Exploiting Chromosomal Instability of PTEN-Deficient Triple-Negative Breast Cancer Cell Lines for the Sensitization against PARP1 Inhibition in a Replication-Dependent Manner

Abstract

Chromosomal instability (CIN) is an emerging hallmark of cancer and its role in therapeutic responses has been increasingly attracting the attention of the research community. To target the vulnerability of tumors with high CIN, it is important to identify the genes and mechanisms involved in the maintenance of CIN. In our work, we recognize the tumor suppressor gene Phosphatase and Tensin homolog (PTEN) as a potential gene causing CIN in triple-negative breast cancer (TNBC) and show that TNBC with low expression levels of PTEN can be sensitized for the treatment with poly-(ADP-ribose)-polymerase 1 (PARP1) inhibitors, independent of Breast Cancer (BRCA) mutations or a BRCA-like phenotype. In silico analysis of mRNA expression data from 200 TNBC patients revealed low expression of PTEN in tumors with a high CIN70 score. Western blot analysis of TNBC cell lines confirm lower protein expression of PTEN compared to non TNBC cell lines. Further, PTEN-deficient cell lines showed cellular sensitivity towards PARP1 inhibition treatment. DNA fiber assays and examination of chromatin bound protein fractions indicate a protective role of PTEN at stalled replication forks. In this study, we recognize PTEN as a potential CIN-causing gene in TNBC and identify its important role in the replication processes.

Keywords: CIN; PARP1 inhibition; PTEN; TNBC; replication fork instability; replication stress.

Figure 1

High chromosomal instability (CIN) scores and low phosphatase and tensin homolog (PTEN) expression in triple-negative breast cancer (TNBC). (a) In silico analysis of PTEN mRNA expression data, comparing 100 TNBC patients with a high CIN70 score to 100 TNBC patients with a low CIN70 score. (Student’s t-test p = 0.0001) (b) Western Blot analysis of PTEN expression in TNBC (MDA231, BT549, BT20, GI101 and HS578T) and non-TNBC breast cancer cell lines (MCF7, T47D, SKBR3, BT474) in relation to ß-actin as loading control. (c) Grouped analysis of quantitative PTEN expression normalized on ß-actin, comparing TNBC and non-TNBC breast cancer cell lines. (Student’s t-test p = 0. 0416). Each value represents the mean of the quantitative analysis of three independent experiments with the SEM indicated.

Figure 2

Replication stress in TNBC compared to non-TNBC. (a) Replication fork progression and (b) activation of replication origins in untreated cell lines of TNBC in comparison to untreated non-TNBC breast cancer cell lines. (Student’s t-test p = 0.98 (a) and p = 0.051 (b). Each value represents the mean of three independent experiments.

Figure 3

Replication fork elongation, stalling and new origin firing after PARP1 inhibition. (a) Overview of DNA fiber assay labelling protocol. Representative picture of reduced IdU-fiber length after treatment, in comparison to full-length IdU fiber in untreated sample (b), of CldU labelled, IdU-negative fiber indicating stalled replication fork (c) and representative picture of IdU labelled, CldU negative fiber, indicating second pulse origin firing (d). Quantitative analysis of elongation tract length (e), replication fork stalling (f) and second pulse origin firing (g) after PARP1 inhibition normalized on untreated controls. Each value represents the mean of at least three independent experiments with SEM indicated.

Figure 4

Cellular sensitivity of TNBC to PARP1 inhibition. Colony-forming assays were performed after treatment, with increasing concentrations of the PARP1 inhibitor Olaparib for 90 min. Each value represents the mean of at least three independent experiments, with SEM indicated.

Figure 5

Fork instability after Hydroxyurea (HU) treatment in TNBC, dependent on PTEN expression. (a) Overview of DNA fiber assay labelling protocol. (b) Representative images of DNA fibers of 5 TNBC cell lines after treatment with 2 mM HU for four hours and untreated cells. Unstable replication fork indicated by shortening in CldU-labelled tract length in comparison to untreated controls. (c) Quantitative analysis of CldU elongation track length after treatment with 2 mM HU in comparison to untreated controls. (red: HU treatment, black: untreated) (d) CldU elongation length correlates negatively with PTEN expression measured at protein level (see Figure 1). Each value represents the mean of three independent experiments with the SEM indicated.

Figure 6

PTEN expression and recruitment of DNA repair proteins to the chromatin in TNBC. (a) Western blot analysis of PTEN, PALB2, PARP1 and CHK1 expression in whole cell extracts (WCE) and chromatin-bound extracts (CBE) of untreated TNBC cell lines. GAPDH and H2B served as a loading control for the WCE and CBE, respectively. (b) Western blot analysis of CHK1, pCHK1, ATR and pATR expression at the chromatin after treatment with 2 mM HU for 4 h in comparison to untreated controls. H2B served as a loading control.