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Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models.

ABSTRACT: Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

SUBMITTER: Becker-Santos DD

PROVIDER: S-EPMC3510737 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models.

Becker-Santos Daiana D DD Guo Yubin Y Ghaffari Mazyar M Vickers Elaine D ED Lehman Melanie M Altamirano-Dimas Manuel M Oloumi Arusha A Furukawa Junya J Sharma Manju M Wang Yuzhuo Y Dedhar Shoukat S Cox Michael E ME

Carcinogenesis 20121001 12

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targ ...[more]

PMID: 23027626

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Project description:Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (?IIb?6B?3) and mutant CHO cells expressing inactive ?IIb?6B?3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and ?-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and ?-parvin, resulting in the restoration of ?IIb?6B?3 activation. In the parental cells expressing active ?IIb?6B?3, ILK, PINCH, and ?-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both ?- and ?-parvin mRNA in the parent cells impaired the ?IIb?6B?3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore ?IIb?6B?3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired ?IIb?6B?3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type ?IIb?3 that is an inactive form, overexpression of a talin head domain (THD) induced ?IIb?3 activation and the THD-induced ?IIb?3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the ?IIb?3-expressing CHO cells further augmented THD-induced ?IIb?3 activation, whereas they did not induce ?IIb?3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.

Project description:Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding ?6?1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of ?6 integrin and E-cadherin in a cell-cell location and ?6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing ?6 integrin (DU145 ?6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without ?6 integrin (DU145 ?6KO) and cells expressing an integrin mutant (DU145 ?6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 ?6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 ?6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 ?mol/L S1P challenge as compared with the DU145 ?6WT cells. The results suggest that gene editing of a specific ?6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific ?6 integrin extracellular region.

Dataset Information

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models.

Publications

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models.

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