Transcriptome-Based Analysis of Molecular Pathways for Clusterin Functions in Kidney Cells.

Clusterin (CLU) is a chaperone-like protein and plays a protective role against renal ischemia-reperfusion injury (IRI); however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU-mediating pathways in kidney cells by using bioinformatics analysis. CLU null renal tubular epithelial cells (TECs) expressing human CLU cDNA (TEC-CLU(hCLU) ) or empty vector (TEC-CLU(-/-) ) were exposed to normoxia or hypoxia (1% O2 ). Transcriptome profiling with a significant twofold change was performed using SurePrint G3 Mouse Gene Expression 8 × 60 K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis. Here, we showed that compared to CLU null controls, ectopic expression of human CLU in CLU null kidney cells promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU expression affected expression of 3864 transcripts (1893 up-regulated) in normoxia and 3670 transcripts (1925 up-regulated) in hypoxia. CLU functions in normoxia were associated mostly with AKT2/PPP2R2B-dependent PI3K/AKT, PTEN, VEGF, and ERK/MAPK signaling and as well with GSK3B-mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU-enhanced cell survival in hypoxia was also associated with PIK3CD/MAPK1-dependent PI3K/AKT, HIF-α, PTEN, VEGF, and ERK/MAPK signaling. In conclusion, our data showed that CLU functions in kidney cells were mainly mediated in a cascade manner by PI3K/AKT, PTEN, VEGF, and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney. J. Cell. Physiol. 231: 2628-2638, 2016. © 2016 Wiley Periodicals, Inc.

Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer.

The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

Use it or lose it? Sablefish, Anoplopoma fimbria, a species representing a fifth teleostean group where the betaNHE associated with the red blood cell adrenergic stress response has been secondarily lost.

Like most teleosts, sablefish (Anoplopoma fimbria Pallas 1814) blood exhibits a moderate Root effect (~35% maximal desaturation), where a reduction in blood pH dramatically reduces O(2) carrying capacity, a mechanism important for oxygenating the eye and filling the swim bladder (SB) in teleosts. Although sablefish lack a SB, we observed a well-defined choroid rete at the eye. The adrenergically mediated cell swelling typically associated with a functional red blood cell (RBC) beta-adrenergic Na(+)/H(+) exchanger (betaNHE), which would normally protect RBC pH, and thus O(2) transport, during a generalized acidosis, was not observed in sablefish blood. Neither isoproterenol (a beta-agonist) nor 8-bromo cAMP could elicit this response. Furthermore, RBC osmotic shrinkage, known to stimulate NHEs in general and betaNHE in other teleosts such as trout and flounder, resulted in no significant regulatory volume increase (RVI), further supporting the absence of a functional RBC betaNHE. The onset of the Root effect occurs at a much lower RBC pH (6.83-6.92) than in other teleosts, and thus RBC betaNHE may not be required to protect O(2) transport during a generalized acidosis in vivo. Phylogenetically, sablefish may represent a fifth group of teleosts exhibiting a secondary reduction or loss of betaNHE activity. However, sablefish have not lost the choroid rete at the eye (unlike in the other four groups), which may still function with the Root effect to oxygenate the retina, but the low pH onset of the Root effect may ensure haemoglobin (Hb)-O(2) binding is not compromised at the respiratory surface during a general acidosis in the absence of RBC betaNHE. The sablefish may represent an anomaly within the framework of Root effect evolution, in that they possess a moderate Root effect and a choroid rete at the eye, but lack the RBC betaNHE and the SB system.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

Angiopoietin-like 4 Enhances the Proliferation and Migration of Tendon Fibroblasts.

PURPOSE: Angiopoietin-like 4 (ANGPTL4) is known to play a variety of roles in the response to exercise, and more recently has been shown to enhance the healing of tendon, a fibrous load-bearing tissue required for efficient movement. The objective of the current study was to further explore the mechanisms of ANGPTL4's effect on tendon cells using a gene array approach. METHODS: Human tendon fibroblasts were treated with recANGPTL4 and their global transcriptome response analyzed after 4 and 24 h. We also conducted functional studies using tendon fibroblasts derived from human subjects, cultured in the presence or absence of applied cyclic stretch and/or siRNA for ANGPTL4, and as confirmation we also used tendon cells from wild type (ANGPTL4 +/+) or knockout (ANGPTL4-/-) mice. RESULTS: The leading functions of ANGPTL4 predicted by the resulting pathway analysis were cell movement and proliferation. The experiments demonstrated that ANGPTL4 significantly enhanced tendon cell proliferation and the cell cycle progression, as well as adhesion and migration. CONCLUSION: Taken together, these findings provide novel molecular insights into the effect of ANGPTL4, a multifunctional protein that regulates the physiological response to exercise, on fundamental tendon cell functions.

Bypassing Drug Resistance Mechanisms of Prostate Cancer with Small Molecules that Target Androgen Receptor-Chromatin Interactions.

Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug target in the treatment of prostate cancer. Current small-molecule AR antagonists, such as enzalutamide, compete with androgens that bind to the steroid-binding pocket of the AR ligand-binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug resistance can manifest through AR-LBD mutations that convert AR antagonists into agonists, or by expression of AR variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development of a series of small molecules that were rationally designed to selectively target the AR DNA-binding domain (DBD) and, hence, to directly interfere with AR-DNA interactions. In the current work, we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of antiandrogen action of the developed AR-DBD inhibitors, establishing proof of principle for targeting DBDs of nuclear receptors in endocrine cancers. Mol Cancer Ther; 16(10); 2281-91. ©2017 AACR.

Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance.

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3-directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936-45. ©2016 AACR.

Dynamic assessment of cell viability, proliferation and migration using real time cell analyzer system (RTCA).

Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.

Orphan nuclear receptors as drug targets for the treatment of prostate and breast cancers.

Nuclear receptors (NRs), a family of 48 transcriptional factors, have been studied intensively for their roles in cancer development and progression. The presence of distinctive ligand binding sites capable of interacting with small molecules has made NRs attractive targets for developing cancer therapeutics. In particular, a number of drugs have been developed over the years to target human androgen- and estrogen receptors for the treatment of prostate cancer and breast cancer. In contrast, orphan nuclear receptors (ONRs), which in many cases lack known biological functions or ligands, are still largely under investigated. This review is a summary on ONRs that have been implicated in prostate and breast cancers, specifically retinoic acid-receptor-related orphan receptors (RORs), liver X receptors (LXRs), chicken ovalbumin upstream promoter transcription factors (COUP-TFs), estrogen related receptors (ERRs), nerve growth factor 1B-like receptors, and ''dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1'' (DAX1). Discovery and development of small molecules that can bind at various functional sites on these ONRs will help determine their biological functions. In addition, these molecules have the potential to act as prototypes for future drug development. Ultimately, the therapeutic value of targeting the ONRs may go well beyond prostate and breast cancers.