Hypoxia disrupts the Fanconi anemia pathway and sensitizes cells to chemotherapy through regulation of UBE2T

AbstractBackground and purposeHypoxia is a common feature of the microenvironment of solid tumors which has been shown to promote malignancy and poor patient outcome through multiple mechanisms. The association of hypoxia with more aggressive disease may be due in part to recently identified links between hypoxia and genetic instability. For example, hypoxia has been demonstrated to impede DNA repair by down-regulating the homologous recombination protein RAD51. Here we investigated hypoxic regulation of UBE2T, a ubiquitin ligase required in the Fanconi anemia (FA) DNA repair pathway.Materials and methodsWe analysed UBE2T expression by microarray, quantitative PCR and western blot analysis in a panel of cancer cell lines as a function of oxygen concentration. The importance of this regulation was assessed by measuring cell survival in response to DNA damaging agents under normoxia or hypoxia. Finally, HIF dependency was determined using knockdown cell lines and RCC4 cells which constitutively express HIF1α.ResultsHypoxia results in rapid and potent reductions in mRNA levels of UBE2T in a panel of cancer cell lines. Reduced UBE2T mRNA expression is HIF independent and was not due to changes in mRNA or protein stability, but rather reflected reduced promoter activity. Exposure of tumor cells to hypoxia greatly increased their sensitivity to treatment with the interstrand crosslinking (ICL) agent mitomycin C.ConclusionsExposure to hypoxic conditions down-regulates UBE2T expression which correlates with an increased sensitivity to crosslinking agents consistent with a defective Fanconi anemia pathway. This pathway can potentially be exploited to target hypoxic cells in tumors

Dual inhibition of PCNA and RNF8 signalling pathways does not mimic the K63RUb phenotype.

<p>(A) Depletion of HLTF or SHPRH in combination with knock-down of RNF8 was achieved using lentiviral shRNA's and was confirmed using real-time PCR. (B) Spontaneous or CPT (20 nM) induced mutations were determined at the <i>HPRT</i> locus, mean ± s.d. (n = 5) per exp). (C) Clonogenic survival of A549 cells after double knock-down of HLTF and RNF8 or SHPRH and RNF8 was determined after treatment with 100 nM CPT (24 h). Data are mean ± s.d. (n = 3), *P<0.05, **P<0.01, ***P<0.001.</p

K63RUb expression induces S-phase specific repair defects.

<p>(A) WT and K63R Ub cells were fixed and immuno-stained for γH2AX and 53BP1 foci at the indicated time points following 2 Gy IR or untreated (Ctrl). Mean values ± sd. of 2 independent exp's, >150 cells were analysed per time-point (B) 30 min EdU (10 µM) incorporation before 2 Gy IR, cells were fixed 30 min or 24 hrs post IR, EdU and 53BP1 foci were visualized by fluorescent staining and quantified. Mean values ± s.e.m. of 2 independent exp's (n>100 per treatment). (C) 30 min EdU (10 µM) incorporation alone (untreated) or before 2.5 h 100 nM CPT treatment. Cells were fixed and immuno-stained for EdU and γH2AX 3 h post treatment. (D) Quantification of 53BP1 foci. Cells were fixed and immuno-stained for 53BP1 directly or 24 h after 1 h 100 nM CPT treatment, >80 cells were analysed per sample. (E) Quantification of γH2AX immuno-staining of untreated (Ctrl) or 1 h 100 nM CPT treated cells. Mean values ± s.e.m. Data is representative exp (n>130) of 3 independent exp's, *P<0.05.</p

Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[a]pyrene-diol-epoxide-induced mutagenicity

Benzo[a]pyrene exerts its mutagenic effects via induction of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLeta), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin (K63R-Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin (WT-Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R-Ub expressing cells, of which the majority (74%) was G-->T transversion. BPDE treatment caused an enhanced recruitment of POLeta to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLeta expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability

K63RUb phenotype is not due to NHEJ.

<p>(A-C) Clonogenic survival of WT and K63R Ub cells after treatment with (A) 1 µM DNA-PKi alone for indicated times, (B) 1 µM DNA-PKi for 24 or 48 hrs, started 1 h before combined 24 hrs treatment with 50 nM CPT or (C) 1 µM DNA-PKi for 24 hrs, started 1 h before 2 Gy IR (A-C) mean ± s.d. (n = 3). (D) Mutations at the <i>HPRT</i> locus were determined after continuous treatment with 1 µM DNA-PKi started 1 h before combined with additional treatments either spontaneous or 20 nM CPT for 6 days total, mean ± s.d. (n = 5).</p

RNF8 depletion does not reproduce K63RUb phenotype.

<p>(A-C) WT and K63R Ub cells were transfected with siRNA against RNF8 or RAP80 on day 1 and 3. (A) Knockdown of RNF8 and RAP80 was assessed by mRNA expression levels determined by real-time PCR. Clonogenic survival was assessed after (B) 4 Gy IR and (C) PARPi (continuous) (B-C) Data are mean ± s.d. of 2 independent exp's (n = 3 per exp). (D) Spontaneous and IR-induced (4 Gy) mutations were determined at the <i>HPRT</i> locus, mean ± s.e.m. of 3 independent exp's (n = 10 per exp), *P<0.05, **P<0.01, ***P<0.001.</p

K63RUb expression sensitizes cells to replication-associated DNA damage.

<p>(A-C) Clonogenic survival of A549 cells expressing empty vector (EV), WTUb or K63RUb was determined after (A) CPT (24h) treatment started following cell attachment. Data are mean ± sd. of 2 independent exp's (n = 3 per exp). (B) Continuous PARPi treatment, data are mean ± sd. of 3 independent exp's (n = 3 per exp). (C) IR, data are mean ± sd. of 3 independent exp's (n = 3 per exp) *P<0.05, **P<0.01, ***P<0.001.</p

Spontaneous, IR- and CPT-induced mutation spectra in K63R ubiquitin expressing cells.

<p>Spontaneous, IR- and CPT-induced mutation spectra in K63R ubiquitin expressing cells.</p