Novel Pharmacologic Targeting of Tight Junctions and Focal Adhesions in Prostate Cancer Cells

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer

Novel pharmacologic targeting of tight junctions and focal adhesions in prostate cancer cells.

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer

ILK and talin mediate DZ-50 action on prostate cancer cell death and migration.

<p>Panel A, right, Western blot revealing that shILK transfectant PC-3 cells exhibit total loss of ILK-1 protein expression relative to vector control cells upon induction with doxycycline (48 hrs). On the left, DU-145 cells in which talin has been silence (shTalin) or overexpressed (Talin+), exhibit significant reduction or marked overexpression of talin protein levels respectively, relative to parental DU-145 cells. Panel B, DZ-50 treatment leads to a significant decrease in prostate cancer cell viability in a time-dependent fashion. Loss of ILK1 enhances the DZ-50 induced loss of cell viability; in contrast talin overexpression confers resistance to DZ-50 induced cell death. Panel C, DZ-50 significantly reduces prostate cancer cell migration. Loss of talin results in a significant reduction of prostate cancer cell migration compared to parental control DU-145 cells. In response to DZ-50, talin overexpression restores cell migratory ability to the levels of untreated cells. Panel D, Functional loss of ILK in PC-3 prostate cancer cells and loss talin in DU-145 cells significantly impairs the ability of the respective prostate cancer cells to adhere to fibronectin (ECM). Talin overexpression markedly enhances prostate cancer cell adhesion to fibronectin, compared to parental DU-145 and DU-145 shTalin cells. Statistical significance was set at *p<0.05.</p

Contribution of Talin-1 to prostate cancer cell anoikis resistance.

<p>DU-145 (panel A) and DU-145 shTalin (panel B) were cultured in the absence or presence of fibronectin-coating and treated with DZ-50; cells were subsequently subjected to confocal microscope for detection of talin (red), ILK (green), and focal adhesions (yellow). Nuclei were detected by DAPI staining (Blue). DZ-50 decreased focal adhesion formation through the targeting of talin and ILK. This effects was abrogated by the presence of fibronectin-ECM, which conferred resistance to DZ-50 (Panel A). Loss of talin resulted in reduced co-localization with ILK and disappearance of focal adhesions in DU-145 shTalin cells (Panel B). Magnification x100. Panels C–E, Western blot and quantitative analysis of the time-dependent effect of DZ-50 on downstream cell survival signaling. DZ-50 leads to dephosphorylation of survival signaling effectors AKT (Panel D) and GSK-3β (Panel E). Talin overexpression confers resistance to DZ-50 anoikis effect by sustaining activation/phosphorylation of AKT and GSK-3β signaling.</p

Disruption of focal adhesions in prostate cancer cells by DZ-50.

<p>PC-3 prostate cancer cells and PC-3 shILK cells harboring loss of ILK (panels A and B, respectively) were treated with DZ-50 (12 hrs, 5 µM) in the absence or presence of fibronectin-ECM. Fluorescent images reveal the co-localization of focal adhesion regulators talin (red) and ILK (green) to be disrupted by DZ-50 treatment, compared to untreated controls. DAPI (blue) is used for nuclear detection. Silencing ILK expression leads to reduced detection of its primary upstream partner, talin and subsequent disruption of focal adhesions (Panel B), relative to parental PC-3 cells (Panel A, composite, focal adhesions identified in yellow). Prostate cancer cells grown on a fibronectin-coated substrate (ECM integrity) stabilize the focal adhesion complex and diminish the targeting ability of DZ-50 on these substrates in both PC-3 parental and PC-shILK cells. Magnification x100.</p

Genes targeted by DZ-50 in prostate cancer cells.

<p>Panel A, Heat map of differentially expressed genes in DU-145 human prostate cancer cells before and after treatment with DZ-50 (9 hrs). We identified 17 markedly downregulated genes following treatment with DZ-50 (9 hrs), including genes encoding for ECM regulators <i>fibronectin</i> and <i>integrin α6</i>, tight junction mediator <i>Claudin-11</i> and angiogenesis signaling effector <i>thrombospondin 1</i>. (fold change >1.5, false discovery rate <20%). Panel B, Validation of gene expression using qRT-PCR after DZ-50 treatment of prostate cancer cells (5 µM) for 3 and 9 hrs. A significant reduction in mRNA relative to untreated cells was detected for genes involved in ECM-focal adhesion signaling components <i>(fibronectin</i>, <i>integrin-α6</i> and <i>talin</i>), tight junctions (<i>claudin-11</i>), angiogenesis (<i>thrombospondin-1</i>) and EMT (<i>Snail</i>). *indicates significant difference at p<0.05. Panel C, Molecular structure of the Doxazosin© derivative, DZ-50.</p

Disruption of tight junctions in human prostate cancer cells by lead agent DZ-50.

<p>Characteristic confocal microscopy images of PC-3 cells (Panel A) and DU-145 cells (Panel B). Treatment with DZ-50 (12 hrs, 5 µM) decreases Claudin-11 expression and inhibits tight junction formation. Tight junctions complexes (arrows), characterized by colocalization of the tight junction proteins Claudin-11 (red) and ZO-1) (green) is completely abrogated by DZ-50. In PC-3 shILK and DU-145shTalin cells there is weak formation of TJ complexes (arrow heads), in response to DZ-50. DAPI (blue) is used for nuclear detection (Panels A and B). Magnification x100. Panels C–F<b>,</b> Western blots and respective densitometric analysis revealing the expression of TJ protein ZO-1 in response to DZ-50 in PC-3 (Panels C and D) and DU-145 (Panels E and F) prostate cancer cell lines.</p

DZ-50 induces anoikis by targeting focal adhesion survival signaling.

<p>DZ-50 targets cell-cell interactions (tight junctions) and cell-ECM interactions (focal adhesions) to decrease intracellular survival signals and disrupt actin cytoskeletal integrity to induce anoikis. Stabilization of focal adhesion complex signaling by ECM components and elevated talin, enhances bidirectional integrin signaling, resulting in cellular resistance to anoikis (right). DZ-50 targets extracellular, intercellular and intracellular adhesions and signaling molecules to induce anoikis (left).</p

Enhanced Inhibition of Prostate Tumor Growth by Dual Targeting the Androgen Receptor and the Regulatory Subunit Type Iα of Protein Kinase A in Vivo

Abstract: Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysisInt. J. Mol. Sci. 2013, 14 1194