The effects of glycemic index on prostate cancer progression in a xenograft mouse model.

BACKGROUND: Previously, we found low-carbohydrate diets slowed prostate cancer (PC) growth and increased survival vs. a Western diet in mice, by inhibiting the insulin/IGF-1 axis. Thus, we tested whether modifying carbohydrate quality to lower glycemic index (GI) without changing quantity results in similar benefits as with reduced quantity. METHODS: Male SCID mice injected with LAPC-4 cells were single-housed and randomized when their tumors reached 200 mm3 on average to a LoGI (48% carbohydrate kcal, from Hylon-VII) or HiGI Western diet (48% carbohydrate kcal, from sucrose). Body weight and tumor volume were measured weekly. Body composition was assessed 35 days after randomization. Blood glucose and serum insulin, IGF-1 and IGFBP3 were measured at study end when tumor volumes reached 800 mm3. We analyzed gene expression of mice tumors by RNA-sequencing and human tumors using the Prostate Cancer Transcriptome Atlas. RESULTS: There were no significant differences in tumor volume (P > 0.05), tumor proliferation (P = 0.29), and overall survival (P = 0.15) between groups. At 35 days after randomization, the LoGI group had 30% lower body fat (P = 0.007) despite similar body weight (P = 0.58). At sacrifice, LoGI mice had smaller livers (P < 0.001) and lower glucose (P = 0.15), insulin (P = 0.11), IGF-1 (P = 0.07) and IGF-1:IGFBP3 ratio (P = 0.05), and higher IGFBP3 (P = 0.09) vs. HiGI, although none of these metabolic differences reached statistical significance. We observed differential gene expression and pathway enrichment in mice tumors by diet. The most upregulated and downregulated gene in the LoGI group showed expression patterns more closely resembling expression in human benign prostate tissue vs. PC. CONCLUSIONS: In this single mouse xenograft model, consuming a low GI diet did not delay PC growth or survival vs. a high GI diet despite suggestions of decreased activation of the insulin/IGF-1 pathway. These data suggest that improving carbohydrate quality alone while consuming a high carbohydrate diet may not effectively slow PC growth.

Racial differences in circulating mitochondria-derived peptides may contribute to prostate cancer health disparities.

INTRODUCTION: The mitochondrial genome has small open reading frames (sORF) which produce measurable mitochondrial-derived peptides (MDPs), including humanin, SHLP2, and MOTS-c. Previously, among men undergoing prostate biopsy, we found higher serum SHLP2 was linked with lower prostate cancer (PC) risk in European American men (EAM), while null associations were found in African American men (AAM). Here, in different patients undergoing prostate biopsy, we tested the link between SHLP2, humanin and MOTS-c and PC risk by race. METHODS: Plasma SHLP2, humanin, and MOTS-c were measured in 198 men (50/49 EAM/AAM cases; 50/49 EAM/AAM controls) undergoing biopsy. Logistic and multinomial regression models tested associations between each MDP and PC diagnosis, low-grade (grade group, GG1) and high-grade (GG2-5). Models were adjusted for age, body mass index, digital rectal examination, and prostate specific antigen (PSA). We tested interactions between MDPs and race. RESULTS: Among controls, humanin was similar by race (p = 0.60), but both SHLP2 (p = 0.007) and MOTS-c (p = 0.026) were lower in AAM controls versus EAM controls. Among EAM, higher MDP values were associated with lower PC risk (all p ≤ 0.001), with null associations in AAM (all p-interactions ≤ 0.01). Similarly, higher MDP expression was associated with decreased risk of low- and high-grade PC in EAM (all p ≤ 0.005) with null associations in AAM. CONCLUSIONS: Higher MDP levels were associated with lower PC risk in EAM but not AAM. Generally, AAM controls had lower MDP levels. These data support MDPs and mitochondrial dysfunction in PC, suggesting greater dysfunction in AAM may contribute to excess PC risk. Future larger studies are needed to confirm these results.

Low Carbohydrate Diets and Estimated Cardiovascular and Metabolic Syndrome Risk in Prostate Cancer.

PURPOSE: A low carbohydrate diet (LCD) was shown to suggestively slow prostate cancer (PC) growth. In noncancer patients, LCDs improve metabolic syndrome (MetS) without weight loss. However, concerns about negative impact on cardiovascular disease (CVD) risk remain. The objective of this secondary analysis is to determine the impact of an LCD on risk of MetS and estimated CVD risk in patients with PC. MATERIALS AND METHODS: Pooled data were analyzed from 2 randomized trials testing LCD vs control on 1) preventing insulin resistance after starting hormone therapy (CAPS1) and 2) slowing PC growth in recurrent PC after failed primary treatment (CAPS2). Both trials included a usual care control vs LCD intervention in which patients were instructed to limit carbohydrate intake to ≤20 gm/day, and in CAPS1 only, to walk for ≥30 minutes/day for ≥5 days/week. MetS components (hypertension, high triglycerides, low high-density lipoprotein cholesterol, central obesity and diabetes), 10-year CVD risk estimated using the Framingham Score with either body mass index (BMI) or lipids, and remnant cholesterol were compared between arms using mixed models adjusting for trial. RESULTS: LCD resulted in a significantly reduced risk of MetS (p=0.004) and remnant cholesterol (p <0.001). Moreover, LCD resulted in significantly lower estimated CVD risk using BMI (p=0.002) over the study with no difference in estimated CVD risk using lipids (p=0.14). CONCLUSIONS: LCD resulted in a significantly reduced risk of MetS and remnant cholesterol, and a significantly lower estimated CVD risk using BMI. By comparison, there was no difference in estimated CVD risk using lipids. Study limitations include small sample size, short followup, and inability to distinguish effects of carbohydrate restriction and weight loss. Long-term studies are needed to confirm this finding.

The influence of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PC) is the second most lethal cancer for men. For metastatic PC, standard first-line treatment is androgen deprivation therapy (ADT). While effective, ADT has many metabolic side effects. Previously, we found in serum metabolome analysis that ADT reduced androsterone sulfate, 3-hydroxybutyric acid, acyl-carnitines but increased serum glucose. Since ADT reduced ketogenesis, we speculate that low-carbohydrate diets (LCD) may reverse many ADT-induced metabolic abnormalities in animals and humans. METHODS: In a multicenter trial of patients with PC initiating ADT randomized to no diet change (control) or LCD, we previously showed that LCD intervention led to significant weight loss, reduced fat mass, improved insulin resistance, and lipid profiles. To determine whether and how LCD affects ADT-induced metabolic changes, we analyzed serum metabolites after 3-, and 6-months of ADT on LCD versus control. RESULTS: We found androsterone sulfate was most consistently reduced by ADT and was slightly further reduced in the LCD arm. Contrastingly, LCD intervention increased 3-hydroxybutyric acid and various acyl-carnitines, counteracting their reduction during ADT. LCD also reversed the ADT-reduced lactic acid, alanine, and S-adenosyl methionine (SAM), elevating glycolysis metabolites and alanine. While the degree of androsterone reduction by ADT was strongly correlated with glucose and indole-3-carboxaldehyde, LCD disrupted such correlations. CONCLUSIONS: Together, LCD intervention significantly reversed many ADT-induced metabolic changes while slightly enhancing androgen reduction. Future research is needed to confirm these findings and determine whether LCD can mitigate ADT-linked comorbidities and possibly delaying disease progression by further lowering androgens.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

27-Hydroxycholesterol Impairs Plasma Membrane Lipid Raft Signaling as Evidenced by Inhibition of IL6-JAK-STAT3 Signaling in Prostate Cancer Cells.

We recently reported that restoring the CYP27A1-27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti-prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6-JAK-STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6-JAK-STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. IMPLICATIONS: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6-JAK-STAT signaling and may synergize with STAT3-targeted compounds.

Prediction of acute coronary syndromes by urinary proteome analysis.

Identification of individuals who are at risk of suffering from acute coronary syndromes (ACS) may allow to introduce preventative measures. We aimed to identify ACS-related urinary peptides, that combined as a pattern can be used as prognostic biomarker. Proteomic data of 252 individuals enrolled in four prospective studies from Australia, Europe and North America were analyzed. 126 of these had suffered from ACS within a period of up to 5 years post urine sampling (cases). Proteomic analysis of 84 cases and 84 matched controls resulted in the discovery of 75 ACS-related urinary peptides. Combining these to a peptide pattern, we established a prognostic biomarker named Acute Coronary Syndrome Predictor 75 (ACSP75). ACSP75 demonstrated reasonable prognostic discrimination (c-statistic = 0.664), which was similar to Framingham risk scoring (c-statistics = 0.644) in a validation cohort of 42 cases and 42 controls. However, generating by a composite algorithm named Acute Coronary Syndrome Composite Predictor (ACSCP), combining the biomarker pattern ACSP75 with the previously established urinary proteomic biomarker CAD238 characterizing coronary artery disease as the underlying aetiology, and age as a risk factor, further improved discrimination (c-statistic = 0.751) resulting in an added prognostic value over Framingham risk scoring expressed by an integrated discrimination improvement of 0.273 ± 0.048 (P < 0.0001) and net reclassification improvement of 0.405 ± 0.113 (P = 0.0007). In conclusion, we demonstrate that urinary peptide biomarkers have the potential to predict future ACS events in asymptomatic patients. Further large scale studies are warranted to determine the role of urinary biomarkers in clinical practice.

Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice.

Nephropathy is among the most frequent complications of diabetes and the leading cause of end-stage renal disease. Despite the success of novel drugs in animal models, the majority of the subsequent clinical trials employing those drugs targeting diabetic nephropathy failed. This lack of translational value may in part be due to an inadequate comparability of human disease and animal models that often capture only a few aspects of disease. Here we overcome this limitation by developing a multimolecular noninvasive humanized readout of diabetic nephropathy based on urinary peptidomics. The disease-modified urinary peptides of 2 type 2 diabetic nephropathy mouse models were identified and compared with previously validated urinary peptide markers of diabetic nephropathy in humans to generate a classifier composed of 21 ortholog peptides. This classifier predicted the response to disease and treatment with inhibitors of the renin-angiotensin system in mice. The humanized classifier was significantly correlated with glomerular lesions. Using a human type 2 diabetic validation cohort of 207 patients, the classifier also distinguished between patients with and without diabetic nephropathy, and their response to renin-angiotensin system inhibition. Thus, a combination of multiple molecular features common to both human and murine disease could provide a significant change in translational drug discovery research in type 2 diabetic nephropathy.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 1. High abundance proteins, acute phase proteins and peptidomics.

A peptidomic investigation of milk from an experimental model of Streptococcus uberis mastitis in dairy cows has incorporated a study of milk high abundance and acute phase (APP) proteins as well as analysis of low molecular weight peptide biomarkers. Intramammary infection (IMI) with S. uberis caused a shift in abundance from caseins, ß-lactoglobulin and α-lactalbumin to albumin, lactoferrin and IgG with the increase in lactoferrin occurring last. The APP response of haptoglobin, mammary associated serum amyloid A3 and C-reactive protein occurred between 30-48 hours post challenge with peak concentrations of APPs at 72-96 hours post challenge and declined thereafter at a rate resembling the fall in bacterial count rather than the somatic cell count. A peptide biomarker panel for IMI based on capillary electrophoresis and mass spectrometry was developed. It comprised 77 identified peptides (IMI77) composed mainly of casein derived peptides but also including peptides of glycosylation dependent cell adhesion molecule and serum amyloid A. The panel had a biomarker classification score that increased from 36 hour to 81 hour post challenge, significantly differentiating infected from non-infected milk, thus suggesting potential as a peptide biomarker panel of bovine mastitis and specifically that of S. uberis origin. The use of omic technology has shown a multifactorial cross system reaction in high and low abundance proteins and their peptide derivatives with changes of over a thousand fold in analyte levels in response to S. uberis infection.

Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers.

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.

Comparison of higher energy collisional dissociation and collision-induced dissociation MS/MS sequencing methods for identification of naturally occurring peptides in human urine.

PURPOSE: The aim of this study is to determine the best fragmentation method for sequence identification of naturally occurring urinary peptides in the field of clinical proteomics. EXPERIMENTAL DESIGN: We used LC-MS/MS analysis of urine samples to determine the analytical performance of higher energy collisional dissociation (HCD), CID with high and low resolution MS/MS for the identification of naturally occurring peptides in the low molecular weight urinary proteome. RESULTS: HCD and CID high-resolution generated a 22% error rate in peptide sequence identifications. CID low-resolution showed significantly higher error rates (37%). Excluding the error rate (i.e rejection of cysteine-containing peptides), we observed a higher degree of overlap between HCD and CID high resolution for identification of peptide sequences of rank 1 and cross-correlation ≥ 1.9 (262 peptide sequences) compared to CID low (208 peptide sequences with HCD and 192 peptide sequences with CID high). Reproducibility of detected peptides in three out of the five replicates was also higher in HCD and CID high in relation to CID low resolution. CONCLUSION AND CLINICAL RELEVANCE: Our data demonstrated that HCD and CID high-resolution performed with better accuracy and reproducibility than CID low resolution in respect to the identification of naturally occurring urinary peptide sequences.

Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics.

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease-associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE-MS), has been increasingly used for the detection of peptide biomarkers (<20 kDa) in body fluids such as urine. This review presents the most relevant urinary proteomic studies addressing the application of CZE-MS in clinically relevant biomarker research between the years 2006 and 2014.

Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration.

Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.