Evaluating circulating tumour cell enrichment techniques to establish an appropriate method for clinical application in glioblastomas.

Brain tumours reduce life expectancy for an average of 20 years per patient, the highest of any cancer. A third of brain tumour patients visit their GP at least five times before diagnosis and many of those are diagnosed late through emergency departments. A possible solution to this challenge is to utilise a "liquid biopsy" blood test designed for circulating tumour cells (CTCs). Such a test could be applied at a primary healthcare centre, contributing to informed decision making for diagnostic imaging referrals. Furthermore, it could also be applied at secondary health care centres for the ongoing monitoring of disease recurrence. There is increased interest in CTC enrichment methods as a potential approach for faster diagnosis and monitoring of disease progression. The aim of this review to compare four CTC enrichment methods - OncoQuick®, Screen Cell®, pluriBead® and Cell Search® - with the objective of identifying a suitable method for application in the clinical setting for the isolation of CTCs from glioblastomas.

Mini-Review: Current Bladder Cancer Treatment-The Need for Improvement.

Bladder cancer is the tenth most common cancer and is a significant burden on health care services worldwide, as it is one of the most costly cancers to treat per patient. This expense is due to the extensive treatment and follow-ups that occur with costly and invasive procedures. Improvement in both treatment options and the quality of life these interventions offer has not progressed at the rates of other cancers, and new alternatives are desperately needed to ease the burden. A more modern approach needs to be taken, with urinary biomarkers being a positive step in making treatments more patient-friendly, but there is still a long way to go to make these widely available and of a comparable standard to the current treatment options. New targets to hit the major signalling pathways that are upregulated in bladder cancer, such as the PI3K/AkT/mTOR pathway, are urgently needed, with only one drug approved so far, Erdafitinib. Immune checkpoint inhibitors also hold promise, with both PD-1 and CDLA-4 antibody therapies approved for use. They effectively block ligand/receptor binding to block the immune checkpoint used by tumour cells. Other avenues must be explored, including drug repurposing and novel biomarkers, which have revolutionised this area in other cancers.

Role of the Insulin-like Growth Factor (IGF) Axis in Diseases.

The insulin-like growth factor axis is a multifaceted, complex system that comprises two ligands, IGF-I and IGF-II, receptors (IGF-1R, IGF-IIR, insulin receptor isoforms IR-A and B, and hybrid receptors) six high affinity IGF-binding proteins (IGFBPs 1-6), and IGFBP proteases [...].

Case report: Molecular characterisation of adipose-tissue derived cells from a patient with ROHHAD syndrome.

There have been over 100 cases of Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) syndrome reported, but there is currently no curative treatment for children with this condition. We aimed to better characterise adipose cells from a child with ROHHAD syndrome. We isolated pre-adipocytes from a 4 year-old female patient with ROHHAD syndrome and assessed proliferation rate of these cells. We evaluated levels of DLP-Pref-1(pre-adipocyte marker) using western blotting, and concentrations of interleukin-6(IL-6) using ELISA. We performed next-generation sequencing (NGS) and bioinformatic analyses on these cells compared to tissue from an age/sex-matched control. The two most up-/down-regulated genes were validated using QPCR. We successfully isolated pre-adipocytes from a fat biopsy, by confirming the presence of Pref-1 and differentiated them to mature adipocytes. Interleukin 6, (Il-6) levels were 5.6-fold higher in ROHHAD cells compared to a control age/sex-matched biopsy. NGS revealed 25,703 differentially expressed genes (DEGs) from ROHHAD cells vs. control of which 2,237 genes were significantly altered. The 20 most significantly up/down-regulated genes were selected for discussion. This paper describes the first transcriptomic analysis of adipose cells from a child with ROHHAD vs. normal control adipose tissue as a first step in identifying targetable pathways/mechanisms underlying this condition with novel therapeutic interventions.

Insulin-like growth factor binding protein-2 and glucose-regulated protein 78 kDa: Potential biomarkers affect prognosis in IDH-wildtype glioblastoma patients.

BACKGROUND: The overall survival of IDH-wildtype glioblastoma patients is poor despite best available treatments. There is an urgent need for new biomarkers to inform more precise disease stratification. Previous studies have identified insulin-like growth factor binding protein-2 (IGFBP-2) as a potential biomarker for glioblastoma diagnosis and therapeutic targeting. Other studies have indicated links between the insulin-like growth factor (IGF) axis and tumorigenic functions of a molecular chaperone glucose related protein of 78 kDa (GRP78). We aimed to interrogate the oncogenic effects of IGFBP-2 and GRP78 in our glioma stem cell (GSC) lines and clinical cohort. METHODS: Immunoblotting, reverse transcription quantitative real-time PCR were used to quantify protein and mRNA levels derived from GSCs and non-malignant neural stem cells (NSCs). Microarray analysis was used to compare the differences in IGFBP-2 (IGFBP-2) and GRP78 (HSPA5) transcript expression between NSCs, GSCs and adult human cortex samples. Immunohistochemistry was used to quantify IGFBP-2 and GRP78 expression in IDH-wildtype glioblastoma tissue sections (n = 92) and clinical implications assessed using survival analysis. Finally, the relationship between IGFBP-2 and GRP78 was further explored molecularly using coimmunoprecipitation. RESULTS: Here, we demonstrate that IGFBP-2 and HSPA5 mRNA is overexpressed in GSCs and NSCs in comparison to non-malignant brain tissue. We also determined a relationship in which G144 and G26 GSCs expressed higher IGFBP-2 protein and mRNA than GRP78, and this was reversed in mRNA isolated from adult human cortex samples. Clinical cohort analysis revealed that Glioblastomas with high IGFBP-2 protein expression paired with low GRP78 protein expression were significantly associated with a much shorter survival time (Median = 4 months, p = 0.019) compared with 12-14 months for any other combination of high/low protein expression. CONCLUSIONS: Inverse levels of IGFBP-2 and GRP78 may be adverse clinical prognostic markers in IDH-wildtype glioblastoma. Further interrogation of the mechanistic link between IGFBP-2 and GRP78 may be important for rationalisation of their potential as biomarkers and therapeutic targets.

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.

Association between genetically proxied PCSK9 inhibition and prostate cancer risk: A Mendelian randomisation study.

BACKGROUND: Prostate cancer (PrCa) is the second most prevalent malignancy in men worldwide. Observational studies have linked the use of low-density lipoprotein cholesterol (LDL-c) lowering therapies with reduced risk of PrCa, which may potentially be attributable to confounding factors. In this study, we performed a drug target Mendelian randomisation (MR) analysis to evaluate the association of genetically proxied inhibition of LDL-c-lowering drug targets on risk of PrCa. METHODS AND FINDINGS: Single-nucleotide polymorphisms (SNPs) associated with LDL-c (P < 5 × 10-8) from the Global Lipids Genetics Consortium genome-wide association study (GWAS) (N = 1,320,016) and located in and around the HMGCR, NPC1L1, and PCSK9 genes were used to proxy the therapeutic inhibition of these targets. Summary-level data regarding the risk of total, advanced, and early-onset PrCa were obtained from the PRACTICAL consortium. Validation analyses were performed using genetic instruments from an LDL-c GWAS conducted on male UK Biobank participants of European ancestry (N = 201,678), as well as instruments selected based on liver-derived gene expression and circulation plasma levels of targets. We also investigated whether putative mediators may play a role in findings for traits previously implicated in PrCa risk (i.e., lipoprotein a (Lp(a)), body mass index (BMI), and testosterone). Applying two-sample MR using the inverse-variance weighted approach provided strong evidence supporting an effect of genetically proxied inhibition of PCSK9 (equivalent to a standard deviation (SD) reduction in LDL-c) on lower risk of total PrCa (odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.76 to 0.96, P = 9.15 × 10-3) and early-onset PrCa (OR = 0.70, 95% CI = 0.52 to 0.95, P = 0.023). Genetically proxied HMGCR inhibition provided a similar central effect estimate on PrCa risk, although with a wider 95% CI (OR = 0.83, 95% CI = 0.62 to 1.13, P = 0.244), whereas genetically proxied NPC1L1 inhibition had an effect on higher PrCa risk with a 95% CI that likewise included the null (OR = 1.34, 95% CI = 0.87 to 2.04, P = 0.180). Analyses using male-stratified instruments provided consistent results. Secondary MR analyses supported a genetically proxied effect of liver-specific PCSK9 expression (OR = 0.90 per SD reduction in PCSK9 expression, 95% CI = 0.86 to 0.95, P = 5.50 × 10-5) and circulating plasma levels of PCSK9 (OR = 0.93 per SD reduction in PCSK9 protein levels, 95% CI = 0.87 to 0.997, P = 0.04) on PrCa risk. Colocalization analyses identified strong evidence (posterior probability (PPA) = 81.3%) of a shared genetic variant (rs553741) between liver-derived PCSK9 expression and PrCa risk, whereas weak evidence was found for HMGCR (PPA = 0.33%) and NPC1L1 expression (PPA = 0.38%). Moreover, genetically proxied PCSK9 inhibition was strongly associated with Lp(a) levels (Beta = -0.08, 95% CI = -0.12 to -0.05, P = 1.00 × 10-5), but not BMI or testosterone, indicating a possible role for Lp(a) in the biological mechanism underlying the association between PCSK9 and PrCa. Notably, we emphasise that our estimates are based on a lifelong exposure that makes direct comparisons with trial results challenging. CONCLUSIONS: Our study supports a strong association between genetically proxied inhibition of PCSK9 and a lower risk of total and early-onset PrCa, potentially through an alternative mechanism other than the on-target effect on LDL-c. Further evidence from clinical studies is needed to confirm this finding as well as the putative mediatory role of Lp(a).

Could Reducing Body Fatness Reduce the Risk of Aggressive Prostate Cancer via the Insulin Signalling Pathway? A Systematic Review of the Mechanistic Pathway.

Excess body weight is thought to increase the risk of aggressive prostate cancer (PCa), although the biological mechanism is currently unclear. Body fatness is positively associated with a diminished cellular response to insulin and biomarkers of insulin signalling have been positively associated with PCa risk. We carried out a two-pronged systematic review of (a) the effect of reducing body fatness on insulin biomarker levels and (b) the effect of insulin biomarkers on PCa risk, to determine whether a reduction in body fatness could reduce PCa risk via effects on the insulin signalling pathway. We identified seven eligible randomised controlled trials of interventions designed to reduce body fatness which measured insulin biomarkers as an outcome, and six eligible prospective observational studies of insulin biomarkers and PCa risk. We found some evidence that a reduction in body fatness improved insulin sensitivity although our confidence in this evidence was low based on GRADE (Grading of Recommendations, Assessment, Development and Evaluations). We were unable to reach any conclusions on the effect of insulin sensitivity on PCa risk from the few studies included in our systematic review. A reduction in body fatness may reduce PCa risk via insulin signalling, but more high-quality evidence is needed before any conclusions can be reached regarding PCa.

The Sirenic Links between Diabetes, Obesity, and Bladder Cancer.

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 and Metabolism: Potential for Novel Therapeutic Strategies.

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.

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.

CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer.

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.

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.

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.