and genomic DNA (Mut) and WT of patient healthy women and

Transcription

1 Supplementary Figure 1 TEKT germline variations in the control group and blood sampless of 84 breast cancerr patients. (a) TEKT44 wild-typee (WT), mutant (Mut) and WT to Mut genotype byy Sanger sequencing of patient genomic DNA samples. (b) The distribution of TEKT4 variations in the blood of 200 healthy women and blood samples of 84 breast cancers of four molecularr subtypes who underwent paclitaxel-based neoadjuvant chemotherapy (NCT). 1 / 21

2 Supplementary Figure 2 Flowchart of case selection, s laser-captured microdissection and pyrosequencing. Mut, mutant; NCT,, neoadjuvant chemotherapy; WT, wild-type. 2 / 21

3 60 P < P < Percentage of mutant allele G (% of A+G) c.a541g c.a547g 0 Blood Pre-NCT tumour Post-NCT tumour Supplementary Figure 3 Quantification of mutant alleles via pyrosequencing. Quantification of the percentage of mutant allele in pre- and post-nct tumours and paired blood samples from the seven cases enriched TEKT4 variations by pyrosequencing (P1, P2 < in two-sided Student s t test). NCT, neoadjuvant chemotherapy. 3 / 21

4 Supplementary Figure 4 The ectopic expression of wild-type (WT) TEKT4 or mutant (Mut) TEKT4. (a) Tektin4 protein levels were determined inn normal breast cell line and various breast cancer cell lines using Westernn blotting. Tektin4 relative protein levels were calculated. (b) The protein levels of tektin4 in mock, WT and Mut MDA-MB-468 and MDA-MB-231cells were determinedd using Western blotting. Relative protein level of tektin4 was calculated and compared (P1, P2 < in 4 / 21

5 two-sided Student s t test). (c) The mrna levels of tektin4 in mock, WT and Mut MDA-MB-468 and MDA-MB-231cells were determined using real-time RT-PCR (P1, P2 < 0.01 in two-sided Student s t test). Results are the mean ± s.d. from three independent experiments. 5 / 21

6 Supplementary Figure 5 TEKT44 germlinee variations inhibit apoptosis in i a CDK1-cyclin B1-dependent manner. (a) Flow cytometric analyses apoptosis in wild-type (WT) and mutant (Mut) MDA-MB-468 and MDA-MB-231 cells. The proportions of apoptotic cells were calculated and compared (P1 = and P2 = in two-sided Student s t test). (b) MDA-MB-468 cells were treated with 50 nm 6 / 21

7 paclitaxel for 48 h, and samples were immunoblotted with the antibodies shown. Relative protein levels of CDK1, cyclin B1, Bcl-2, Bcl-xL, Mcl-1, cleaved PARP were calculated and compared (P1 <0.01, P2 = 0.016, P3 < 0.001, P4 < 0.01, P5 = and P6 < in two-sided Student s t test). Results are the mean ± s.d. from three independent experiments. 7 / 21

8 Supplementary Figure 6 Effect of TEKT4 variations on cell invasion, cell adhesion, celll cycle distribution and cell proliferation. (a) Representative microscopic images of Transwell invasion assay for mock, wild-type (WT) light and mutant (Mut) analysis off the invasion assay MDA-MB-468 and MDA-MB-231 cells. Quantitative on Matrigel was performed. Scale bar, 50 µm. (b) Cell adhesion on extracellular matrix (ECM) assay for mock, WT and Mut MDA-MB-468 and MDA-MB-231 cells. (c) Flow cytometric analyses of cell cycle distribution for mock, WT and Mut MDA-MB-468 andd MDA-MB-231 cells were performed. The proportions of G2-M, S and G0-G11 cells were calculatedd and compared. (d) Cell 8 / 21

9 proliferation assay for mock, WT and Mut MDA-MB-468 and MDA-MB-231 cells. Results are the mean ± s.d. from three independent experiments. 9 / 21

10 Supplementary Figure 7 The co-localisation of endogenous α-tubulin (red) and overexpressed tektin4 (green) in WT and Mut MDA-MB-231 cells. Scale bar, 10 µm. 10 / 21

11 Supplementary Figure 8 Full-length images of immunoblots in the main paper. 11 / 21

12 Supplementary Tables Supplementary Table 1 Summary of exome sequencing data from pre-, post-treatment tumour tissues and paired blood DNA of P1 and P2. P1 P2 Sample Pre-NCT Post-NCT Paired Pre-NCT Post-NCT Paired tumour tumour blood tumour tumour blood Initial bases on target Initial bases near target Initial bases on or near target Total effective reads Total effective yield(mb) Average read length (bp) Effective sequences on target (Mb) Effective sequences near target(mb) Effective sequences on or near target (Mb) Number of reads uniquely mapped to target Number of reads uniquely mapped to genome Fraction of effective bases on target 36.50% 36.00% 56.80% 34.20% 29.60% 31.70% Fraction of uniquely mapped on target 44.30% 43.70% 69.30% 44.50% 39.00% 41.90% Fraction of effective bases on or near target 50.70% 50.00% 78.80% 52.80% 48.30% 51.20% Average sequencing depth on target Average sequencing depth near target Mismatch rate in target region 0.23% 0.24% 0.23% 0.56% 0.36% 0.53% Mismatch rate in all effective sequence 0.26% 0.27% 0.22% 0.49% 0.34% 0.47% Base covered on target Coverage of target region 99.20% 99.20% 98.90% 99.50% 99.40% 99.50% Base covered near target Coverage of flanking 97.20% 97.50% 94.90% 99.10% 99.10% 99.20% 12 / 21

13 region Fraction of target covered with at least % 86.20% 85.60% 94.80% 94.50% 94.90% Fraction of target covered with at least % 92.50% 91.80% 97.50% 97.50% 97.50% Fraction of target covered with at least % 97.00% 96.30% 98.80% 98.70% 98.80% Fraction of flanking region covered with at least % 39.70% 40.00% 56.60% 67.60% 68.50% Fraction of flanking region covered with at least % 59.90% 58.40% 80.60% 88.10% 88.40% Fraction of flanking region covered with at 83.80% 84.40% 79.40% 95.50% 96.90% 97.00% least 4 Mapping rate 98.45% 98.32% 99.53% 98.71% 98.78% 98.41% Duplicate rate 15.53% 12.81% 12.77% 10.43% 9.01% 10.79% Abbreviations: NCT, neoadjuvant chemotherapy. 13 / 21

14 Supplementary Table 2 Validated SNVs comparing pre- and post-treatment tumour tissues of P1 and P2. Function Gene Exonic function CDS mutation dbsnp132 Chr Position Patient TEKT4 nonsynony mous SNV c.a541g rs chr P1, P2 TEKT4 nonsynony mous SNV c.a547g rs chr P1, P2 TRAF3IP3 nonsynony mous SNV c.c1117g rs chr P1 OR4M1 nonsynony mous SNV c.c347t rs chr P1 KCNJ12,K CNJ18 nonsynony mous SNV c.a167c rs chr P1 CAPN13 stopgain SNV c.c361t NA chr P1 KIF1A synonymou s SNV c.g3840a rs chr P1 SLC6A1 synonymou s SNV c.g651t rs6344 chr P2 GPRIN2 synonymou s SNV c.g1017a rs chr P2 TTC7B synonymou s SNV c.g1803a rs chr P2 DRD5 synonymou s SNV c.c252t rs chr P2 ELN synonymou s SNV c.t681g NA chr P2 ASMTL synonymou s SNV c.a228g rs chrx P2 ATP6V1A synonymou s SNV c.t108g NA chr P2 ncrna_ Chr KRT16P3 NA NA NA P1 ncrna_ LOC10013 intronic 3920 NA NA rs chr P1 NBPF11(di st=137608), intergenic LOC72898 NA NA NA chr P1 9(dist= ) intergenic NBPF11(di NA NA NA chr P2 14 / 21

15 st=363886), LOC (dist=4437 6) intergenic OVCH2(dis t=66709),o R5P2(dist= NA NA rs chr P ) intronic NBPF11 NA NA NA chr P1 intronic NBPF11 NA NA NA chr P1 intronic OGG1 NA NA NA chr P1 intronic DNAH1 NA NA NA chr P1 intronic WHSC1 NA NA NA chr P1 intronic RGSL1 NA NA rs chr P1 intronic CAPN8 NA NA rs chr P1 intronic TARBP1 NA NA rs chr P2 intronic CAPN13 NA NA rs chr P2 intronic EHBP1 NA NA NA chr P2 intronic SPINK5 NA NA rs chr P2 intronic LRP4 NA NA rs chr P2 intronic ATP5S NA NA rs chr P2 intronic PDILT NA NA rs chr P2 intronic EFCAB13 NA NA rs chr P2 intronic MUC16 NA NA rs chr P2 UTR3 PATL1 NA NA rs chr P1 UTR3 PATL1 NA NA rs chr P2 UTR5 PARP1 NA NA rs chr P2 UTR5 MYBPC1 NA NA rs chr P2 Abbreviations: CDS, coding DNA sequence; Chr, chromosome; NA, not applicable; nc RNA, non-coding RNA; SNV, single nucleotide variant; UTR, untranslated region. 15 / 21

16 Supplementary Table 3 Validation of TEKT4 c.a541g and c.a547g in 84 cases using Sanger sequencing and pyrosequencing. TEKT4 genotype TEKT4 genotype Cases by Sanger sequencing by pyrosequencing LCM Pre-NCT Post-NCT Paired Pre-NCT Post-NCT Paired tumour tumour blood tumour tumour blood NCT106 WT WT WT No NA NA NA NCT124 WT WT WT No NA NA NA NCT129 WT WT WT No NA NA NA NCT178 WT Mut Mut No NA NA NA NCT196 WT WT WT No NA NA NA NCT213 WT WT WT No NA NA NA NCT219 WT WT WT No NA NA NA NCT221 Mut Mut Mut Yes Mut Mut Mut NCT229 WT WT WT No NA NA NA NCT246 Mut Mut Mut No NA NA NA NCT290 WT WT WT No NA NA NA NCT308 WT WT WT No NA NA NA NCT345 WT WT WT No NA NA NA NCT356 Mut Mut Mut Yes Mut Mut Mut NCT360 WT Mut Mut No NA NA NA NCT372 Mut Mut Mut Yes Mut Mut Mut NCT388 WT WT WT No NA NA NA NCT425 Mut Mut Mut Yes Mut Mut Mut NCT455 Mut Mut Mut No NA NA NA NCT462 WT WT WT No NA NA NA NCT476 WT Mut Mut Yes WT Mut Mut NCT483 WT WT WT No NA NA NA NCT499 WT WT WT No NA NA NA NCT521 Mut Mut Mut No NA NA NA NCT530 WT WT WT No NA NA NA NCT541 WT WT WT No NA NA NA NCT552 WT WT WT Yes WT WT WT NCT554 WT Mut Mut No NA NA NA NCT562 WT WT WT No NA NA NA NCT567 WT WT WT Yes WT WT WT NCT581 WT WT WT Yes WT WT WT NCT584 WT WT WT Yes WT WT WT NCT590 WT WT WT Yes WT WT WT NCT591 WT WT WT Yes WT WT WT NCT599 Mut Mut Mut Yes Mut Mut Mut NCT608 WT WT WT Yes WT WT WT 16 / 21

17 NCT619 WT WT WT Yes WT WT WT NCT622 WT WT WT Yes WT WT WT NCT638 WT WT WT Yes WT WT WT NCT641 WT WT WT Yes WT WT WT NCT655 WT WT WT Yes WT WT WT NCT660 Mut Mut Mut Yes Mut Mut Mut NCT661 WT Mut Mut Yes WT Mut Mut NCT666 WT WT WT Yes WT WT WT NCT671 WT WT WT Yes WT WT WT NCT679 WT WT WT Yes WT WT WT NCT685 WT WT WT Yes WT WT WT NCT699 WT WT WT Yes WT WT WT NCT708 WT WT WT Yes WT WT WT NCT712 WT WT WT Yes WT WT WT NCT719 WT WT WT Yes WT WT WT NCT722 WT WT WT Yes WT WT WT NCT730 WT WT WT No NA NA NA NCT736 WT WT WT Yes WT WT WT NCT741 WT WT WT Yes WT WT WT NCT746 WT Mut Mut Yes WT Mut Mut NCT749 WT WT WT Yes WT WT WT NCT753 WT WT WT Yes WT WT WT NCT769 WT WT WT Yes WT WT WT NCT782 WT WT WT Yes WT WT WT NCT790 WT WT WT No NA NA NA NCT795 WT WT WT Yes WT WT WT NCT817 Mut Mut Mut Yes Mut Mut Mut NCT819 WT Mut Mut Yes WT Mut Mut NCT824 WT WT WT Yes WT WT WT NCT842 WT WT WT Yes WT WT WT NCT861 WT WT WT Yes WT WT WT NCT878 WT WT WT Yes WT WT WT NCT888 WT WT WT No NA NA NA NCT895 WT WT WT Yes WT WT WT NCT918 WT WT WT Yes WT WT WT NCT920 WT WT WT Yes WT WT WT NCT926 WT Mut Mut Yes WT Mut Mut NCT945 WT WT WT Yes WT WT WT NCT976 WT WT WT Yes WT WT WT NCT985 WT WT WT No NA NA NA NCT993 WT WT WT Yes WT WT WT NCT1002 WT Mut Mut Yes WT Mut Mut NCT1011 WT WT WT Yes WT WT WT NCT1025 Mut Mut Mut No NA NA NA 17 / 21

18 NCT1039 WT Mut Mut Yes WT Mut Mut NCT1046 WT WT WT Yes WT WT WT NCT1058 WT WT WT Yes WT WT WT NCT1072 WT WT WT Yes WT WT WT Abbreviations: LCM, laser-captured microdissection; Mut, mutant; NCT, neoadjuvant chemotherapy; WT, wild-type. 18 / 21

19 Supplementary Table 4 TEKT4 genotype determined by Sanger sequencing or pyrosequencing in pre- and post-treatment tumour tissues after laser-captured microdissection (n=56). TEKT4 genotype by TEKT4 genotype by Sanger sequencing pyrosequencing WT-to-Mut Mut-to-Mut WT-to-WT WT-to-Mut Mut-to-Mut WT-to-WT Abbreviations: Mut, mutant; WT, wild-type. P < in Chi-square test. 19 / 21

20 Supplementary Table 5 IC 50 profile of wild-type and mutant MDA-MB-231 cells for different kinds of agents. Compound Microtubule-polymerising agents Paclitaxel Docetaxel Epothilone B IC 50 (nm) * WT ± ± ± 28.9 IC 50 (nm) Mut ± ± ± 40.6 Relative resistance P < Microtubule-depolymerising agents Vinblastine 1.24 ± ± DNA-damaging agents Doxorubicin ± ± *Mean IC 50 (nm) ± SD from five independent determinations. Relative resistance = ratio of IC 50 of the Mut cell line to the WT cell line. Two-sided Student s t test. Abbreviations: IC 50, half maximal inhibitory concentrations; Mut, mutant; WT, wild-type. 20 / 21

21 Supplementary Table 6 Relationship between tumour genotype and clinical responses to treatment in the neoadjuvant chemotherapy group. Neoadjuvant chemotherapy Total n = 84 Clinical response Patient No (%) P * TEKT4 WT n = 63 TEKT4 Mut n = 11 TEKT4 WT to Mut n = 10 PR 38 (60.3) 3 (27.3) 3 (30.0) SD/PD 25 (39.7) 8 (72.7) 7 (70.0) Abbreviations: Mut, mutant; PR, partial response; SD/PD, stable disease or progression of disease; WT, wild-type. *Chi-square test. 21 / 21