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Supplementary Fig. S1. Effect by caffeine or caffeic acid on apoptotic mediators

Background: Human, animal, and cell experimental studies; human biomarker studies; and genetic studies complement epidemiologic findings and can offer insights into biological plausibility and pathways between exposure and disease, but methods for synthesizing such studies are lacking. We, therefore, developed a methodology for identifying mechanisms and carrying out systematic reviews of mechanistic studies that underpin exposure–cancer associations.Methods: A multidisciplinary team with expertise in informatics, statistics, epidemiology, systematic reviews, cancer biology, and nutrition was assembled. Five 1-day workshops were held to brainstorm ideas; in the intervening periods we carried out searches and applied our methods to a case study to test our ideas.Results: We have developed a two-stage framework, the first stage of which is designed to identify mechanisms underpinning a specific exposure–disease relationship; the second stage is a targeted systematic review of studies on a specific mechanism. As part of the methodology, we also developed an online tool for text mining for mechanism prioritization (TeMMPo) and a new graph for displaying related but heterogeneous data from epidemiologic studies (the Albatross plot).Conclusions: We have developed novel tools for identifying mechanisms and carrying out systematic reviews of mechanistic studies of exposure–disease relationships. In doing so, we have outlined how we have overcome the challenges that we faced and provided researchers with practical guides for conducting mechanistic systematic reviews.Impact: The aforementioned methodology and tools will allow potential mechanisms to be identified and the strength of the evidence underlying a particular mechanism to be assessed. Cancer Epidemiol Biomarkers Prev; 26(11); 1667–75. ©2017 AACR.

Purpose: Epidemiologic studies indicate that dietary factors, such as coffee, may influence breast cancer and modulate hormone receptor status. The purpose of this translational study was to investigate how coffee may affect breast cancer growth in relation to estrogen receptor-α (ER) status.Experimental Design: The influence of coffee consumption on patient and tumor characteristics and disease-free survival was assessed in a population-based cohort of 1,090 patients with invasive primary breast cancer in Sweden. Cellular and molecular effects by the coffee constituents caffeine and caffeic acid were evaluated in ER+ (MCF-7) and ER− (MDA-MB-231) breast cancer cells.Results: Moderate (2–4 cups/day) to high (≥5 cups/day) coffee intake was associated with smaller invasive primary tumors (Ptrend = 0.013) and lower proportion of ER+ tumors (Ptrend = 0.018), compared with patients with low consumption (≤1 cup/day). Moderate to high consumption was associated with lower risk for breast cancer events in tamoxifen-treated patients with ER+ tumors (adjusted HR, 0.51; 95% confidence interval, 0.26–0.97). Caffeine and caffeic acid suppressed the growth of ER+ (P ≤ 0.01) and ER− (P ≤ 0.03) cells. Caffeine significantly reduced ER and cyclin D1 abundance in ER+ cells. Caffeine also reduced the insulin-like growth factor-I receptor (IGFIR) and pAkt levels in both ER+ and ER− cells. Together, these effects resulted in impaired cell-cycle progression and enhanced cell death.Conclusions: The clinical and experimental findings demonstrate various anticancer properties of caffeine and caffeic acid against both ER+ and ER− breast cancer that may sensitize tumor cells to tamoxifen and reduce breast cancer growth. Clin Cancer Res; 21(8); 1877–87. ©2015 AACR.

A guide to using the methodology

Introduction Neoadjuvant cisplatin-based combination chemotherapy improves survival in muscle-invasive bladder cancer. However, response rates and survival remain suboptimal. We evaluated the efficacy, safety, and tolerability of cisplatin plus cabazitaxel. Methods A phase II single-arm trial was designed to recruit at least 26 evaluable patients. This would give 80% power to detect the primary endpoint, an objective response rate defined as a pathologic complete response plus partial response (pathologic downstaging), measured by pathologic staging at cystectomy (p0 = 0.35 and p1 = 0.60, α = 0.05). Results Objective response was seen in 15 of 26 evaluable patients (57.7%) and more than one- third of patients achieved a pathologic complete response (9/26; 34.6%). Seventy-eight percent of the patients (21/27) completed all cycles of treatment, with only 6.7% of the reported adverse events being graded 3 or 4. There were 6 treatment-related serious adverse event reported, but no suspected unexpected serious adverse reactions. In the patients who achieved an objective response, the median progression-free survival and overall survival were not reached (median follow-up of 41.5 months). In contrast, the median progression-free survival (7.2 months) and overall survival (16.9 months) were significantly worse (P = .001, log-rank) in patients who did not achieve an objective response. Conclusion Cabazitaxel plus cisplatin for neoadjuvant treatment of muscle-invasive bladder cancer can be considered a well-tolerated and effective regimen before definitive therapy with higher rates (57.7%) of objective response, comparing favorably to that with of cisplatin/gemcitabine (23%–26%). These results warrant further evaluation in a phase III study.

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Background: Circulating lipids and insulin-like growth factor 1 (IGF-I) have been reliably associated with breast cancer. Observational studies suggest an interplay between lipids and IGF-I, however, whether these relationships are causal and if pathways from these phenotypes to breast cancer overlap is unclear. Methods: Mendelian randomization (MR) was conducted to estimate the relationship between lipids or IGF-I and breast cancer risk using genetic summary statistics for lipids (low-density lipoprotein cholesterol, LDL-C; high-density lipoprotein cholesterol, HDL-C; triglycerides, TGs), IGF-I and breast cancer from GLGC/UKBB (N = 239,119), CHARGE/UKBB (N = 252,547), and Breast Cancer Association Consortium (N = 247,173), respectively. Cross-sectional observational and MR analyses were conducted to assess the bi-directional relationship between lipids and IGF-I in SHIP (N = 3,812) and UKBB (N = 422,389), and using genetic summary statistics from GLGC (N = 188,577) and CHARGE/UKBB (N = 469,872). Results: In multivariable MR (MVMR) analyses, the OR for breast cancer per 1-SD increase in HDL-C and TG was 1.08 [95% confidence interval (CI), 1.04–1.13] and 0.94 (95% CI, 0.89–0.98), respectively. The OR for breast cancer per 1-SD increase in IGF-I was 1.09 (95% CI, 1.04–1.15). MR analyses suggested a bi-directional TG–IGF-I relationship (TG–IGF-I β per 1-SD: −0.13; 95% CI, −0.23 to −0.04; and IGF-I–TG β per 1-SD: −0.11; 95% CI, −0.18 to −0.05). There was little evidence for a causal relationship between HDL-C and LDL-C with IGF-I. In MVMR analyses, associations of TG or IGF-I with breast cancer were robust to adjustment for IGF-I or TG, respectively. Conclusions: Our findings suggest a causal role of HDL-C, TG, and IGF-I in breast cancer. Observational and MR analyses support an interplay between IGF-I and TG; however, MVMR estimates suggest that TG and IGF-I may act independently to influence breast cancer. Impact: Our findings should be considered in the development of prevention strategies for breast cancer, where interventions are known to modify circulating lipids and IGF-I.

There is considerable evidence of a positive association between the incidence of type 2 diabetes mellitus (T2DM) and obesity with bladder cancer (BCa), with the link between T2DM and obesity having already been established. There also appear to be potential associations between Pleckstrin homology domain containing S1 (PLEKHS1) and the Insulin-like Growth Factor (IGF) axis. Seven literature searches were carried out to investigate the backgrounds of these potential links. PLEKHS1 is a candidate biomarker in BCa, with mutations that are easily detectable in urine and increased expression seemingly associated with worse disease states. PLEKHS1 has also been implicated as a potential mediator for the onset of T2DM in people with obesity. The substantial evidence of the involvement of IGF in BCa, the role of the IGF axis in obesity and T2DM, and the global prevalence of T2DM and obesity suggest there is scope for investigating the links between these components. Preliminary findings on the relationship between PLEKHS1 and the IGF axis signal possible associations with BCa progression. This indicates that PLEKHS1 plays a role in the pathogenesis of BCa that may be mediated by members of the IGF axis. Further detailed research is needed to establish the relationship between PLEKHS1 and the IGF axis in BCa and determine how these phenomena overlap with T2DM and obesity.

Glioma stem-like cells (GSCs) were first described as a population which may in part be resistant to traditional chemotherapeutic therapies and responsible for tumour regrowth. Knowledge of the underlying metabolic complexity governing GSC growth and function may point to potential differences between GSCs and the tumour bulk which could be harnessed clinically. There is an increasing interest in the direct/indirect targeting or reprogramming of GSC metabolism as a potential novel therapeutic approach in the adjuvant or recurrent setting to help overcome resistance which may be mediated by GSCs. In this review we will discuss stem-like models, interaction between metabolism and GSCs, and potential current and future strategies for overcoming GSC resistance.

There are mixed reports on the role that IGFBP-2 plays in cancer progression, with some indicating a tumour suppressive role and others showing that IGFBP-2 may act as an oncogene. These apparent contradictions may be context and tissue specific. In this study we determined the role that IGFBP-2 played on the phenotype and chemosensitivity of a selection of bladder cancer cell lines and investigated how the abundance of IGFBP-2 was regulated. We found that IGFBP-2 was more abundant in the epithelial bladder cancer cells, RT4 and UMUC3 and absent in the more mesenchymal T24 and TCCSUP cells. Silencing IGFBP-2 using siRNA in epithelial RT4 cells promoted cell proliferation, invasion, colony formation, resulted in a reduction in epithelial (E-cadherin) and an increase in mesenchymal (N-cadherin) markers and increased sensitivity to cisplatin-induced cell death. Conversely, we observed the opposite effects when adding exogenous IGFBP-2 to the mesenchymal T24 cells. We determined that IGFBP-2 was epigenetically silenced via DNA methylation as the cells adopted a mesenchymal phenotype. Collectively these data suggest that IGFBP-2 acts as a tumour suppressor and marker of chemosensitivity in epithelial bladder cancer cells and that IGFBP-2 is epigenetically silenced by methylation to promote bladder cancer progression.

Background Capecitabine and oxaliplatin (CAPOX) chemotherapy is a standard treatment for stage 2/3 colorectal cancer. Treatment is associated with dose-limiting toxicities such as neutropenia, vomiting, diarrhoea, and stomatitis. Short-term fasting prior to chemotherapy may help protect normal cells from the toxic effects of chemotherapy by allowing them to conserve energy for maintenance and repair. However, there is a lack of evidence to support the efficacy of short-term fasting in protecting against chemotherapy-related toxicities in humans, and it is not known whether people due to undergo chemotherapy will be willing and able to follow a short-term fast. Preliminary data confirming this is feasible are required before adequately powered trials can be designed and conducted. Methods The short-term, water only, fasting trial (SWiFT) is a two-armed feasibility randomised controlled trial, aiming to recruit 30 people scheduled to begin routine treatment with CAPOX chemotherapy for stage 2/3 colorectal cancer. Participants will be randomly allocated, in a 1:1 ratio, to either a 36-h fast or standard dietary advice prior to chemotherapy administration for the first 3 cycles of chemotherapy. The primary outcome measures will assess the feasibility of the trial and include: adherence to intervention, recruitment, retention, and data completion rates as well as the acceptability of the intervention which will be qualitatively assessed. The secondary outcome measures aim to provide further information on possible outcomes of interest for a definitive trial and include side effects of chemotherapy, quality of life, markers of cellular metabolism and inflammation, appetite, and sarcopenia. Discussion It is not known whether it is possible to recruit to a trial of short-term fasting in this population, or whether participants would be able to adhere to the intervention. Therefore, we aim to test the feasibility of a pre-chemotherapy, 36-h, water-only fast in people receiving CAPOX chemotherapy for stage 2/3 colorectal cancer. Trial registration This trial has been registered with the ISRCTN Registry. Trial registration no: ISRCTN17994717. Date of registration: 23 October 2018. URL: http://www.isrctn.com/ISRCTN17994717

Background Diets that restrict energy or macronutrient intake (e.g. fasting/ketogenic diets (KDs)) may selectively protect non-tumour cells during cancer treatment. Previous reviews have focused on a subset of dietary restrictions (DR) or have not performed systematic searches. We conducted a systematic scoping review of DR at the time of cancer treatment. Methods MEDLINE, Embase, CINAHL, AMED and Web of Science databases were searched for studies of adults undergoing DR alongside treatment for cancer. Search results were screened against inclusion/exclusion criteria. Data from included studies were extracted by two independent reviewers. Results were summarised narratively. Results Twenty-three independent studies (34 articles), with small sample sizes, met the inclusion criteria. Four categories were identified: KDs (10 studies), fasting (4 studies), protein restriction (5 studies) and combined interventions (4 studies). Diets were tolerated well, however adherence was variable, particularly for KDs. Biomarker analysis in KDs and fasting resulted in the expected increase in ketones or reduction in insulin-like growth factors, respectively, however they did not reduce glucose. Conclusions Future research with adequately powered studies is required to test the effects of each DR intervention on treatment toxicities and outcomes. Further research into improving adherence to DR may improve the feasibility of larger trials. Electronic supplementary material The online version of this article (10.1186/s12885-019-5931-7) contains supplementary material, which is available to authorized users.

Prostate cancer is the second major cause of male cancer deaths. Obesity, type 2 diabetes, and cancer risk are linked. Insulin-like growth factor II (IGF-II) is involved in numerous cellular events, including proliferation and survival. The IGF-II gene shares its locus with the lncRNA, H19. IGF-II/H19 was the first gene to be identified as being “imprinted”—where the paternal copy is not transcribed—a silencing phenomenon lost in many cancer types. We disrupted imprinting behaviour in vitro by altering metabolic conditions and quantified it using RFLP, qPCR and pyrosequencing, changes to peptide were measured using RIA. Prostate tissue samples were analysed using ddPCR, pyrosequencing and IHC. We compared with in silico data, provided by TGCA on the cBIO Portal. We observed disruption of imprinting behaviour, in vitro, with a significant increase in IGF-II and a reciprocal decrease in H19 mRNA, the increased mRNA was not translated into peptides. In vivo, most specimens retained imprinting status apart from a small subset which showed reduced imprinting. A positive correlation was seen between IGF-II and H19 mRNA expression, which concurred with findings of larger Cancer Genome Atlas (TGCA) cohorts. This positive correlation did not affect IGF-II peptide. Our findings show that type 2 diabetes and/or obesity, can directly affect regulation growth factors involved in carcinogenesis, indirectly suggesting a modification of lifestyle habits may reduce cancer risk.

Aims/hypothesis Podocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes. Methods IGFBP expression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging. Results Data from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerular IGFBP-1 in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)–forkhead box O1 (FoxO1) activity. In vitro, IGFBP-1 signalled to podocytes via β1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability. Conclusions/interpretation This work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression of IGFBP-1 is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD. Graphical abstract

Background: Prostate cancer is the most frequently diagnosed cancer type and the second major cause of cancer deaths amongst men. A link exists between obesity, type 2 diabetes, and cancer risk. Insulin-like growth factor II (IGF-II) plays a role in numerous cellular events, including proliferation and survival. The IGF-II gene shares its locus with the lncRNA, H19. IGF-II/H19 was also the first gene to be identified as being ‘imprinted’ – where the paternal copy is not transcribed. This silencing phenomenon is lost in many cancer types. Methods: We disrupted imprinting behaviour in vitro through the alteration of metabolic conditions and quantified it using RFLP, qPCR and pyrosequencing; changes to peptide were measured using RIA. Prostate tissue samples were analysed using ddPCR, pyrosequencing and IHC. We then compared with in silico data, provided by TGCA on the cBIO Portal.Results: Disruption of imprinting behaviour, in vitro, occurs at the molecular level with no changes to peptide. In vivo, most specimens primarily retained imprinting status, apart from a small subset which showed reduced imprinting. A positive correlation was seen between IGF-II and H19 mRNA expression, which concurred with findings of larger Cancer Genome Atlas (TGCA) cohorts. This positive correlation did not affect IGF-II peptide. Conclusions: Type 2 diabetes and / or obesity directly affect regulation growth factors involved in carcinogenesis.Trial registration: Prostate Cancer Evidence of Exercise and Nutrition Trial: nutritional and physical activity interventions for men with localised prostate cancer – feasibility study (ISRCTN99048944). Registered on 17 November 2014

Insulin-like growth factor binding protein 3 (IGFBP-3) plays a key role in breast cancer progression and was recently shown to bind to the chaperone protein glucose-regulated protein 78 (GRP78), however, the clinical significance of this association remains poorly investigated. Here we report a direct correlation between the expression of GRP78 and IGFBP-3 in breast cancer cell lines and tumour sections. Kaplan&ndash, Meier survival plots revealed that patients with low GRP78 expression that are positive for IGFBP-3 had poorer survival rates than those with low IGFBP-3 levels, and we observed a similar trend in the publicly available METABRIC gene expression database. With breast cancer cells, in vitro IGFBP-3 enhanced induced apoptosis, however when GRP78 expression was silenced the actions of IGFBP-3 were switched from increasing to inhibiting ceramide (C2)-induced cell death and promoted cell invasion. Using immunofluorescence and cell surface biotinylation, we showed that knock-down of GRP78 negated the entry of IGFBP-3 into the cells. Together, our clinical and experimental results suggest that loss of GRP78 reduces IGFBP-3 entry into cells switching its actions to promote tumorigenesis and predicts a poor prognosis in breast cancer patients.

Hyperglycaemia is a common metabolic alteration associated with breast cancer risk and progression. We have previously reported that BRCA1 restrains metabolic activity and proliferative response to IGF-I anabolic actions in breast cancer cells cultured in high glucose. Here, we evaluated the impact of normal physiological glucose on these tumour suppressive roles of BRCA1. Human breast cancer cells cultured in normal physiological and high glucose were treated with IGF-I (0&ndash, 500 ng/mL). Cellular responses were evaluated using immunoblotting, co-immunoprecipitation, and cell viability assay. As we previously reported, IGF-I induced ACCA dephosphorylation by reducing the association between BRCA1 and phosphorylated ACCA in high glucose, and upregulated FASN abundance downstream of ACCA. However, these effects were not observed in normal glucose. Normal physiological glucose conditions completely blocked IGF-I-induced ACCA dephosphorylation and FASN upregulation. Co-immunoprecipitation studies showed that normal physiological glucose blocked ACCA dephosphorylation by increasing the association between BRCA1 and phosphorylated ACCA. Compared to high glucose, the proliferative response of breast cancer cells to IGF-I was reduced in normal glucose, whereas no difference was observed in normal mammary epithelial cells. Considering these results collectively, we conclude that normal physiological glucose promotes the novel function of BRCA1 as a metabolic restraint of IGF-I actions. These data suggest that maintaining normal glucose levels may improve BRCA1 function in breast cancer and slow down cancer progression.

The pandemic of COVID-19, caused by the coronavirus, SARS-CoV-2, has had a global impact not seen for an infectious disease for over a century. This acute pandemic has spread from the East and has been overlaid onto a slow pandemic of metabolic diseases of obesity and diabetes consequent from the increasing adoption of a Western-lifestyle characterized by excess calorie consumption with limited physical activity. It has become clear that these conditions predispose individuals to a more severe COVID-19 with increased morbidity and mortality. There are many features of diabetes and obesity that may accentuate the clinical response to SARS-CoV-2 infection: including an impaired immune response, an atherothrombotic state, accumulation of advanced glycation end products and a chronic inflammatory state. These could prime an exaggerated cytokine response to viral infection, predisposing to the cytokine storm that triggers progression to septic shock, acute respiratory distress syndrome, and multi-organ failure. Infection leads to an inflammatory response and tissue damage resulting in increased metabolic activity and an associated increase in the mechanisms by which cells ingest and degrade tissue debris and foreign materials. It is becoming clear that viruses have acquired an ability to exploit these mechanisms to invade cells and facilitate their own life-cycle. In obesity and diabetes these mechanisms are chronically activated due to the deteriorating metabolic state and this may provide an increased opportunity for a more profound and sustained viral infection.

Localized prostate cancer (PCa) is a manageable disease but for most men with metastatic disease, it is often fatal. A western diet has been linked with PCa progression and hyperglycaemia has been associated with the risk of lethal and fatal prostate cancer.Using PCa cell lines, we examined the impact of IGF-I and glucose on markers of epithelial-to-mesenchymal transition (EMT), migration and invasion. We examined the underlying mechanisms using cell lines and tumour tissue samples.IGF-I had differential effects on the process of EMT: inhibiting in normal and promoting in cancer cells, whereas hyperglycamia alone had a stimulatory effect in both. These effects were independent of IGF and in both cases, hyperglycaemia induced an increase IGFBP-2(tumour promoter) and FOXA1. A positive correlation existed between levels of IGFBP-2 and FOXA1 in benign and cancerous prostate tissue samples and in vitro and in vivo data indicated that FOXA1 strongly interacted with the IGFBP2 gene in normal prostate epithelial cells that was associated with a negative regulation of IGFBP-2, whereas in cancer cells the level of FOXA1 associating with the IGFBP2 gene was minimal, suggesting loss of this negative regulation. IGF-I and hyperglycaemia-induced FOXA1/IGFBP-2 play important roles in EMT.

Circulating lipids have been associated with breast cancer (BCa). This association may, in part, be due to an effect of lipids on insulin-like growth factors (IGFs), which have been reliably associated with BCa. In two-sample Mendelian randomization (MR) analyses, we found that low density lipoprotein (LDL-C) was associated with IGFBP-3 (beta:0.08 SD; 95%CI:0.02,0.15; p = 0.01, per SD increase in LDL-C) and IGFBP-3 was associated with postmenopausal BCa (OR:1.09; 95%CI:1.00,1.19; p = 0.05, per SD increase in IGFBP-3). We also found that triglycerides were associated with IGF-I (beta:-0.13SD; 95%CI:-0.22,-0.03, per SD increase in triglycerides) and that IGF-I was associated with overall BCa (OR:1.10;95%CI:1.02,1.18, per SD increase in IGF-I). Taken together, these results suggest that IGFBP-3 may be a potential causal step between LDL-C and postmenopausal BCa and IGF-I a potential causal for triglycerides. Our two-step MR results build on evidence linking circulating lipids and IGFs with BCa, however, multivariable MR analyses are currently unable to support this relationship due to weak instruments.

The cytosolic branched chain aminotransferase (BCATc) protein has been found to be highly expressed in breast cancer subtypes, including triple negative breast cancer (TNBC), compared with normal breast tissue. The catabolism of branched-chain amino acids (BCAAs) by BCATc leads to the production of glutamate and key metabolites which further drive the TCA cycle, important for cellular metabolism and growth. Upregulation of BCATc has been associated with increased cell proliferation, cell cycle progression and metastasis in several malignancies including breast, gliomas, ovarian and colorectal cancer but the underlying mechanisms are unclear. As nutrient levels of BCAAs, substrates of BCATc, regulate the PI3K/Akt pathway we hypothesized that increased expression of BCATc would contribute to tumour cell growth through upregulation of the insulin/IGF-1 signalling pathway. This pathway is known to potentiate proliferation and metastasis of malignant cells through the activation of PI3K/Akt and the RAS/ERK signalling cascades. Here we show that knockdown of BCATc significantly reduced insulin and IGF-1-mediated proliferation, migration and invasion of TNBC cells. An analysis of this pathway showed that when overexpressed BCATc regulates proliferation through the PI3K/Akt axis, whilst simultaneously attenuating the Ras/Erk pathway indicating that BCATc acts as a conduit between these two pathways. This ultimately led to an increase in FOXO3a, a key regulator of cell proliferation and Nrf2, which mediates redox homeostasis. Together this data indicates that BCATc regulates TNBC cell proliferation, migration and invasion through the IGF-1/insulin PI3K/Akt pathway, culminating in the upregulation of FOXO3a and Nrf2, pointing to a novel therapeutic target for breast cancer treatment.

Introduction: Preclinical, clinical, and population studies have provided robust evidence for an important role for the insulin-like growth factor (IGF) system in the development of prostate cancer.Areas covered: An overview of the IGF system is provided. The evidence implicating the IGF system in the development of prostate cancer is summarized. The compelling evidence culminated in a number of clinical trials of agents targeting the system; the reasons for the failure of these trials are discussed.Expert opinion: Clinical trials of agents targeting the IGF system in prostate cancer were terminated due to limited objective clinical responses and are unlikely to be resumed unless a convincing predictive biomarker is identified that would enable the selection of likely responders. The aging population and increased screening will lead to greater diagnosis of prostate cancer. Although the vast majority will be indolent disease, the epidemics of obesity and diabetes will increase the proportion that progress to clinical disease. The increased population of worried men will result in more trials aimed to reduce the risk of disease progression; actual clinical endpoints will be challenging and the IGFs remain the best intermediate biomarkers to indicate a response that could alter the course of disease.

Epithelial to mesenchymal transition (EMT) is a necessary process in the conversion of benign tumor to aggressive and highly invasive cancer. Dysregulation of the IGF system and impaired metabolic regulation have been implicated in the progression of prostate cancer. However, the mechanisms underlying these effects require further investigation. We used normal prostate epithelial cells PNT2 and DU145 prostate cancer cells. Western immunoblotting was used to determine changes in protein abundance. Trypan blue dye exclusion assay was employed to assess cell proliferation and transwell migration assays to assess cells migration. Under normal glucose conditions, IGF-I inhibited EMT in PNT2 cells demonstrated by an upregulation in the epithelial marker E-cadherin together with loss of mesenchymal markers; vimentin and fibronectin. In contrast to PNT2 cells, IGF-I induced EMT in DU145 cells, as shown by the reduction of E-cadherin level and upregulation of vimentin and fibronectin. We observed that exposure to hyperglycaemia (25mM glucose concentration) alone induced EMT in both PNT2 and DU145 cells. The changes in EMT markers induced by hyperglycaemia (loss of epithelial marker and increase of mesenchymal markers) associated with increased cell proliferation and migration. In high glucose conditions, IGF-I was still able to inhibit EMT in PNT2 cells, whereas in DU145 cancer cells, the addition of IGF-I could not enhance EMT any further. In conclusion, IGF-I and hyperglycaemia play important roles in promoting prostate cancer cell progression through the regulation of EMT programme.

When originally discovered, one of the initial observations was that, when all of the insulin peptide was depleted from serum, the vast majority of the insulin activity remained and this was due to a single additional peptide, IGF-II. The IGF-II gene is adjacent to the insulin gene, which is a result of gene duplication, but has evolved to be considerably more complicated. It was one of the first genes recognised to be imprinted and expressed in a parent-of-origin specific manner. The gene codes for IGF-II mRNA, but, in addition, also codes for antisense RNA, long non-coding RNA, and several micro RNA. Recent evidence suggests that each of these have important independent roles in metabolic regulation. It has also become clear that an alternatively spliced form of the insulin receptor may be the principle IGF-II receptor. These recent discoveries have important implications for metabolic disorders and also for cancer, for which there is renewed acknowledgement of the importance of metabolic reprogramming.

There has been a resurgence of interest in cancer metabolism; primarily in the resetting of metabolism within malignant cells. Metabolism within cells has always been a tightly regulated process; initially in protozoans due to metabolic enzymes, and the intracellular signalling pathways that regulate these, being directly sensitive to the availability of nutrients. With the evolution of metazoans many of these controls had been overlaid by extra-cellular regulators that ensured coordinated regulation of metabolism within the community of cells that comprised the organism. Central to these systemic regulators is the insulin/insulin-like growth factor (IGF) system that throughout evolution has integrated the control of tissue growth with metabolic status. Oncological interest in the main systemic metabolic regulators greatly subsided when pharmaceutical strategies designed to treat cancers failed in the clinic. During the same period, however the explosion of new information from genetics has revealed the complexity and heterogeneity of advanced cancers and helped explain the problems of managing cancer when it reaches such a stage. Evidence has also accumulated implying that the setting of the internal environment determines whether cancers progress to advanced disease and metabolic status is clearly an important component of this local ecology. We are in the midst of an epidemic of metabolic disorders and there is considerable research into strategies for controlling metabolism. Integrating these new streams of information suggests new possibilities for cancer prevention; both primary and secondary.

Recent evidence from epidemiology indicates that inter-individual variations in the growth hormone (GH)/IGF-I pathway affect the risk of individuals developing common epithelial cancers. This is supported by associations between normal common variants within genes from the pathway and these cancers, which excludes many potential confounding issues, such as reverse causality. This raises concern for the increasing numbers of patients treated with GH; although replacement therapy for GH-deficiency should aim to restore normality, which should then only incur a normal risk. The links with cancer also offers promising new opportunities. Clinical trials treating cancer patients with pharmaceuticals targeting the IGF-I receptor are well advanced with promising initial findings. In the future, there has to be much more emphasis within oncology on prevention and the GH/IGF-I pathway is one of few identified risk factors that are modifiable, not just by pharmaceutical, but also nutritional, interventions that may, in the long term, be more appropriate. Assessing the status of the GH/IGF-I pathway in individuals may also provide a means for targeting screening programs and preventative measures.

There has been a resurgence of interest in cancer metabolism; primarily in the resetting of metabolism within malignant cells. Metabolism within cells has always been a tightly regulated process; initially in protozoans due to metabolic enzymes, and the intracellular signaling pathways that regulate these, being directly sensitive to the availability of nutrients. With the evolution of metazoans many of these controls had been overlaid by extra-cellular regulators that ensured coordinated regulation of metabolism within the community of cells that comprised the organism. Central to these systemic regulators is the insulin/insulin-like growth factor (IGF) system that throughout evolution has integrated the control of tissue growth with metabolic status. Oncological interest in the main systemic metabolic regulators greatly subsided when pharmaceutical strategies designed to treat cancers failed in the clinic. During the same period, however the explosion of new information from genetics has revealed the complexity and heterogeneity of advanced cancers and helped explain the problems of managing cancer when it reaches such a stage. Evidence has also accumulated implying that the setting of the internal environment determines whether cancers progress to advanced disease and metabolic status is clearly an important component of this local ecology. We are in the midst of an epidemic of metabolic disorders and there is considerable research into strategies for controlling metabolism. Integrating these new streams of information suggests new possibilities for cancer prevention; both primary and secondary.

Background:Circulating insulin-like growth factor binding protein 3 (IGFBP-3) has been associated with prostate cancer. Preclinical studies found that vitamin D regulates IGFBP-3 expression, although evidence from epidemiologic studies is conflicting.Methods:Mendelian randomization analyses (MR) were conducted to reassess associations between IGFBP-3 and prostate cancer risk and advanced prostate cancer using summary statistics from the PRACTICAL consortium (44,825 cases; 27,904 controls). Observational and MR analyses were conducted to assess the relationship between inactive vitamin D [25(OH)D] and IGFBP-3 using data from the ProtecT study (1,366 cases;1,071 controls) and summary statistics from the CHARGE consortium (n = 18,995).Results:The OR for prostate cancer per SD unit increase in circulating IGFBP-3 was 1.14 [95% confidence interval (CI), 1.02–1.28]. The OR for advanced prostate cancer per SD unit increase in IGFBP-3 was 1.22 (95% CI, 1.07–1.40). Observationally, a SD increase in 25(OH)D was associated with a 0.1SD (95% CI, 0.05–0.14) increase in IGFBP-3. MR analyses found little evidence for a causal relationship between circulating 25(OH)D and IGFBP-3 in the circulation.Conclusions:This study provided confirmatory evidence that IGFBP-3 is a risk factor for prostate cancer risk and progression. Observationally, there was evidence that 25(OH)D is associated with IGFBP-3, but MR analyses suggested that these findings were unlikely to be causal. Findings may be limited by the nature of instrumentation of 25(OH)D and IGFBP-3 and the utility of circulating measures. 25(OH)D appears unlikely to be causally related to IGFBP-3 in the circulation, however, our findings do not preclude causal associations at the tissue level.Impact:IGFBP-3 is a prostate cancer risk factor but 25(OH)D are unlikely to be causally related to IGFBP-3 in the circulation.

The IGF system has an important role in growth and development. IGF-II is a recognized fetal growth promoter. However, its physiological postnatal role remains uncertain, although it is maintained in the circulation at a substantially high level throughout life. IGF-II has been strongly linked to obesity in genetic studies, and more recent evidence suggests a metabolic role. We examined fat depot differences in IGF-II’s action on differentiation and metabolism. We speculate a specific effect on visceral adipocytes in relation to the differential distribution of insulin receptors between visceral and subcutaneous fat depots. We used a previously established adipocyte, cell culture system of matched pairs of visceral and subcutaneous fat biopsies from 20 normal weight children undergoing routine surgery for nonmalignant, nonseptic conditions. Preadipocytes were differentiated for 14 days in the presence or absence of IGF-II. Oil Red O staining, Western blotting, and reverse transcription polymerase chain reaction techniques were employed to assess levels of adipogenesis markers and levels of the insulin receptor and insulin receptor isoforms. Our data indicate that IGF-II promotes preadipocyte differentiation in subcutaneous preadipocytes but showed a protective, opposing effect restricting visceral preadipocyte differentiation, confirmed by reductions in the differentiation markers peroxisome proliferator-activated receptor gamma and adiponectin and in triglyceride staining. Additionally, IGF-II reduced mRNA expression of the insulin receptor in adipocytes and downregulated insulin receptor isoform A and glucose transporter 4 abundance and corresponding glucose uptake in visceral adipocytes. In conclusion, IGF-II is a regulator of preadipocyte differentiation and metabolism by acting as a differential modulator of fat accumulation favoring less visceral fat deposition in children.

IGFBP-2 is an important modulator of IGF availability and activity. It is the second most abundant of the IGFBPs in the circulation and its levels are increased in a variety of tumours and associated with progression and poor prognosis. PTEN is a phosphatase that returns the PI3K/AKT/mTOR pathway to its inactivated state and is therefore a critical modulator of one of the main intracellular signalling pathways activated by the IGFs. Recent evidence has indicated that IGFBP-2 regulates PTEN in a variety of normal and malignant cell types. This review summarises the recent evidence that these extracellular and intracellular modulators are linked to provide a synchronous system for cell regulation with coordinated control of both the ‘accelerator’ and the ‘brake’.

Purpose: Epidemiologic studies indicate that dietary factors, such as coffee, may influence breast cancer and modulate hormone receptor status. The purpose of this translational study was to investigate how coffee may affect breast cancer growth in relation to estrogen receptor-α (ER) status. Experimental Design: The influence of coffee consumption on patient and tumor characteristics and disease-free survival was assessed in a population-based cohort of 1,090 patients with invasive primary breast cancer in Sweden. Cellular and molecular effects by the coffee constituents caffeine and caffeic acid were evaluated in ER+ (MCF-7) and ER− (MDA-MB-231) breast cancer cells. Results: Moderate (2–4 cups/day) to high (≥5 cups/day) coffee intake was associated with smaller invasive primary tumors (Ptrend = 0.013) and lower proportion of ER+ tumors (Ptrend = 0.018), compared with patients with low consumption (≤1 cup/day). Moderate to high consumption was associated with lower risk for breast cancer events in tamoxifen-treated patients with ER+ tumors (adjusted HR, 0.51; 95% confidence interval, 0.26–0.97). Caffeine and caffeic acid suppressed the growth of ER+ (P ≤ 0.01) and ER− (P ≤ 0.03) cells. Caffeine significantly reduced ER and cyclin D1 abundance in ER+ cells. Caffeine also reduced the insulin-like growth factor-I receptor (IGFIR) and pAkt levels in both ER+ and ER− cells. Together, these effects resulted in impaired cell-cycle progression and enhanced cell death. Conclusions: The clinical and experimental findings demonstrate various anticancer properties of caffeine and caffeic acid against both ER+ and ER− breast cancer that may sensitize tumor cells to tamoxifen and reduce breast cancer growth. Clin Cancer Res; 21(8); 1877–87. ©2015 AACR.

Evidence indicates that for most human cancers the problem is not that gene mutations occur but is more dependent upon how the body deals with damaged cells. It has been estimated that only about 1% of human cancers can be accounted for by unmistakable hereditary cancer syndromes, only up to 5% can be accounted for due to high-penetrance, single-gene mutations, and in total only 5%-15% of all cancers may have a major genetic component. The predominant contribution to the causation of most sporadic cancers is considered to be environmental factors contributing between 58% and 82% toward different cancers. A nutritionally poor lifestyle is associated with increased risk of many cancers, including those of the breast. As nutrition, energy balance, macronutrient composition of the diet, and physical activity levels are major determinants of insulin-like growth factor (IGF-I) bioactivity, it has been proposed that, at least in part, these increases in cancer risk and progression may be mediated by alterations in the IGF axis, related to nutritional lifestyle. Localized breast cancer is a manageable disease, and death from breast cancer predominantly occurs due to the development of metastatic disease as treatment becomes more complicated with poorer outcomes. In recent years, epithelial-to-mesenchymal transition has emerged as an important contributor to breast cancer progression and malignant transformation resulting in tumor cells with increased potential for migration and invasion. Furthermore, accumulating evidence suggests a strong link between components of the IGF pathway, epithelial-to-mesenchymal transition, and breast cancer mortality. Here, we highlight some recent studies highlighting the relationship between IGFs, IGF-binding protein 3, and epithelial-to-mesenchymal transition.

Background: epidemiologic evidence shows that obesity is associated with a greater risk of aggressive prostate cancer and prostate cancer-specific mortality and this is observed mainly in men with the TMPRSS2-ERG gene fusion. Obesity is often associated with co-morbid conditions such as type 2 diabetes and hyperglycaemia: we investigated whether some of the exposures associated with disturbed metabolism can also affect the frequency of this gene fusion.Methods: fusion was induced in LNCaP prostate cancer cells in normal or high levels of glucose, with or without insulin-like growth factor binding protein-2 (IGFBP-2) silenced or the presence of IGF-I, insulin or epidermal growth factor (EGF). RNA was extracted for analysis by nested PCR. Abundance of IGFBP-2, H2AX, DNA-dependent protein kinase catalytic subunit (DNAPKcs) and β-actin were analysed by Western immunoblotting.Results: our data suggests that hyperglycaemia-induced IGFBP-2 increased the frequency of the gene fusion that was accompanied by decreased levels of DNAPKcs implying that they were mediated by alterations in the rate of repair of double strand breaks. In contrast insulin, IGF-I and EGF all decreased gene fusion events. Conclusions: these novel observations may represent a further mechanism by which obesity can exert an effect aggravating prostate cancer progression.

Background: Human, animal, and cell experimental studies; human biomarker studies; and genetic studies complement epidemiologic findings and can offer insights into biological plausibility and pathways between exposure and disease, but methods for synthesizing such studies are lacking. We, therefore, developed a methodology for identifying mechanisms and carrying out systematic reviews of mechanistic studies that underpin exposure–cancer associations. Methods: A multidisciplinary team with expertise in informatics, statistics, epidemiology, systematic reviews, cancer biology, and nutrition was assembled. Five 1-day workshops were held to brainstorm ideas; in the intervening periods we carried out searches and applied our methods to a case study to test our ideas. Results: We have developed a two-stage framework, the first stage of which is designed to identify mechanisms underpinning a specific exposure–disease relationship; the second stage is a targeted systematic review of studies on a specific mechanism. As part of the methodology, we also developed an online tool for text mining for mechanism prioritization (TeMMPo) and a new graph for displaying related but heterogeneous data from epidemiologic studies (the Albatross plot). Conclusions: We have developed novel tools for identifying mechanisms and carrying out systematic reviews of mechanistic studies of exposure–disease relationships. In doing so, we have outlined how we have overcome the challenges that we faced and provided researchers with practical guides for conducting mechanistic systematic reviews. Impact: The aforementioned methodology and tools will allow potential mechanisms to be identified and the strength of the evidence underlying a particular mechanism to be assessed. Cancer Epidemiol Biomarkers Prev; 26(11); 1667–75. ©2017 AACR.

// Athba Al Qahtani 1 , Jeff Holly 1 and Claire Perks 1 1 IGFs and Metabolic Endocrinology Group, School of Clinical Sciences, University of Bristol, Learning and Research Building, Southmead Hospital, Bristol BS10 1TD, UK Correspondence to: Claire Perks, email: claire.m.perks@bristol.ac.uk Keywords: hyperglycaemia, hypoxia, estrogen receptor, insulin-like growth factor binding protein 2, chemo-resistance Abbreviations: CoCl 2 , cobalt chloride; ERα, estrogen receptor α; FASN, fatty acid synthase; HIF-I, hypoxia-inducible factor-I; IGFBP-2, insulin-like growth factor binding protein-2 Received: May 05, 2017 Accepted: July 25, 2017 Published: August 16, 2017 ABSTRACT Background: Women who suffer from breast cancer and type II diabetes with associated hyperglycaemia respond less well to chemotherapy. We have shown that hyperglycaemia induces resistance to chemotherapy through upregulation of fatty acid synthase (FASN) in breast cancer cells and increased insulin-like binding protein 2 (IGFBP-2) in prostate cancer cells. As a tumour develops the tumour mass can outgrow the blood supply resulting in the cancer cells being exposed to hypoxia that stimulates many tumorigenic signalling pathways. Methods: We used MCF-7 and T47D breast cancer cell lines. Trypan blue dye exclusion assay was employed to assess cell death and Western immunoblotting was used to determine changes in protein abundance. Hypoxia was induced both chemically by the addition of cobalt chloride (CoCl 2 ) and using a hypoxia chamber. Results: IGFBP-2 abundance increased with increasing concentrations of glucose (0-25 mM) that contributed to hyperglycaemia-induced chemo-resistance as it was abrogated by downregulating IGFBP-2 using siRNA. Production of IGFBP-2 is ER dependent: pre-treatment of MCF-7 cells with β-estradiol increased IGFBP-2 and induced chemo-resistance to doxorubicin. The hyperglycaemia-induced chemo-resistance and increases in FASN and IGFBP-2 were negated in a hypoxic environment, with levels of cell death unaffected by glucose concentrations. Conclusions: The sensitivity of breast cancer cells to chemotherapy is reduced in hyperglycaemic conditions but this effect is negated by hypoxia. These effects appear to be mediated via regulation of IGFBP-2 and FASN. Understanding the role of FASN and IGFBP-2 in chemo-resistance could provide a novel target for improving the effectiveness of breast cancer treatment.

Objectives: To investigate the association between PTEN loss and IGFBP2 expression in a series of triple-negative breast cancers and to relate this expression to basal cytokeratin expression and clinicopathologic features. Methods: One hundred and one formalin-fixed and paraffin-processed triple-negative breast cancer cases from the University of Malaya Medical Centre were tested immunohistochemically for cytokeratins 5/6 and 14, PTEN, and IGFBP2. The resulting slides were scored for proportion and intensity of staining. Results: Loss of tumor nuclear and cytoplasmic staining for PTEN occurred in 48.3% of cases and was significantly associated with younger age at diagnosis (47 years compared with 57 years in those without PTEN loss; P = .005). Independent predictors of PTEN loss were late stage at presentation (P = .026), cytokeratin 5/6 positivity (P = .028), and IGFBP2 expression (P = .042). High levels of IGFBP2 expression were seen in 32% of cases; an independent predictor of high levels was cytokeratin 14 negativity (P = .005). PTEN loss and high levels of IGFBP2 expression were associated with poorer survival, but neither of these trends was significant. Conclusions: PTEN loss is a frequent event in triple-negative breast cancers and is significantly associated with younger age at onset of breast cancer, late stage, and IGFBP2 expression.

The age-related epithelial cancers of the breast, colorectum and prostate are the most prevalent and are increasing in our aging populations. Epithelial cells turnover rapidly and mutations naturally accumulate throughout life. Most epithelial cancers arise from this normal mutation rate. All elderly individuals will harbour many cells with the requisite mutations and most will develop occult neoplastic lesions. Although essential for initiation, these mutations are not sufficient for the progression of cancer to a life-threatening disease. This progression appears to be dependent on context: the tissue ecosystem within individuals and lifestyle exposures across populations of individuals. Together, this implies that the seeds may be plentiful but they only germinate in the right soil. The incidence of these cancers is much lower in Eastern countries but is increasing with Westernisation and increases more acutely in migrants to the West. A Western lifestyle is strongly associated with perturbed metabolism, as evidenced by the epidemics of obesity and diabetes: this may also provide the setting enabling the progression of epithelial cancers. Epidemiology has indicated that metabolic biomarkers are prospectively associated with cancer incidence and prognosis. Furthermore, within cancer research, there has been a rediscovery that a switch in cell metabolism is critical for cancer progression but this is set within the metabolic status of the host. The seed may only germinate if the soil is fertile. This perspective brings together the different avenues of investigation implicating the role that metabolism may play within the context of post-genomic concepts of cancer.

Breast cancer progression is associated with loss of estrogen receptor (ER-α), often due to epigenetic silencing. IGFBP genes have consistently been identified among the most common to be aberrantly methylated in tumours. To understand the impact of losing IGFBP-3 tumour expression via DNA methylation, we treated four breast cancer cell lines (MCF-7, T47D, Hs578T and MDA-MB-231) with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (AZA) to determine IGFBP-3's role in the effects of AZA on total cell number and survival relative to changes in the ER. AZA induced cell growth inhibition, death and a reduction in the formation of colonies, despite increasing ER-α expression in ER-negative cells but reducing ER-α in ER-positive cells. Regardless of the differential effects on the ER-α, AZA consistently increased the abundance of IGFBP-3 and negating this increase in IGFBP-3 with siRNA reduced the AZA-induced growth inhibition and induction of cell death and virtually negated the AZA-induced inhibition of colony formation. With ER-α positive cells AZA increased the abundance of the tumour suppressor gene, p53 and induced demethylation of the IGFBP-3 promoter, whereas with ER negative cells, AZA epigenetically increased the transcription factor AP2-α, which when silenced prevented the increase in IGFBP-3. IGFBP-3 plays an important role in the anti-tumorigenic effects of AZA on breast cancer cells.

Background: A growing body of observational evidence suggests that nutritional and physical activity interventions are associated with beneficial outcomes for men with prostate cancer, including brisk walking, lycopene intake, increased fruit and vegetable intake and reduced dairy consumption. However, randomised controlled trial data are limited. The 'Prostate Cancer: Evidence of Exercise and Nutrition Trial' investigates the feasibility of recruiting and randomising men diagnosed with localised prostate cancer and eligible for radical prostatectomy to interventions that modify nutrition and physical activity. The primary outcomes are randomisation rates and adherence to the interventions at 6 months following randomisation. The secondary outcomes are intervention tolerability, trial retention, change in prostate specific antigen level, change in diet, change in general physical activity levels, insulin-like growth factor levels, and a range of related outcomes, including quality of life measures. Methods/design: The trial is factorial, randomising men to both a physical activity (brisk walking or control) and nutritional (lycopene supplementation or increased fruit and vegetables with reduced dairy consumption or control) intervention. The trial has two phases: men are enrolled into a cohort study prior to radical prostatectomy, and then consented after radical prostatectomy into a randomised controlled trial. Data are collected at four time points (cohort baseline, true trial baseline and 3 and 6 months post-randomisation). Discussion: The Prostate Cancer: Evidence of Exercise and Nutrition Trial aims to determine whether men with localised prostate cancer who are scheduled for radical prostatectomy can be recruited into a cohort and subsequently randomised to a 6-month nutrition and physical activity intervention trial. If successful, this feasibility trial will inform a larger trial to investigate whether this population will gain clinical benefit from long-term nutritional and physical activity interventions post-surgery. Prostate Cancer: Evidence of Exercise and Nutrition Trial (PrEvENT) is registered on the ISRCTN registry, ref number ISRCTN99048944. Date of registration 17 November 2014.