Significant chronic airway abnormalities in never-smoking HIV-infected patients.

OBJECTIVES: The aim of the study was to describe chronic lung disease in HIV-infected never-smokers by looking at clinical, structural and functional abnormalities. METHODS: This comparative cross-sectional study included 159 HIV-infected never-smoking patients [mean (± standard deviation) age 54.6 ± 9.1 years; 13.2% female; 98.1% with undetectable viral load] and 75 nonmatched never-smoking controls [mean (± standard deviation) age 52.6 ± 6.9 years; 46.7% female]. We examined calcium scoring computer tomography (CT) scans or chest CT scans, all with a lung-dedicated algorithm reconstruction, to assess emphysema and airway disease (respiratory bronchiolitis and/or bronchial wall thickening), tested pulmonary function using spirometry, lung volumes and the diffusion lung capacity of carbon monoxide (DLCO), and assessed respiratory symptoms using the Chronic Obstructive Pulmonary Disease (COPD) Assessment Test (CAT). RESULTS: Twenty-five (17.2%) of the HIV-infected patients versus two (2.7%) of the controls had a CAT score > 10. Only 5% of the HIV-infected patients showed FEV1% < 80%, and 25% had DLCO < 75% of the predicted value. Based on the CT scans, they had increased prevalences, compared with the controls, of airway disease (37% versus 7.9%, respectively) and emphysema (18% versus 4%, respectively), with more severe and more frequent centrilobular disease. After correction for age, sex and clinical factors, HIV infection was significantly associated with CAT > 10 [odds ratio (OR) 7.7], emphysema (OR 4), airway disease (OR 4.5) and DLCO < 75% of predicted (OR 4). CONCLUSIONS: Although comparisons were limited by the different enrolment methods used for HIV-infected patients and controls, the results suggest that never-smoking HIV-infected patients may present with chronic lung damage characterized by CT evidence of airway disease. A minority of them showed respiratory symptoms, without significant functional abnormalities.

Independent association of subclinical coronary artery disease and emphysema in HIV-infected patients.

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) and coronary artery disease are inflammatory states with a significant clinical impact. The relationship between them has not been investigated in patients with HIV infection. We assessed the presence of subclinical emphysema and coronary artery disease using chest computed tomography (CT) imaging in a cohort of HIV-infected patients receiving antiretroviral therapy. METHODS: Gated chest CT scans were performed in 1446 consecutive patients to assess the presence and severity of coronary artery calcium (CAC) (classified as a score of 0, 1-100 or > 100) and emphysema (classified using a visual semiquantitative scale: 0, absent; 1-4, mild to moderate; > 4, severe). Univariable and multivariable logistic regression analyses were performed to identify factors independently associated with CAC and emphysema. RESULTS: The emphysema score was significantly higher in patients with CAC scores of 1-100 and > 100 compared with those with a CAC score of 0. After adjustments for age, sex, smoking status, pack-years of smoking, visceral adiposity and duration of HIV infection, the presence of any emphysema was significantly associated with a CAC score > 0 [odds ratio (OR) 1.43; 95% confidence interval (CI) 1.08-1.88; P = 0.012]. The association persisted after adjustment for the Framingham risk score (OR 1.52; 95% CI 1.16-1.99; P = 0.002). There was a dose-dependent effect in the association between emphysema score and CAC score. CONCLUSIONS: In this cross-sectional study of HIV-infected patients, there was an independent association between emphysema and CAC, after adjustment for traditional cardiovascular risk factors, suggesting a common pathogenesis of these chronic inflammatory conditions in a chronic inflammatory disease such as HIV infection.

Serum total estradiol, but not testosterone is associated with reduced bone mineral density (BMD) in HIV-infected men: a cross-sectional, observational study.

SUMMARY: By investigating the relationship between serum testosterone, estradiol, and bone mineral density (BMD) in a large cohort of HIV-infected men, estradiol was associated with BMD, relative estrogen deficiency being involved in bone loss in men with hypogonadism, in addition to all HIV-related factors. Increased aromatization in adipose tissue does not counteract HIV-related bone loss. INTRODUCTION: The purpose of this study is to evaluate the relationship between serum testosterone, estradiol, and BMD in a large cohort of HIV-infected men. METHODS: We investigated biochemical, hormonal parameters, and BMD in 1204 HIV-infected men (age 45.64 ± 7.33 years) participating in a cross-sectional, observational study. Among other parameters, the main outcome measures were serum total testosterone and estradiol, gonadotropins, 25-hydroxyvitamin D [25(OH)D], parathormone (PTH), calcium, phosphorous, femoral, and lumbar BMD. RESULTS: In men with HIV, the prevalence of osteoporosis and osteopenia is 15.1 and 63.2% with 25(OH)D insufficiency being very common (60.1%). After age adjustment, BMD is positively associated with estradiol, but not testosterone, at linear (p < 0.001) and stepwise (p < 0.05) multiple regression. Lumbar BMD significantly increases across the estradiol quartiles but not among testosterone quartiles. Femoral and lumbar BMD are significantly higher in men with estradiol ≥ 27 pg/mL than in those with estradiol <27 pg/mL. Apart from estradiol, only age, calcium, and BMI predict BMD at stepwise linear multiple regression, but the strength of this association is weak. CONCLUSIONS: Estradiol, but not testosterone, is associated with BMD in HIV-infected men and exerts a protective role on bone especially when it is above 27 pg/mL. Relative estrogen deficiency is a potential mechanism involved in bone loss in hypogonadal HIV-infected men, in addition to all HIV-related factors. Increased aromatization in adipose tissue does not counteract HIV-related bone loss. Finally, reduced BMD in young-to-middle-aged HIV-infected men might be considered a peculiar hallmark of HIV infection due to its relevant prevalence, representing one of the several pieces composing the complicated puzzle of premature aging related to HIV infection.

Incidence and factors associated with the risk of sexually transmitted diseases in HIV-infected people seen for care in Italy: data from the Icona Foundation cohort.

OBJECTIVES: The aims of this study were to identify temporal trends in the incidence of sexually transmitted diseases (STDs) in a cohort of HIV-infected people and to evaluate factors associated with the risk of a new STD diagnosis. METHODS: All HIV-infected patients in the Icona Foundation Study cohort enrolled after 1998 were included in this study. STD incidence rates (IRs) were calculated and stratified by calendar period. Predictors of STDs were identified using a Poisson regression model with sandwich estimates for standard errors. RESULTS: Data for 9168 participants were analysed [median age 37.3 (range 18-81) years; 74% male; 30% men who have sex with men (MSM)]. Over 46 736 person-years of follow-up (PYFU), 996 episodes of STDs were observed [crude IR 21.3/1000 PYFU; 95% confidence interval (CI) 20.0-22.6/1000 PYFU]. In multivariable Poisson regression analysis, MSM [rate ratio (RR) 3.03; 95% CI 2.52-3.64 versus heterosexuals], calendar period (RR 1.67; 95% CI 1.42-1.97 for 2008-2012 versus 1998-2002), HIV RNA > 50 HIV-1 RNA copies/mL (RR 1.44; 95% CI 1.19-1.74 versus HIV RNA ≤ 50 copies/mL) and a current CD4 count < 100 cells/µL (RR 4.66; 95% CI 3.69-5.89; P < 0.001 versus CD4 count > 500 cells/µL) were associated with an increased risk of STDs. In contrast, older age (RR 0.82 per 10 years older; 95% CI 0.77-0.89) and being currently on ART (RR 0.38; 95% CI 0.33-0.45) compared with being ART-naïve or on a treatment interruption were associated with a lower risk of developing STDs. CONCLUSIONS: An increase in the incidence of STDs was observed in more recent years. Interventions to prevent STDs and potential spread of HIV should target the younger population, MSM and people currently not receiving ART.

Surrogate markers of visceral adipose tissue in treated HIV-infected patients: accuracy of waist circumference determination.

OBJECTIVES: The accuracy of the use of anthropometrics to quantify visceral adipose tissue (VAT) in treated HIV-infected patients is unknown. We evaluated the predictive accuracy of waist circumference (WC) with and without dual-energy X-ray absorptiometry (DXA)-derived trunk : limb fat ratio [fat mass ratio (FMR)] as surrogates for VAT determined using computerized axial tomography (CT-determined VAT). METHODS: We performed a retrospective cohort analysis of treated HIV-infected male patients followed at the Modena HIV Clinic. We developed prediction equations for VAT using linear regression analysis and Spearman correlations. Receiver operating characteristic (ROC) analysis evaluated the accuracy of WC alone or with FMR at discrete VAT thresholds. RESULTS: The 1500 Caucasian male patients had a median age of 45 years, body mass index (BMI) of 24, WC of 87 cm, VAT area of 127 cm(2) and body fat percentage of 14%. The correlation between WC-predicted VAT and CT-VAT was 0.613, and this increased significantly if FMR was added. The WC-associated R(2) of 0.35 increased to 0.51 if the prediction equation included WC plus FMR. The area under the ROC curve (AUC) using WC was 0.795-0.820 at all VAT thresholds. The positive predictive value (PPV) and negative predictive value (NPV) changed reciprocally at CT-VAT thresholds from 75 to 200 cm(2) and ranged from 0.72 to 0.74, respectively, at a representative VAT of 125 cm(2). Adding the FMR to the predictive equations increased the AUC in the range of 0.854-0.889 with the PPV and NPV increasing minimally, ranging from 0.780 to 0.821. Limits of precision were wide, especially at the highest CT-VAT levels, and varied from 24 to 68 cm(2). CONCLUSIONS: WC is a limited surrogate for CT-VAT in this population and DXA-derived parameters do not improve performance indices to a clinically relevant level. These findings should inform the applicability of WC to predict VAT in treated HIV-infected male patients.

The natural history of HIV-associated lipodystrophy in the changing scenario of HIV infection.

OBJECTIVES: In long-term HIV-infected patients, peripheral lipoatrophy (LA) and central lipohypertrophy (LH) appear to be related to the same insults (virus and antiretroviral drugs), but are likely to be associated with different fat depot physiologies. The objective of this study was to describe the natural history of lipodystrophy assessed using dual energy X-ray absorptiometry (DEXA) and computed tomography (CT) in a large HIV out-patients metabolic clinic. METHODS: An observational retrospective study was carried out including HIV-infected patients recruited at the Metabolic Clinic of Modena, Modena, Italy, who were assessed for lipodystrophy and had at least two anthropometric evaluations using DEXA for leg fat per cent mass and abdominal CT for visceral adipose tissue (VAT). Factors associated with leg fat per cent and VAT changes were analysed using multivariable generalized estimating equation (GEE) regression models. RESULTS: A total of 6789 DEXAs and 7566 CT scans were evaluated in the observation period. A total of 1840 patients were included; the mean age was 45.2 ± 7.2 (standard deviation) years, 621 (34%) were women, and the median HIV infection duration was 176 (interquartile range 121-232) years. According to the GEE multivariable regression analysis, leg fat per cent evaluated with DEXA appeared to increase over calendar years (ß = 0.92; P < 0.001); moreover, a progressive increase in VAT was observed in the cohort (ß = 5.69; P < 0.001). No association with antiretroviral drugs was found. CONCLUSIONS: In our study, neither LA nor LH appeared to be associated with antiretroviral drug exposure. We observed a progressive increase in LH in HIV-infected patients over calendar years. This anthropometric change, together with loss of appendicular lean mass, could describe a physiological aging process in HIV-infected patients.

Randomized trial to evaluate cardiometabolic and endothelial function in patients with plasma HIV-1 RNA suppression switching to darunavir/ritonavir with or without nucleoside analogues.

BACKGROUND: We performed a study to evaluate change in cardiometabolic and endothelial function in HIV-infected patients switching to darunavir/ritonavir (DRV/r) monotherapy versus triple therapy. METHODS: The MONARCH trial recruited 30 patients who were taking triple combination therapy and with HIV RNA<40 copies/ mL. Patients were randomized to either DRV/r 800/100 mg once daily (OD) monotherapy or DRV/r 800/100 mg OD plus 2 nucleoside reverse transcriptase inhibitors (NRTIs). The primary objective was to assess endothelial function change from baseline to 24 and 48 weeks in brachial artery flow-mediated dilation (FMD) test; changes in endothelial precursor cells (EPCs) and circulating endothelial cells (CECs) were secondary objectives. RESULTS: At baseline, the median age of participants was 43 years, 77% were men, and median CD4 cell count was 585 cells/µL. The median FMD (%) decreased in both arms in the study period (P ≯ .05), with no statistically significant difference between arms (10.7% at baseline and 6.7% at week 48 in the DRV/r + 2 NRTIs arm; 11.1% at baseline and 8.8% at week 48 in the DRV/r arm). The changes at week 48 were similar in the 2 arms for EPCs and CECs. Total cholesterol and low-density lipoprotein (LDL) cholesterol showed larger rises to week 48 in the DRV/r arm monotherapy group than in the triple-therapy group (+26 vs +9 mg/dL for total cholesterol and +14 vs +5 mg/dL for LDL cholesterol). CONCLUSIONS: In the MONARCH trial, switching from triple combination treatment to DRV/r, with or without nucleoside analogues, did not translate into clinically meaningful reductions in endothelial function as measured by FMD.

Differential dose adjustments of immunosuppressants after resuming boosted versus unboosted HIV-protease inhibitors postliver transplant.

Pharmacokinetic (PK) interactions between protease inhibitors (PI(s)) and immunosuppressive agents (IS) are critical elements in the management of HIV-infected patients who undergo liver transplantation (LT(x)). The primary objective of this study was to evaluate the decreases in IS dosages necessary to maintain an appropriate therapeutic window (TW) after initiating PI-based antiretroviral therapy regimens post-LT(x). Single-center, PK cross-sectional study of consecutive HIV-infected adult patients who underwent LT(x) was done. Blood trough concentrations (C(t)) of IS were obtained using a commercial MEIA test; plasma C(t) of PI(s) were measured using HPLC. Twelve consecutive HIV-infected adult patients (11 males, 1 female) were enrolled. More rapid increases in IS plasma C(t) were observed 48 h after initiating ritonavir (RTV)-boosted PI therapy post-LT(x) than when using unboosted PI(s). Seven patients developed acute renal failure. The median fold decrease in IS dosages required to regain IS concentrations that were in the TW was 7.5 (range 6-14) after resuming boosted PI(s) and 2.9 (range 2-4) after unboosted PI(s). The overall median time necessary to reach IS TW after dose adjustment was 3.5 days (range 0-15). Unboosted PI(s) exhibited lesser PK interactions with IS than did RTV-boosted PI(s) and were thus more amenable to use in the post-LT(x) setting.

Hyperhomocysteinaemia in HIV-infected patients: determinants of variability and correlations with predictors of cardiovascular disease.

OBJECTIVE: We evaluated hyperhomocysteinaemia (HHcy) in a cohort of HIV-infected patients in order to assess its relation to cardiovascular risk (CVR) and identify determinants of HHcy variability. METHODS: Cross-sectional observational study. HIV-infected patients on stable highly active antiretroviral therapy (ART) were evaluated for the presence of the metabolic syndrome, lipodystrophy and traditional CVR factors. Plasma homocysteine levels were measured using high-performance liquid chromatography. RESULTS: Five hundred and sixty-seven patients (38% female) with a median age of 44 years were included in the study. Homocysteine (Hcy) was significantly higher in patients with the metabolic syndrome and lipodystrophy. No significant association was found between Hcy levels and the use of ART. However, Hcy was associated with higher blood pressure, waist circumference and waist-to-hip ratio, total lean body mass, visceral adipose tissue (VAT), VAT/total adipose tissue, homeostasis model assessment of insulin resistance (HOMA-IR), triglycerides, high-density lipoprotein cholesterol, apolipoprotein A1, B, and creatinine. All 10-year CVR assessment scores were significantly associated with Hcy. In a multivariate regression model, systolic blood pressure, vitamin supplementation and HOMA-IR were significantly and independently related to Hcy. CONCLUSIONS: Hcy is elevated in HIV-infected patients and is significantly associated with increased CVR. Measurement of Hcy might be useful in identifying particularly high-risk populations at whom therapeutic interventions could be targeted.

FOXM1 recruits nuclear Aurora kinase A to participate in a positive feedback loop essential for the self-renewal of breast cancer stem cells.

Substantial evidence suggests that breast cancer initiation, recurrence and drug resistance is supported by breast cancer stem cells (BCSCs). Recently, we reported a novel role of Aurora kinase A (AURKA) in BCSCs, as a transactivating co-factor in the induction of the c-Myc oncoprotein. However, the mode of action and transcriptional network of nuclear AURKA in BCSCs remain unknown. Here, we report that nuclear AURKA can be recruited by Forkhead box subclass M1 (FOXM1) as a co-factor to transactivate FOXM1 target genes in a kinase-independent manner. In addition, we show that AURKA and FOXM1 participate in a tightly coupled positive feedback loop to enhance BCSC phenotype. Indeed, kinase-dead AURKA can effectively transactivate the FOXM1 promoter through a Forkhead response element, whereas FOXM1 can activate AURKA expression at the transcriptional level in a similar manner. Consistently, breast cancer patient samples portrayed a strong and significant correlation between the expression levels of FOXM1 and AURKA. Moreover, both FOXM1 and AURKA were essential for maintaining the BCSC population. Finally, we demonstrated that the AURKA inhibitor AKI603 and FOXM1 inhibitor thiostrepton acted synergistically to inhibit cytoplasmic AURKA activity and disrupt the nuclear AURKA/FOXM1-positive feedback loop, respectively, resulting in a more effective inhibition of the tumorigenicity and self-renewal ability of BCSCs. Collectively, our study uncovers a previously unknown tightly coupled positive feedback signalling loop between AURKA and FOXM1, crucial for BCSC self-renewal. Remarkably, our data reveal a novel potential therapeutic strategy for targeting both the cytoplasmic and nuclear AURKA function to effectively eliminate BCSCs, so as to overcome both breast cancer and drug resistance.

RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment.

The forkhead box M1 (FOXM1) transcription factor has a central role in genotoxic agent response in breast cancer. FOXM1 is regulated at the post-translational level upon DNA damage, but the key mechanism involved remained enigmatic. RNF168 is a ubiquitination E3-ligase involved in DNA damage response. Western blot and gene promoter-reporter analyses showed that the expression level and transcriptional activity of FOXM1 reduced upon RNF168 overexpression and increased with RNF168 depletion by siRNA, suggesting that RNF168 negatively regulates FOXM1 expression. Co-immunoprecipitation studies in MCF-7 cells revealed that RNF168 interacted with FOXM1 and that upon epirubicin treatment FOXM1 downregulation was associated with an increase in RNF168 binding and conjugation to the protein degradation-associated K48-linked polyubiquitin chains. Consistently, RNF168 overexpression resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide. Conversely, RNF168, knockdown significantly enhanced the half-life of FOXM1 in both absence and presence of epirubicin. Using a SUMOylation-defective FOXM1-5x(K>R) mutant, we demonstrated that SUMOylation is required for the recruitment of RNF168 to mediate FOXM1 degradation. In addition, clonogenic assays also showed that RNF168 mediates epirubicin action through targeting FOXM1, as RNF168 could synergise with epirubicin to repress clonal formation in wild-type but not in FOXM1-deficient mouse embryo fibroblasts (MEFs). The physiological relevance of RNF168-mediated FOXM1 repression is further emphasized by the significant inverse correlation between FOXM1 and RNF168 expression in breast cancer patient samples. Moreover, we also obtained evidence that RNF8 recruits RNF168 to FOXM1 upon epirubicin treatment and cooperates with RNF168 to catalyse FOXM1 ubiquitination and degradation. Collectively, these data suggest that RNF168 cooperates with RNF8 to mediate the ubiquitination and degradation of SUMOylated FOXM1 in breast cancer genotoxic response.

OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.

Low testosterone is associated with poor health status in men with human immunodeficiency virus infection: a retrospective study.

Men with human immunodeficiency virus (HIV) infection are often hypogonadal and develop several HIV-associated non-acquired immunodeficiency syndrome (AIDS) (HANA) conditions that impair overall health status. No studies explored the relationship between health status and serum testosterone (T) in HIV-infected men. This study aims to investigate the association between total serum T and HANA, multimorbidity, and frailty in a large cohort of 1359 HIV-infected men and to explore the relationship between patients' overall health status and serum T. Among biochemical and hormonal measurement performed the main are serum total T, free triiodothyronine (fT3), and luteinizing hormone. Other outcome measurements include anthropometry, assessment of comorbidities and disabilities, overall health status defined as the number of HANA and by the 38-item multimorbidity frailty index, anthropometry, and bone mineral density. The cumulative relative risk of comorbidities is increased in HIV-infected men with hypogonadism (p < 0.001) and hypogonadism is associated with several comorbidities. The prevalence of hypogonadism increases progressively with the increase of the number of comorbidities. Frailty index is inversely related to serum total T (age-adjusted r = 0.298, r(2) = 0.089, p < 0.0001). Serum fT3 levels are significantly lower in hypogonadal than eugonadal men (p = 0.022). This suggests that low serum T could be considered a sensitive marker of frailty and poor health status and that the latter might induce hypogonadism. The more HIV-infected men are frail the more they are hypogonadal. This suggests that hypogonadism might be a naturally occurring condition in unhealthy HIV-infected men and raises concern about the safety of T treatment. In conclusion, low serum T is associated with multimorbidity, HANA, and frailty in HIV-infected men and this association seems to be bidirectional. Given the wide attitude to offer T treatment to HIV-infected men, caution is needed when prescribing T to HIV-infected male patients, especially if the patient is unhealthy or frail.

SUMOylation inhibits FOXM1 activity and delays mitotic transition.

The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.

FOXM1 targets NBS1 to regulate DNA damage-induced senescence and epirubicin resistance.

FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and mouse embryonic fibroblasts (MEFs) into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated ß-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an forkhead response element on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7Epi(R) cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ataxia-telangiectasia mutated (ATM)phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7Epi(R) cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated homologous recombination (HR) DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.