Limits...
The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin-β8 in prostate cancer cells.

Mertens-Walker I, Fernandini BC, Maharaj MS, Rockstroh A, Nelson CC, Herington AC, Stephenson SA - BMC Cancer (2015)

Bottom Line: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA).Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed.Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia. inga.mertenswalker@qut.edu.au.

ABSTRACT

Background: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways.

Methods: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion.

Results: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion.

Conclusions: These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Show MeSH

Related in: MedlinePlus

Knockdown ofITGB8results in reduced metastatic potential in prostate cancer cells. A) Quantitative real-time PCR was carried out to determine knockdown levels of siRNA against ITGB8. 100 nM negative non-silencing or ITGB8 targeting siRNA were transiently transfected into PC-3 cells. RNA was isolated 48 h after transfection, transcribed into cDNA and analyzed for gene expression. ITGB8 expression is reduced by approximately 60-70%. B) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and 24 h later a scratch wound was applied using the IncuCyte (Essen Bioscience) system and migration was monitored for a further 24 h. Cell migration was reduced following knockdown of ITGB8. C) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and subjected to a Matrigel transwell invasion assay. After 22 h of incubation, invaded cells were stained and counted. Cell invasion was reduced following knockdown of ITGB8. D) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with 100 nM siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si). An invasion assay was carried out using the Matrigel invasion system and cells were allowed to invade for 22 h. Cells containing the ITGB8 siRNA showed significantly reduced ability to invade. E) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si) and subjected to an adhesion assay to vitronectin. No significant changes were seen. n = 3 * p < 0.01 vs VO negative; # p < 0.001 vs B4 negative.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4389669&req=5

Fig2: Knockdown ofITGB8results in reduced metastatic potential in prostate cancer cells. A) Quantitative real-time PCR was carried out to determine knockdown levels of siRNA against ITGB8. 100 nM negative non-silencing or ITGB8 targeting siRNA were transiently transfected into PC-3 cells. RNA was isolated 48 h after transfection, transcribed into cDNA and analyzed for gene expression. ITGB8 expression is reduced by approximately 60-70%. B) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and 24 h later a scratch wound was applied using the IncuCyte (Essen Bioscience) system and migration was monitored for a further 24 h. Cell migration was reduced following knockdown of ITGB8. C) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and subjected to a Matrigel transwell invasion assay. After 22 h of incubation, invaded cells were stained and counted. Cell invasion was reduced following knockdown of ITGB8. D) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with 100 nM siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si). An invasion assay was carried out using the Matrigel invasion system and cells were allowed to invade for 22 h. Cells containing the ITGB8 siRNA showed significantly reduced ability to invade. E) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si) and subjected to an adhesion assay to vitronectin. No significant changes were seen. n = 3 * p < 0.01 vs VO negative; # p < 0.001 vs B4 negative.

Mentions: Integrin β8 has been implicated in tumor cell invasiveness [11]. To analyse functional effects of integrin β8 in prostate cancer cells, we employed siRNA knockdown. In PC-3 cells, ITGB8 knockdown resulted in a 70% decrease in ITGB8 mRNA (Figure 2A). PC-3 cells transfected with ITGB8-targeted siRNA showed a significant decrease in in vitro wound healing migration and in Matrigel invasion, when compared with PC-3 cells transfected with negative siRNA (Figure 2B & C). Furthermore, transfection of 22Rv1-VO and 22Rv1-B4 cells with the ITGB8 siRNA resulted in a significant decrease in Matrigel invasion (Figure 2D). There was no significant difference in the adhesion to vitronectin of 22Rv1-VO or 22Rv1–B4 cells transfected with ITGB8 siRNA (Figure 2E). Together, these results highlight that integrin β8 plays an important role in prostate cancer cell migration and invasion in both endogenous and exogenously over-expressing EphB4 cell models.Figure 2


The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin-β8 in prostate cancer cells.

Mertens-Walker I, Fernandini BC, Maharaj MS, Rockstroh A, Nelson CC, Herington AC, Stephenson SA - BMC Cancer (2015)

Knockdown ofITGB8results in reduced metastatic potential in prostate cancer cells. A) Quantitative real-time PCR was carried out to determine knockdown levels of siRNA against ITGB8. 100 nM negative non-silencing or ITGB8 targeting siRNA were transiently transfected into PC-3 cells. RNA was isolated 48 h after transfection, transcribed into cDNA and analyzed for gene expression. ITGB8 expression is reduced by approximately 60-70%. B) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and 24 h later a scratch wound was applied using the IncuCyte (Essen Bioscience) system and migration was monitored for a further 24 h. Cell migration was reduced following knockdown of ITGB8. C) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and subjected to a Matrigel transwell invasion assay. After 22 h of incubation, invaded cells were stained and counted. Cell invasion was reduced following knockdown of ITGB8. D) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with 100 nM siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si). An invasion assay was carried out using the Matrigel invasion system and cells were allowed to invade for 22 h. Cells containing the ITGB8 siRNA showed significantly reduced ability to invade. E) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si) and subjected to an adhesion assay to vitronectin. No significant changes were seen. n = 3 * p < 0.01 vs VO negative; # p < 0.001 vs B4 negative.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389669&req=5

Fig2: Knockdown ofITGB8results in reduced metastatic potential in prostate cancer cells. A) Quantitative real-time PCR was carried out to determine knockdown levels of siRNA against ITGB8. 100 nM negative non-silencing or ITGB8 targeting siRNA were transiently transfected into PC-3 cells. RNA was isolated 48 h after transfection, transcribed into cDNA and analyzed for gene expression. ITGB8 expression is reduced by approximately 60-70%. B) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and 24 h later a scratch wound was applied using the IncuCyte (Essen Bioscience) system and migration was monitored for a further 24 h. Cell migration was reduced following knockdown of ITGB8. C) PC-3 cells were transiently transfected with 100 nM negative non-silencing (neg si) or ITGB8 targeting siRNA (β8 si) and subjected to a Matrigel transwell invasion assay. After 22 h of incubation, invaded cells were stained and counted. Cell invasion was reduced following knockdown of ITGB8. D) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with 100 nM siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si). An invasion assay was carried out using the Matrigel invasion system and cells were allowed to invade for 22 h. Cells containing the ITGB8 siRNA showed significantly reduced ability to invade. E) 22Rv1-VO (vector only) or 22Rv1–B4 (EphB4 over-expressing) cells were transiently transfected with siRNA against ITGB8 (β8 si) or negative non-silencing siRNA (neg si) and subjected to an adhesion assay to vitronectin. No significant changes were seen. n = 3 * p < 0.01 vs VO negative; # p < 0.001 vs B4 negative.
Mentions: Integrin β8 has been implicated in tumor cell invasiveness [11]. To analyse functional effects of integrin β8 in prostate cancer cells, we employed siRNA knockdown. In PC-3 cells, ITGB8 knockdown resulted in a 70% decrease in ITGB8 mRNA (Figure 2A). PC-3 cells transfected with ITGB8-targeted siRNA showed a significant decrease in in vitro wound healing migration and in Matrigel invasion, when compared with PC-3 cells transfected with negative siRNA (Figure 2B & C). Furthermore, transfection of 22Rv1-VO and 22Rv1-B4 cells with the ITGB8 siRNA resulted in a significant decrease in Matrigel invasion (Figure 2D). There was no significant difference in the adhesion to vitronectin of 22Rv1-VO or 22Rv1–B4 cells transfected with ITGB8 siRNA (Figure 2E). Together, these results highlight that integrin β8 plays an important role in prostate cancer cell migration and invasion in both endogenous and exogenously over-expressing EphB4 cell models.Figure 2

Bottom Line: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA).Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed.Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia. inga.mertenswalker@qut.edu.au.

ABSTRACT

Background: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways.

Methods: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion.

Results: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion.

Conclusions: These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Show MeSH
Related in: MedlinePlus