Evaluation of CDK12 Protein Expression as a Potential Novel Biomarker for DNA Damage Response-Targeted Therapies in Breast Cancer

Abstract

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer-specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306-15. ©2017 AACR.

Figure 1. Distribution of CDK12 protein expression in breast cancer

A, Modified CONSORT diagram depicting the distribution of CDK12 positive and negative breast cancers in each of the cohorts analysed. B, Representative micrographs of CDK12 protein expression in i, MCF7 cell line treated with non-targeting siRNA controls; ii, MCF7 cell line treated with previously validated siRNA against CDK12 (13); iii. BT474 CDK12 amplified cells all at x400 magnification; iv, tonsil positive control (x 200 magnification). v-viii, Representative images of staining intensity in primary breast cancers, where CDK12 expression was quantified using a modified Allred score, which assessed both intensity (highest score = 3) and percentage positivity (highest score = 5): (v) negative; (vi) 1+; (vii) 2+; and (viii) 3+; all images at 200x magnification. A score of 0 was considered absent and a score of 7 or 8, as high expression.

Figure 2. CDK12 positive breast cancer has a poorer survival in univariate analysis

Kaplan-Meier curves showing breast cancer specific survival for CDK12 high (7-8) versus negative (0) breast cancers asses by IHC in A, Nottingham unselected primary breast cancer series (n= 203); B, tumors from METABRIC (n= 140); C, Gene expression correlations of CDK12 low versus high from METABRIC (n= 1961) and D, HER2-positive tumors treated with Herceptin (n= 75).

Figure 3. CDK12 amplified cells are not dependent on CDK12 expression for their survival

A, Scatter dot plot depicting a significant association of CDK12 transcript expression with gene amplification (n= 208) versus no amplification (n= 1769) (error bars represent median with the interquartile range). B, bar-chart depicting a significant increase of CDK12 protein expression as measured by IHC in CDK12 amplified tumors (n= 16) compared to non-amplified (n= 103). C-D, Relative cell viability after CDK12 silencing in CDK12 amplified (Campbell, n= 8; Marcotte n= 14) versus non-amplified cell lines (Campbell, n= 19; Marcotte n= 40), showing no significant difference in cell survival from c) Marcotte et al (34) and d) Campbell at al (35).

Figure 4. CDK12 protein loss is associated with DNA repair defects in breast cancer

A, Box and whisker plot (min-max) showing a significant decrease in CDK12 transcript levels in HER2-amplified tumors with breakpoints (n= 30) in CDK12 compared with no breakpoints (n= 178) from METABRIC. B, Lollipop diagram depicting the distribution of CDK12 mutations in breast cancer (red= frameshift and nonsense mutations, green= non-synonymous coding mutations). C, Scatter dot plot diagrams showing significant associations between CDK12 absent (n= 74) (IHC score 0) versus CDK12 high (n= 51) (IHC score 7-8) in unselected METABRIC tumors with large tandem duplication score, indicative of gross genomic defects. D, Scatter dot plot diagrams showing significant associations between CDK12 absent (n= 8) (IHC score 0) versus CDK12 high (n= 4) (IHC score 7-8) in TNBC from METABRIC, with large tandem duplication score, indicative of gross genomic defects.