Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents
DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DNA repair efficiency is regulated by both intracellular and extracellular chemical signals. However, it is largely unknown whether this process is regulated by physical stimuli such as extracellular mechanical signals. Here, we report that DSB repair is regulated by extracellular mechanical signals. Low extracellular matrix (ECM) stiffness impairs DSB repair and renders cells sensitive to genotoxic agents. Mechanistically, we found that the MAP4K4/6/7 kinases are activated and phos- phorylate ubiquitin in cells at low stiffness. Phosphorylated ubiquitin impairs RNF8-mediated ubiquitin signaling at DSB sites, leading to DSB repair deficiency. Our results thus demonstrate that ECM stiffness regulates DSB repair efficiency and genotoxic sensitivity through MAP4K4/6/7 kinase–mediated ubiquitin phosphorylation, providing a previously unidentified regulation in DSB-induced ubiquitin signaling.
INTRODUCTION
DNA double-strand breaks (DSBs) pose a potent threat to genomic integrity (1). Unrepaired DSBs cause genomic instability and cellular transformation. The capability to repair DSBs also contributes to the efficacy of radiotherapy and many cancer chemotherapeutics. The DSB elicits a signaling cascade that modifies the chromatin surrounding the break, first by ataxia telangiectasia mutated (ATM) kinase–dependent phosphorylation of the histone H2AX (2–4) and recruitment of mediator of DNA damage checkpoint protein 1 (MDC1) (5–7), followed by ring finger protein 8 (RNF8)– and ring finger protein 168 (RNF168)-dependent regulatory ubiquitination (8–10). The DSB-induced ubiquitination cascade is initiated by the RNF8-dependent conjugation of ubiquitin (10–12), which promotes the accumulation of RNF168. RNF8 and RNF168 cooperate to catalyze the formation of K63-linked ubiquitin (Ub) chains on chromatin- bound substrates that include histones H2A and H2AX (8, 9, 13). This regulatory ubiquitination event promotes the independent recruitment of the receptor-associated protein 80 (RAP80)–BRCA1 complex and TP53-binding protein 1 (53BP1) to the damaged chromatin (14–17), which are critical for DSB repair and the G2-M checkpoint. Deficiency of ubiquitination in the DSB response leads to impaired DSB repairand enhanced sensitivity of tumor cells to genotoxic agents, such as ionizing radiation (IR) and chemotherapy.Signaling from the extracellular matrix (ECM) is a fundamental cellular input that sustains proliferation, opposes cell death, and regulates differentiation (18). Through integrins, cells perceive both the chemical composition and physical properties of the ECM (19, 20).
In particular, cell behavior is profoundly influenced by themechanical elasticity or stiffness of the ECM, which regulates the ability of cells to develop forces through their contractile actomyosin cytoskeleton and to mature focal adhesions. This mechanosensing ability affects fundamental cellular functions such that alterations of ECM stiffness are nowadays considered to be not only a simple consequence of pathology but also a causative input driving aberrant cellular behavior. For example, ECM stiffness can regulate YAP/TAZ (yes1 associated transcriptional regulator/transcriptional coactivator with PDZ-binding motif)–mediated transcription through RAP2. At low stiffness, active RAP2 binds to and stimulates the kinases MST1/2 and MAP4K, resulting in activation of large tumor suppressor kinase 1 (LATS1) and LATS2 and inhibition of YAP and TAZ (21, 22).Tissue-level matrix stiffness ranges from elastic modulus Ebrain ~ 0.1to 1 kPa to Emuscle ~ 8 to 17 kPa to Eosteoid ~ 25 to 40 kPa (23, 24). While the effects of stiffness on tumor progression are extensively studied (25, 26), it is largely unknown whether ECM stiffness affects DSB repair pathway and chemotherapy.Here, we demonstrate that ECM stiffness is a key extracellular regulator of the DSB repair pathway. Low stiffness activates the RAP2- MAP4K4/6/7 signaling, which phosphorylates ubiquitin at Thr66. The phosphorylated ubiquitin blocks RNF8-mediated ubiquitin chain formation, resulting in deficiency of BRCA1 and 53BP1 recruitment and impaired DSB repair. Thus, ECM stiffness plays a key role in DNA repair and controls cellular sensitivity to genotoxic agents.
RESULTS
We first analyzed the relationship between ECM component andchemotherapy sensitivity in breast cancer. We found that the ex- pression level of several ECM components such as connective tissue growth factor (CTGF), lysyl oxidase (LOX), vascular endothelial growth factor receptor (VEGFR), lysyl oxidase like 2 (LOXL2), and actin alpha 2 (ACTA2) are closely correlated with chemotherapy outcome. As shown in fig. S1 (A to F), high expression of any of these genes or combination of these multiple genes correlated with lower survival rate in patients with chemotherapy treatment but notin untreated patients or endocrine-treated patients. As CTGF, LOX, VEGFR, LOXL2, and ACTA2 were correlated with fibrosis and high stiffness (27), we thus questioned whether ECM stiffness might regulate chemotherapy efficiency. To test this possibility, we treated mouse mammary tumor 4 T1 xenograft with LOX inhibitor β-aminopropionitrile (BAPN) to reduce tumor stiffness (28). BAPN treatment markedly sensitized tumor to chemotherapy drug cisplatin (fig. S1, G to I). Furthermore, combination of BAPN and cisplatin also induced much more apoptosis than cisplatin alone (fig. S1J). These results indicate that ECM stiffness might play a direct role in DNA repair regulation. To find out whether ECM stiffness directly regulates the DSB re- pair pathway, we monitored DSB repair events in human embryonic kidney–293 (HEK293) cells grown on fibronectin-coated acrylamide hydrogels of varying stiffness from 0.5 to 30 kPa, which match the physiological elasticities of natural tissues and tumor (Fig. 1, A to C) (29–33). We first examined how different stiffness regulates cellular response to genotoxic stress. HEK293 cells were treated with IR, cisplatin, etoposide, or neocarzinostatin (NCS). Cells at low stiffness (0.5 and 1 kPa) exhibited a significantly increased sensitivity to all four genotoxic agents compared to cells grown on high stiffness ECM (10, 20, and 30 kPa; Fig. 1, D to G). We found that cells at low stiffness (0.5 kPa and 1 kPa) exhibited a significantly decreased sensitivity to the cyclin-dependent kinase 4/6 inhibitor Palbociclib (34), suggesting that low stiffness specifically increases cellular sensitivity to DNA damaging agents (Fig. 1H).
To further confirm this, we detected the ratio of apoptotic cells at different stiffness after genotoxic treatment. As shown in Fig. 1I, cells at low stiffness (0.5 and 1 kPa) exhibited a significantly increased apoptosis after four genotoxic agents com- pared to cells grown on high stiffness ECM (10, 20, and 30 kPa).Together, our results indicate that low stiffness affects DNA repair efficiency.We next assessed the clearance of DNA lesions following IR ex- posure under different ECM stiffness conditions. We used automated immunofluorescence microscopy to quantify nuclear y-H2AX foci as a surrogate for unrepaired DNA lesions. In response to IR, we observed a significantly delayed clearance of y-H2AX foci in cells grown on low stiffness ECM (0.5 and 1 kPa) compared to cells cul- tured on high stiffness ECM (10, 20, and 30 kPa; Fig. 1J), suggesting delayed DSB repair in cells at low stiffness. To further confirm this result, we monitored DSB in cells by neutral comet assay. In re- sponse to IR, we observed a significantly delayed clearance of DSB in cells grown on low stiffness ECM (0.5 and 1 kPa) compared to cells cultured on high stiffness ECM (10, 20, and 30 kPa; Fig. 1K), suggesting delayed DSB repair in cells under low stiffness.Eukaryotic cells use two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), as well as branches of these pathways, to repair DSBs. To assess DSB repair efficiency, we monitored HR and NHEJ using the DR-GFP and EJ5- GFP reporters. The efficiency of both HR and NHEJ was inhibited in cells at low stiffness (0.5 and 1 kPa; Fig. 1, L and M).To test whether the effect of stiffness on DSB repair is cell type dependent, we next performed colony formation assay and apop- tosis analysis in different cell lines, including U2OS, MDA-MB-231, MCF7, A549 and MCF10A. When grown on low-stiff ECM, all five cell lines showed increased sensitivity to genotoxic agents [U2OS (fig. S2, A and B), MDA-MB-231 (fig. S2, C and D), MCF7 (fig. S2,E and F), A549 (fig. S2, G and H), and MCF10A (fig. S2, I and J)], suggesting that the effect of stiffness on cellular DSB repair is a com- mon mechanism that is shared in different cell types.
To further confirm the effects of stiffness on DNA repair, we used two additional cell culture models to manipulate ECM stiff- ness. First, we used Matrigel-coated plates (stiff) and gelled Matrigel thick layer (soft) system (fig. S2K). Compared to cells grown on Matrigel-coated plates (stiff), cells grown on gelled Matrigel thick layer (soft) were much more sensitive to genotoxic agents (fig. S2, L and M).Second, we also used a three-dimensional (3D) culture system with soft or stiff hydrogels (35) to assay the effects of stiffness on DNA repair (ig. S2N). Similar to our 2D culture system, cells grown on soft hydrogel in 3D culture system were more sensitive to geno- toxic agents than cells grown on stiff hydrogel (Fig. 2O).To confirm the effect of stiffness on DNA repair in xenograft models, we used semisynthetic hyaluronan-derived hydrogels (36) (soft, 0.4 kPa; stiff, 9 kPa) to mimic the solid tumor stiffness. ECM stiffness did not affect tumor exposure to chemotherapy, as tumor samples from both gels (0.4 and 9 kPa) showed similar tail moment 6 hours after cisplatin treatment (fig. S2P). With this mouse model, we further checked whether stiffness affects cisplatin sensitivity. As shown in fig. S2 (Q to S), tumors at low stiffness were more sensitive to cisplatin. Furthermore, cisplatin also induced much more apoptosis in tumor under soft ECM than under stiff ECM (fig. S2T), indicating that ECM stiffness affects tumor response to chemotherapy.Low stiffness inhibits DSB repair at the level of RNF8 in the DSB repair pathwayIn the DSB repair process, DSB repair proteins are recruited to the DSB site. This process seems to occur in a step-wise fashion with upstream proteins showing faster kinetics. We sought to pinpoint the step during DSB repair signaling that is sensitive to low stiffness. Low stiffness did not have a marked effect on the earliest steps of DNA damage response, as the foci assembly of y-H2AX, MDC1, and RNF8 were largely unchanged (Fig. 2, A to C, and fig. S3, A to B). However, the ubiquitin conjugation at the DSB site was blocked in cells at low stiffness (Fig. 2D and fig. S3B).
The recruitment of RNF168, 53BP1, and BRCA1 to DNA damage sites was also blocked at low stiffness (Fig. 2, E to G, and fig. S3, C to D). As the recruitment of RNF168, 53BP1, and BRCA1 is dependent on RNF8-mediated ubiquitin signaling, we thus concluded that low stiffness inhibits DSB repair through modulating RNF8-mediated ubiquitin signaling (Fig. 2H).Inhibition of DSB repair by low stiffness is dependent on Rap2We next sought to investigate how ECM stiffness affects the RNF8- mediated ubiquitin signaling. It is known that Hippo-YAP pathway is regulated by ECM stiffness (24). Recent studies have demonstrated that the RAP2-Hippo pathway is a direct link between ECM stiffness and intracellular processes. Low stiffness can activate RAP2 and the downstream Hippo kinase MST1/2 and MAP4K4/6/7 kinases (fig. S4A). We thus tested whether Rap2 is involved in DSB repair regu- lation by quantifying the IR-induced foci. Using Rap2 knockout (KO) cells, we further confirmed the effects of Rap2 in DNA repair inhibition by low stiffness. As shown in fig. S4 (B to J), Rap2 KO cells at low stiffness restored FK2/53BP1/BRCA1 foci. Furthermore, at low stiffness, Rap2 KO cells became more resistant to IR when compared to control cells (fig. S4K), indicating that Rap2 is required for DNA repair inhibition by low stiffness.We further tested whether downstream Hippo kinases MST1/2 and MAP4K4/6/7 kinases regulate DNA repair. To test the role of MST1/2 and MAP4K kinases in regulation of DSB repair, we used MST1/2 knockout (MM2KO), MAP4K4/6/7 knockout (MM3KO), and MST1/2 and MAP4K4/6/7 knockout (MM5KO) cell lines to specify the contribution of Hippo kinases to DNA repair regulation (Fig. 3A). At low stiffness, wild-type cells exhibited decreased FK2, 53BP1, and BRCA1 foci at DSB sites. MM3KO and MM5KO cells,but not MM2KO cells, restored FK2, 53BP1, and BRCA1 foci at low stiffness (Fig. 3, B to J), indicating that MAP4K4/6/7 kinases mediate ubiquitin signaling deficiency at low stiffness. Depletion of MAP4K4/6/7 kinases, but not MST1/2, resulted in a near-complete restoration of HR and NHEJ in cells at low stiffness (Fig. 3, K and L).
The survival rate after irradiation was also restored in MM3KO cells at low stiffness (Fig. 3M). These data indicate that MAP4K4/6/7, but not MST1/2, mediate DNA repair inhibition by low stiffness. We thus asked whether MAP4/6/7 regulate DNA repair through their downstream signaling LATS-YAP pathway. To test this, weused LATS1/2 knockout cells and YAP/TAZ knockout cells (fig. S5, A and B). As shown in fig. S5 (C to K), in LATS1/2 or YAP/TAZ knockout cells, low stiffness still blocked FK2/53BP1/BRCA1 foci formation. Furthermore, low stiffness also affected HR and NHEJ efficiency (fig. S5, L and M) and cellular sensitivity to IR (fig. S5N) in LATS1/2 knockout and YAP/TAZ knockout cells as in control cells.These results suggest that the inhibition of DNA repair by MAP4K4/6/7 kinases at low stiffness is independent of LATS1/2-YAP.MAP4K4/6/7 kinases phosphorylate ubiquitin at Thr66The direct effect of MAP4K4/6/7 kinases on FK2 foci formation spurred us to test whether these kinases suppress RNF8 E3 ligase activity in afully recombinant system. Ubiquitination reactions were assembled with RNF8 in the presence or absence of MAP4K4/6/7 kinases. MAP4K4/6/7 kinases efficiently inhibited RNF8-mediated ubiquitin chain assembly (fig. S6, A and B), indicating that one or more of the components of the ubiquitin cascade might be phosphorylated by MAP4K4/6/7 kinases.We next sought to determine substrates of MAP4K4/6/7 kinases in the ubiquitination reaction. In vitro kinase assays were performed with E1, E2, E3, or ubiquitin mixed with MAP4K4/6/7 kinases, and samples were subjected to phos-tag polyacrylamide gel electro- phoresis (PAGE). No notable shift was found on E1, E2, or E3 using phos-tag gel (fig. S6, C to F), whereas one retarded mobility form of ubiquitin was specifically seen (Fig. 4A), indicating that mono ubiquitin itself is phosphorylated by MAP4K4 kinases. To deter- mine the phosphorylation site, the phosphorylated ubiquitin was trypsinized and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
One phosphorylated ubiquitin peptide 64 to 72 (ESpTLHLVLR) was identified (Fig. 4B), suggest- ing Thr66 phosphorylation. The phosphorylated ubiquitin can be specifically recognized by our homemade anti-pT66 antibody (Fig. 4C). Besides monomer Ub, we found that different ubiquitin chains can also be phosphorylated by MAP4K4 kinases in vitro (Fig. 4D). To confirm the phosphorylation site on ubiquitin, we mutated Thr66 on ubiquitin to alanine (T66A). Mutation of Thr66 prevented ubiquitin phosphorylation by MAP4K4 kinases (Fig. 4E).We next checked whether phosphorylation of ubiquitin is regu- lated by ECM stiffness in cells. As shown in fig. S7A, low stiffness markedly induced phosphorylation of ubiquitin. Although the Hippo pathway can be regulated by multiple signals such as cell density, serum starvation, and energy stress, phosphorylation of ubiquitin was only slightly induced by these signals when compared to low stiffness condition (fig. S7A). We also found that the phosphorylation of ubiquitin was induced in multiple cell lines at low stiffness, indi- cating that this phosphorylation is a general event across different cell lines (fig. S7B). Furthermore, we found phosphorylated ubiquitin to be mainly localized in the nucleus, indicating that it might mainly regulate nuclear events (fig. S7C). To demonstrate the phosphoryla- tion of Ub in cells, lysates from cells at low stiffness were trypsinized and subjected to LC-MS/MS analysis. Sixty-four to 72 pT66 were identified with high confidence, suggesting pT66 phosphorylation in cells (fig. S7D).Since MAP4K4/6/7 kinases phosphorylated ubiquitin in vitro, we checked whether MAP4K4/6/7 regulate ubiquitin phosphorylation level in cells. Overexpression of MAP4K4/6/7 kinases increased the level of ubiquitin phosphorylation (Fig. 4F). Knockout MAP4/6/7 kinases decreased ubiquitin phosphorylation in cells at low stiffness, while knockout MST1/2 kinases had no notable effect (Fig. 4G). To test whether the kinase activity of MAP4K4/6/7 is required for ubiquitin phosphorylation, we reconstituted MAP4K4 wild-type (MAP4K4 WT) and kinase dead (MAP4K4 KD) mutant into MM3KO cell lines. As shown in Fig. 4H, phosphorylation of ubiq- uitin at low stiffness was restored in cells expressing MAP4K4 WT, but not in cells expressing MAP4K4 KD.
Consistent with the up- stream role of Rap2, knocking out Rap2 blocked ubiquitin phos- phorylation in cells at low stiffness (Fig. 4I), However, knocking out LATS1/2 or YAP/TAZ had no notable effect on ubiquitin phos- phorylation (Fig. 4J), indicating that Rap2-MAP4K4/6/7 pathway, but not LATS1/2-YAP signal, is required for low stiffness–induced ubiquitin phosphorylation. Phosphorylation of ubiquitin blocks RNF8 mediated ubiquitin conjugation in vitro and in cellsWe next examined the role of ubiquitin phosphorylation in RNF8- mediated ubiquitin chain assembly. We first examined whether phosphorylated ubiquitin affects RNF8-mediated ubiquitin chain assembly. We first purified phosphorylated ubiquitin via a biosyn- thesis system (37). With the purified phosphorylated ubiquitin (Fig. 5, A and B), we performed in vitro ubiquitination assays. The phosphorylated ubiquitin blocked ubiquitin chain assembly cata- lyzed by UbcH5C/RNF8 (Fig. 5C). RNF8 has been reported to work with several other E2s such as Ubc13, UBE2E1, UBE2E2, and UBE2E3 (38). We found that phosphorylation of ubiquitin blocked chain formation when different E2s were used together with RNF8 (Fig. 5D).We next sought to determine the step in the ubiquitination reac- tion that is sensitive to phosphorylated ubiquitin. Not unexpectedly, phosphorylated ubiquitin was charged to E2 to a similar extent as nonphosphorylated ubiquitin (fig. S8A), indicating a similar effi- ciency of conjugation of E2. We thus hypothesized that phosphoryl- ation of ubiquitin might affect RNF8 activity through disrupting the receptor function of ubiquitin. As shown in fig. S8B, discharging of UbcH5c ~ Ub by RNF8 was inhibited by phosphorylated ubiquitin. These results indicate that phosphorylation of ubiquitin inhibits RNF8-mediated UbcH5c discharging. Phosphorylated ubiquitin did not affect MDM2- or X-linked inhibitor of apoptosis (XIAP)– mediated ubiquitin chain assembly (extended data; fig. S8, C and D), indicating that the effect of phosphorylated ubiquitin might be lim- ited to some specific E3 ligases and not the global ubiquitin system.
To study the role of ubiquitin phosphorylation in DNA repair regulation, we first used a phosphomimetic ubiquitin mutant T66E. The phosphomimetic ubiquitin mutant T66E totally blocked RNF8- mediated ubiquitin chain assembly (Fig. 5E), indicating that phos- phomimetic ubiquitin has similar function as real phosphorylated ubiquitin. We thus generated cell lines stably expressing ubiquitin wild-type (WT), T66A, or T66E mutant and assessed their effect on the DSB repair pathway (Fig. 5F). We observed blocking effect of ubiquitin phosphorylation on histone H2A and H2Ax ubiquitina- tion by T66E mutant (Fig. 5G). Furthermore, we also observed a drastic suppression of DNA damage–induced ubiquitin conjugation on chromatin (FK2 foci) and 53BP1 and BRCA1 foci formation in cells expressing T66E mutant (Fig. 5, H to P). These results strongly suggest that ubiquitin phosphorylation at Thr66 impairs the ubiquitinconjugation at DSB sites.To study the physiological function of Ub phosphorylation in cells at low stiffness, we used a Ub replacement strategy in HEK293 cells, wherein all endogenous copies of Ub were depleted by doxycycline (DOX)–inducible RNA interference while simultaneously express- ing short hairpin RNA (shRNA)–resistant Ub WT and Ub T66A fused to the N terminus of ribosomal proteins L40 and S27a from a DOX-responsive promoter (fig. S9A) (39). Immunoblotting of ex- tracts demonstrated similar levels of Ub WT, Ub T66A, and T66E proteins after DOX induction for 3 days (fig. S9B).
In this Ub re- placement system, low stiffness induced Ub phosphorylation only in cells expressing WT Ub, but not Ub T66A or Ub T66E (fig. S9C), further confirming that low stiffness leads to the phosphorylation of Ub at T66 in cells.Low stiffness reduced FK2, 53BP1, and BRCA1 foci in ubiquitin replacement HEK293 cells expressing wild-type ubiquitin (Fig. 6, A to F, and fig. S9, D to F). However, low stiffness had little effect on FK2, 53BP1, and BRCA1 foci in ubiquitin replacement HEK293 cells expressing T66A mutant ubiquitin, suggesting that Ub-T66 phosphorylation is responsible for the impaired DSB repair under low stiffness. Notably, in ubiquitin replacement HEK293 cells ex- pressing T66E mutant ubiquitin, FK2, 53BP1, and BRCA1 foci were blocked no matter whether the cells were cultured on stiff or soft hydrogel. These results indicated that phosphorylation of ubiquitin mediated DNA repair deficiency in cells at low stiffness condition. We further tested the effects of ubiquitin phosphorylation on HR and NHEJ. Both HR and NHEJ were inhibited in WT cells under low stiffness but were not affected in cells expressing ubiquitin T66A (Fig. 6, G and H). The lower survival rates in response to genotoxic agents were also reversed in cells expressing ubiquitin T66A at low stiffness (Fig. 6, I to L). Overall, we propose that MAP4K4/6/7- mediated phosphorylation of ubiquitin leads to DSB repair deficiency in cells at low stiffness conditions (fig. S9G).
DISCUSSION
Here, we conclude that cells limit DNA repair under low stiffness, which matches the proapoptosis status in soft tissue. This might represent an evolutional strategy for cell to maintain genomic sta- bility. Under low stiffness conditions, the Hippo-LATS pathway is activated (40), which promotes apoptosis and inhibits the prosur- vival signaling. In the meanwhile, low stiffness also inhibits DNA re- pair pathway through Rap2-MAP4K4/6/7-ubiquitin signaling, which causes slow clearance of damaged DNA and further amplifies the proapoptotic signaling. These two effects together limit the expan- sion of genomically unstable cell populations by triggering their death.
ECM stiffness controls the HIPPO-YAP pathway to affect cell growth. As shown in fig. S5B, cell cycle was partially arrested at low stiffness. The percentage of G1 cells increased around 8% from stiff to soft matrix. This change in cell cycle might contribute to decreased HR efficiency. However, we do not think that this 8% change would dominate the marked change of HR and NHEJ decrease in Fig. 1 L and M). To exclude the effect of HIPPO pathway and cell cycle, we further used LATS1/2 KO cells and TAZ/YAP KO cells to test the effect of ECM stiffness on HR and NHEJ. As shown in fig. S5B, the effect of stiffness on cell cycle was blocked in LATS1/2 KO cells and TAZ/YAP KO cells. However, in these cells, low stiffness also affected HR and NHEJ efficiency (fig. S5, L and M) and cellular sen- sitivity to IR (fig. S5N), indicating that ECM stiffness can regulate DNA repair independent of cell cycle effect.
We also noticed a slight decrease in y-H2AX foci number at low stiffness (Fig. 2A). This indicated that the initial steps of DNA damage such as activation of ATM and meiotic recombination 11 homolog A (MRE11) might also be affected by ECM stiffness. However, this effect seems to be very mild. As shown in Fig. 2 (B and C), the slight change in y-H2AX foci did not significantly affect the downstream effectors such as MDC1 and RNF8 recruitment. We thus mainly focused on the ubiquitination defect in our current study. Ubiquitin signaling plays an important role in coordinating the recruitment of DSB repair factors such as BRCA1 and 53BP1. Ex- tensive studies have identified components of the ubiquitin system that regulate ubiquitin signaling after DNA damage (8, 9, 13–17, 38). Here, we showed that phosphorylation of ubiquitin at T66 blocked this ubiquitin signaling, providing a previously unknown layer of ubiquitin regulation in the DSB repair process. Further structural analysis will provide more detailed information on the mechanism regarding how this phosphorylation affects the RNF8-mediated ubiquitin system. Besides its role in DSB repair, ubiquitin is also involved in multiple cellular processes. Although phosphorylation of ubiquitin at T66 does not affect several E3 ubiquitin system such as MDM2 and XIAP (extended data; fig. S8, C and D), we could not exclude the possibility that phosphorylation of ubiquitin at T66 has additional effects within and outside of the DNA damage response (DDR) pathway.
In our current study, we found that ECM stiffness might be an important factor that regulates genotoxic sensitivity. Tumor stiff- ness thus might be used as a potential biomarker to predict chemo- therapy and radiotherapy outcome. Furthermore, we found that the inhibition of DNA repair under low stiffness is dependent of MAP4K4/6/7 kinases. Given that the phosphorylation of ubiquitin is reversible, some phosphatase(s) in cells should be able to remove the phosphorylation GNE-495 of ubiquitin and promote DSB repair process. Pharmacological activating MAP4K4/6/7 kinases or inhibiting the potent phosphatase might promote ubiquitin phosphorylation and inhibit DSB repair, providing potential therapeutic strategies for the enhancement of the response to DSBs.