53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients

Abstract

Evidence suggests that the DNA end-binding protein p53-binding protein 1 (53BP1) is down-regulated in subsets of breast cancer. Circulating tumor cells (CTCs) provide accessible "biopsy material" to track cell traits and functions and their alterations during treatment. Here, we prospectively monitored the 53BP1 status in CTCs from 67 metastatic breast cancer (MBC) patients with HER2-CTCs and known hormone receptor (HR) status of the primary tumor and/or metastases before, during, and at the end of chemotherapeutic treatment with Eribulin. Nuclear 53BP1 staining and genomic integrity were evaluated by immunocytochemical and whole-genome-amplification-based polymerase chain reaction (PCR) analysis, respectively. Comparative analysis of CTCs from patients with triple-negative and HR+ tumors revealed elevated 53BP1 levels in CTCs from patients with HR+ metastases, particularly following chemotherapeutic treatment. Differences in nuclear 53BP1 signals did not correlate with genomic integrity in CTCs at baseline or with nuclear γH2AX signals in MBC cell lines, indicating that 53BP1 detected features beyond DNA damage. Kaplan-Meier analysis revealed an increasing association between nuclear 53BP1-positivity and progression-free survival (PFS) during chemotherapy until the final visit. Our data suggest that 53BP1 detection in CTCs could be a useful marker to capture dynamic changes of chemotherapeutic responsiveness in triple-negative and HR+ MBC.Cancers 2020, 12, 930 2 of 18 13.3 months after diagnosis of metastatic sites, TNBC has a particularly poor prognosis. Often patients develop a resistance to first-line chemotherapy very rapidly, resulting in a median duration of first-line palliative chemotherapy of 11.9 weeks [1].Triple-negative tumors are characterized by the lack of HR and HER2 expression, but aside from this common feature this subgroup includes very heterogeneous tumors. A mutation in BRCA1 or 2 is found in up to 20% of triple-negative metastatic patients [2]. The proteins encoded by BRCA genes are critically involved in DNA double-strand break (DSB) repair, more specifically, in the error-free pathway of homologous recombination repair (HRR) [3]. In TNBC, a high prevalence of gene mutations as well as epigenetic changes result in BRCAness, compromising safe DNA repair through HRR [2,4,5]. The DNA damage response factor 53BP1 is crucial in protecting DNA ends in BRCA1-defective cells from resection and entry into error-prone repair pathways [6][7][8]. It has been demonstrated that 53BP1 expression in breast cancer is associated with poor prognosis, particularly in TNBC frequently showing BRCA1 dysfunction [7,9].Due to the lack of predictive targets, chemotherapy was the only treatment option available for a long time. This results in an urgent clinical need for new therapies. During the last years, new drugs such as the Poly(ADP-ribose) polymerase (PARP)-inhibitors targeting HRR-defective tumors were studied in several clinical trials. Two different phase III trials (OlympiaAD and EMBRACA) showed an impro...

Dual PARP and RAD51 Inhibitory Drug Conjugates Show Synergistic and Selective Effects on Breast Cancer Cells

“…Therefore, it is conceivable that MDA-MB-436 cells quickly adapted to BRCA1 loss by rewiring their RAD51 status similar to the pre-existing resistance mechanisms described for HCC-1937 cells [18]. Further resistance mechanisms, such as reduced DNA end protection by 53BP1 seen in all the TNBC cell lines investigated in our study [35], may explain why Olaparib sensitivities were in general similar in the TNBC as compared with non-TNBC cell lines.…”

Circulating Tumor Cells in Breast Cancer Patients: A Balancing Act between Stemness, EMT Features and DNA Damage Responses

“…This study revealed upregulation of MMEJ activity in cells displaying EMT and derived from BC patients with high-risk tumors. Along this line, recent data from CTCs of triple-negative MBC patients demonstrated a downregulation of 53BP1, which protects DNA ends from processing for error-prone DSB repair [ 111 ]. Manifestation of such mutagenic repair in TNBC was indeed demonstrated by comprehensive genomic analysis of BC subtypes engaging Next Generation Sequencing (NGS) [ 112 ].…”

“…With the advent of synthetically lethal treatment strategies, phenotypic analysis of tumor cells has become an extremely promising approach to capture single and combined defects resulting from genetic or epigenetic mechanisms [ 142 , 143 ]. In recent years, major efforts were made to establish methods for the analysis of DNA repair functions in freshly isolated BC tissues [ 6 , 144 ] and CTCs from metastatic cancer patients [ 111 , 145 , 146 , 147 , 148 , 149 ]. Regarding the functional analysis of primary BC, so far, the most promising approach is immunofluorescence microscopy of RAD51 in S-phase nuclei of tumor slices ex vivo, which provides a biomarker for the response to PARP inhibitory drugs with superior predictive value as compared to a genomic signature correlating with HR-deficiency [ 150 ].…”

“…Regardless of the relative CTC numbers, all three BC patients showed a rise in the percentage of γH2AX+ CTCs on day 2 post-treatment, from as low as 0% to up to 64%, and in two out of the three cases, a subsequent drop down to baseline on day 5 post-treatment. Recently, our team provided evidence that CTCs display dynamic and treatment-inducible DNA damage responses during chemotherapy of MBC patients with Eribulin [ 111 ]. In this study, we included 67 MBC patients with HER2-negative CTCs in the DETECT trial program before, during and after chemotherapy.…”

Emerging Roles of Liquid–Liquid Phase Separation in Cancer: From Protein Aggregation to Immune-Associated Signaling

“…Following PAR signaling studies, p53-binding protein 1 (53BP1) has also received attention for its important role in maintaining genomic stability ( Figure 2B ). By binding with p53, 53BP1 directly regulates the stability of p53 and affects the expression of p53 target genes, and it has been reported to regulate tumor cell behaviors in a variety of malignancies such as esophageal, colorectal, and breast cancers ( Misteli and Soutoglou, 2009 ; Bi et al, 2015 ; Mirza-Aghazadeh-Attari et al, 2019 ; Yang et al, 2019 ; Schochter et al, 2020 ). And reduction of 53BP1 expression was reported to be associated with cell cycle arrest in esophageal cancer cells ( Yang et al, 2019 ).…”