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EpCAM is overexpressed in local and metastatic prostate cancer, suppressed by chemotherapy and modulated by MET-associated miRNA-200c/205.

Massoner P, Thomm T, Mack B, Untergasser G, Martowicz A, Bobowski K, Klocker H, Gires O, Puhr M - Br. J. Cancer (2014)

Bottom Line: Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells.Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro.In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression.

View Article: PubMed Central - PubMed

Affiliation: 1] Experimental Urology, Department of Urology, Innsbruck Medical University, Innsbruck, Austria [2] Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT

Background: Expression of epithelial cell adhesion molecule (EpCAM) is deregulated in epithelial malignancies. Beside its role in cell adhesion, EpCAM acts as signalling molecule with tumour-promoting functions. Thus, EpCAM is part of the molecular network of oncogenic receptors and considered an interesting therapeutic target.

Methods: Here, we thoroughly characterised EpCAM expression on mRNA and protein level in comprehensive tissue studies including non-cancerous prostate specimens, primary tumours of different grades and stages, metastatic lesions, and therapy-treated tumour specimens, as well as in prostate cancer cell lines.

Results: Epithelial cell adhesion molecule was overexpressed at mRNA and at protein level in prostate cancer tissues and cell lines. Altered EpCAM expression was an early event in prostate carcinogenesis with an upregulation in low-grade cancers and further induction in high-grade tumours and metastatic lesions. Interestingly, EpCAM was repressed upon induction of epithelial-to-mesenchymal transition (EMT) following chemotherapeutic treatment with docetaxel. Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells. Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro.

Conclusions: In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression. Epithelial cell adhesion molecule displays a dynamic, heterogeneous expression and associates with epithelial cells rather than mesenchymal, chemoresistant cells along with processes of EMT and MET.

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EpCAM has no proliferative effect on PCa cells in vitro. EpCAM knockdown was determined at mRNA level by qRT–PCR (A) and at protein level by immunoblot (B). Uniform transduction efficiency in shRNA cells was controlled by GFP expression (C). Neither transient EpCAM knockdown by siRNA for 72 h nor stable EpCAM knockdown by shRNA for a long time period (up to passage 20 after transduction) had any effect on PCa cell proliferation (D). Inhibition of EpCAM cleavage (first step of EpCAM signalling) by DAPT (γ-secretase inhibitor, E) or overexpression of EpICD-YFP (YFP-stabilised intracellular domain of EpCAM, F) had no influence on prostate cell proliferation.
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fig5: EpCAM has no proliferative effect on PCa cells in vitro. EpCAM knockdown was determined at mRNA level by qRT–PCR (A) and at protein level by immunoblot (B). Uniform transduction efficiency in shRNA cells was controlled by GFP expression (C). Neither transient EpCAM knockdown by siRNA for 72 h nor stable EpCAM knockdown by shRNA for a long time period (up to passage 20 after transduction) had any effect on PCa cell proliferation (D). Inhibition of EpCAM cleavage (first step of EpCAM signalling) by DAPT (γ-secretase inhibitor, E) or overexpression of EpICD-YFP (YFP-stabilised intracellular domain of EpCAM, F) had no influence on prostate cell proliferation.

Mentions: Epithelial cell adhesion molecule expression was reported to have a major impact on proliferation of cellular models deriving from several carcinoma entities including breast (Martowicz et al, 2012), colorectal (Maaser and Borlak, 2008), lung (Maaser and Borlak, 2008) and various forms of head and neck cancer (Maetzel et al, 2009; Chaves-Perez et al, 2012; Driemel et al, 2013). To test whether EpCAM has a proliferative effect also on PCa cells, we transiently or stably knocked down EpCAM by siRNA or shRNA, respectively. Epithelial cell adhesion molecule knockdown at mRNA and protein level was controlled by qRT–PCR and immunoblot (Figure 5A and B). As our shRNA vector contained a GFP gene, we controlled uniform transduction efficiency of shRNA cells by measuring GFP expression using flow cytometry (Figure 5C). Next, we investigated the impact of EpCAM knockdown on cell proliferation using two different PCa cell lines. We found that neither transient EpCAM knockdown for 72 h nor stable EpCAM knockdown for a long time period (cells observed from passage 2–20=up to10 weeks) had any measurable effect on cell proliferation (Figure 5D). In addition, inhibition of EpCAM signalling via RIP by inhibiting EpCAM cleavage using the chemical γ-secretase inhibitor DAPT had no inhibitory effect on prostate cell proliferation. For this experiment, we used three different PCa cell lines (Du145, DuCAP and PC3) and a non-cancerous prostate cell line (RWPE-1) with comparable results (Figure 5E). Finally, we tested whether overexpression of the signalling moiety EpICD mediates proliferative effects since EpICD was described to have the highest impact on cell proliferation (Maetzel et al, 2009; Chaves-Perez et al, 2012). Overexpression of EpICD-YFP had no influence on PCa cell proliferation or on proliferation of non-cancerous prostate cells (RWPE-1; Figure 5F). In summary EpCAM did not exert proliferative actions in PCa cells in vitro indicating that PCa cells are – in contrasts to other cellular cancer models – not dependent on high EpCAM expression levels for fast cell proliferation.


EpCAM is overexpressed in local and metastatic prostate cancer, suppressed by chemotherapy and modulated by MET-associated miRNA-200c/205.

Massoner P, Thomm T, Mack B, Untergasser G, Martowicz A, Bobowski K, Klocker H, Gires O, Puhr M - Br. J. Cancer (2014)

EpCAM has no proliferative effect on PCa cells in vitro. EpCAM knockdown was determined at mRNA level by qRT–PCR (A) and at protein level by immunoblot (B). Uniform transduction efficiency in shRNA cells was controlled by GFP expression (C). Neither transient EpCAM knockdown by siRNA for 72 h nor stable EpCAM knockdown by shRNA for a long time period (up to passage 20 after transduction) had any effect on PCa cell proliferation (D). Inhibition of EpCAM cleavage (first step of EpCAM signalling) by DAPT (γ-secretase inhibitor, E) or overexpression of EpICD-YFP (YFP-stabilised intracellular domain of EpCAM, F) had no influence on prostate cell proliferation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150273&req=5

fig5: EpCAM has no proliferative effect on PCa cells in vitro. EpCAM knockdown was determined at mRNA level by qRT–PCR (A) and at protein level by immunoblot (B). Uniform transduction efficiency in shRNA cells was controlled by GFP expression (C). Neither transient EpCAM knockdown by siRNA for 72 h nor stable EpCAM knockdown by shRNA for a long time period (up to passage 20 after transduction) had any effect on PCa cell proliferation (D). Inhibition of EpCAM cleavage (first step of EpCAM signalling) by DAPT (γ-secretase inhibitor, E) or overexpression of EpICD-YFP (YFP-stabilised intracellular domain of EpCAM, F) had no influence on prostate cell proliferation.
Mentions: Epithelial cell adhesion molecule expression was reported to have a major impact on proliferation of cellular models deriving from several carcinoma entities including breast (Martowicz et al, 2012), colorectal (Maaser and Borlak, 2008), lung (Maaser and Borlak, 2008) and various forms of head and neck cancer (Maetzel et al, 2009; Chaves-Perez et al, 2012; Driemel et al, 2013). To test whether EpCAM has a proliferative effect also on PCa cells, we transiently or stably knocked down EpCAM by siRNA or shRNA, respectively. Epithelial cell adhesion molecule knockdown at mRNA and protein level was controlled by qRT–PCR and immunoblot (Figure 5A and B). As our shRNA vector contained a GFP gene, we controlled uniform transduction efficiency of shRNA cells by measuring GFP expression using flow cytometry (Figure 5C). Next, we investigated the impact of EpCAM knockdown on cell proliferation using two different PCa cell lines. We found that neither transient EpCAM knockdown for 72 h nor stable EpCAM knockdown for a long time period (cells observed from passage 2–20=up to10 weeks) had any measurable effect on cell proliferation (Figure 5D). In addition, inhibition of EpCAM signalling via RIP by inhibiting EpCAM cleavage using the chemical γ-secretase inhibitor DAPT had no inhibitory effect on prostate cell proliferation. For this experiment, we used three different PCa cell lines (Du145, DuCAP and PC3) and a non-cancerous prostate cell line (RWPE-1) with comparable results (Figure 5E). Finally, we tested whether overexpression of the signalling moiety EpICD mediates proliferative effects since EpICD was described to have the highest impact on cell proliferation (Maetzel et al, 2009; Chaves-Perez et al, 2012). Overexpression of EpICD-YFP had no influence on PCa cell proliferation or on proliferation of non-cancerous prostate cells (RWPE-1; Figure 5F). In summary EpCAM did not exert proliferative actions in PCa cells in vitro indicating that PCa cells are – in contrasts to other cellular cancer models – not dependent on high EpCAM expression levels for fast cell proliferation.

Bottom Line: Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells.Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro.In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression.

View Article: PubMed Central - PubMed

Affiliation: 1] Experimental Urology, Department of Urology, Innsbruck Medical University, Innsbruck, Austria [2] Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT

Background: Expression of epithelial cell adhesion molecule (EpCAM) is deregulated in epithelial malignancies. Beside its role in cell adhesion, EpCAM acts as signalling molecule with tumour-promoting functions. Thus, EpCAM is part of the molecular network of oncogenic receptors and considered an interesting therapeutic target.

Methods: Here, we thoroughly characterised EpCAM expression on mRNA and protein level in comprehensive tissue studies including non-cancerous prostate specimens, primary tumours of different grades and stages, metastatic lesions, and therapy-treated tumour specimens, as well as in prostate cancer cell lines.

Results: Epithelial cell adhesion molecule was overexpressed at mRNA and at protein level in prostate cancer tissues and cell lines. Altered EpCAM expression was an early event in prostate carcinogenesis with an upregulation in low-grade cancers and further induction in high-grade tumours and metastatic lesions. Interestingly, EpCAM was repressed upon induction of epithelial-to-mesenchymal transition (EMT) following chemotherapeutic treatment with docetaxel. Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells. Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro.

Conclusions: In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression. Epithelial cell adhesion molecule displays a dynamic, heterogeneous expression and associates with epithelial cells rather than mesenchymal, chemoresistant cells along with processes of EMT and MET.

Show MeSH
Related in: MedlinePlus