Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1α expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy. Cancer Res; 70(10); 3925-34. ©2010 AACR.

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51.

The current preclinical and clinical findings demonstrate that, in addition to the conventional clinical and pathological indicators that have a prognostic value in radiation oncology, the number of cancer stem cells (CSCs) and their inherent radioresistance are important parameters for local control after radiotherapy. In this review, we discuss the molecular mechanisms of CSC radioresistance attributable to DNA repair mechanisms and the development of CSC-targeted therapies for tumor radiosensitization. We also discuss the current challenges in preclinical and translational CSC research including the high inter- and intratumoral heterogeneity, plasticity of CSCs, and microenvironment-stimulated tumor cell reprogramming.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3′-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

RAD51 is the central protein in the homologous recombination pathway and is therefore of great relevance in terms of both therapy resistance as well as genomic stability. By using a tissue microarray analysis of 1,213 biopsies taken from colorectal adenocarcinomas (CRCs), we investigated whether RAD51 expression can be used as a prognostic marker as well as potential associations between this and the expression of other proteins known to be related to CRC. Strong RAD51 expression was observed in 1% of CRC, moderate in 11%, weak in 34% and no expression in 44%. No correlation was found between RAD51 expression and clinicopathological parameters. RAD51 expression correlated significantly (p 5 0.001) with overall survival, with a median survival of 11 months for patients with strong, 46 with moderate, 76 with weak and 68 with negative expression. Multivariate analyses revealed that in addition to tumor stage (p < 0.0001) and nodal status (p < 0.0001), RAD51 expression is also an independent prognostic parameter (p 5 0.011). Strong RAD51 expression was found to be associated with the loss of the two DNA mismatch repair proteins MSH (p 5 0.0003), MLH (p 5 0.002) and b-catenin (p 5 0.012) as well as with elevated p21 (p 5 0.003) and EGFR expression (p 5 0.0001). However, a correlation with overall survival could only be found for EGFR expression (p 5 0.008), although no added benefit in risk stratification could be determined when evaluated together with RAD51. Overexpression of RAD51 is a predictor of poor outcome in CRC. This finding indicated the promise of future studies using RAD51 as a prognostic marker and therapeutic target.Colorectal cancer (CRC) is one of the most frequent cancers, with over 1 million new cases worldwide each year and a disease-specific mortality of 33-40%.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC‐derived breast cancer cell line, designated CTC‐ITB‐01, established from a patient with metastatic estrogen receptor‐positive (ER+) breast cancer, resistant to endocrine therapy. CTC‐ITB‐01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC‐ITB‐01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA‐sequencing data indicate that CTC‐ITB‐01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC‐ITB‐01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC‐ITB‐01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

Besides mutations in BRCA1/BRCA2, heterozygous defects in PALB2 are important in breast cancer predisposition. PALB2 heterozygosity increases the risk of malignancy about sixfold. PALB2 interacts with BRCA1 and BRCA2 to regulate homologous recombination and mediate DNA damage response. Here we show, by analysing lymphoblastoid cell lines from heterozygous female PALB2 mutation carriers, that PALB2 haploinsufficiency causes aberrant DNA replication/damage response. Mutation carrier cells show increased origin firing and shorter distance between consecutive replication forks. Carrier cell lines also show elevated ATR protein, but not phosphorylation levels, and a majority of them display aberrant Chk1-/Chk2-mediated DNA damage response. Elevated chromosome instability is observed in primary blood lymphocytes of PALB2 mutation carriers, indicating that the described mechanisms of genome destabilization operate also at the organism level. These findings provide a new mechanism for early stages of breast cancer development that may also apply to other heterozygous homologous recombination signalling pathway gene mutations in hereditary cancer predisposition.

β1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of β1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of β1 integrin targeting on the repair of radiation-induced DNA double-strand breaks (DSBs). β1 Integrin inhibition was accomplished using the monoclonal antibody AIIB2 and experiments were performed in three-dimensional cell cultures and tumor xenografts of human head and neck squamous cell carcinoma (HNSCC) cell lines. AIIB2, X-ray irradiation, small interfering RNA-mediated knockdown and Olaparib treatment were performed and residual DSB number, protein and gene expression, non-homologous end joining (NHEJ) activity as well as clonogenic survival were determined. β1 Integrin targeting impaired repair of radiogenic DSB (γH2AX/53BP1, pDNA-PKcs T2609 foci) in vitro and in vivo and reduced the protein expression of Ku70, Rad50 and Nbs1. Further, we identified Ku70, Ku80 and DNA-PKcs but not poly(ADP-ribose) polymerase (PARP)-1 to reside in the β1 integrin pathway. Intriguingly, combined inhibition of β1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Here, we support β1 integrins as potential cancer targets and highlight a regulatory role for β1 integrins in the repair of radiogenic DNA damage via classical NHEJ. Further, the data suggest combined targeting of β1 integrin and PARP as promising approach for radiosensitization of HNSCC.