Kristopher Kuchenbecker

Chromodomain-Mediated Oligomerization of HP1 Suggests a Nucleosome-Bridging Mechanism for Heterochromatin Assembly

HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9-methylated chromatin in vitro relies on an interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl mark recognition is highly sensitive to internucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

Inhibition of Wnt-2 and galectin-3 synergistically destabilizes β-catenin and induces apoptosis in human colorectal cancer cells

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt‐2 is well known for its overexpression in colorectal cancer. Galectin‐3 (Gal‐3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with β‐catenin. In this study, we investigated roles of Wnt‐2 and Gal‐3 in the regulation of canonical Wnt/β‐catenin signaling. We found that siRNA silencing of either Wnt‐2 or Gal‐3 expression inhibited TCF‐reporter activity, decreased cytosolic β‐catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt‐2 and Gal‐3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/β‐catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt‐2 and Gal‐3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.

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.

Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma.

Introduction: Heat 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. Methods: We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM). Results: We 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. Conclusion: These 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.

The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1.

Significance We previously reported that lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP2 and PIP3 bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIPn headgroups. We find that stabilization by the PIP3 ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP3 increases SF-1 activity. This newly created surface harbors a cluster of human mutations that lead to endocrine disorders, thus explaining how these puzzling mutations cripple SF-1 activity. We propose that this new surface acts as a PIP3-regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides. 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.

Frizzled-8 receptor is activated by the Wnt-2 ligand in non-small cell lung cancer

BackgroundWnt-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.MethodsSemi-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.ResultsAmong 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.ConclusionsWe 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.

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 Ki and 20-fold improvement in IC50 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 not 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.

Abstract 3238: Frizzled-8 receptor is activated by the Wnt-2 ligant in non-small cell lung cancer

Introduction: Lung cancer is the leading cause of cancer-associated mortality throughout the world. We previously showed that the Wnt signaling pathway is aberrantly activated in lung cancer. In this report, we investigated the role of interactions between different Frizzled receptors and the Wnt-2 ligant in non-small cell lung cancer (NSCLC). Methods: Quantitative RT-PCR was used to measure expression of Frz-8 and Wnt-2 from fifty freshly resected lung tumors and matched normal lung tissue. A human dominant-negative Wnt-2 mutant (dnhWnt-2) was designed to target the interactions between Fzd-8 and Wnt-2 in vitro and in vivo. pTOPflash/pFOPflash luciferase activity assay was used to detect TCF/β-catenine transcription activity. Crystal violet staining assay was used to determine the colony formation. A mouse xenogrant model using A549 cells transfected with dnhWnt-2 vector or control vector was used to detect tumorigenic effects. Results: RT-PCR analysis showed that Wnt-2 overexpression is in correlation with overexpression of Fzd-8 in 45/50 (90%) tumor samples analyzed. Coexpression of the dnhWnt-2 construct together with Wnt-2 and Fzd8 expression vectors in 293T, 293 and A549 cells strongly reduced TCF-dependent transcriptional activity (TOPflash assay) as compared with cells transfected with control vectors. In lung cancer cell line A549, naturally overexpressing both Wnt-2 and Fzd8, the dnhWnt-2 construct induced cell death, inhibited cell proliferation. Results from colony formation assay showed 50% reduction in colonies formed from the dnhWnt-2 transfected cells as compared to control vector. A tumor xenograft model of A549 cells expressing the dnhWnt-2 mutant, demonstrated a marked reduction in tumor formation 43 days after cell inoculation (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3238. doi:1538-7445.AM2012-3238