low expression of microRNA-145 to induce aggressive TNBC-like lesions in mice. To systematically identify microRNAs that cooperate with PTEN-loss to induce aggressive human BC, we screened for miRNAs whose expression correlated with PTEN mRNA levels and determined the prognostic power of each PTEN-miRNA pair alone and in combination with other miRs. METHODS: Publically available data sets with mRNA, microRNA, genomics, and clinical outcome were interrogated to identify miRs that correlate with PTEN expression and predict poor clinical outcome. Alterations in genomic landscape and signaling pathways were identified in most aggressive TNBC subgroups. Connectivity mapping was used to predict response to therapy. RESULTS: In TNBC, PTEN loss cooperated with reduced expression of hsa-miR-4324, hsa-miR-125b, hsa-miR-381, hsa-miR-145, and has-miR136, all previously implicated in metastasis, to predict poor prognosis. A subgroup of TNBC patients with PTEN-low and reduced expression of four or five of these miRs exhibited the worst clinical outcome relative to other TNBCs (hazard ratio (HR) = 3.91; P < 0.0001), and this was validated on an independent cohort (HR = 4.42; P = 0.0003). The PTEN-low/miR-low subgroup showed distinct oncogenic alterations as well as TP53 mutation, high RB1-loss signature and high MYC, PI3K, and ß-catenin signaling. This lethal subgroup almost completely overlapped with TNBC patients selected on the basis of Pten-low and RB1 signature loss or ß-catenin signaling-high. Connectivity mapping predicted response to inhibitors of the PI3K pathway. CONCLUSIONS: This analysis identified microRNAs that define a subclass of highly lethal TNBCs that should be prioritized for aggressive therapy.

Polypharmacological Profiles Underlying the Antitumor Property of Salvia miltiorrhiza Root (Danshen) Interfering with NOX-Dependent Neutrophil Extracellular Traps.

Danshen, the dried root of Salvia miltiorrhiza, one of the most investigated medicinal plants with well-defined phytochemical constituents, has shown prominent clinical outcomes for antioxidant, anti-inflammatory, and anticoagulant activities to attain vascular protection and additional benefits for cancer therapy. More recently, activation of neutrophil and excessive formation of neutrophil extracellular traps (NETs) have been observed in pathological conditions of metastatic cancers; thus, we hypothesized that suppression of NETs could account for an essential cellular event underlying Danshen-mediated reduction of the incidence of metastasis. Using an experimental pulmonary metastases model of red fluorescent protein- (RFP-) labeled gastric cancer cells in combination with macroscopic ex vivo live-imaging system, our data indicated that Danshen impaired the fluorescent intensity and quantity of metastatic nodules. Moreover, Danshen could prevent neutrophil trafficking to the metastatic sites with decreased plasma levels of neutrophil elastase (NE) and procoagulant potential featured by fibrinogen. We further established phorbol 12-myristate 13-acetate- (PMA-) induced NET formation of human neutrophils and screened representative active compounds derived from the hydrophilic and hydrophobic fractions of Danshen using qualitative and quantitative methods. As a result, we found that salvianolic acid B (Sal B) and 15,16-dihydrotanshinone I (DHT I) exhibited superior inhibitory activities on NET formation and significantly attenuated the levels of citrullinated histone H3 (citH3), a biomarker for NET formation. Multitarget biochemical assays demonstrated that Sal B and DHT I distinctly modulated the enzymatic cascade involved in NET formation. Sal B and DHT I could disrupt NET formation at the earlier stage by blocking the activities of myeloperoxidase (MPO) and NADPH oxidase (NOX), respectively. Lastly, combining treatment of Sal B and DHT I under subED50 doses displayed remarkable synergism effect on NET inhibition. Altogether, these data provide insight into how promiscuous compounds from herbal medicine can be effectively targeted NETs towards hematogenous metastasis of certain tumors.

Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer.

CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.