11 research outputs found
Inhibition of Hsp90 Leads to Cell Cycle Arrest and Apoptosis in Human Malignant Pleural Mesothelioma
IntroductionHeat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials.MethodsWe investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM).ResultsWe found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines.ConclusionThese results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM
Design of a Photoswitchable Cadherin
There is a growing
interest in engineering proteins whose function
can be controlled with the spatial and temporal precision of light.
Here, we present a novel example of a functional light-triggered switch
in the Ca-dependent cell–cell adhesion protein E-cadherin,
created using a mechanism-based design strategy. We report an 18-fold
change in apparent Ca<sup>2+</sup> binding affinity upon illumination.
Our results include a detailed examination of functional switching
via linked changes in Ca<sup>2+</sup> binding and cadherin dimerization.
This design opens avenues toward controllable tools that could be
applied to many long-standing questions about cadherin’s biological
function in cell–cell adhesion and downstream signaling
Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism
The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism--enabled by the functional coupling between a reader and a catalytic domain in KDM5A--suggests a model for the spread of demethylation on chromatin
Frizzled-8 receptor is activated by the Wnt-2 ligand in non-small cell lung cancer
BACKGROUND: Wnt-2 plays an oncogenic role in cancer, but which Frizzled receptor(s) mediates the Wnt-2 signaling pathway in lung cancer remains unclear. We sought to (1) identify and evaluate the activation of Wnt-2 signaling through Frizzled-8 in non-small cell lung cancer, and (2) test whether a novel expression construct dominant negative Wnt-2 (dnhWnt-2) reduces tumor growth in a colony formation assay and in a xenograft mouse model. METHODS: Semi-quantitative RT-PCR was used to identify the expression of Wnt-2 and Frizzled-8 in 50 lung cancer tissues from patients. The TCF reporter assay (TOP/FOP) was used to detect the activation of the Wnt canonical pathway in vitro. A novel dnhWnt-2 construct was designed and used to inhibit activation of Wnt-2 signaling through Frizzled-8 in 293T, 293, A549 and A427 cells and in a xenograft mouse model. Statistical comparisons were made using Student’s t-test. RESULTS: Among the 50 lung cancer samples, we identified a 91% correlation between the transcriptional increase of Wnt-2 and Frizzled-8 (p<0.05). The Wnt canonical pathway was activated when both Wnt-2 and Frizzled-8 were co-expressed in 293T, 293, A549 and A427 cells. The dnhWnt-2 construct we used inhibited the activation of Wnt-2 signaling in 293T, 293, A549 and A427 cells, and reduced the colony formation of NSCLC cells when β-catenin was present (p<0.05). Inhibition of Wnt-2 activation by the dnhWnt-2 construct further reduced the size and mass of tumors in the xenograft mouse model (p<0.05). The inhibition also decreased the expression of target genes of Wnt signaling in these tumors. CONCLUSIONS: We demonstrated an activation of Wnt-2 signaling via the Frizzled-8 receptor in NSCLC cells. A novel dnhWnt-2 construct significantly inhibits Wnt-2 signaling, reduces colony formation of NSCLC cells in vitro and tumor growth in a xenograft mouse model. The dnhWnt-2 construct may provide a new therapeutic avenue for targeting the Wnt pathway in lung cancer
The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1
The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As
Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen
Resistance
to clinical antiandrogens has plagued the evolution
of effective therapeutics for advanced prostate cancer. As with the
first-line therapeutic bicalutamide (Casodex), resistance to newer
antiandrogens (enzalutamide, ARN-509) develops quickly in patients,
despite the fact that these drugs have ∼10-fold better affinity
for the androgen receptor than bicalutamide. Improving affinity alone
is often not sufficient to prevent resistance, and alternative strategies
are needed to improve antiandrogen efficacy. Covalent and reversible
covalent drugs are being used to thwart drug resistance in other contexts,
and activated aryl nitriles are among the moieties being exploited
for this purpose. We capitalized on the presence of an aryl nitrile
in bicalutamide, and the existence of a native cysteine residue (Cys784)
in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide,
a cysteine-reactive antiandrogen. 5N-bicalutamide exhibits a 150-fold
improvement in <i>K</i><sub><i>i</i></sub> and
20-fold improvement in IC<sub>50</sub> over the parent compound. We
attribute the marked improvement in affinity and activity to the formation
of a covalent adduct with Cys784, a residue that is <i>not</i> among the more than 160 androgen receptor point mutations associated
with prostate cancer. Increasing the residence time of bound antiandrogen
via formation of a covalent adduct may forestall the drug resistance
seen with current clinical antiandrogens
Chromodomain-Mediated Oligomerization of HP1 Suggests a Nucleosome-Bridging Mechanism for Heterochromatin Assembly
Design of a Photoswitchable Cadherin
[Image: see text] There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell–cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca(2+) binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca(2+) binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin’s biological function in cell–cell adhesion and downstream signaling