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

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.


Knockdown of GAS6 or L1CAM reverses drug resistance.(aā€“c) Validation of siRNAs targeting genes of interest. qRT-PCR confirms knockdown of L1CAM (46%) and GAS6 (90%) in Ov8GFP-TWIST1 cells treated with corresponding siRNAs, and 91% knockdown of HMGA2 in Ov8GFP-sh492 cells treated with HMGA2 siRNAs. (d) Western blot confirms knockdown of L1CAM and HMGA2 at the protein level (normalized results from three independent experiments, pā€‰=ā€‰0.0276 for HMGA2, pā€‰=ā€‰0.0042 for L1CAM). (e) SRB assay demonstrates that knockdown of HMGA2 in Ov8GFP-sh492 cells is not sufficient to confer an increased resistance to cisplatin. (f) Knockdown of either GAS6 or L1CAM in Ov8GFP-TWIST1 cells sensitizes this line to cisplatin, compared to treatment with non-targeting siRNA. Upper asterisks, siGAS6 (1ā€‰Ī¼M, pā€‰=ā€‰0.0108, 3ā€‰Ī¼M pā€‰=ā€‰0.00077, 9ā€‰Ī¼M pā€‰=ā€‰0.054); lower, siL1CAM (1ā€‰Ī¼M, pā€‰=ā€‰0.00077, 3ā€‰Ī¼M pā€‰<ā€‰0.0001, 9ā€‰Ī¼M pā€‰=ā€‰0.0064). qPCR error bars represent minimum and maximum values calculated by the StepOne software analysis. SRB error bars represent standard error of the mean.
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f3: Knockdown of GAS6 or L1CAM reverses drug resistance.(aā€“c) Validation of siRNAs targeting genes of interest. qRT-PCR confirms knockdown of L1CAM (46%) and GAS6 (90%) in Ov8GFP-TWIST1 cells treated with corresponding siRNAs, and 91% knockdown of HMGA2 in Ov8GFP-sh492 cells treated with HMGA2 siRNAs. (d) Western blot confirms knockdown of L1CAM and HMGA2 at the protein level (normalized results from three independent experiments, pā€‰=ā€‰0.0276 for HMGA2, pā€‰=ā€‰0.0042 for L1CAM). (e) SRB assay demonstrates that knockdown of HMGA2 in Ov8GFP-sh492 cells is not sufficient to confer an increased resistance to cisplatin. (f) Knockdown of either GAS6 or L1CAM in Ov8GFP-TWIST1 cells sensitizes this line to cisplatin, compared to treatment with non-targeting siRNA. Upper asterisks, siGAS6 (1ā€‰Ī¼M, pā€‰=ā€‰0.0108, 3ā€‰Ī¼M pā€‰=ā€‰0.00077, 9ā€‰Ī¼M pā€‰=ā€‰0.054); lower, siL1CAM (1ā€‰Ī¼M, pā€‰=ā€‰0.00077, 3ā€‰Ī¼M pā€‰<ā€‰0.0001, 9ā€‰Ī¼M pā€‰=ā€‰0.0064). qPCR error bars represent minimum and maximum values calculated by the StepOne software analysis. SRB error bars represent standard error of the mean.

Mentions: We next knocked down each of these three genes to observe their individual effects on TWIST1-driven cell survival. qRT-PCR showed that siRNA against L1CAM and GAS6 produced 46% and 90% knockdown of their target mRNAs, respectively, in Ov8GFP-TWIST1 cells compared to non-targeting control siRNA (siQ) (Fig. 3a,b). An siRNA pool against HMGA2 reduced HMGA2 mRNA levels by 91% on average in Ov8GFP-sh492 cells (Fig. 3c). Knockdown of L1CAM and HMGA2 by their respective siRNA sequences was also confirmed at the protein level via western blot (Fig. 3d).


TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6 , L1CAM , and Akt signalling
Knockdown of GAS6 or L1CAM reverses drug resistance.(aā€“c) Validation of siRNAs targeting genes of interest. qRT-PCR confirms knockdown of L1CAM (46%) and GAS6 (90%) in Ov8GFP-TWIST1 cells treated with corresponding siRNAs, and 91% knockdown of HMGA2 in Ov8GFP-sh492 cells treated with HMGA2 siRNAs. (d) Western blot confirms knockdown of L1CAM and HMGA2 at the protein level (normalized results from three independent experiments, pā€‰=ā€‰0.0276 for HMGA2, pā€‰=ā€‰0.0042 for L1CAM). (e) SRB assay demonstrates that knockdown of HMGA2 in Ov8GFP-sh492 cells is not sufficient to confer an increased resistance to cisplatin. (f) Knockdown of either GAS6 or L1CAM in Ov8GFP-TWIST1 cells sensitizes this line to cisplatin, compared to treatment with non-targeting siRNA. Upper asterisks, siGAS6 (1ā€‰Ī¼M, pā€‰=ā€‰0.0108, 3ā€‰Ī¼M pā€‰=ā€‰0.00077, 9ā€‰Ī¼M pā€‰=ā€‰0.054); lower, siL1CAM (1ā€‰Ī¼M, pā€‰=ā€‰0.00077, 3ā€‰Ī¼M pā€‰<ā€‰0.0001, 9ā€‰Ī¼M pā€‰=ā€‰0.0064). qPCR error bars represent minimum and maximum values calculated by the StepOne software analysis. SRB error bars represent standard error of the mean.
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f3: Knockdown of GAS6 or L1CAM reverses drug resistance.(aā€“c) Validation of siRNAs targeting genes of interest. qRT-PCR confirms knockdown of L1CAM (46%) and GAS6 (90%) in Ov8GFP-TWIST1 cells treated with corresponding siRNAs, and 91% knockdown of HMGA2 in Ov8GFP-sh492 cells treated with HMGA2 siRNAs. (d) Western blot confirms knockdown of L1CAM and HMGA2 at the protein level (normalized results from three independent experiments, pā€‰=ā€‰0.0276 for HMGA2, pā€‰=ā€‰0.0042 for L1CAM). (e) SRB assay demonstrates that knockdown of HMGA2 in Ov8GFP-sh492 cells is not sufficient to confer an increased resistance to cisplatin. (f) Knockdown of either GAS6 or L1CAM in Ov8GFP-TWIST1 cells sensitizes this line to cisplatin, compared to treatment with non-targeting siRNA. Upper asterisks, siGAS6 (1ā€‰Ī¼M, pā€‰=ā€‰0.0108, 3ā€‰Ī¼M pā€‰=ā€‰0.00077, 9ā€‰Ī¼M pā€‰=ā€‰0.054); lower, siL1CAM (1ā€‰Ī¼M, pā€‰=ā€‰0.00077, 3ā€‰Ī¼M pā€‰<ā€‰0.0001, 9ā€‰Ī¼M pā€‰=ā€‰0.0064). qPCR error bars represent minimum and maximum values calculated by the StepOne software analysis. SRB error bars represent standard error of the mean.
Mentions: We next knocked down each of these three genes to observe their individual effects on TWIST1-driven cell survival. qRT-PCR showed that siRNA against L1CAM and GAS6 produced 46% and 90% knockdown of their target mRNAs, respectively, in Ov8GFP-TWIST1 cells compared to non-targeting control siRNA (siQ) (Fig. 3a,b). An siRNA pool against HMGA2 reduced HMGA2 mRNA levels by 91% on average in Ov8GFP-sh492 cells (Fig. 3c). Knockdown of L1CAM and HMGA2 by their respective siRNA sequences was also confirmed at the protein level via western blot (Fig. 3d).

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.