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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.

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Related in: MedlinePlus

Integrins are significantly de-regulated in response to changing EphB4 levels in prostate cancer cells. A) Relative gene expression of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as identified on the cDNA microarray, compared to control negative siRNA. Dotted line indicates normalized level of negative siRNA control. B) Relative gene expression normalized to GAPDH of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as determined by qRT-PCR (independent experiments from results shown in A). Dotted line indicates normalized level of negative siRNA control. C) Western blotting analysis showing integrin β8 and EphB4 protein levels in LNCaP cells that have been transfected with two different siRNAs (#2 and #5) targeting EPHB4. GAPDH was used to normalize for loading. D) Relative gene expression of ITGA3, ITGA10 and ITGB8, normalized to GAPDH, in stably over-expressing 22Rv1-B4 cells as determined by qRT-PCR. Dotted line indicates normalized level of negative siRNA control. E) Western blotting analysis showing integrin β8 and EphB4 protein levels in 22Rv1-B4 over-expressing prostate cancer cells. GAPDH was used as a loading control. F) Relative gene expression of ITGAV in stably over-expressing 22Rv1-B4 compared to VO (vector only) cells as determined by qRT-PCR. QRT-PCR experiments were carried out in triplicate and with three biological replicates. Western blotting experiments were carried out three times and representative cropped blots are shown. Graphs are presented with ± SD. *** p < 0.005, ** p < 0.01, * p < 0.05.
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Fig1: Integrins are significantly de-regulated in response to changing EphB4 levels in prostate cancer cells. A) Relative gene expression of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as identified on the cDNA microarray, compared to control negative siRNA. Dotted line indicates normalized level of negative siRNA control. B) Relative gene expression normalized to GAPDH of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as determined by qRT-PCR (independent experiments from results shown in A). Dotted line indicates normalized level of negative siRNA control. C) Western blotting analysis showing integrin β8 and EphB4 protein levels in LNCaP cells that have been transfected with two different siRNAs (#2 and #5) targeting EPHB4. GAPDH was used to normalize for loading. D) Relative gene expression of ITGA3, ITGA10 and ITGB8, normalized to GAPDH, in stably over-expressing 22Rv1-B4 cells as determined by qRT-PCR. Dotted line indicates normalized level of negative siRNA control. E) Western blotting analysis showing integrin β8 and EphB4 protein levels in 22Rv1-B4 over-expressing prostate cancer cells. GAPDH was used as a loading control. F) Relative gene expression of ITGAV in stably over-expressing 22Rv1-B4 compared to VO (vector only) cells as determined by qRT-PCR. QRT-PCR experiments were carried out in triplicate and with three biological replicates. Western blotting experiments were carried out three times and representative cropped blots are shown. Graphs are presented with ± SD. *** p < 0.005, ** p < 0.01, * p < 0.05.

Mentions: The microarray analysis revealed significant quantitative changes in ITGB8 and ITGA10 (down-regulated) and ITGA3 (up-regulated) (Figure 1A). To confirm these data we employed real-time PCR analysis on a different set of LNCaP transfectants. Upon EPHB4 knockdown, ITGB8 and ITGA10 were significantly down-regulated (Figure 1B). ITGA3 however, was unchanged (Figure 1B). Knockdown of EPHB4 with two different siRNAs also resulted in a reduction of integrin β8 protein expression (Figure 1C), confirming the results seen at the gene level. Conversely, to investigate whether EphB4 over-expression would result in a parallel increase in ITGB8 expression, and to ensure that these data were not due to siRNA off-target effects, the expression of ITGB8 in stable 22Rv1-EphB4 over-expressing cells was also determined using qRT-PCR and compared with 22Rv1 cells containing the empty vector (22Rv1-VO). EphB4 over-expression resulted in a significant 2.5 fold increase in ITGB8 gene levels, but no significant effect was seen on ITGA3 or ITGA10 expression (Figure 1D). Again, the increase in gene expression of ITGB8 correlated with an increase in protein level (Figure 1E). Together, these data suggest that EPHB4 and ITGB8 are co-regulated in prostate cancer cells. As integrin β8 has only one known heterodimer partner, integrin αV (ITGAV) [10], we also sought to determine whether over-expression of EphB4 increases the expression of this integrin subunit. EPHB4 overexpression in 22Rv1 cells significantly increased ITGAV gene expression by 1.7 fold (Figure 1F) suggesting an overall increase in the integrin αvβ8 heterodimer complex.Figure 1


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)

Integrins are significantly de-regulated in response to changing EphB4 levels in prostate cancer cells. A) Relative gene expression of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as identified on the cDNA microarray, compared to control negative siRNA. Dotted line indicates normalized level of negative siRNA control. B) Relative gene expression normalized to GAPDH of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as determined by qRT-PCR (independent experiments from results shown in A). Dotted line indicates normalized level of negative siRNA control. C) Western blotting analysis showing integrin β8 and EphB4 protein levels in LNCaP cells that have been transfected with two different siRNAs (#2 and #5) targeting EPHB4. GAPDH was used to normalize for loading. D) Relative gene expression of ITGA3, ITGA10 and ITGB8, normalized to GAPDH, in stably over-expressing 22Rv1-B4 cells as determined by qRT-PCR. Dotted line indicates normalized level of negative siRNA control. E) Western blotting analysis showing integrin β8 and EphB4 protein levels in 22Rv1-B4 over-expressing prostate cancer cells. GAPDH was used as a loading control. F) Relative gene expression of ITGAV in stably over-expressing 22Rv1-B4 compared to VO (vector only) cells as determined by qRT-PCR. QRT-PCR experiments were carried out in triplicate and with three biological replicates. Western blotting experiments were carried out three times and representative cropped blots are shown. Graphs are presented with ± SD. *** p < 0.005, ** p < 0.01, * p < 0.05.
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Related In: Results  -  Collection

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Fig1: Integrins are significantly de-regulated in response to changing EphB4 levels in prostate cancer cells. A) Relative gene expression of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as identified on the cDNA microarray, compared to control negative siRNA. Dotted line indicates normalized level of negative siRNA control. B) Relative gene expression normalized to GAPDH of EPHB4, ITGA3, ITGA10 and ITGB8 after siRNA knockdown of EPHB4 in LNCaP cells as determined by qRT-PCR (independent experiments from results shown in A). Dotted line indicates normalized level of negative siRNA control. C) Western blotting analysis showing integrin β8 and EphB4 protein levels in LNCaP cells that have been transfected with two different siRNAs (#2 and #5) targeting EPHB4. GAPDH was used to normalize for loading. D) Relative gene expression of ITGA3, ITGA10 and ITGB8, normalized to GAPDH, in stably over-expressing 22Rv1-B4 cells as determined by qRT-PCR. Dotted line indicates normalized level of negative siRNA control. E) Western blotting analysis showing integrin β8 and EphB4 protein levels in 22Rv1-B4 over-expressing prostate cancer cells. GAPDH was used as a loading control. F) Relative gene expression of ITGAV in stably over-expressing 22Rv1-B4 compared to VO (vector only) cells as determined by qRT-PCR. QRT-PCR experiments were carried out in triplicate and with three biological replicates. Western blotting experiments were carried out three times and representative cropped blots are shown. Graphs are presented with ± SD. *** p < 0.005, ** p < 0.01, * p < 0.05.
Mentions: The microarray analysis revealed significant quantitative changes in ITGB8 and ITGA10 (down-regulated) and ITGA3 (up-regulated) (Figure 1A). To confirm these data we employed real-time PCR analysis on a different set of LNCaP transfectants. Upon EPHB4 knockdown, ITGB8 and ITGA10 were significantly down-regulated (Figure 1B). ITGA3 however, was unchanged (Figure 1B). Knockdown of EPHB4 with two different siRNAs also resulted in a reduction of integrin β8 protein expression (Figure 1C), confirming the results seen at the gene level. Conversely, to investigate whether EphB4 over-expression would result in a parallel increase in ITGB8 expression, and to ensure that these data were not due to siRNA off-target effects, the expression of ITGB8 in stable 22Rv1-EphB4 over-expressing cells was also determined using qRT-PCR and compared with 22Rv1 cells containing the empty vector (22Rv1-VO). EphB4 over-expression resulted in a significant 2.5 fold increase in ITGB8 gene levels, but no significant effect was seen on ITGA3 or ITGA10 expression (Figure 1D). Again, the increase in gene expression of ITGB8 correlated with an increase in protein level (Figure 1E). Together, these data suggest that EPHB4 and ITGB8 are co-regulated in prostate cancer cells. As integrin β8 has only one known heterodimer partner, integrin αV (ITGAV) [10], we also sought to determine whether over-expression of EphB4 increases the expression of this integrin subunit. EPHB4 overexpression in 22Rv1 cells significantly increased ITGAV gene expression by 1.7 fold (Figure 1F) suggesting an overall increase in the integrin αvβ8 heterodimer complex.Figure 1

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