Siu Chiu Chan

Androgen Receptor Splice Variants Mediate Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cell Lines

Persistent androgen receptor (AR) transcriptional activity underlies resistance to AR-targeted therapy and progression to lethal castration-resistant prostate cancer (CRPC). Recent success in retargeting persistent AR activity with next generation androgen/AR axis inhibitors such as enzalutamide (MDV3100) has validated AR as a master regulator during all stages of disease progression. However, resistance to next generation AR inhibitors limits therapeutic efficacy for many patients. One emerging mechanism of CRPC progression is AR gene rearrangement, promoting synthesis of constitutively active truncated AR splice variants (AR-V) that lack the AR ligand-binding domain. In this study, we show that cells with AR gene rearrangements expressing both full-length and AR-Vs are androgen independent and enzalutamide resistant. However, selective knock-down of AR-V expression inhibited androgen-independent growth and restored responsiveness to androgens and antiandrogens. In heterogeneous cell populations, AR gene rearrangements marked individual AR-V-dependent cells that were resistant to enzalutamide. Gene expression profiling following knock-down of full-length AR or AR-Vs showed that AR-Vs drive resistance to AR-targeted therapy by functioning as constitutive and independent effectors of the androgen/AR transcriptional program. Further, mitotic genes deemed previously to be unique AR-V targets were found to be biphasic targets associated with a proliferative level of signaling output from either AR-Vs or androgen-stimulated AR. Overall, these studies highlight AR-Vs as key mediators of persistent AR signaling and resistance to the current arsenal of conventional and next generation AR-directed therapies, advancing the concept of AR-Vs as therapeutic targets in advanced disease.

Androgen Receptor Splice Variants Activate Androgen Receptor Target Genes and Support Aberrant Prostate Cancer Cell Growth Independent of Canonical Androgen Receptor Nuclear Localization Signal

Background: Truncated AR splice variants support castration-resistant prostate cancer. Results: The AR NTD/DBD core is sufficient for AR variants to access the nucleus, activate AR target genes, and support androgen-independent prostate cancer cell growth. Conclusion: Diverse truncated AR variants are constitutively active transcription factors. Significance: These novel biochemical properties could lead to the development of new prostate cancer therapies. Synthesis of truncated androgen receptor (AR) splice variants has emerged as an important mechanism of prostate cancer (PCa) resistance to AR-targeted therapy and progression to a lethal castration-resistant phenotype. However, the precise role of these factors at this stage of the disease is not clear due to loss of multiple COOH-terminal AR protein domains, including the canonical nuclear localization signal (NLS) in the AR hinge region. Despite loss of this NLS, we show that diverse truncated AR variant species have a basal level of nuclear localization sufficient for ligand-independent transcriptional activity. Whereas full-length AR requires Hsp90 and importin-β for active nuclear translocation, basal nuclear localization of truncated AR variants is independent of these classical signals. For a subset of truncated AR variants, this basal level of nuclear import can be augmented by unique COOH-terminal sequences that reconstitute classical AR NLS activity. However, this property is separable from ligand-independent transcriptional activity. Therefore, the AR splice variant core consisting of the AR NH2-terminal domain and DNA binding domain is sufficient for nuclear localization and androgen-independent transcriptional activation of endogenous AR target genes. Indeed, we show that truncated AR variants with nuclear as well as nuclear/cytoplasmic localization patterns can drive androgen-independent growth of PCa cells. Together, our data demonstrate that diverse truncated AR species with varying efficiencies of nuclear localization can contribute to castration-resistant PCa pathology by driving persistent ligand-independent AR transcriptional activity.

Axin is a scaffold protein in TGF‐β signaling that promotes degradation of Smad7 by Arkadia

TGF‐β signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF‐β pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF‐β signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF‐β signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF‐β signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse‐chase experiments further illustrated that Axin significantly decreased the half‐life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF‐β signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt‐1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF‐β signaling.

Effects of the anti-bacterial peptide cecropin B and its analogs, cecropins B-1 and B-2, on liposomes, bacteria, and cancer cells

Custom designed analogs of the natural anti-bacterial peptide cecropin B (CB) have been synthesized; cecropin B-1 (CB-1) was constructed by replacing the C-terminal segment (residues 26 to 35) with the N-terminal sequence of CB (positions 1 to 10 which include five lysine residues). The second analog, CB-2, is identical to CB-1 except for the insertion of a Gly-Pro residue pair between Pro-24 and Ala-25. These peptides were used to investigate their anti-liposome, anti-bacterial and anti-cancer activities. The strength of anti-liposome activity is reduced two- to three-fold when the analogs are used instead of natural CB based on DL50 analysis. Similarly, the potency of these analogs on certain bacteria is about two- to four-fold lower than those of CB based on LC measurements. In contrast, on leukemia cancer cells, the potency of CB-1 and CB-2 is about two- to three-fold greater than that of natural CB based on IC50 measurements. All CB, CB-1 and CB-2 peptides have comparable helix contents according to CD measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killing bacteria but more effective in lysing cancer cells. The possible interpretations for these observations are discussed.

Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies

Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa.

beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death

Tumor necrosis factor (TNF)-α induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, β-actin was disrupted by viral insertion, and subsequent reconstitution of β-actin expression levels in the mutant line Actinmut restored its sensitivity to TNF. Resistance to TNF in Actinmut cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-κB activation and p38 phosphorylation in TNF-treated wild-type and Actinmut cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actinmut cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.

Microscopic observations of the different morphological changes caused by anti-bacterial peptides on Klebsiella pneumoniae and HL-60 leukemia cells

Natural anti‐bacterial peptides cecropin B (CB) and its analogs cecropin B‐1 (CB‐1), cecropin B‐2 (CB‐2) and cecropin B‐3 (CB‐3) were prepared. The different characteristics of these peptides, with amphipathic/hydrophobic α‐helices for CB, amphipathic/amphipathic α‐helices for CB‐1/CB‐2, and hydrophobic/hydrophobic α‐helices for CB‐3, were used to study the morphological changes in the bacterial cell, Klebsiella pneumoniae and the leukemia cancer cell, HL‐60, by scanning and transmission electron microscopies. The natural and analog peptides have comparable secondary structures as shown by circular dichroism measurements. This indicates that the potency of the peptides on cell membranes is dependent of the helical characteristics rather than the helical strength. The microscopic results show that the morphological changes of the cells treated with CB are distinguishably different from those treated with CB‐1/CB‐2, which are designed to have enhanced anti‐cancer properties by having an extra amphipathic α‐helix. The morphological differences may be due to their different modes of action on the cell membranes resulting in the different potencies with lower lethal concentration and higher concentration of 50% inhibition (IC50) of CB on bacterium and cancer cell, respectively, as compared with CB‐1/CB‐2 (Chen et al. 1997. Biochim. Biophys. Acta 1336, 171–179). In contrast, CB‐3 has little effect on either the bacterium or the cancer cell. These results provide microscopic evidence that different killing pathways are involved with the peptides.

Constitutive Activity of the Androgen Receptor

Prostate cancer (PCa) is the most frequently diagnosed cancer in the United States. The androgen receptor (AR) signaling axis is central to all stages of PCa pathophysiology and serves as the main target for endocrine-based therapy. The most advanced stage of the disease, castration-resistant prostate cancer (CRPC), is presently incurable and accounts for most PCa mortality. In this chapter, we highlight the mechanisms by which the AR signaling axis can bypass endocrine-targeted therapies and drive progression of CRPC. These mechanisms include alterations in growth factor, cytokine, and inflammatory signaling pathways, altered expression or activity of transcriptional coregulators, AR point mutations, and AR gene amplification leading to AR protein overexpression. Additionally, we will discuss the mechanisms underlying the synthesis of constitutively active AR splice variants (AR-Vs) lacking the COOH-terminal ligand-binding domain, as well as the role and regulation of AR-Vs in supporting therapeutic resistance in CRPC. Finally, we summarize the ongoing development of inhibitors targeting discrete AR functional domains as well as the status of new biomarkers for monitoring the AR signaling axis in patients.

Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway

Cidea, one of three members of the CIDE (cell-death-inducing DNA-fragmentation-factor-45-like effector) family of proteins, is highly enriched in brown adipose tissue, in which it plays a critical role in adaptive thermogenesis and fat accumulation. Cidea-null mice have increased energy expenditure with resistance to high-fat-diet-induced obesity and diabetes. However, little is known as to how the Cidea protein is regulated. In the present study we show that Cidea is a short-lived protein as measured by cycloheximide-based protein chase experiments in different cell lines or in differentiated brown adipocytes. Proteasome inhibitors specifically increased the stability of both transfected and endogenous Cidea protein. Furthermore, Cidea protein was found to be polyubiquitinated when overexpressed in different culture cells as well as in differentiated mature brown adipocytes. Extensive mutational analysis of individual lysine residues revealed that ubiquitinated lysine residues are located in the N-terminal region of Cidea, as alteration of these lysine residues to alanine (N-5KA mutant) renders Cidea much more stable when compared with wild-type or C-terminal lysine-less mutant (C-5KA). Furthermore, K23 (Lys23) within the N-terminus of the Cidea was identified as the major contributor to its polyubiquitination signal and the protein instability. Taken together, the results of our study demonstrated that the ubiquitin-proteasome system confers an important post-translational modification that controls the protein stability of Cidea.

Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding

Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of protein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower ΔHcal values than the wild‐type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge–charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three‐dimensional structure of the protein.