The Role Of Glutaminase In Cancer

Increased glutamine metabolism (glutaminolysis) is a hallmark of cancer and is recognised as a key metabolic change in cancer cells. As a heterogeneous disease with different morpholog- ical and molecular subtypes and response to therapy, breast cancer cells are known to rewire glutamine metabolism to support survival and proliferation. Glutaminase isoenzymes (GLS and GLS2) are key enzymes for glutamine metabolism. Interestingly, GLS and GLS2 display contrasting functions in tumourigenesis. In this review, we explore the role of glutaminase in cancer, primarily focussing on breast cancer, address the role played by oncogenes and tu- mour suppressor genes in regulating glutaminase, and discuss current therapeutic approaches in targeting glutaminase

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues

The solute carrier SLC7A8 is a marker of favourable prognosis in ER-positive low proliferative invasive breast cancer

Purpose: Breast cancer (BC) is a heterogeneous disease consisting of various subtypes, withdifferent prognostic and therapeutic outcomes. The amino acid transporter, SLC7A8, is over expressed in estrogen receptor positive BC. However the consequences of this overexpression, it terms of disease prognosis, is still obscure. This study aimed to evaluate the biological and prognostic value of SLC7A8 in BC with emphasis on the intrinsic molecular subtypes.Methods: SLC7A8 was assessed at the genomic, using METABRIC data (n=1,980), and proteomic, using immunohistochemistry and TMA (n=1,562), levels in well-characterised primary BC cohorts. SLC7A8 expression was examined with clinicopathological parameters, molecular subtypes, and patient outcome.Results: SLC7A8 mRNA and SLC7A8 protein expression were strongly associated with good prognostic features, including small tumour size, low tumour grade and good Nottingham Prognostic Index (NPI) (all

CDC20 expression in oestrogen receptor positive breast cancer predicts poor prognosis and lack of response to endocrine therapy

PurposeEndocrine therapy is the standard treatment for oestrogen receptor positive (ER+) breast cancer. Despite its efficacy, around half of patients will develop resistance to this treatment and eventually relapse. Identification of effective and reliable biomarkers to predict the efficacy of endocrine therapy is of crucial importance in the management of ER+ breast cancer. Emerging evidence has revealed that the cell division regulator CDC20 exhibits an oncogenic function and plays important roles in tumourigenesis and progression of solid tumours. In this study, we investigated the prognostic and predictive role of CDC20 in early ER+ breast cancer patients.MethodsThe biological and clinical impact of CDC20 expression was assessed in large clinical annotated cohort of ER+ breast cancer with long-term follow-up at the mRNA level, using METABRIC and KM-Plotter datasets, and the protein level using immunohistochemistry on patients presenting at Nottingham. CDC20 expression was correlated with clinico-pathological parameters, molecular subtypes, clinical outcome and efficacy of endocrine therapy.ResultsHigh CDC20 mRNA expression was associated with poor clinico-pathological parameters including large tumour size and high tumour grade (P

PPFIA1 expression associates with poor response to endocrine treatment in luminal breast cancer

BackgroundPPFIA1 is an important regulator of cell migration and invasion, regulating focal adhesion signalling and disassembly. PPFIA1 is frequently amplified in breast cancer, and recent functional studies indicate that PPFIA1 is an important promoter of migration and invasion in breast cancer. This study aims to evaluate the utility of PPFIA1 expression in the luminal breast cancer as a prognostic marker to predict the response to endocrine therapy.MethodsLarge, well-characterised cohorts of primary luminal breast cancer patients with long-term follow-up was assessed for the clinical impact of PPFIA1 expression at the transcriptomic and proteomic levels. Prognostic significance of PPFIA1 and its relationship with clinical outcome and benefit of endocrine therapy were analysed. In addition, its association with other related-genes was analysed.ResultsThere was significant association between PPFIA1 expression and a member of the liprin family that involves in cell invasion (PPFIBPI), and the cell cycle regulator (CCND1), whereas a negative association was observed with the tumour suppressor gene (CD82). Patients with high PPFIA1 expression were associated with high risk of recurrence, distant metastasis and death from breast cancer (P< 0.05). Importantly, high PPFIA1 expression predicted relapse in a subset of patients who were subject to endocrine treatment alone, and was an independent prognostic marker of unfavourable outcome in these patients (P< 0.05).ConclusionsThese findings support the proposed role for PPFIA1 as a regulator of cell migration in breast cancer and provides definitive evidence for the clinical utility of PPFIA1 expression in patients with luminal breast cancer. Most importantly, our data suggests that PPFIA1 might be a potential predictive marker for poor benefit from endocrine therapy

Co-Expression Effect of SLC7A5/SLC3A2 to Predict Response to Endocrine Therapy in Oestrogen-Receptor-Positive Breast Cancer

The majority of breast cancers are oestrogen receptor positive (ER+) and are subject to endocrine therapy however, an unpredictable subgroup of patients will develop resistance to endocrine therapy. SLC7A5/SLC3A2 complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Alterations in the expression and function of those amino acid transporters lead to metabolic reprogramming, which contributing to the tumorigenesis and drug resistance. This study aims to assess the effects and roles of SLC7A5/SLC3A2 co-expression in predicting response to endocrine therapy in patients with ER+ breast cancer. The biological and clinical impact of SLC7A5/SLC3A2 co-expression was assessed in large annotated cohorts of ER+/HER2- breast cancer with long-term follow-up at the mRNA and protein levels. In vitro experiments were conducted to investigate the effect of SLC7A5/SLC3A2 knockdown in the proliferation of cancer cells and to the sensitivity to tamoxifen. We found that proliferation-related genes are highly expressed in subgroup of patients with high SLC7A5/SLC3A2, and knockdown of SLC7A5/SLC3A2 decreased proliferation of ER+ breast cancer cells. In patients treated with endocrine therapy, high SLC7A5/SLC3A2 co-expression was associated with poor patient outcome, and depletion of SLC7A5/SLC3A2 using siRNA increased the sensitivity of breast cancer cells to tamoxifen. On the basis of our findings, SLC7A5/SLC3A2 co-expression has the potential of identifying a subgroup of ER+/HER2- breast cancer patients who fail to benefit from endocrine therapy and could guide the choice of other alternative therapy

Glutamate dehydrogenase (GLUD1) expression in breast cancer

Dysregulated cellular metabolism is regarded as one of the hallmarks of cancer with some tumours utilising the glutamine metabolism pathway for their sustained proliferation and survival. Glutamate dehydrogenase (GLUD1) is a key enzyme in glutaminolysis converting glutamate to α-Ketoglutarate for entry into the TCA cycle. Breast cancer (BC) comprises a heterogeneous group of tumours in terms of molecular biology and clinical behaviour, and we have previously shown that altered glutamine metabolism varies substantially among the different molecular subtypes. We hypothesise that the prognostic value of GLUD1 expression will differ between the BC molecular subtypes and may act as a potential therapeutic target for BC tumours.Methods: GLUD1 was assessed at the DNA, mRNA (n=1,980) and protein (n=1,300) levels in large and well-characterised cohorts and correlated with clinicopathological parameters, molecular subtypes, patient outcome and treatments. Results: There was a correlation between GLUD1 mRNA and GLUD1 protein expression which were highly expressed in low grade Luminal/ER+ BC (

FOXP1 expression correlates with better prognosis in invasive breast cancer including the ER-positive luminal subtype

Background: Fork head box P1 (FOXP1) is a FOX family transcription factor influencing ERα;-regulated transcription by interaction with the ERα; related pioneer factor FOXA1. Altered FOXP1 expression is seen in breast and prostate cancers. This study investigated FOXP1 at the protein level in breast cancer (BC) and examined associations with clinicopathological and molecular features. Methods: FOXP1 mRNA expression was investigated in the METABRIC BC cohort (n=1980) and validated using online expression datasets [bc-GenExMiner v4.0]. Protein expression was studied in a well characterised BC primary series (n=621) using immunohistochemistry and correlations made with clinicopathological parameters and outcome. Results: High FOXP1 mRNA and protein expression was significantly associated with low grade, low NPI, positive ER/PR status, lobular BCs, low Ki67 and negative Her2 status (p<0.001). Within PAM50 subtypes, high FOXP1 expression was associated with Luminal A BCs and good prognosis integrative clusters (IC3 and IC8). Nuclear FOXP1 (n-FOXP1) protein positively associated with luminal markers: CARM1, RERG and FOXA1 (p<0.05). Negative association with PIK3 (p=0.023) indicates a possible dual role whereby n-FOXP1 reduces EGFR-mediated ligand-independent ER activation, but enhances AKT mediated activation. Positive correlations with GATA3, STAT3 and CDC42 (p<0.001), suggest interacting pathways. On univariate analysis, n-FOXP1 overexpression showed better long term outcome in the whole cohort and ER+ subgroups (p<0.05). Pooled FOXP1 gene expression data in the ER+ external validation cohort showed similar association with better outcome even when adjusted for NPI/proliferation (p<0.001). Conclusions: Higher n-FOXP1 correlated with low grade ER positive BCs and spelt better prognosis with increased long-term survival. The marker may be helpful to distinguish between good versus poor prognosis luminal A tumours

FOXP1 expression correlates with better prognosis in invasive breast cancer including the ER-positive luminal subtype

Background: Fork head box P1 (FOXP1) is a FOX family transcription factor influencing ERα;-regulated transcription by interaction with the ERα; related pioneer factor FOXA1. Altered FOXP1 expression is seen in breast and prostate cancers. This study investigated FOXP1 at the protein level in breast cancer (BC) and examined associations with clinicopathological and molecular features. Methods: FOXP1 mRNA expression was investigated in the METABRIC BC cohort (n=1980) and validated using online expression datasets [bc-GenExMiner v4.0]. Protein expression was studied in a well characterised BC primary series (n=621) using immunohistochemistry and correlations made with clinicopathological parameters and outcome. Results: High FOXP1 mRNA and protein expression was significantly associated with low grade, low NPI, positive ER/PR status, lobular BCs, low Ki67 and negative Her2 status (p<0.001). Within PAM50 subtypes, high FOXP1 expression was associated with Luminal A BCs and good prognosis integrative clusters (IC3 and IC8). Nuclear FOXP1 (n-FOXP1) protein positively associated with luminal markers: CARM1, RERG and FOXA1 (p<0.05). Negative association with PIK3 (p=0.023) indicates a possible dual role whereby n-FOXP1 reduces EGFR-mediated ligand-independent ER activation, but enhances AKT mediated activation. Positive correlations with GATA3, STAT3 and CDC42 (p<0.001), suggest interacting pathways. On univariate analysis, n-FOXP1 overexpression showed better long term outcome in the whole cohort and ER+ subgroups (p<0.05). Pooled FOXP1 gene expression data in the ER+ external validation cohort showed similar association with better outcome even when adjusted for NPI/proliferation (p<0.001). Conclusions: Higher n-FOXP1 correlated with low grade ER positive BCs and spelt better prognosis with increased long-term survival. The marker may be helpful to distinguish between good versus poor prognosis luminal A tumours

Multicomponent analysis of the tumour microenvironment reveals low CD8 T cell number, low stromal caveolin-1 and high tenascin-C and their combination as significant prognostic markers in non-small cell lung cancer

The complex interplay of the tumour microenvironment (TME) and its role in disease progression and response to therapy is poorly understood. The majority of studies to date focus on individual components or molecules within the TME and so lack the power correlative analysis. Here we have performed a multi-parameter analysis of the TME in 62 resectable non-small cell lung cancer (NSCLC) specimens detailing number and location of immune infiltrate, assessing markers of cancer-associated fibroblasts, caveolin-1 and tenascin-C, and correlating with clinicopathological details, as well as markers of disease progression such as epithelial-to-mesenchymal transition (EMT). The influence of individual parameters on overall survival was determined in univariate and multivariate analysis and the combination of risk factors and interplay between components analysed. Low numbers of CD8 T cells, low stromal levels of caveolin-1 or high levels of tenascin-C were significant prognostic markers of decreased overall survival in both univariate and multivariate analysis. Patients with two or more risk factors had dramatically reduced overall survival and those with all three a median survival of just 7.5 months. In addition, low levels of tumour E-cadherin correlated with reduced immune infiltrate into the tumour nests, possibly linking EMT to the avoidance of CD8 T cell control. The multicomponent approach has allowed identification of the dominant influences on overall survival, and exploration of the interplay between different components of the TME in NSCLC