The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34 degrees C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34 degrees C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited

Leptinemia is Associated With Peripheral Artery Disease

BackgroundLeptin, adiponectin, and resistin are in a class of hormones called adipokines that are produced by adipocytes and have been implicated in the causal pathway of atherosclerosis. We examined the association between adipokine levels and peripheral artery disease (PAD), hypothesizing that after adjusting for fat mass, leptin and resistin would be higher, whereas adiponectin would be lower, in patients with PAD.MethodsA cross-sectional sample of 179 predominately male (97%) vascular surgery outpatients was recruited from the San Francisco Veterans Affairs Medical Center (SFVAMC). PAD was defined as either an ankle-brachial index < 0.9 plus symptoms of claudication or prior revascularization for symptomatic PAD (n = 141). Controls had an ankle-brachial index ≥0.9 and no history of atherosclerotic disease (n = 38). Adipokines were assayed using commercially available ELISA kits and values were log-transformed. Fat mass was measured using bioelectrical impedance.ResultsIn an analysis adjusting for body mass index (BMI) and atherosclerotic risk factors, higher serum leptin was associated with PAD (OR 2.54, 95% CI 1.07-6.01, P = 0.03), whereas high molecular weight adiponectin was inversely associated, though not significantly (OR 0.60, 95% CI 0.33-1.08, P = 0.09). Resistin was not associated with PAD. Sensitivity analyses using fat mass/height2 rather than BMI yielded similar results.ConclusionsThese results indicate that after adjusting for BMI or fat mass, serum leptin levels are positively and independently associated with PAD, whereas high molecular weight adiponectin might be inversely associated. Using a more representative, nonveteran sample, further investigations should focus on the potential role of adipokines in the pathophysiology of PAD as well as determine whether leptin levels have clinical utility in predicting PAD outcomes

Serum resistin is associated with impaired endothelial function and a higher rate of adverse cardiac events in patients with peripheral artery disease

ObjectiveResistin is a hormone that has been associated with metabolic syndrome and cardiovascular disease. The role of resistin in patients with peripheral artery disease (PAD) has not been fully explored. This study seeks to understand the relationship between serum resistin, vascular function, and cardiovascular outcomes in patients with PAD.MethodsThere were 106 patients with PAD who were recruited between 2011 and 2016. Patients attended a baseline visit during which a comprehensive vascular physiology assessment including medical and surgical history, radial artery tonometry, and flow mediated-vasodilation (FMD) was completed. A blood sample was drawn, and serum resistin was assayed using enzyme-linked immunosorbent assay kits. Using the time of study enrollment as the time of origin, incident major adverse cardiac events (MACEs) were identified by subsequent chart review and defined as a composite end point of myocardial infarction, coronary revascularization, transient ischemic attack, stroke, or death from a cardiac cause.ResultsPatients had a mean age of 68 ± 8 years, were largely white (75%), and had comorbidities commonly associated with PAD including hypertension (92%), hyperlipidemia (87%), coronary artery disease (37%), and diabetes mellitus (38%). After stratification by resistin quartile, higher resistin quartiles were significantly associated with an older age, a greater number of pack-years smoked, and a lower estimated glomerular filtration rate. Despite similar comorbidities and medication use, endothelial function, as measured by FMD, was significantly lower with increasing resistin quartile (I, 9.1% ± 3.3%; II, 7.1% ± 3.5%; III, 5.8% ± 4.0%; IV, 5.6% ± 3.5%; P = .002). In multivariable linear regression, higher resistin quartiles (III and IV) were associated with lower FMD relative to quartile I after adjusting for several patient characteristics, medications, and comorbidities (III, -2.26 [95% confidence interval (CI), -4.51 to -0.01; P = .05]; IV, -2.53 [95% CI, -4.87 to -0.20; P = .03]). During a median follow-up period of 36 months (interquartile range, 29-45 months), 21 patients experienced the primary end point. In a Cox proportional hazards model adjusted for smoking status, coronary artery disease, and age, each 1 ng/mL increase in resistin was associated with a 10% increased risk of MACEs (hazard ratio, 1.10; 95% CI, 1.00-1.20; P = .04).ConclusionsIn patients with PAD, higher levels of resistin were associated with impaired endothelial function and an increased rate of MACEs. These results suggest that resistin may be a marker or effector of impaired vascular physiology and adverse cardiac outcomes in patients with PAD. Further research is needed to determine the potential mechanisms by which resistin may impair endothelial function and increase MACEs in this population

Adaptive and Reversible Resistance to Kras Inhibition in Pancreatic Cancer Cells

Activating mutations in KRAS are the hallmark genetic alterations in pancreatic ductal adenocarcinoma (PDAC) and the key drivers of its initiation and progression. Longstanding efforts to develop novel KRAS inhibitors have been based on the assumption that PDAC cells are addicted to activated KRAS, but this assumption remains controversial. In this study, we analyzed the requirement of endogenous Kras to maintain survival of murine PDAC cells, using an inducible shRNA-based system that enables temporal control of Kras expression. We found that the majority of murine PDAC cells analyzed tolerated acute and sustained Kras silencing by adapting to a reversible cell state characterized by differences in cell morphology, proliferative kinetics, and tumor-initiating capacity. While we observed no significant mutational or transcriptional changes in the Kras-inhibited state, global phosphoproteomic profiling revealed significant alterations in cell signaling, including increased phosphorylation of focal adhesion pathway components. Accordingly, Kras-inhibited cells displayed prominent focal adhesion plaque structures, enhanced adherence properties, and increased dependency on adhesion for viability in vitro. Overall, our results call into question the degree to which PDAC cells are addicted to activated KRAS, by illustrating adaptive nongenetic and nontranscriptional mechanisms of resistance to Kras blockade. However, by identifying these mechanisms, our work also provides mechanistic directions to develop combination strategies that can help enforce the efficacy of KRAS inhibitors. Keywords: Pancreatic cancer; cell adhesion; cell signaling; protein tyrosine kinases; animal models of cancer; gene expression profiling; oncogenes; tumor suppressor genes; gene products as targets for therapy; novel mechanismsNCIK08 AwardKL2/Catalyst MeRIT awardCCF/ASCO Young Investigator AwardLustgarten Foundation Consortium grantBlum-Kovler Pancreatic Cancer Action Network-AACR Innovative grantDepartment of Defense Congressionally-Directed Medical Research Program (Grant W81XWH-12-043)National Cancer Institute. Cancer Center Support (Grant P30-CA14051

Abstract P3-04-03: PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors

Abstract Triple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses androgen receptor (AR+). TNBC cells derived from these luminal AR+ tumors have high frequency PIK3CA mutations. The purpose of this study was to determine if targeting PI3K alone or in combination with an AR antagonist is effective in AR+ TNBC. Methods: We determined the frequency of activating PIK3CA mutations in AR+ and AR- TNBC clinical cases. Using AR+ TNBC cell lines and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability. Results: PIK3CA kinase mutations were highly clonal, more frequent in AR+ vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR+ TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects. Conclusions: While approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with androgen receptor positive (AR+) TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR+ TNBC; and, the growth and viability of AR+ TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors. Citation Format: Brian D Lehmann, Joshua A Bauer, Johanna M Schafer, Christopher S Pendleton, Luojia Tang, Kimberly C Johnson, Xi Chen, Justin M Balko, Henry Gomez, Carlos L Arteaga, Gordon B Mills, Melinda E Sanders, Jennifer A Pietenpol. PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-03.</jats:p

PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors

INTRODUCTION: Triple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC. METHODS: We determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability. RESULTS: PIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects. CONCLUSIONS: While approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-014-0406-x) contains supplementary material, which is available to authorized users

Impact of potential modifications to Alzheimer's disease clinical trials in response to disruption by COVID-19: a simulation study.

BackgroundThe COVID-19 pandemic disrupted Alzheimer disease randomized clinical trials (RCTs), forcing investigators to make changes in the conduct of such trials while endeavoring to maintain their validity. Changing ongoing RCTs carries risks for biases and threats to validity. To understand the impact of exigent modifications due to COVID-19, we examined several scenarios in symptomatic and disease modification trials that could be made.MethodsWe identified both symptomatic and disease modification Alzheimer disease RCTs as exemplars of those that would be affected by the pandemic and considered the types of changes that sponsors could make to each. We modeled three scenarios for each of the types of trials using existing datasets, adjusting enrollment, follow-ups, and dropouts to examine the potential effects COVID-19-related changes. Simulations were performed that accounted for completion and dropout patterns using linear mixed effects models, modeling time as continuous and categorical. The statistical power of the scenarios was determined.ResultsTruncating both symptomatic and disease modification trials led to underpowered trials. By contrast, adapting the trials by extending the treatment period, temporarily stopping treatment, delaying outcomes assessments, and performing remote assessment allowed for increased statistical power nearly to the level originally planned.DiscussionThese analyses support the idea that disrupted trials under common scenarios are better continued and extended even in the face of dropouts, treatment disruptions, missing outcomes, and other exigencies and that adaptations can be made that maintain the trials' validity. We suggest some adaptive methods to do this noting that some changes become under-powered to detect the original effect sizes and expected outcomes. These analyses provide insight to better plan trials that are resilient to unexpected changes to the medical, social, and political milieu