Extended Data Fig. 10: Endogenous hFWE in human cancer cell lines and tumorigenic potential.

a, Culture experiments were conducted to examine the dynamics of hFWE^Win and hFWE^Lose isoform expression. MCF-7 hFWE^KO cells expressing hFWE2–RFP were co-cultured with wild-type MCF-7 cells expressing GFP for 24 h and sorted for analysis of hFWE isoform expression. n = 3 biologically independent experiments with similar results. b, Co-culture of MCF-7 hFWE^KO cells expressing hFWE2–RFP with wild-type MCF-7 cells expressing GFP caused upregulation of hFWE^Lose isoforms in wild-type MCF-7 cells. qPCR analysis of the expression of hFWE^Win and hFWE^Lose isoforms in GFP⁺ wild-type MCF-7 cells sorted from co-culture shows a significant increase in hFWE^Lose isoforms (bar 4) when compared with monocultured wild-type MCF-7 cells (bar 2). n = 3 biologically independent experiments with similar results; fold change calculated relative to the expression of hFWE^Win isoforms in monocultured GFP⁺ wild-type MCF-7 cells, all statistically significant P values shown, two-tailed t-test, mean ± s.d. c, MCF-7 cells expressing hFWE2 were xenografted into Fwe^WT mice to assess their tumorigenic potential and the host expression of endogenous hFWE isoforms compared to control. The mouse tissue adjacent to the tumour showed a significant increase in expression of hFWE^Lose isoforms at 21 days post-xenograft. n = 3 biologically independent experiments with similar results; all statistically significant P values shown, one-tailed t-test, mean ± s.d. d, The effect of anti-hFWE shRNA cocktail on the tumorigenic potential of HCT-116 cells. Row 1, growth potential of HCT-116 hFWE^WT cells. Row 2, knockout of hFWE in these cells significantly reduced tumour growth. Row 3, treatment of HCT-116 hFWE^WT cells with anti-hFWE shRNA reduced tumour volume. Row 4, Rescue experiment in which similar hFWE shRNA-treated tumours to row 3 were infected with lentivirus overexpressing hFWE2 14 days after implantation. These tumours are significantly larger than those in row 3. e, Tumour volumes for experiments shown in Fig. 4a were measured weekly, and growth patterns were analysed over 42 days for groups shown in a. Growth curves show the reduced growth of tumours from HCT-116 hFWE^KO cells (blue) and hFWE shRNA-treated HCT-116 hFWE^WT cells (red). Green line shows rescue experiment and growth pattern changes in hFWE shRNA-treated HCT-116 hFWE^WT tumours expressing with hFWE2 (n = 5, P values shown, one-tailed t-test, mean ± s.d.). f, All mice used in the study were examined for the presence or absence of metastases in ILN, ALN, colon, pancreas, prostate, lung and liver. Heat map scale indicates the probability of metastasis. Metastatic potential was reduced by knockout or knockdown of hFWE in HCT-116 cells (compare column 1 with columns 2 and 3). The rescue of tumour growth by re-introduction of hFWE2 cDNA was accompanied by an increase in metastasis of these cells (compare column 3 with column 4; n = 5 each group). g, A cocktail of shRNAs were designed to knock down all four isoforms of hFWE. All shRNAs were checked for off-target effects. h, Gene expression analysis confirmed deletion of total hFWE in HCT-116 hFWE^KO cells. Exogenous expression of hFWE2 cDNA was detectable as the total hFWE expression in wild-type HCT-116 cells co-treated with anti-hFWE shRNA (observed in resected tumours). n = 3 biologically independent experiments, fold change calculated relative to expression of hFWE^Win isoforms in MCF-10A cells, P values shown, two-tailed t-test, mean ± s.d. i, Tumorigenic potential of wild-type CCL-218 cells and CCL-218 cells overexpressing hFWE2. Xenografts overexpressing hFWE2 cDNA showed increased tumour volume at 28 days. Photos of resected tumours are shown. n = 3 biologically independent experiments with similar results, all statistically significant P values shown, ANOVA, mean ± s.d. Control qPCR experiment demonstrates the overexpression of hFWE2 in CCL-218 tumours at day 28. n = 3 biologically independent experiments, fold change calculated relative to expression of hFWE^Win isoforms in MCF-10A cells, P values shown, two-tailed t-test, mean ± s.d. j, Endogenous expression of the four hFWE isoforms in 19 cancer cell lines of multiple origins (n = 3 biologically independent experiments, fold change is calculated relative to expression of hFWE^Win isoforms in MCF-10A cells, P values shown, ANOVA, mean ± s.d.). k, Gene expression analysis shows efficient shRNA-mediated knockdown of total hFWE in HCT-116, DU-145, CCL-218 and OVCAR-8 tumours (observed in resected tumours). n = 3 biologically independent experiments, fold change calculated relative to expression of hFWE^Win isoforms in MCF-10A cells, P values shown, ANOVA, mean ± s.d.

Source Data