Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline

Abstract

Cetrorelix, a luteinising hormone-releasing hormone (LHRH) analogue, has been shown to limit growth of the human androgenindependent prostate cell line DU-145, although other inhibitory actions may also be affected. Both growth and invasion of DU-145 cells are linked to autocrine epidermal growth factor receptor (EGFR) signalling. Invasiveness requires not only cells to migrate to conduits, but also reduced adhesiveness between tumour cells to enable separation from the tumour mass. Thus, we investigated whether Cetrorelix alters the DU-145 cell -cell adhesion and if this occurs via altered EGFR signalling. Pharmacologic levels of Cetrorelix limited the invasiveness of a highly invasive DU-145 subline overexpressing full-length EGFR (DU-145 WT). Extended exposure of the cells to Cetrorelix resulted in increased levels of the cell -cell adhesion complex molecules E-cadherin, a-and bcatenin, and p120. Puromycin blocked the increases in E-cadherin and b-catenin levels, suggesting that de novo protein synthesis is required. The Cetrorelix effect appears to occur via transmodulation of EGFR by a protein kinase C (PKC)-dependent mechanism, as there were no changes in DU-145 cells expressing EGFR engineered to negate the PKC transattenuation site (DU-145 A654); downregulation of EGFR signalling produced a similar upregulation in adhesion complex proteins, further suggesting a role for autocrine signalling. Cetrorelix increased the cell -cell adhesiveness of DU-145 WT cells to an extent similar to that seen when autocrine EGFR signalling is blocked; as expected, DU-145 A654 cell -cell adhesion also was unaffected by Cetrorelix. The increased adhesiveness is expected as the adhesion complex molecules moved to the cells' periphery. These data offer direct insight into the possible crosstalk pathways between the LHRH and EGFR receptor signalling. The ability of Cetrorelix to downregulate EGFR signalling and subsequently reverse the antiadhesiveness found in metastatic prostate cancer highlights a novel potential target for therapeutic strategies.

Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin

“…This suggested that autocrine EGFR signalling present in prostate carcinomas may contribute to E-cadherin repression in these tumours. This was supported by our recent finding that pharmacological abrogation of EGFR signalling in prostate carcinoma cells reverses decreased E-cadherin expression rendering these cells less invasive and more cohesive (Jawhari et al, 1999;Yates et al, 2005). This re-expression of E-cadherin is similarly observed by close contacts of DU-145 prostate carcinoma cells when cultured within the liver microenvironment (Yates et al, 2007).…”

“…As this was reminiscent of the situation with LHRH receptor transmodulation of autocrine EGFR signalling in DU-145 cells (Yates et al, 2005), we asked whether a similar situation might be functioning in this setting. We expressed the PKC transmodulation-resistant EGFR A654 variant (Welsh et al, 1991) in the DU-145 cells.…”

“…As the cell -cell adhesion of the prostate carcinoma cells was limited, though real, we sought to increase the levels of Ecadherin on these cells. For this we used the EGFR kinase inhibitor, PD153035, that we had shown earlier to promote E-cadherin expression in DU-145 cells within 24 -48 h (Yates et al, 2005), and confirmed in PC-3 cells at 48 h ( Figure 3C). This 48-h pretreatment with PD153035 increased prostate carcinoma cell binding to the hepatocyte monolayer ( Figure 3A -E).…”

“…A transmodulation-resistant EGFR construct was generated by replacing the PKC-target threonine at amino acid 654 with alanine (A654) and transfected into DU-145 cells (Yates et al, 2005). Prostate cancer cells lines were cultured as described previously (Yates et al, 2005).…”

Mechanism of the Mesenchymal–Epithelial Transition and Its Relationship with Metastatic Tumor Formation

“…They are always overexpressed in malignant tumors and are thought to contribute to tumor progression (34,35). Yates and colleagues (36,37) found that in vitro inhibition of autocrine EGFR signaling increased E-cadherin expression and cell-cell heterotypic adhesion and that E-cadherin upregulation was prevented by expressing a downregulation-resistant EGFR variant. E-cadherin and catenins, but not activated EGFR, were also found in human prostate cancer metastases to the liver, supporting the notion that the inverse relationship between E-cadherin expression and EGFR also exists in de novo human tumors (36).…”