Epimorphin-Induced MET Sensitizes Ovarian Cancer Cells to Platinum

<div><p>Distinctive genotypic and phenotypic features of ovarian cancer via epithelial-mesenchymal transition (EMT) have been correlated with drug resistance and disease recurrence. We investigated whether therapeutic reversal of EMT could re-sensitize ovarian cancer cells (OCCs) to existing chemotherapy. We report that epimorphin, a morphogenic protein, has pivotal control over mesenchymal versus epithelial cell lineage decision of the putative OCCs. Exposure to epimorphin induced morphological changes reminiscent of mesenchymal-to-epithelial transition (MET), but in a dose dependent manner, i.e., at 10 µg/mL of epimorphin cells obtain a more mesenchymal-like morphology while at 20 µg/mL of epimorphin cells display an epithelial morphology. The latter changes were accompanied by suppression of mesenchymal markers, such as vimentin (∼8-fold↓, <i>p</i><0.02), Twist1 (∼7-fold↓, <i>p</i><0.03), dystroglycan (∼4-fold↓, <i>p</i><0.01) and palladin (∼3-fold↓, <i>p</i><0.01). Conversely, significant elevations of KLF4 (∼28-fold↑, <i>p</i><0.002), β-catenin (∼6-fold↑, <i>p</i><0.004), EpCAM (∼6-fold↑, <i>p</i><0.0002) and occludin (∼15-fold↑, <i>p</i><0.004) mRNAs as part of the commitment to the epithelial cell lineage were detected in response to 20 µg/mL of exogenous epimorphin. Changes in occludin mRNA levels were accompanied by a parallel, albeit weaker expression at the protein level (∼5-fold↑, <i>p</i><0.001). Likewise, acquisition of epithelial-like properties, including mucin1, CK19, and β-catenin gene expression, was also obtained following epimorphin treatment. Further, MMP3 production was found to be reduced whereas laminin secretion was strongly amplified upon epimorphin-induced MET. These results suggest there is a dosage window for actions of epimorphin on cellular differentiation, wherein it can either suppress or enhance epithelial differentiation of OCCs. Importantly, induction of epithelial-like phenotypes by epimorphin led to an enhanced sensitivity to carboplatin. Overall, we demonstrate that epimorphin can revert OCCs away from their mesenchymal phenotype and toward an epithelial phenotype, thereby enhancing their sensitivity to a front-line chemotherapeutic agent.</p></div

Quantitative effects of epimorphin on expression of epithelial and mesenchymal genes in ovarian cancer cells.

<p>A and B: αV-integrin receptor (A) and C/EBPβ (B) showed marked elevation of mRNA levels in response to 20 µg/mL of epimorphin. C–F: Epithelial markers such as KLF4 (C), β-catenin (D), occludin (E), and EpCAM (F) were found to be upregulated by exogenous 20 µg/mL of epimorphin. G–J: Expression of mesenchymal markers TWIST1 (G), vimentin (H), dystroglycan (I) and palladin (J) were downregulated following treatment with epimorphin (20 µg/ml). However, all four mesenchymal markers (G–J) were found to be upregulated by 10 µg/mL of epimorphin (means ± S.D., n = 3) [*<i>p</i><0.05 compared with control].</p

Analysis of expression of MET-associated proteins.

<p>Protein expression of the epithelial-associated markers, e.g., mucin-1, CK-19 and occludin were evaluated by immunoblotting and band densities were quantified by Image-J software. Fold increases in protein expression of treated (20 µg/ml epimorphin) versus untreated A1847: 2.5-fold increase for CK-19; 1.5-fold increase for mucin-1, and 4.5-fold increase for occludin. β-actin served as the loading control. (means ± S.D., n = 3). [*<i>p</i><0.05 compared with control].</p

Evaluation of laminin and MMP-3 following epimorphin induced MET.

<p>Secretion of laminin (epithelial marker) and MMP-3 (mesenchymal marker) was measure by ELISA following treatment of A1847 ovarian cancer cells with epimorphin for 3 days (at 10 or 20 µg/ml). Exposure to epimorphin (20 µg/ml) significantly increased laminin production and lead to a decrease in MMP-3 secretion when compared to control. In comparison, MMP-3 release was enhanced when A1847 treated with the lower concentration of epimorphin (10 µg/ml), again suggesting epimorphin can induce phenotypic changes in ovarian cancer cells in a dose-dependent manner (means ± S.D., n = 3) [*<i>p</i><0.05 compared with control].</p

Carboplatin-induced changes in cell viability and apoptosis following epimorphin-induced MET.

<p>A-F: A1847, A2780, and OVCAR10 were treated with 20 µg/mL epimorphin for 3 days. After 3 days, epimorphin-treated and untreated OCCs were cultured in triplicate with serial doses of carboplatin for an additional 3 days. Cell viability was quantified using a CellTiter Blue® assay (A–C). Apoptosis was quantitated using a Guava Nexin assay (D–F). A–C: IC₅₀ values indicate carboplatin induced more cell viability loss in all three epimophin-treated OCCs than the untreated controls in a dose-dependent manner. D–F: Detection of apoptotic responses was found to increase in all three epimorphin-treated OCCs with increasing concentrations of cisplatin compared to those of the untreated controls. Data were normalized to the controls and are represented as means ± S.D. [*<i>p</i><0.05 compared with control]. Images of apoptotic cells were captured at 10X magnification, with at least 3 images per well, using an upright phase-contrast microscope (T3.15A; Fisher Scientific).</p

β-catenin activation of A1847 in response to epimorphin.

<p>Untreated and 20 µg/mL epimorphin-treated A1847 and OVCAR10 were analyzed for β-catenin, a marker of epithelial differentiation, by immunostaining. β-catenin (green), DAPI (blue) and merge (neon green) in untreated A1847 (A–C) and OVCAR10 (G–I); epimorphin-treated A1847 (D–F) and OVCAR10 (J–L). Immunostaining analysis showed increased expression of β-catenin-positive cells in epimorphin-treated A1847 (D&F) and epimorphin-treated OVCAR10 (J&L). There were abundant β-catenin positive cells at and around the cell-cell junctions of epimorphin-induced A1847 and OVCAR10 (F&L) compared to untreated controls (C&I).</p