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TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6 , L1CAM , and Akt signalling

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ABSTRACT

Epithelial ovarian cancer (EOC) is the most deadly gynaecologic malignancy due to late onset of symptoms and propensity towards drug resistance. Epithelial-mesenchymal transition (EMT) has been linked to the development of chemoresistance in other cancers, yet little is known regarding its role in EOC. In this study, we sought to determine the role of the transcription factor TWIST1, a master regulator of EMT, on cisplatin resistance in an EOC model. We created two Ovcar8-derived cell lines that differed only in their TWIST1 expression. TWIST1 expression led to increased tumour engraftment in mice, as well as cisplatin resistance in vitro. RNA sequencing analysis revealed that TWIST1 expression resulted in upregulation of GAS6 and L1CAM and downregulation of HMGA2. Knockdown studies of these genes demonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin, but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, in part via GAS6 and L1CAM, led to higher expression and activation of Akt upon cisplatin treatment, and inhibition of Akt activation sensitized cells to cisplatin. These results suggest TWIST1- and EMT-driven increase in Akt activation, and thus tumour cell proliferation, as a potential mechanism of drug resistance in EOC.

No MeSH data available.


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TWIST1, GAS6, and L1CAM expression lead to upregulation of Akt signalling following cisplatin treatment.(a) Quantification of western blot data shows that over the course of 24 hr, 5 μM cisplatin treatment leads to increased levels of Akt in Ov8GFP-TWIST1 cells (blue), but not in Ov8GFP-sh492 cells (green). Knockdown of GAS6 (brown) or L1CAM (yellow) in Ov8GFP-TWIST1 cells partially abrogates the increase in Akt. NT, not treated with cisplatin. (b) Western blot also reveals an increase in activation of Akt via phosphorylation at Ser 473 over 24 hr of 5 μM cisplatin in TWIST1 expressing cells (blue). The opposite is true in Ov8GFP-sh492 cells, in which Akt activity is reduced over the same time period (green). Knockdown of GAS6 in Ov8GFP-TWIST1 cells (brown) maintains a constant pAkt/Akt ratio, while L1CAM knockdown (yellow) partially prevents Akt activation. (c) Ov8GFP-TWIST1 cells further increase their TWIST1 expression up to 2.3 fold over 24 hr of exposure to 5 μM cisplatin (p=0.0827), whereas Ov8GFP-sh492 cells show no increase. (d) Treatment of Ov8GFP-TWIST1 cells with the PI3K inhibitor LY294002 to prevent Akt activation sensitized cells to cisplatin, compared to DMSO only control, supporting the assertion that Akt signalling is central to TWIST1-driven cisplatin resistance. p < 0.0001 for both concentrations. Error bars represent standard error of the mean.
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f4: TWIST1, GAS6, and L1CAM expression lead to upregulation of Akt signalling following cisplatin treatment.(a) Quantification of western blot data shows that over the course of 24 hr, 5 μM cisplatin treatment leads to increased levels of Akt in Ov8GFP-TWIST1 cells (blue), but not in Ov8GFP-sh492 cells (green). Knockdown of GAS6 (brown) or L1CAM (yellow) in Ov8GFP-TWIST1 cells partially abrogates the increase in Akt. NT, not treated with cisplatin. (b) Western blot also reveals an increase in activation of Akt via phosphorylation at Ser 473 over 24 hr of 5 μM cisplatin in TWIST1 expressing cells (blue). The opposite is true in Ov8GFP-sh492 cells, in which Akt activity is reduced over the same time period (green). Knockdown of GAS6 in Ov8GFP-TWIST1 cells (brown) maintains a constant pAkt/Akt ratio, while L1CAM knockdown (yellow) partially prevents Akt activation. (c) Ov8GFP-TWIST1 cells further increase their TWIST1 expression up to 2.3 fold over 24 hr of exposure to 5 μM cisplatin (p=0.0827), whereas Ov8GFP-sh492 cells show no increase. (d) Treatment of Ov8GFP-TWIST1 cells with the PI3K inhibitor LY294002 to prevent Akt activation sensitized cells to cisplatin, compared to DMSO only control, supporting the assertion that Akt signalling is central to TWIST1-driven cisplatin resistance. p < 0.0001 for both concentrations. Error bars represent standard error of the mean.

Mentions: Following treatment of cells with siQ control siRNA or pooled siRNAs against GAS6 or L1CAM, western blotting of total Akt showed that while Ov8GFP-TWIST1 cells have lower initial Akt expression compared to OvGFP-sh492, continued exposure to 5 μM cisplatin led to a 150% increase in Akt levels in Ov8GFP-TWIST1 cells over the course of 24 hr (Fig. 4a). In Ov8GFP-sh492 cells, total Akt levels remain relatively constant over 24 hr of cisplatin exposure (Fig. 4a). Interestingly, the proportion of Akt in its active form (i.e. phosphorylated at Ser 473) increases 128% over the course of 24 hr in Ov8GFP-TWIST1 cells, even when normalized to total Akt expression at each time point (Fig. 4b). Conversely, OvGFP-sh492 cells show a 63% reduction in phosphorylated Akt over the same 24 hr period (Fig. 4b). The pattern of Akt activation in these cell lines mirrors the activation of TWIST1 itself in Ov8GFP-TWIST1 cells over 24 hours of cisplatin treatment, which is absent in sh492 cells (Fig. 4c).


TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6 , L1CAM , and Akt signalling
TWIST1, GAS6, and L1CAM expression lead to upregulation of Akt signalling following cisplatin treatment.(a) Quantification of western blot data shows that over the course of 24 hr, 5 μM cisplatin treatment leads to increased levels of Akt in Ov8GFP-TWIST1 cells (blue), but not in Ov8GFP-sh492 cells (green). Knockdown of GAS6 (brown) or L1CAM (yellow) in Ov8GFP-TWIST1 cells partially abrogates the increase in Akt. NT, not treated with cisplatin. (b) Western blot also reveals an increase in activation of Akt via phosphorylation at Ser 473 over 24 hr of 5 μM cisplatin in TWIST1 expressing cells (blue). The opposite is true in Ov8GFP-sh492 cells, in which Akt activity is reduced over the same time period (green). Knockdown of GAS6 in Ov8GFP-TWIST1 cells (brown) maintains a constant pAkt/Akt ratio, while L1CAM knockdown (yellow) partially prevents Akt activation. (c) Ov8GFP-TWIST1 cells further increase their TWIST1 expression up to 2.3 fold over 24 hr of exposure to 5 μM cisplatin (p=0.0827), whereas Ov8GFP-sh492 cells show no increase. (d) Treatment of Ov8GFP-TWIST1 cells with the PI3K inhibitor LY294002 to prevent Akt activation sensitized cells to cisplatin, compared to DMSO only control, supporting the assertion that Akt signalling is central to TWIST1-driven cisplatin resistance. p < 0.0001 for both concentrations. Error bars represent standard error of the mean.
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f4: TWIST1, GAS6, and L1CAM expression lead to upregulation of Akt signalling following cisplatin treatment.(a) Quantification of western blot data shows that over the course of 24 hr, 5 μM cisplatin treatment leads to increased levels of Akt in Ov8GFP-TWIST1 cells (blue), but not in Ov8GFP-sh492 cells (green). Knockdown of GAS6 (brown) or L1CAM (yellow) in Ov8GFP-TWIST1 cells partially abrogates the increase in Akt. NT, not treated with cisplatin. (b) Western blot also reveals an increase in activation of Akt via phosphorylation at Ser 473 over 24 hr of 5 μM cisplatin in TWIST1 expressing cells (blue). The opposite is true in Ov8GFP-sh492 cells, in which Akt activity is reduced over the same time period (green). Knockdown of GAS6 in Ov8GFP-TWIST1 cells (brown) maintains a constant pAkt/Akt ratio, while L1CAM knockdown (yellow) partially prevents Akt activation. (c) Ov8GFP-TWIST1 cells further increase their TWIST1 expression up to 2.3 fold over 24 hr of exposure to 5 μM cisplatin (p=0.0827), whereas Ov8GFP-sh492 cells show no increase. (d) Treatment of Ov8GFP-TWIST1 cells with the PI3K inhibitor LY294002 to prevent Akt activation sensitized cells to cisplatin, compared to DMSO only control, supporting the assertion that Akt signalling is central to TWIST1-driven cisplatin resistance. p < 0.0001 for both concentrations. Error bars represent standard error of the mean.
Mentions: Following treatment of cells with siQ control siRNA or pooled siRNAs against GAS6 or L1CAM, western blotting of total Akt showed that while Ov8GFP-TWIST1 cells have lower initial Akt expression compared to OvGFP-sh492, continued exposure to 5 μM cisplatin led to a 150% increase in Akt levels in Ov8GFP-TWIST1 cells over the course of 24 hr (Fig. 4a). In Ov8GFP-sh492 cells, total Akt levels remain relatively constant over 24 hr of cisplatin exposure (Fig. 4a). Interestingly, the proportion of Akt in its active form (i.e. phosphorylated at Ser 473) increases 128% over the course of 24 hr in Ov8GFP-TWIST1 cells, even when normalized to total Akt expression at each time point (Fig. 4b). Conversely, OvGFP-sh492 cells show a 63% reduction in phosphorylated Akt over the same 24 hr period (Fig. 4b). The pattern of Akt activation in these cell lines mirrors the activation of TWIST1 itself in Ov8GFP-TWIST1 cells over 24 hours of cisplatin treatment, which is absent in sh492 cells (Fig. 4c).

View Article: PubMed Central - PubMed

ABSTRACT

Epithelial ovarian cancer (EOC) is the most deadly gynaecologic malignancy due to late onset of symptoms and propensity towards drug resistance. Epithelial-mesenchymal transition (EMT) has been linked to the development of chemoresistance in other cancers, yet little is known regarding its role in EOC. In this study, we sought to determine the role of the transcription factor TWIST1, a master regulator of EMT, on cisplatin resistance in an EOC model. We created two Ovcar8-derived cell lines that differed only in their TWIST1 expression. TWIST1 expression led to increased tumour engraftment in mice, as well as cisplatin resistance in vitro. RNA sequencing analysis revealed that TWIST1 expression resulted in upregulation of GAS6 and L1CAM and downregulation of HMGA2. Knockdown studies of these genes demonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin, but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, in part via GAS6 and L1CAM, led to higher expression and activation of Akt upon cisplatin treatment, and inhibition of Akt activation sensitized cells to cisplatin. These results suggest TWIST1- and EMT-driven increase in Akt activation, and thus tumour cell proliferation, as a potential mechanism of drug resistance in EOC.

No MeSH data available.


Related in: MedlinePlus