The WD40 domain-containing protein WRAP53b (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53b as an essential regulator of DNA double-strand break (DSB) repair. WRAP53b rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53b targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53b facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53b controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53b impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53b as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.Therapy resistance represents a major hurdle in the management of cancer patients. Several resistance mechanisms have been described (e.g., overexpression of efflux pumps, mutations in the DNA repair system, activation of the antiapoptotic machinery). Multicellular resistance, suggested to be important for the resistance of advanced solid tumors, 1 is due both to limited penetration of drugs into tumor parenchyma 2 and to the presence of quiescent cell populations in hypoxic regions. These populations are insensitive to clinically used drugs and are believed to repopulate tumors between therapy cycles ("regrowth resistance"). 3 Most experimental studies of resistance mechanisms and anticancer drug testing are performed with monolayer cell cultures, which is not an optimal experimental system and has limitations. Three-dimensional (3D) cell cultures, as the well-established multicellular spheroid (MCS) system, reflect the in vivo structure of solid tumors and more adequately reproduce the metabolic microenvironment of tumor tissue. The formation of a 3D structure by tumor cells is accompanied by changes in cells and their microenvironment: the cells are more tightly packed than in 2D and have differences in nutrient supply, oxygen tension, pH, proliferation, resistance to apoptosis. 4,5 MCS are more resistant to chemotherapy than cells grown in 2D. 6,7 To examine the mechanisms of drug resistance, we performed a gene expression profiling of a colon cancer cell line MCS. Among highly upregulated genes in MCS we found a subset of type I interferonstimulated genes (ISGs). A similar signature designated the interferon-related DNA damage signature (IRDS) was previously associated with resistance to radiotherapy. 8,9 The full set of ISGs is activated by the transcription factor complex ISGF3, consisting of tyrosine-phosphorylated STAT1 and STAT2, and the DNA-binding protein IRF9. However, ISGF3 lacking tyrosine phosphorylation of STAT1 and STAT2 (unphosphorylated ISGF3, U-ISGF3) can ...

Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells.

We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence.

Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling Iryna Kolosenko1, Mårten Fryknäs2, Hanif Rassoolzadeh1, Paola Pellegrini1, Slavica Brnjic1, Per Johnsson1, Giuseppe Di Lernia1, Dan Grandér1, Rolf Larsson2, Katja Pokrovskaja Tamm1, Stig Linder1, Angelo De Milito1 1 Cancer Center Karolinska, Dep. Oncology-Pathology, Karolinska Institute, Stockholm, Sweden 2 Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, 751 85 Uppsala, Sweden Solid tumors contain microenvironments characterized by hypoxia and limited availability of nutrients. Such metabolically compromised environments show an increased level of resistance to many chemotherapeutic agents. Multicellular tumor spheroids (MCS) contain hypoxic and nutrient-starved cell populations and provide a model to study multicellular therapy-resistance. We here used 3D cultures of HCT116 colon cancer cells as a model to identify factors related to multicellular drug-resistance. Microarray analysis revealed a dramatic increase in the expression of interferon-alpha (IFNα)-regulated genes in the 3D as compared to 2D cultures. This finding is of significance since an IFNα-related gene signature has previously been associated with therapy resistance in several human cancers. IFNα is known to regulate gene transcription through transcription complex ISGF3 consisting of STAT1, STAT2 and IRF9, as well as through STAT1 homodimers. We found that IRF9, but not STAT1, was consistently induced in colon cancer cell lines grown in 3D as compared to the 2D cultures. Upregulation of IFN-induced genes was observed during crowding of monolayer HCT116 cultures, providing both mechanistic insight as well as a convenient experimental model. STAT1 knock-down experiments showed that induction of IRF9 occurred independently of STAT1. Interestingly, over-expression of IRF9 rendered monolayer 2D HCT116 cells significantly more resistant to cisplatin, docetaxel and etoposide. Vice versa, inhibition of IRF9 expression significantly increased drug-sensitivity of monolayer HCT116 cells to cisplatin. Our data further support an association of IFNα-related gene signature and drug resistance and suggest an important role for the STAT1-independent IRF9 upregulation associated with therapy resistance in cancer. Citation Format: Iryna Kolosenko, Mårten Fryknäs, Hanif Rassoolzadeh, Paola Pellegrini, Slavica Brnjic, Per Johnsson, Giuseppe Di Lernia, Dan Grander, Rolf Larsson, Katja Pokrovskaja, Stig Linder, Angelo De Milito De Milito. Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3999. doi:10.1158/1538-7445.AM2013-3999

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