Associations of CTCF and FOXA1 with androgen and IGF pathways in men with localized prostate cancer.

AIMS: To examine associations between the transcription factors CCCTC-binding factor (CTCF) and forkhead box protein A1 (FOXA1) and the androgen receptor (AR) and their association with components of the insulin-like growth factor (IGF)-pathway in a cohort of men with localized prostate cancer. METHODS: Using prostate tissue samples collected during the Prostate cancer: Evidence of Exercise and Nutrition Trial (PrEvENT) trial (N = 70 to 92, depending on section availability), we assessed the abundance of CTCF, FOXA1, AR, IGFIR, p-mTOR, PTEN and IGFBP-2 proteins using a modified version of the Allred scoring system. Validation studies were performed using large, publicly available datasets (TCGA) (N = 489). RESULTS: We identified a strong correlation between CTCF and AR staining with benign prostate tissue. CTCF also strongly associated with the IGFIR, with PTEN and with phospho-mTOR. FOXA1 was also correlated with staining for the IGF-IR, with IGFBP-2 and with staining for activated phosphor-mTOR. The staining for the IGF-IR was strongly correlated with the AR. CONCLUSION: Our findings emphasise the close and complex links between the endocrine controls, well known to play an important role in prostate cancer, and the transcription factors implicated by the recent genetic evidence.

Declining Levels and Bioavailability of IGF-I in Cardiovascular Aging Associate With QT Prolongation-Results From the 1946 British Birth Cohort.

Background: As people age, circulating levels of insulin-like growth factors (IGFs) and IGF binding protein 3 (IGFBP-3) decline. In rat cardiomyocytes, IGF-I has been shown to regulate sarcolemmal potassium channel activity and late sodium current thus impacting cardiac repolarization and the heart rate-corrected QT (QTc). However, the relationship between IGFs and IGFBP-3 with the QTc interval in humans, is unknown. Objectives: To examine the association of IGFs and IGFBP-3 with QTc interval in an older age population-based cohort. Methods: Participants were from the 1946 Medical Research Council (MRC) National Survey of Health and Development (NSHD) British birth cohort. Biomarkers from blood samples at age 53 and 60-64 years (y, exposures) included IGF-I/II, IGFBP-3, IGF-I/IGFBP-3 ratio and the change (Δ) in marker levels between the 60-64 and 53y sampled timepoints. QTc (outcome) was recorded from electrocardiograms at the 60-64y timepoint. Generalized linear multivariable models with adjustments for relevant demographic and clinical factors, were used for complete-cases and repeated after multiple imputation. Results: One thousand four hundred forty-eight participants were included (48.3% men; QTc mean 414 ms interquartile range 26 ms). Univariate analysis revealed an association between low IGF-I and IGF-I/IGFBP-3 ratio at 60-64y with QTc prolongation [respectively: ß -0.30 ms/nmol/L, (95% confidence intervals -0.44, -0.17), p < 0.001; ß-28.9 ms/unit (-41.93, -15.50), p < 0.001], but not with IGF-II or IGFBP-3. No association with QTc was found for IGF biomarkers sampled at 53y, however both ΔIGF-I and ΔIGF-I/IGFBP-3 ratio were negatively associated with QTc [ß -0.04 ms/nmol/L (-0.08, -0.008), p = 0.019; ß -2.44 ms/unit (-4.17, -0.67), p = 0.007] while ΔIGF-II and ΔIGFBP-3 showed no association. In fully adjusted complete case and imputed models (reporting latter) low IGF-I and IGF-I/IGFBP-3 ratio at 60-64y [ß -0.21 ms/nmol/L (-0.39, -0.04), p = 0.017; ß -20.14 ms/unit (-36.28, -3.99), p = 0.015], steeper decline in ΔIGF-I [ß -0.05 ms/nmol/L/10 years (-0.10, -0.002), p = 0.042] and shallower rise in ΔIGF-I/IGFBP-3 ratio over a decade [ß -2.16 ms/unit/10 years (-4.23, -0.09), p = 0.041], were all independently associated with QTc prolongation. Independent associations with QTc were also confirmed for other previously known covariates: female sex [ß 9.65 ms (6.65, 12.65), p < 0.001], increased left ventricular mass [ß 0.04 ms/g (0.02, 0.06), p < 0.001] and blood potassium levels [ß -5.70 ms/mmol/L (-10.23, -1.18) p = 0.014]. Conclusion: Over a decade, in an older age population-based cohort, declining levels and bioavailability of IGF-I associate with prolongation of the QTc interval. As QTc prolongation associates with increased risk for sudden death even in apparently healthy people, further research into the antiarrhythmic effects of IGF-I on cardiomyocytes is warranted.

Investigation of the Interplay between Circulating Lipids and IGF-I and Relevance to Breast Cancer Risk: An Observational and Mendelian Randomization Study.

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 ß per 1-SD: -0.13; 95% CI, -0.23 to -0.04; and IGF-I-TG ß 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.

Insulin-like growth factors: Ligands, binding proteins, and receptors.

BACKGROUND: The insulin-like growth factor family of ligands (IGF-I, IGF-II, and insulin), receptors (IGF-IR, M6P/IGF-IIR, and insulin receptor [IR]), and IGF-binding proteins (IGFBP-1-6) play critical roles in normal human physiology and disease states. SCOPE OF REVIEW: Insulin and insulin receptors are the focus of other chapters in this series and will therefore not be discussed further. Here we review the basic components of the IGF system, their role in normal physiology and in critical pathology's. While this review concentrates on the role of IGFs in human physiology, animal models have been essential in providing understanding of the IGF system, and its regulation, and are briefly described. MAJOR CONCLUSIONS: IGF-I has effects via the circulation and locally within tissues to regulate cellular growth, differentiation, and survival, thereby controlling overall body growth. IGF-II levels are highest prenatally when it has important effects on growth. In adults, IGF-II plays important tissue-specific roles, including the maintenance of stem cell populations. Although the IGF-IR is closely related to the IR it has distinct physiological roles both on the cell surface and in the nucleus. The M6P/IGF-IIR, in contrast, is distinct and acts as a scavenger by mediating internalization and degradation of IGF-II. The IGFBPs bind IGF-I and IGF-II in the circulation to prolong their half-lives and modulate tissue access, thereby controlling IGF function. IGFBPs also have IGF ligand-independent cell effects.

Alteration of Metabolic Conditions Impacts the Regulation of IGF-II/H19 Imprinting Status in Prostate Cancer.

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.

IGFBP-1 expression is reduced in human type 2 diabetic glomeruli and modulates ß1-integrin/FAK signalling in human podocytes.

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 ß1-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.

Circulating adiponectin and leptin and risk of overall and aggressive prostate cancer: a systematic review and meta-analysis.

Obesity is associated with an increased risk of advanced, recurrent and fatal prostate cancer. Adipokines may mediate this relationship. We conducted a systematic review and meta-analysis of associations of leptin and adiponectin with overall and aggressive prostate cancer. Bibliographic databases were systematically searched up to 1st April 2017. Log Odds Ratios (ORs) per 2.5 unit increase in adiponectin or leptin levels were derived and pooled. All analyses were stratified by study type (cross-sectional/prospective). 746 papers were retrieved, 34 eligible studies identified, 31 of these could be included in the meta-analysis. Leptin was not consistently associated with overall prostate cancer (pooled OR 1.00, 95%CI 0.98-1.02, per 2.5 ng/ml increase, prospective study OR 0.97, 95%CI 0.95-0.99, cross-sectional study OR 1.19, 95%CI 1.13-1.26) and there was weak evidence of a positive association with aggressive disease (OR 1.03, 95%CI 1.00-1.06). There was also weak evidence of a small inverse association of adiponectin with overall prostate cancer (OR 0.96, 95%CI 0.93-0.99, per 2.5 µg/ml increase), but less evidence of an association with aggressive disease (OR 0.98, 95%CI 0.94-1.01). The magnitude of any effects are small, therefore levels of circulating adiponectin or leptin alone are unlikely to be useful biomarkers of risk or prognosis.

A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

Cholesterol-in particular, high levels of low-density lipoprotein (LDL) and its metabolite, 27-hydroxycholesterol (27-OHC)-is correlated with increases in the risks of breast cancer and obesity. Although the high expression of LDL/27-OHC has been reported in breast cancer, its effects and mechanism of action remain to be fully elucidated. In this study, we found that the effects of LDL on cell proliferation were mediated by the activation of the cytochrome P450 enzyme, sterol 27 hydroxylase, and cholesterol 27-hydroxylase (CYP27A1) in both ER-α-positive and ER-α-negative breast cancer cells. We found that treatment with 27-OHC only increased cell growth in oestrogen receptor-α (ER-α)-positive breast cancer cells in an ER-α-dependent manner, but, interestingly, the effects of 27-OHC on cell migration and invasion were independent of ER-α. Using ER-α-negative MDA-MB-231 cells, we found that 27-OHC similarly promoted cell invasion and migration, and this was mediated by oestrogen receptor ß (ER-ß). These results suggest that 27-OHC promotes breast cancer cell proliferation in ER-α-positive breast cancer cells via ER-α, but migration and invasion are mediated via ER-ß in ER-α positive and negative cell lines. The addition of LDL/27OHC increased the production of IGF-I and the abundance of IGF-IR in TNBC. We further found that modulating ER-ß using an agonist or antagonist increased or decreased, respectively, levels of the IGF-I and EGF receptors in TNBC. The inhibition of the insulin-like growth factor receptor blocked the effects of cholesterol on cell growth and the migration of TNBC. Using TCGA and METABRIC microarray expression data from invasive breast cancer carcinomas, we also observed that higher levels of ER-beta were associated with higher levels of IGF-IR. Thus, this study shows novel evidence that ER-ß is central to the effects of LDL/27OHC on invasion, migration, and the IGF and EGF axes. Our data suggest that targeting ER-ß in TNBC could be an alternative approach for downregulating IGF/EGF signalling and controlling the impact of LDL in breast cancer patients.

Interaction between GRP78 and IGFBP-3 Affects Tumourigenesis and Prognosis in Breast Cancer Patients.

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-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.

Glucose Concentration in Cell Culture Medium Influences the BRCA1-Mediated Regulation of the Lipogenic Action of IGF-I in Breast Cancer Cells.

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 (0-500 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.

Obesity, Diabetes and COVID-19: An Infectious Disease Spreading From the East Collides With the Consequences of an Unhealthy Western Lifestyle.

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.

Mini Review: Opposing Pathologies in Cancer and Alzheimer's Disease: Does the PI3K/Akt Pathway Provide Clues?

This minireview is a brief overview examining the roles of insulin-like growth factors (IGFs) and the PI3K/Akt pathway in two apparently unconnected diseases: Alzheimer's dementia and cancer. For both, increased age is a major risk factor, and, in accord with the global rise in average life expectancy, their prevalence is also increasing. Cancer, however, involves excessive cell proliferation and metastasis, whereas Alzheimer's disease (AD) involves cell death and tissue destruction. The apparent "inverse" nature of these disease states is examined here, but also some important commonalities in terms of the PI3K/Akt pathway, glucose utilization and cell deregulation/death. The focus here is on four key molecules associated with this pathway; notably, the insulin receptor substrate 1 (IRS-1), cellular tumor antigen p53 (p53), peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) and low-density lipoprotein receptor-related protein-1 (LRP1), all previously identified as potential therapeutic targets for both diseases. The insulin-resistant state, commonly reported in AD brain, results in neuronal glucose deprivation, due to a dampening down of the PI3K/Akt pathway, including overactivity of the mammalian target of rapamycin 1 (mTORC1) complex, hyperphosphorylation of p53 and neuronal death. This contrasts with cancer, where there is overstimulation of the PI3K/Akt pathway and the suppression of mTORC1 and p53, enabling abundant energy and unrestrained cell proliferation. Although these disease states appear to be diametrically opposed, the same key molecules are controlling pathology and, with differential targeting of therapeutics, may yet provide a beneficial outcome for both.

The role of insulin-like growth factors in the development of prostate cancer.

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.

Phase II randomised control feasibility trial of a nutrition and physical activity intervention after radical prostatectomy for prostate cancer.

OBJECTIVE: Dietary factors and physical activity may alter prostate cancer progression. We explored the feasibility of lifestyle interventions following radical prostatectomy for localised prostate cancer. DESIGN: Patients were recruited into a presurgical observational cohort; following radical prostatectomy, they were offered randomisation into a 2×3 factorial randomised controlled trial (RCT). SETTING: A single National Health Service trust in the South West of England, UK. PARTICIPANTS: Those with localised prostate cancer and listed for radical prostatectomy were invited to participate. RANDOMISATION: Random allocation was performed by the Bristol Randomised Trial Collaboration via an online system. INTERVENTIONS: Men were randomised into both a modified nutrition group (either increased vegetable and fruit, and reduced dairy milk; or lycopene supplementation; or control) and a physical activity group (brisk walking or control) for 6 months. BLINDING: Only the trial statistician was blind to allocations. PRIMARY OUTCOME MEASURES: Primary outcomes were measures of feasibility: randomisation rates and intervention adherence at 6 months. Collected at trial baseline, three and six months, with daily adherence reported throughout. Our intended adherence rate was 75% or above, the threshold for acceptable adherence was 90%. RESULTS: 108 men entered the presurgical cohort, and 81 were randomised into the postsurgical RCT (randomisation rate: 93.1%) and 75 completed the trial. Of 25 men in the nutrition intervention, 10 (40.0%; 95% CI 23.4% to 59.3%) adhered to the fruit and vegetable recommendations and 18 (72.0%; 95% CI 52.4% to 85.7%) to reduced dairy intake. Adherence to lycopene (n=28), was 78.6% (95% CI 60.5% to 89.8%), while 21/39 adhered to the walking intervention (53.8%; 95% CI 38.6% to 68.4%). Most men were followed up at 6 months (75/81; 92.6%). Three 'possibly related' adverse events were indigestion, abdominal bloating and knee pain. CONCLUSIONS: Interventions were deemed feasible, with high randomisation rates and generally good adherence. A definitive RCT is proposed. TRIAL REGISTRATION NUMBER: ISRCTN 99048944.

The Neglected Insulin: IGF-II, a Metabolic Regulator with Implications for Diabetes, Obesity, and Cancer.

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.