The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an ...intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.
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•MAP3K4 promotes HDAC6 ubiquitination and degradation to maintain the epithelial state•Trophoblast stem cell differentiation and EMT induce HDAC6 expression and activity•Nuclear HDAC6 promotes EMT by direct deacetylation of tight junction gene promoters•MAP3K4 inhibits HDAC6 to control tight junctions during EMT
Mobley et al. find that HDAC6 is regulated by MAP3K4 during trophoblast stem cell differentiation and EMT. MAP3K4 promotes HDAC6 ubiquitination and degradation, maintaining the epithelial state. During EMT, HDAC6 directly deacetylates histones on epithelial gene promoters such as claudin6 and occludin, promoting the dissolution of tight junctions.
Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give ...rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.
Metastasis suppression by high-dose, multi-drug targeting is unsuccessful due to network heterogeneity and compensatory network activation. Here, we show that targeting driver network signaling ...capacity by limited inhibition of core pathways is a more effective anti-metastatic strategy. This principle underlies the action of a physiological metastasis suppressor, Raf Kinase Inhibitory Protein (RKIP), that moderately decreases stress-regulated MAP kinase network activity, reducing output to transcription factors such as pro-metastastic BACH1 and motility-related target genes. We developed a low-dose four-drug mimic that blocks metastatic colonization in mouse breast cancer models and increases survival. Experiments and network flow modeling show limited inhibition of multiple pathways is required to overcome variation in MAPK network topology and suppress signaling output across heterogeneous tumor cells. Restricting inhibition of individual kinases dissipates surplus signal, preventing threshold activation of compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of driver networks represents a transformative, clinically relevant strategy for anti-metastatic treatment.
CHRISTIAN WIDMANN ,
SPENCER GIBSON ,
MATTHEW B. JARPE , AND
GARY L. JOHNSON
Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, ...Colorado
Widmann, Christian, Spencer Gibson, Matthew B. Jarpe, and Gary L. Johnson. Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human. Physiol. Rev . 79: 143-180, 1999. Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. Mitogen-activated protein kinases are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK. Currently, there have been 14 MKKK, 7 MKK, and 12 MAPK identified in mammalian cells. The mammalian MAPK can be subdivided into five families: MAPK erk1/2 , MAPK p38 , MAPK jnk , MAPK erk3/4 , and MAPK erk5 . Each MAPK family has distinct biological functions. In Saccharomyces cerevisiae , there are five MAPK pathways involved in mating, cell wall remodelling, nutrient deprivation, and responses to stress stimuli such as osmolarity changes. Component members of the yeast pathways have conserved counterparts in mammalian cells. The number of different MKKK in MAPK modules allows for the diversity of inputs capable of activating MAPK pathways. In this review, we define all known MAPK module kinases from yeast to humans, what is known about their regulation, defined MAPK substrates, and the function of MAPK in cell physiology.
We develop a dependent Dirichlet process model for survival analysis data. A major feature of the proposed approach is that there is no necessity for resulting survival curve estimates to satisfy the ...ubiquitous proportional hazards assumption. An illustration based on a cancer clinical trial is given, where survival probabilities for times early in the study are estimated to be lower for those on a high-dose treatment regimen than for those on the low dose treatment, while the reverse is true for later times, possibly due to the toxic effect of the high dose for those who are not as healthy at the beginning of the study.
Introduction Approximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in ...the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy. Methods To compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition. Results RNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3 . Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells. Conclusions The findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.
BACKGROUND:Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not ...targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium.
METHODS:To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining.
RESULTS:First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKACh by a mechanism involving frequency-dependent activation of PKCε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA–treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling.
CONCLUSIONS:NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.
4‐Anilinoquinolines were identified as potent and narrow‐spectrum inhibitors of the cyclin G associated kinase (GAK), an important regulator of viral and bacterial entry into host cells. Optimization ...of the 4‐anilino group and the 6,7‐quinoline substituents produced GAK inhibitors with nanomolar activity, over 50 000‐fold selectivity relative to other members of the numb‐associated kinase (NAK) subfamily, and a compound (6,7‐dimethoxy‐N‐(3,4,5‐trimethoxyphenyl)quinolin‐4‐amine; 49) with a narrow‐spectrum kinome profile. These compounds may be useful tools to explore the therapeutic potential of GAK in prevention of a broad range of infectious and systemic diseases.
Targeting GAK: The availability of chemical probes with improved selectivity for cyclin G associated kinase (GAK) or a different spectrum of off‐targets would be useful in target validation studies. Herein we report the synthesis and characterization of 4‐anilinoquinolines and 4‐anilinoquinazolines as potent narrow‐spectrum GAK inhibitors. Several of these compounds have the potential for development into high‐quality chemical probes for the study of GAK biology.
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