Chromosomal instability determines taxane response

Abstract

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these "CIN-survival" genes is associated with poor outcome in estrogen receptor-positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.

Fig. 1.

Genes overexpressed in tumors with CIN are repressed by MTS treatment in vitro and in vivo. (A) The CIN27wp gene expression signature, which correlates with total functional aneuploidy in several cancer types (9), was analyzed in 5 gene expression data sets measuring response to MTS treatment. The distribution of changes induced by MTS was lower in the CIN27wp genes (red) compared with the set of all measured genes (black) (P = 3.3e-7). (B) As part of the OV01 clinical trial, expression profiling was performed on ovarian carcinomas before and after 3 cycles of paclitaxel (Px) treatment. For each gene in the CIN70 signature, the median expression in the pretreatment and posttreatment tumors was compared, with the red line indicating equivalence.

Fig. 2.

Identification of 22 CIN-survival genes. (A) siRNA silencing of MTS-repressed genes impairs cell viability. Genes repressed within the 2 MTS expression signatures that are overexpressed in CIN tumors significantly altered HCT-116 cell viability when targeted by siRNA. An Acumen eX3 cytometer (Acumen TTP Labtech) was used to quantify viable cells 72 h after siRNA transfection. SDs are displayed for 3 independent experiments. P values are shown for Student 2-sided t-tests in all cases: *P < 0.05; **P < 0.005; ***P < 0.0005. (B) siRNA silencing of MTS-repressed genes promotes cell death. FACS analysis was used to quantify the mean subG1 fraction 72 h after transfection of siRNA. SDs and P values are displayed for 3 independent experiments. (C and D) Identification of 22 CIN-survival genes. Cells were transfected with siRNA targeting the 50 genes overexpressed in CIN tumors and repressed by MTS agents. Cell viability was quantified relative to scrambled control siRNA by a CellTiter-Blue assay 4 days after siRNA transfection in triplicate. Shown are the 22 genes that significantly impaired viability in the MDA-MB-468 and A549 cell lines. (E) Flowchart of analysis resulting in the derivation of 22 CIN-survival genes. The binomial test with probability corrections (BTPC) and rank methods were used to derive 2 MTS expression signatures. A total of 50 genes were repressed in the MTS signatures and overexpressed in CIN tumors. Of these 50 genes, 22 impaired cancer cell viability and/or induced apoptosis when silenced by RNA interference in 3 cancer cell lines: HCT-116 (colon), A549 (NSCLC), and MDA-MB-468 (breast).

Fig. 3.

Paclitaxel cytotoxicity is associated with CIN-survival gene repression. (A) Quantification of gene repression following paclitaxel treatment of cell lines with increasing chromosomal numerical heterogeneity. Fold change in gene expression post-paclitaxel treatment (24 h) relative to expression in vehicle control treated cells was determined by qPCR analysis (normalization to 18S and GAPDH) after 24 h of paclitaxel treatment (50% of the Gi50 concentration) in 3 biological replicate experiments in cell lines with increasing CIN. Color-coding represents the mean fold change in gene expression of 3 biological replicates relative to cells treated with vehicle alone + 1 SD. CIN-survival genes are highlighted in gray. Gene expression following paclitaxel treatment is compared with the HCT-116 cell line displaying the lowest CIN. The significance of the differences in gene expression were determined using the unpaired 2-sided Student t-test: *P <0.05; **P <0.005. (B) CIN-survival genes are relatively overexpressed in CIN^high COLO205 and SW620 cells compared with near-diploid CIN^low HCT-116 cells. Shown is the relative quantification of gene expression normalized to 18S and GAPDH in COLO205 and SW620 cells compared with near-diploid HCT-116 cells. The graph indicates the mean fold change in gene expression compared with HCT-116 cells of 3 biological replicates (+ 1 SD). (C) Colorectal cancer cell lines with increasing chromosomal numerical heterogeneity uncouple mitotic arrest from cell death. Cell lines were treated with paclitaxel relative to the Gi50, Gi50/2, and Gi50/4 concentrations. Quantification of dying cells (subG1 fraction) and cells arrested in mitosis (MPM2-positive fraction) were assessed by FACS analysis. The MPM2:subG1 ratio was calculated by assessing the percentage of MPM2 positive cells relative to the percentage of subG1 cells following 24 h of paclitaxel treatment. The mean ratio of 3 independent experiments is presented (+1 SD). Cell lines are represented in ascending order of CIN status (21). (D and E) Silencing CIN-survival genes in the SW620 CIN^high cell line promotes cell death after 24 h of paclitaxel Gi50 treatment. At 48 h after transfection of CIN-survival siRNAs, SW620 cells were treated with paclitaxel for 24 h, and cells were prepared and analyzed as in C. In D, the mean MPM2:subG1 ratio of 2 independent experiments is presented (+ 1 SD). E shows that silencing of NUP205, H2AFX, CDC6, and RPA1 promoted a significant increase in subG1 cells after paclitaxel exposure compared with DMSO-treated cells (P <0.05; Student t-test). (F) Impaired repression of CIN-survival genes following paclitaxel treatment in an isogenic model of CIN. Quantification of gene repression by qPCR analysis of 21 CIN-survival genes following treatment of HCT-116 wild-type parental cells and isogenic Mad2^+/- cells after 24 h of paclitaxel treatment (50 nM) normalized to 18s. Color-coding represents the mean fold repression + 1 SD from 3 biological replicates. P values indicate significantly greater gene repression in the HCT-116 parental cell line (Student 1-sided t-test). (G) CIN-survival gene repression correlates with cytotoxic response and stable tumor karyotype.

Fig. 4.

CIN70 predicts paclitaxel sensitivity and is a surrogate for CIN in breast cancer. (A) Expression of CIN70 genes determines sensitivity to paclitaxel and carboplatin. The figure contrasts basal median gene expression for each CIN70 gene in tumors with differing responses to paclitaxel and carboplatin. Paclitaxel-resistant tumors exhibited a higher median log-intensity of the CIN70 signature compared with paclitaxel-sensitive tumors (P = 0.043). CIN70 gene expression differed significantly between tumors subsequently resistant to paclitaxel and tumors resistant to carboplatin (P = 0.044; Student 2-sided t-test). (B) Expression of CIN and CIN-survival genes was greater in the aGU breast cancers. DNA image cytometry was used to classify breast cancers as dGS, aGS, or aGU. Boxplots summarize the expression of the CIN70, CIN27wp, and CIN-survival signatures within each group. P values were calculated using the Student 2-tailed t-test.