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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 cleavage in PCa. (A) Schematic representation of EpCAM cleavage. (B) Endogenous EpCAM is cleaved into EpCAM CTF in PCa cells. Membranes of prostate cancer cells were enriched by centrifugation and incubated at 37 °C or on ice. EpCAM FL and EpCAM C-terminal fragment (CTF) were detected using an anti-EpICD antibody. EpCAM CTF was not detected by an anti-EpEX antibody (lower blot, negative control for EpCAM CTF). (C) EpCAM-YFP is cleaved to EpCAM-CTF-YFP and EpICD-YFP in PCa (PC3) and benign prostate (RWPE-1) cells. Cleavage to EpICD-YFP was inhibited using DAPT, a γ-secretase inhibitor. Loading, 2 μg DuCAP and PC3, 5 μg Du145, LNCAP and RWPE-1. (D) EpCAM was visualised by immunofluorescence using either an anti-EpEx or an anti-EpICD antibody. Abbreviations: CTF, C-terminal fragment; EpICD, EpCAM intracellular domain; EpEx, EpCAM extracellular domain; FL, full length.
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fig4: EpCAM cleavage in PCa. (A) Schematic representation of EpCAM cleavage. (B) Endogenous EpCAM is cleaved into EpCAM CTF in PCa cells. Membranes of prostate cancer cells were enriched by centrifugation and incubated at 37 °C or on ice. EpCAM FL and EpCAM C-terminal fragment (CTF) were detected using an anti-EpICD antibody. EpCAM CTF was not detected by an anti-EpEX antibody (lower blot, negative control for EpCAM CTF). (C) EpCAM-YFP is cleaved to EpCAM-CTF-YFP and EpICD-YFP in PCa (PC3) and benign prostate (RWPE-1) cells. Cleavage to EpICD-YFP was inhibited using DAPT, a γ-secretase inhibitor. Loading, 2 μg DuCAP and PC3, 5 μg Du145, LNCAP and RWPE-1. (D) EpCAM was visualised by immunofluorescence using either an anti-EpEx or an anti-EpICD antibody. Abbreviations: CTF, C-terminal fragment; EpICD, EpCAM intracellular domain; EpEx, EpCAM extracellular domain; FL, full length.

Mentions: Besides its role as an adhesion molecule, EpCAM functions as a signalling molecule and transcription regulator. This EpCAM function is based on RIP; Maetzel et al, 2009; Figure 4A). To study whether EpCAM exerts potential signalling functions in PCa, we investigated EpCAM RIP. Epithelial cell adhesion molecule is cleaved by proteases present in cell membranes (sheddases) from EpCAM full length (EpCAM-FL) to EpCAM C-terminal fragment (EpCAM-CTF), which is a substrate for γ-secretase that is further processed to the intracellular signalling molecule EpICD Maetzel et al, 2009; Figure 4A). To visualise EpCAM-FL, EpCAM-CFT and EpICD, we performed an immunoblot using antibodies, which selectively recognise the extracellular (anti-EpEX) or intracellular (anti-EpICD) domain of EpCAM. Cell membranes of EpCAM-positive PCa cell lines were enriched as previously described (Hachmeister et al, 2013) and incubated at 4 °C and 37 °C to repress or allow protease activity. In all four investigated PCa cell lines the first step of EpCAM cleavage (i.e., generation of EpCAM-CTF) was observed (Figure 4B). Visualisation of the second step of EpCAM cleavage (i.e., EpCAM-CTF to EpICD) is challenging in this setting because EpICD has a low apparent molecular weight of 5–6 kDa and a short half-live owing to degradation by the proteasome (Hachmeister et al, 2013). To facilitate the detection of EpICD, we stabilised and enlarged EpICD by fusion to yellow fluorescence protein (YFP), as described (Maetzel et al, 2009; Hachmeister et al, 2013). In PCa cells overexpressing EpCAM-YFP, cleavage to EpCAM-YFP-CTF (first cleavage) and EpICD-YFP (second cleavage) was observed. Cleavage of EpCAM-CTF-YFP to EpICD-YFP was inhibited by DAPT, a chemical γ-secretase inhibitor (Figure 4C). Interestingly, also cells, which have very low endogenous EpCAM levels, such as the non-cancerous prostate cells RWPE-1, process EpCAM-YFP to EpICD-YFP, suggesting that the required processing machinery is present even in the absence of high levels of EpCAM protein. Next, EpCAM and its cleavage products were visualised using anti-EpEX and anti-EpICD antibodies in immunofluorescence stainings. The anti-EpEX antibody recognised full length EpCAM, localised at the membrane, while the anti-EpICD antibody recognised both, full length EpCAM localised at the cell membrane as well as cleaved EpICD in the cytoplasm (Figure 4D). Collectively these data reveal that EpCAM cleavage and generation of the intracellular signalling molecule EpICD is detectable in PCa cells, indicating that EpCAM RIP is active in PCa.


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 cleavage in PCa. (A) Schematic representation of EpCAM cleavage. (B) Endogenous EpCAM is cleaved into EpCAM CTF in PCa cells. Membranes of prostate cancer cells were enriched by centrifugation and incubated at 37 °C or on ice. EpCAM FL and EpCAM C-terminal fragment (CTF) were detected using an anti-EpICD antibody. EpCAM CTF was not detected by an anti-EpEX antibody (lower blot, negative control for EpCAM CTF). (C) EpCAM-YFP is cleaved to EpCAM-CTF-YFP and EpICD-YFP in PCa (PC3) and benign prostate (RWPE-1) cells. Cleavage to EpICD-YFP was inhibited using DAPT, a γ-secretase inhibitor. Loading, 2 μg DuCAP and PC3, 5 μg Du145, LNCAP and RWPE-1. (D) EpCAM was visualised by immunofluorescence using either an anti-EpEx or an anti-EpICD antibody. Abbreviations: CTF, C-terminal fragment; EpICD, EpCAM intracellular domain; EpEx, EpCAM extracellular domain; FL, full length.
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fig4: EpCAM cleavage in PCa. (A) Schematic representation of EpCAM cleavage. (B) Endogenous EpCAM is cleaved into EpCAM CTF in PCa cells. Membranes of prostate cancer cells were enriched by centrifugation and incubated at 37 °C or on ice. EpCAM FL and EpCAM C-terminal fragment (CTF) were detected using an anti-EpICD antibody. EpCAM CTF was not detected by an anti-EpEX antibody (lower blot, negative control for EpCAM CTF). (C) EpCAM-YFP is cleaved to EpCAM-CTF-YFP and EpICD-YFP in PCa (PC3) and benign prostate (RWPE-1) cells. Cleavage to EpICD-YFP was inhibited using DAPT, a γ-secretase inhibitor. Loading, 2 μg DuCAP and PC3, 5 μg Du145, LNCAP and RWPE-1. (D) EpCAM was visualised by immunofluorescence using either an anti-EpEx or an anti-EpICD antibody. Abbreviations: CTF, C-terminal fragment; EpICD, EpCAM intracellular domain; EpEx, EpCAM extracellular domain; FL, full length.
Mentions: Besides its role as an adhesion molecule, EpCAM functions as a signalling molecule and transcription regulator. This EpCAM function is based on RIP; Maetzel et al, 2009; Figure 4A). To study whether EpCAM exerts potential signalling functions in PCa, we investigated EpCAM RIP. Epithelial cell adhesion molecule is cleaved by proteases present in cell membranes (sheddases) from EpCAM full length (EpCAM-FL) to EpCAM C-terminal fragment (EpCAM-CTF), which is a substrate for γ-secretase that is further processed to the intracellular signalling molecule EpICD Maetzel et al, 2009; Figure 4A). To visualise EpCAM-FL, EpCAM-CFT and EpICD, we performed an immunoblot using antibodies, which selectively recognise the extracellular (anti-EpEX) or intracellular (anti-EpICD) domain of EpCAM. Cell membranes of EpCAM-positive PCa cell lines were enriched as previously described (Hachmeister et al, 2013) and incubated at 4 °C and 37 °C to repress or allow protease activity. In all four investigated PCa cell lines the first step of EpCAM cleavage (i.e., generation of EpCAM-CTF) was observed (Figure 4B). Visualisation of the second step of EpCAM cleavage (i.e., EpCAM-CTF to EpICD) is challenging in this setting because EpICD has a low apparent molecular weight of 5–6 kDa and a short half-live owing to degradation by the proteasome (Hachmeister et al, 2013). To facilitate the detection of EpICD, we stabilised and enlarged EpICD by fusion to yellow fluorescence protein (YFP), as described (Maetzel et al, 2009; Hachmeister et al, 2013). In PCa cells overexpressing EpCAM-YFP, cleavage to EpCAM-YFP-CTF (first cleavage) and EpICD-YFP (second cleavage) was observed. Cleavage of EpCAM-CTF-YFP to EpICD-YFP was inhibited by DAPT, a chemical γ-secretase inhibitor (Figure 4C). Interestingly, also cells, which have very low endogenous EpCAM levels, such as the non-cancerous prostate cells RWPE-1, process EpCAM-YFP to EpICD-YFP, suggesting that the required processing machinery is present even in the absence of high levels of EpCAM protein. Next, EpCAM and its cleavage products were visualised using anti-EpEX and anti-EpICD antibodies in immunofluorescence stainings. The anti-EpEX antibody recognised full length EpCAM, localised at the membrane, while the anti-EpICD antibody recognised both, full length EpCAM localised at the cell membrane as well as cleaved EpICD in the cytoplasm (Figure 4D). Collectively these data reveal that EpCAM cleavage and generation of the intracellular signalling molecule EpICD is detectable in PCa cells, indicating that EpCAM RIP is active in PCa.

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