Transcriptional profiling identifies an androgen receptor activity-low, stemness program associated with enzalutamide resistance.

The androgen receptor (AR) antagonist enzalutamide is one of the principal treatments for men with castration-resistant prostate cancer (CRPC). However, not all patients respond, and resistance mechanisms are largely unknown. We hypothesized that genomic and transcriptional features from metastatic CRPC biopsies prior to treatment would be predictive of de novo treatment resistance. To this end, we conducted a phase II trial of enzalutamide treatment (160 mg/d) in 36 men with metastatic CRPC. Thirty-four patients were evaluable for the primary end point of a prostate-specific antigen (PSA)50 response (PSA decline ≥50% at 12 wk vs. baseline). Nine patients were classified as nonresponders (PSA decline <50%), and 25 patients were classified as responders (PSA decline ≥50%). Failure to achieve a PSA50 was associated with shorter progression-free survival, time on treatment, and overall survival, demonstrating PSA50's utility. Targeted DNA-sequencing was performed on 26 of 36 biopsies, and RNA-sequencing was performed on 25 of 36 biopsies that contained sufficient material. Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis (GSEA) to identify pathways whose activity state correlated with de novo resistance. TP53 gene alterations were more common in nonresponders, although this did not reach statistical significance (P = 0.055). AR gene alterations and AR expression were similar between groups. Importantly, however, transcriptional measurements demonstrated that specific gene sets-including those linked to low AR transcriptional activity and a stemness program-were activated in nonresponders. Our results suggest that patients whose tumors harbor this program should be considered for clinical trials testing rational agents to overcome de novo enzalutamide resistance

Chromium supplementation in non-obese non-diabetic subjects is associated with a decline in insulin sensitivity

<p>Abstract</p> <p>Background</p> <p>The use of chromium supplements is widespread for the prevention and treatment of diabetes mellitus but there are conflicting reports on efficacy, possibly reflecting discrepant effects across different populations. In the present studies, we test the hypothesis that chromium supplementation raises serum chromium levels and correspondingly improves insulin sensitivity.</p> <p>Methods</p> <p>A double blind placebo-controlled randomized trial was conducted on 31 non-obese, normoglycemic subjects. After baseline studies, the subjects were randomized to placebo or chromium picolinate 500 μg twice a day. The primary endpoint was change in insulin sensitivity as measured by euglycemic hyperinsulinemic clamp. Pre-specified secondary endpoints included fasting lipids, blood pressure, weight, body composition measured by DXA scan.</p> <p>Results</p> <p>After 16 weeks of chromium picolinate therapy there was no significant change in insulin sensitivity between groups (p=0.83). There was, however, a strong association between serum chromium and change in insulin resistance (β = -0.83, p=0.01), where subjects with the highest serum chromium had a worsening of insulin sensitivity. This effect could not be explained by changes in physiological parameters such as body weight, truncal fat and serum lipids with chromium therapy.</p> <p>Conclusions</p> <p>Chromium therapy did not improve insulin sensitivity in non-obese normoglycemic individuals. Further, subjects who have high serum chromium levels paradoxically had a decline in insulin sensitivity. Caution therefore should be exercised in recommending the use of this supplement.</p> <p>Trial registration</p> <p>The study was registered on the NIH registry (clinicaltrials.gov) and the identifier is NCT00846248</p

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle.

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and (1)H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H(2)O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle.

BackgroundThe pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.Methodology/principal findingsTo explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and (1)H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P&lt;0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P&lt;0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P&lt;0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H(2)O peak, P&lt;0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.ConclusionsThis is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population

The IR remains membrane localized following chronic insulin stimulation of HTC-IR-YFP cells.

<p>(<b>A</b>) YFP fused to the IR fluoresces green and co-localizes with the membrane-specific stain (orange) and not with nuclei (blue). (<b>B</b>) Total IR tyrosine phosphorylation in HTC-IR-YFP cells, detected by ELISA, mirrors that of the unfused similar to HTC-IR in response to insulin stimulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">Fig. 1D</a>). The fluorescence intensity of (<b>C</b>) YFP (IR) and (<b>D</b>) the membrane marker were quantified at the membrane and in the cytosol of HTC-IR-YFP cells.</p

Differential down-regulation of downstream signaling pathways following chronic insulin stimulation.

<p>(<b>A</b>) phospho-ERK, (<b>B</b>) total ERK (<b>C</b>) phospho-AKT, (<b>D</b>) total AKT, (<b>E</b>) phospho-GSKβ, and (<b>F</b>) total GSKβ proteins quantified by western blot using the same extracts analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>. The fluorescence intensities of pERK and ERK, or pGSK and GSK, were quantified on the same blot with the phospho-specific and total protein antibodies (of rabbit and mouse origin) conjugated to specific-specific secondary antibodies with distinguishable fluorophores. The pAKT and total AKT antibodies both originated from rabbits and were probed in separate blots. Data normalization, number of studies, symbols and representative westerns are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>.</p