Fig 1: Knockdown of GAS6 or L1CAM reverses drug resistance.(a–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, p = 0.0276 for HMGA2, p = 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 (1 µM, p = 0.0108, 3 µM p = 0.00077, 9 µM p = 0.054); lower, siL1CAM (1 µM, p = 0.00077, 3 µM p < 0.0001, 9 µM p = 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.
Fig 2: Schematic representation of our proposed model.TWIST1 upregulates expression of GAS6 and L1CAM. TWIST1, GAS6, and L1CAM facilitate phosphorylation and activation of Akt, leading to increased proliferation. This in turn results in cisplatin resistance and greater tumour cell engraftment in vivo.
Fig 3: 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.
Fig 4: RNA sequencing reveals differential expression of GAS6, L1CAM, and HMGA2.(a) RNA sequencing showed approximately 2-fold increases in GAS6 and L1CAM and a 2-fold decrease in HMGA2 mRNA when TWIST1 is overexpressed. FPKM, fragments per kilobase per million reads. (b) Western blot confirms differential expression of L1CAM and HMGA2 found by RNA-seq. Blots cropped for clarity; full blots are shown in Supplementary Fig. S5. (c) No western blot was possible for GAS6, as the protein is secreted, but qRT-PCR shows on average a 50% decrease in GAS6 mRNA level upon TWIST1 knockdown. p = 0.31, although a clear trend is present. Error bars represent standard error of the mean.
Supplier Page from Santa Cruz Biotechnology, Inc. for NCAM-L1 siRNA (h)