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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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Integrin expression levels across disease progression. A) cDNA from several different prostate-derived cell lines was analyzed using semi-quantitative RT-PCR. GAPDH amplification was used as a loading control. B) Gene expression omnibus dataset GDS3289 investigating prostate cancer progression in LCM-captured clinical samples was interrogated for EPHB4 and ITGB8 expression using the Oncomine clinical database (www.oncomine.org). Both genes are significantly elevated in prostatic intraepithelial neoplasia (PIN) and EPHB4 is significantly upregulated in carcinoma samples compared to benign. * p < 0.001, mets = metastatic disease.
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Fig3: Integrin expression levels across disease progression. A) cDNA from several different prostate-derived cell lines was analyzed using semi-quantitative RT-PCR. GAPDH amplification was used as a loading control. B) Gene expression omnibus dataset GDS3289 investigating prostate cancer progression in LCM-captured clinical samples was interrogated for EPHB4 and ITGB8 expression using the Oncomine clinical database (www.oncomine.org). Both genes are significantly elevated in prostatic intraepithelial neoplasia (PIN) and EPHB4 is significantly upregulated in carcinoma samples compared to benign. * p < 0.001, mets = metastatic disease.

Mentions: To investigate whether there is a correlation between ITGB8 and EPHB4 expression in different prostate cancer cells representing different stages of the disease, several cell lines were subjected to RT-PCR (Figure 3A). All prostate cell lines tested expressed EPHB4 as expected [5,7]. ITGB8 was most highly expressed in PC-3 and DU145 cells, followed by BPH-1 and LNCaP cells. The metastatic subclonal line C4-2B derived from LNCaP cells [12] showed no ITGB8 mRNA expression. To further assess whether ITGB8 expression could be indicative of clinical disease progression we surveyed the Oncomine database. Analysis of the prostate cancer progression study conducted by Tomlins et al. [13] revealed that in benign samples both EPHB4 and ITGB8 are expressed at a low level and this increases dramatically and significantly in prostatic intraepithelial neoplasia (PIN) (ITGB8: 9.5 fold, p = 1.24 x 10-4 and EPHB4 2.9 fold, p = 0.001), the precursor for prostate carcinoma [14]. In carcinoma samples the expression of both genes is elevated, with EPHB4 being significantly up-regulated in comparison to benign tissue samples, but expression of both ITGB8 and EPHB4 is much lower than in PIN tissues. Metastatic samples show expression levels similar to benign tissue for both EPHB4 and ITGB8. These results indicate that EPHB4 and ITGB8 are concurrently up-regulated in PIN and their expression is progressively lowered with disease advancement. This suggests that both genes may play a role in the onset of prostate cancer.Figure 3


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)

Integrin expression levels across disease progression. A) cDNA from several different prostate-derived cell lines was analyzed using semi-quantitative RT-PCR. GAPDH amplification was used as a loading control. B) Gene expression omnibus dataset GDS3289 investigating prostate cancer progression in LCM-captured clinical samples was interrogated for EPHB4 and ITGB8 expression using the Oncomine clinical database (www.oncomine.org). Both genes are significantly elevated in prostatic intraepithelial neoplasia (PIN) and EPHB4 is significantly upregulated in carcinoma samples compared to benign. * p < 0.001, mets = metastatic disease.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Integrin expression levels across disease progression. A) cDNA from several different prostate-derived cell lines was analyzed using semi-quantitative RT-PCR. GAPDH amplification was used as a loading control. B) Gene expression omnibus dataset GDS3289 investigating prostate cancer progression in LCM-captured clinical samples was interrogated for EPHB4 and ITGB8 expression using the Oncomine clinical database (www.oncomine.org). Both genes are significantly elevated in prostatic intraepithelial neoplasia (PIN) and EPHB4 is significantly upregulated in carcinoma samples compared to benign. * p < 0.001, mets = metastatic disease.
Mentions: To investigate whether there is a correlation between ITGB8 and EPHB4 expression in different prostate cancer cells representing different stages of the disease, several cell lines were subjected to RT-PCR (Figure 3A). All prostate cell lines tested expressed EPHB4 as expected [5,7]. ITGB8 was most highly expressed in PC-3 and DU145 cells, followed by BPH-1 and LNCaP cells. The metastatic subclonal line C4-2B derived from LNCaP cells [12] showed no ITGB8 mRNA expression. To further assess whether ITGB8 expression could be indicative of clinical disease progression we surveyed the Oncomine database. Analysis of the prostate cancer progression study conducted by Tomlins et al. [13] revealed that in benign samples both EPHB4 and ITGB8 are expressed at a low level and this increases dramatically and significantly in prostatic intraepithelial neoplasia (PIN) (ITGB8: 9.5 fold, p = 1.24 x 10-4 and EPHB4 2.9 fold, p = 0.001), the precursor for prostate carcinoma [14]. In carcinoma samples the expression of both genes is elevated, with EPHB4 being significantly up-regulated in comparison to benign tissue samples, but expression of both ITGB8 and EPHB4 is much lower than in PIN tissues. Metastatic samples show expression levels similar to benign tissue for both EPHB4 and ITGB8. These results indicate that EPHB4 and ITGB8 are concurrently up-regulated in PIN and their expression is progressively lowered with disease advancement. This suggests that both genes may play a role in the onset of prostate cancer.Figure 3

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