Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

Long-term treatment with the PARP inhibitor niraparib does not increase the mutation load in cell line models and tumour xenografts.

BACKGROUND: Poly-ADP ribose polymerase (PARP) inhibitor-based cancer therapy selectively targets cells with deficient homologous recombination repair. Considering their long-term use in maintenance treatment, any potential mutagenic effect of PARP inhibitor treatment could accelerate the development of resistance or harm non-malignant somatic cells. METHODS: We tested the mutagenicity of long-term treatment with the PARP inhibitor niraparib using whole-genome sequencing of cultured cell clones and whole-exome sequencing of patient-derived breast cancer xenografts. RESULTS: We observed no significant increase in the number and alteration in the spectrum of base substitutions, short insertions and deletions and genomic rearrangements upon niraparib treatment of human DLD-1 colon adenocarcinoma cells, wild-type and BRCA1 mutant chicken DT40 lymphoblastoma cells and BRCA1-defective SUM149PT breast carcinoma cells, except for a minor increase in specific deletion classes. We also did not detect any contribution of in vivo niraparib treatment to subclonal mutations arising in breast cancer-derived xenografts. CONCLUSIONS: The results suggest that long-term inhibition of DNA repair with PARP inhibitors has no or only limited mutagenic effect. Mutagenesis due to prolonged use of PARP inhibitors in cancer treatment is therefore not expected to contribute to the genetic evolution of resistance, generate significant immunogenic neoepitopes or induce secondary malignancies.

Increased frequency of CHD1 deletions in prostate cancers of African American men is associated with rapid disease progression without inducing homologous recombination deficiency.

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. We found that subclonal deletion of CHD1 is nearly three times as frequent in prostate tumors of African American men than in men of European ancestry and it associates with rapid disease progression. We further showed that CHD1 deletion is not associated with homologous recombination deficiency associated mutational signatures in prostate cancer. In prostate cancer cell line models CHD1 deletion did not induce HR deficiency as detected by RAD51 foci formation assay or mutational signatures, which was consistent with the moderate increase of olaparib sensitivity. CHD1 deficient prostate cancer cells, however, showed higher sensitivity to talazoparib. CHD1 loss may contribute to worse outcome of prostate cancer in African American men. A deeper understanding of the interaction between CHD1 loss and PARP inhibitor sensitivity will be needed to determine the optimal use of targeted agents such as talazoparib in the context of castration resistant prostate cancer.

Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma.

Purpose: Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. Experimental Design: We used functional assays that detect UV-induced 6-4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Results: Functional assays showed NER deficiency in ccRCC cells. Irofulven sensitivity increased in some cell lines. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. Conclusions: ccRCC cell line based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.

Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma.

Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. We used functional assays that detect UV-induced 6-4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Functional assays showed NER deficiency in ccRCC cells. Some cell lines showed irofulven sensitivity at a concentration that is well tolerated by patients. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ccRCC cell line-based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements.

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.

Assessing the significance of consistently mis-regulated genes in cancer associated gene expression matrices.

MOTIVATION: The simplest level of statistical analysis of cancer associated gene expression matrices is aimed at finding consistently up- or down-regulated genes within a given set of tumor samples. Considering the high level of gene expression diversity detected in cancer, one needs to assess the probability that the consistent mis-regulation of a given gene is due to chance. Furthermore, it is important to determine the required sample number that will ensure the meaningful statistical analysis of massively parallel gene expression measurements. RESULTS: The probability of consistent mis-regulation is calculated in this paper for binarized gene expression data, using combinatorial considerations. For practical purposes, we also provide a set of accurate approximate formulas for determining the same probability in a computationally less intensive way. When the pool of mis-regulatable genes is restricted, the probability of consistent mis-regulation can be overestimated. We show, however, that this effect has little practical consequences for cancer associated gene expression measurements published in the literature. Finally, in order to aid experimental design, we have provided estimates on the required sample number that will ensure that the detected consistent mis-regulation is not due to chance. Our results suggest that less than 20 sufficiently diverse tumor samples may be enough to identify consistently mis-regulated genes in a statistically significant manner. AVAILABILITY: An implementation using Mathematica (tm) of the main equation of the paper, (4), is available at www.me.chalmers.se/~mwahde/bioinfo.html.