The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide ...production. Cyclin D1 abundance is rate-limiting in normal G1 phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-κB (NF-κB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IκB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (RacLeu-61, RacVal-12) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-κB and an ATF-2 binding site. Inhibiting NF-κB by overexpression of an NF-κB trans-dominant inhibitor (nonphosphorylatable IκBα) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-κB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of RacLeu-61 had comparable effects on NF-κB transcriptional activity and activation of the cyclin D1 promoter. The NF-κB factors Rel A (p65) and NF-κB1 (p50) induced the cyclin D1 promoter, requiring both the NF-κB binding site and the ATF-2 site. Stable overexpression of RacLeu-61 increased binding of Rel A and NF-κB1 to the cyclin D1 promoter NF-κB site. Activation of Rac1 in NIH 3T3 cells induces both NF-κB binding and activity and enhances expression of cyclin D1 through an NF-κB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-κB in cell cycle regulation through cyclin D1 and Rac1.
The Wnt/β-catenin/Tcf and IκB/NF-κB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/β-catenin signaling occurs in ...certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear β-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IκB kinase complex (IKK) that phosphorylates IκB contains IKKα, IKKβ, and IKKγ. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/β-catenin and IκB pathways in mitogenic signaling. Mitogenic induction of G
1
-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1
−/−
cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IκB/IKKα or β-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKα. In IKKα
−/−
cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKα restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKα
−/−
cells, β-catenin phosphorylation was decreased and purified IKKα was sufficient for phosphorylation of β-catenin through its N-terminus in vitro. Because IKKα but not IKKβ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKα and IKKβ may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.
The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20–30% of human breast tumors and ...is induced both by oncogenes including those for Ras, Neu, and Src, and by the β-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of β1 and β3integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide −54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3β (GSK-3β) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.
Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. ...p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs in < or =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.
Antiretroviral therapy (ART) during pregnancy is important for both maternal health and prevention of perinatal HIV-1 transmission; however adequate data on the safety and efficacy of different ART ...regimens that are likely to be used by pregnant women are scarce. In this trial we compared the safety and efficacy of three antiretroviral regimens started in pregnancy: dolutegravir, emtricitabine, and tenofovir alafenamide fumarate; dolutegravir, emtricitabine, and tenofovir disoproxil fumarate; and efavirenz, emtricitabine, and tenofovir disoproxil fumarate.
This multicentre, open-label, randomised controlled, phase 3 trial was done at 22 clinical research sites in nine countries (Botswana, Brazil, India, South Africa, Tanzania, Thailand, Uganda, the USA, and Zimbabwe). Pregnant women (aged ≥18 years) with confirmed HIV-1 infection and at 14–28 weeks' gestation were eligible. Women who had previously taken antiretrovirals in the past were excluded (up to 14 days of ART during the current pregnancy was permitted), as were women known to be pregnant with multiple fetuses, or those with known fetal anomaly or a history of psychiatric illness. Participants were randomly assigned (1:1:1) using a central computerised randomisation system. Randomisation was done using permuted blocks (size six) stratified by gestational age (14–18, 19–23, and 24–28 weeks' gestation) and country. Participants were randomly assigned to receive either once-daily oral dolutegravir 50 mg, and once-daily oral fixed-dose combination emtricitabine 200 mg and tenofovir alafenamide fumarate 25 mg; once-daily oral dolutegravir 50 mg, and once-daily oral fixed-dose combination emtricitabine 200 mg and tenofovir disoproxil fumarate 300 mg; or once-daily oral fixed-dose combination of efavirenz 600 mg, emtricitabine 200 mg, and tenofovir disoproxil fumarate 300 mg. The primary efficacy outcome was the proportion of participants with viral suppression, defined as an HIV-1 RNA concentration of less than 200 copies per mL, at or within 14 days of delivery, assessed in all participants with an HIV-1 RNA result available from the delivery visit, with a prespecified non-inferiority margin of −10% in the combined dolutegravir-containing groups versus the efavirenz-containing group (superiority was tested in a pre-planned secondary analysis). Primary safety outcomes, compared pairwise among treatment groups, were the occurrence of a composite adverse pregnancy outcome (ie, either preterm delivery, the infant being born small for gestational age, stillbirth, or spontaneous abortion) in all participants with a pregnancy outcome, and the occurrence of grade 3 or higher maternal and infant adverse events in all randomised participants. This trial was registered with ClinicalTrials.gov, NCT03048422.
Between Jan 19, 2018, and Feb 8, 2019, we enrolled and randomly assigned 643 pregnant women: 217 to the dolutegravir, emtricitabine, and tenofovir alafenamide fumarate group, 215 to the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group, and 211 to the efavirenz, emtricitabine, and tenofovir disoproxil fumarate group. At enrolment, median gestational age was 21·9 weeks (IQR 18·3–25·3), the median HIV-1 RNA concentration among participants was 902·5 copies per mL (152·0–5182·5; 181 28% of 643 participants had HIV-1 RNA concentrations of <200 copies per mL), and the median CD4 count was 466 cells per μL (308–624). HIV-1 RNA concentrations at delivery were available for 605 (94%) participants. Of these, 395 (98%) of 405 participants in the combined dolutegravir-containing groups had viral suppression at delivery compared with 182 (91%) of 200 participants in the efavirenz, emtricitabine, and tenofovir disoproxil fumarate group (estimated difference 6·5% 95% CI 2·0 to 10·7, p=0·0052; excluding the non-inferiority margin of −10%). Significantly fewer participants in the dolutegravir, emtricitabine, and tenofovir alafenamide fumarate group (52 24% of 216) had a composite adverse pregnancy outcome than those in the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group (70 33% of 213; estimated difference −8·8% 95% CI −17·3 to −0·3, p=0·043) or the efavirenz, emtricitabine, and tenofovir disoproxil fumarate group (69 33% of 211; −8·6% –17·1 to −0·1, p=0·047). The proportion of participants or infants with grade 3 or higher adverse events did not differ among the three groups. The proportion of participants who had a preterm delivery was significantly lower in the dolutegravir, emtricitabine, and tenofovir alafenamide fumarate group (12 6% of 208) than in the efavirenz, emtricitabine, and tenofovir disoproxil fumarate group (25 12% of 207; −6·3% –11·8 to −0·9, p=0·023). Neonatal mortality was significantly higher in the efavirenz, emtricitabine, and tenofovir disoproxil fumarate group (ten 5% of 207 infants) than in the dolutegravir, emtricitabine, and tenofovir alafenamide fumarate group (two 1% of 208; p=0·019) or the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group (three 2% of 202; p=0·050).
When started in pregnancy, dolutegravir-containing regimens had superior virological efficacy at delivery compared with the efavirenz, emtricitabine, and tenofovir disoproxil fumarate regimen. The dolutegravir, emtricitabine, and tenofovir alafenamide fumarate regimen had the lowest frequency of composite adverse pregnancy outcomes and of neonatal deaths.
National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health.
Background. Maraviroc (MVC) is a candidate for human immunodeficiency virus (HIV) pre-exposure prophylaxis. Methods. Phase 2 48-week safety/tolerability study was conducted, comparing 4 regimens: MVC ...alone, MVC plus emtricitabine (FTC), MVC plus tenofovir disoproxil fumarate (TDF), and TDF plus FTC. Eligible participants were HIV-uninfected men and transgender women reporting condomless anal intercourse with ≥1 HIV-infected or unknown-serostatus man within 90 days. At each visit, assessments, laboratory testing, and counseling were done. Analyses were intention to treat. Results. Among 406 participants, 84% completed follow-up, 7% stopped early, and 9% were lost to follow-up; 9% discontinued their regimen early. The number discontinuing and the time to discontinuation did not differ among study regimens (P = .60). Rates of grade 3-4 adverse events did not differ among regimens (P = .37). In a randomly selected subset, 77% demonstrated detectable drug concentrations at week 48. Five participants acquired HIV infection (4 MVC alone, 1 MVC + TDF; overall annualized incidence, 1.4% 95% confidence interval, .5%-3.3%, without differences by regimen; P = .32); 2 had undetectable drug concentrations at every visit, 2 had low concentrations at the seroconversion visit, and 1 had variable concentrations. Conclusions. MVC-containing regimens were safe and well tolerated compared with TDF + FTC; this study was not powered for efficacy. Among those acquiring HIV infection, drug concentrations were absent, low, or variable. MVC-containing regimens may warrant further study for pre-exposure prophylaxis. Clinical Trials Registration. NCT01505114.
Background Previous studies have raised concerns that video-assisted thoracoscopic (VATS) lobectomy may compromise nodal evaluation. The advantages or limitations of robotic lobectomy have not been ...thoroughly evaluated. Methods Perioperative outcomes and survival of patients who underwent open versus minimally-invasive surgery (MIS VATS and robotic) lobectomy and VATS versus robotic lobectomy for clinical T1-2, N0 non-small cell lung cancer from 2010 to 2012 in the National Cancer Data Base were evaluated using propensity score matching. Results Of 30,040 lobectomies, 7,824 were VATS and 2,025 were robotic. After propensity score matching, when compared with the open approach (n = 9,390), MIS (n = 9,390) was found to have increased 30-day readmission rates (5% versus 4%, p < 0.01), shorter median hospital length of stay (5 versus 6 days, p < 0.01), and improved 2-year survival (87% versus 86%, p = 0.04). There were no significant differences in nodal upstaging and 30-day mortality between the two groups. After propensity score matching, when compared with the robotic group (n = 1,938), VATS (n = 1,938) was not significantly different from robotics with regard to nodal upstaging, 30-day mortality, and 2-year survival. Conclusions In this population-based analysis, MIS (VATS and robotic) lobectomy was used in the minority of patients for stage I non-small cell lung cancer. MIS lobectomy was associated with shorter length of hospital stay and was not associated with increased perioperative mortality, compromised nodal evaluation, or reduced short-term survival when compared with the open approach. These results suggest the need for broader implementation of MIS techniques.
The objective of this study was to compare HIV-negative cisgender women (CGW) with MSM for mucosal tissue differences in pharmacokinetics, HIV infectivity and cell phenotype.
A substudy of HPTN ...069/ACTG A5305, 48-week study of three oral candidate preexposure prophylaxis regimens: maraviroc, maraviroc/emtricitabine and maraviroc/tenofovir disoproxil fumarate (TDF) compared with a TDF/emtricitabine control group.
Plasma, peripheral blood mononuclear cells and cervical and colorectal tissue biopsies were collected at Baseline (no drug), Week 24 and 48 (on drug), and Week 49 (1-week postdrug). Drug concentrations were assessed in all matrices. HIV infectivity was assessed using tissue biopsy 'explants' challenged with HIV ex vivo followed by HIV p24 measurement. Flow cytometry evaluated colorectal cell phenotype.
Thirty-seven CGW and 54 MSM participated. CGW's colorectal explant p24 was higher than MSM before (0.31 log10, P = 0.046), during (1.01-1.19 log10, P = 0.016) and one week after (0.61 log10, P = 0.011) study drug dosing. Pooling regimens, cervical explant p24 did not differ among visits. CGW had higher plasma maraviroc and colorectal tissue tenofovir diphosphate and lower colorectal tissue emtricitabine (all P < 0.005) compared with MSM. Each study drug's cervical tissue concentrations were more than 10-fold below paired colorectal concentrations (P < 0.001). Cell phenotype sex differences included 4% higher CD38+/CD8+ cells at baseline and 3-7% higher CD69+/CD8+ cells throughout Weeks 24-49 in CGW compared with MSM (P < 0.05).
Colorectal explants in CGW demonstrated greater HIV infectivity than MSM with and without study drugs. Small differences in adherence, drug concentration and colorectal tissue flow cytometry cannot fully explain this difference.
HPTN 069/ACTG 5305 was designed to evaluate potential new PrEP regimens that included maraviroc, tenofovir disoproxil fumarate, and/or emtricitabine. The current analyses assessed antiretroviral ...(ARV) plasma concentrations in relation to sexual behavior in 224 cisgender men who have sex with men and 2 transgender women at risk for HIV. Poisson generalized estimating equations (GEE) regression were used to test for associations between self-reported sexual behavior, sociodemographic, behavioral variables, and study drug levels The median (IQR) age was 30 25, 37 years old; 48.2% had completed college; 27.4% were Black and 21.7% Latino. At weeks 24 and 48, one third of participants reported condomless anal sex (CAS) in the prior month with more than one partner. CAS was associated with daily ARV drug use (χ
2
= 12.64, p = 0.002). Older individuals and those with greater education were more likely to ingest ARV drugs daily (χ
2
= 9.36, p = 0.009 and χ
2
= 8.63, p = 0.013, respectively), while neither race nor ethnicity was associated with daily ARV drug use. Participants who reported recent condomless anal sex and/or advanced education had higher rates of daily ARV drug use. These data support the need for ongoing adherence counseling in clinical trials of new PrEP modalities.
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