Search

Additional file 1: Supplementary Figure 1. Identification of age and breast cancer associated genes. (A) Workflow of in silico analyses. Volcano plots show the genes expressions in association with age in the matched normal samples from either all 82 breast cancer patients (B) or 54 post-menopausal patients (C). Venn Diagrams show the difference in up-regulated (D) and down-regulated (E) age-associated genes identified from all 82 patients versus from 54 post-menopausal patients. Venn diagrams show the number of genes up-regulated (F) and down-regulated (G) from four models in the tumor-adjacent normal samples comparing post-menopausal to pre-menopausal patients. Venn diagrams show the overlapping and difference in genes that are up-regulated (H) and down-regulated (I) with age and menopause. Venn diagrams show the number of genes up-regulated (J) and down-regulated (K) from four models comparing tumors to their matched normal tissues in pairs. Venn diagrams show 14 up-regulated (L) and 24 down-regulated (M) genes by both age and tumorigenesis.

Additional file 4: Supplementary Figure 4. Growth inhibition of breast cancer cell lines by siRNAs targeting selected upregulated ABC genes. Knockdown by siRNAs were performed to study the effect of loss-of-function on 14 up-regulated genes. Knockdowns of seven genes (DYNLT3, P4HA3, CLEC3A, CTHRC1, RNASE2, LPAR5, LRRC15) showed different inhibitory effects on cell proliferation of seven breast cancer cell lines. Three control conditions (green: regular culture; blue: plus transfection reagent; Yellow: plus transfection reagent and a scrambled siRNA) and three siRNAs (black, gray, and red lines) to each gene are included in the experiment.

Additional file 8: Supplementary Figure 8. P4HA3 knockdown in BT-474 cells reduced their lung metastatic potential in NSG mice. Completely excised lung tissue from each mouse was placed in the well of 24-well place. Gray images were taken to show the whole lung tissue. Appropriate color scale was overlaid on top of gray images to depict the total flux signal received from luciferase activity. Top two rows are images of ten lungs from the control cell-inoculated mice and bottom two rows are images of ten lungs from the P4HA3 knockdown cell-inoculated mice.

Additional file 7: Supplementary Figure 7. Confirmation of DYNLT3 knockdown in tumors formed by DYNLT3 Knockdown BT-474 cells. Protein expression level of DYNLT3 were measured by Western blotting in the tumors formed by control and DYNLT3 Knockdown BT-474 cells in NSG mice.

Additional file 3: Supplementary Figure 3. Scatter Heatmap plot on mutations of ABC genes. CTHRC1 and ETV3L had over 10% alterations in 1074 breast cancer patients (analyzed by cBioPortal, as of August 22nd 2017). Each vertical bar represent one patient. Light gray bars with no red, blue, dark gray or green color represent patients without any genetic alteration in consideration. The color bars at the top depicts patient status, including ER, HER2 and menopause status. The color legends are shown at the bottom.

Additional file 6: Supplementary Figure 6. DYNLT3 knockdown in BT-474 cells showed no effect on their lung metastatic potential from subcutaneous tumors in NSG Mice. Completely excised lung tissue from each mouse was placed in the well of 24-well place. Gray images were taken to show the whole lung tissue. Appropriate color scale was overlaid on top of gray images to depict the total flux signal received from luciferase activity. Top two rows are images of ten lungs from the control cell-inoculated mice and bottom two rows are images of ten lungs from the DYNLT3 knockdown cell-inoculated mice.

Additional file 5: Supplementary Figure 5. Confirmation of knockdown and overexpression of the depicted gene proteins with Western blotting. Knockdown of DYNLT3 and P4HA3 proteins and overexpression of ALX4 and WDR86 proteins were confirmed in both BT-474 and MDA-MB-231 cell lines with Western blotting.

Additional file 1. Supplementary methods, figures and tables.

Additional file 2: Supplementary Figure 2. Matched tumor samples do not show age dependent expression. In all 82 matched tumor samples which were ordered by age at diagnosis as indicated by the arrow, no expression pattern of up or down regulated ABC genes was observed. Row side color bar represents genes that were upregulated (red) or downregulated (blue).

Additional file 9: Supplementary Figure 9. Confirmation of P4HA3 knockdown in tumors formed by P4HA3 Knockdown BT-474 cells. Protein expression level of P4HA3 were measured by Western blotting in the tumors formed by control and P4HA3 Knockdown BT-474 cells in NSG mice.

Additional file 1: Figure S1. The expression profile of breast tumors with PAM50 subtypes and normal controls of the 96 amplification-associated transcription coupling (ATC) loci from the five regions on 17q (A, 17q11.1-q11.2; B, 17q12-q21.2; C, 17q21.2-q21.31; D, 17q21.31-q21.33; E, 17q22-q24.1). The locations of the tumors of different subtypes are displayed by the solid lines with designated colors on the top of the heat map. Figure S2. Circos plots of inter- and intra- chromosomal rearrangements related to three 17q regions. Figure S3. Nanopore sequencing of inter-chromosomal rearrangements involving the 17q23 region. a. 17q23-associated rearrangements identified in MCF-7. Rearrangement frequencies were determined using previously generated whole-genome mate-pair sequencing data. b. Nanopore sequencing of the MCF-7 genome. A schematic flow chart (left) indicates the principle of Nanopore long-read sequencing. Unique molecular barcodes were incorporated into amplicons individually by PCR to enable multiplex sequencing of samples and the resultant reactions were then pooled. After end repair and A-tail reactions, leader and the hairpin adapters, each containing a motor protein (orange), were ligated to the end prepared DNA, followed by His-tag purification. On the MinION device, DNA molecules are pulled through a protein pore (gray) with motor proteins, producing 2D reads, which were consensus calls of the combined template and complement strands (red). PCR amplicons (right) spanning the 18 chromosomal breakpoints between chromosomes 17q23 and 20q13 were individually barcoded and pooled together for sequencing. The image of the desktop sequencing process on the MinION device (left bottom) was captured in our laboratory. c. Screen captures of representative NCBI-BLAST search outputs. DNA sequences from the 17q23 and 20q13 regions are shown in orange and blue, respectively.