Amino acid transporters in breast cancer: promising prognostic markers and emerging targets across molecular subtypes

Background: To maintain the necessary energy and cellular building blocks, tumour cells selectively upregulate expression of cell-surface nutrienttransporters. One of the most prominent are amino acid transporters, which catalyse the uptake of vital amino acids, particularly glutamine. Glutamine promotes cell growth by supporting bioenergetic and biosynthetic metabolism, maintaining redox balance and activating the mTORC1 signalling pathway, which supports protein translation and prevents apoptosis in cancer cells. These metabolic alterations are mediated by mutations in oncogenes and/or tumour suppressors that result in sustained cancer cell growth and proliferation. Cellular interaction with a tumour microenvironment, at the metabolic level, can also promote cancer aggressiveness and metastasis. Breast cancer (BC) is a heterogeneous disease with various subtypes that differ in terms of biology and clinical behaviour. There is growing evidence that differences in amino acid metabolism exist between BC molecular subtypes. However, the expression of amino acid transporters and the subsequent prognostic implications in BC remain elusive, especially with regard to the BC molecular subtypes. It was therefore hypothesised that amino acid transporters involved in the glutamine pathway are overexpressed in BC and they have prognostic implications that vary between BC molecular subtypes. This study aimed to determine the prognostic significance of the eminent amino acid transporters and their downstream signal in different BC molecular subtypes and to investigate the usability of the key amino acid transporters as potential targets for therapeutic interventions in BC. Furthermore, this study sought to find whether upregulation of amino acid uptake in BC participates in shaping the tumour microenvironment through attracting specific immune cell subtypes, including the subsequent impact on patient outcome. Methodologies: The solute carriers SLC1A5, SLC7A5, SLC3A2, SLC38A2, SLC7A11 and SLC7A8 and the downstream signal,p70S6K, were each assessed at the genomic level using METABRIC data (n=1980) and Breast Cancer GeneExpression Miner dataset (n= 4904), and at the proteomic level using immunohistochemical analysis and tissue microarray (TMA) sections constructed from a large, well-characterised primary BC cohort (n=2770). Their expression was correlated with clinicopathological parameters, BC molecular subtypes, relevant biological markers and patient outcome. K-mean and PAM clustering algorithms were applied to stratify the BC cohort into accredited “SLC clusters” based on the protein expression data of the key solute carriers (SLC1A5, SLC7A5 and SLC3A2). The clinicopathological relevance of the SLC clusters was assessed, including associations with patient outcome. In addition, SLC clusters were correlated with various immune cell markers (CD3, CD8, FOXP3, CD20, CD68, PD1 and PDL1) as components of tumour microenvironment. The co-expression of the two compartments was associated with patient outcome, considering the BC molecular subtypes. In a different approach, in vitro studies using different BC cell lines were conducted to investigate the effect of transient siRNA knockdown of SLC1A5 and SLC7A5 on cell proliferation, cell invasion and amino acid uptake. The effect of silencing the two solute carriers on PDL1 expression was also assessed. Results: All biomarkers included in this study, apart from SLC7A8, showed a strong correlation with poor clinicopathological parameters and oestrogen receptor negative (ER-) status. The results of the survival analysis differed among BC molecular subtypes. While high SLC1A5, SLC7A5 and SLC3A2 expression shared the characteristics of being predictive of patient outcome only in the ER+ highly proliferative/luminal B subtype, SLC38A2 showed similar associations in triple negative (TN) tumours, and p70S6K and SLC7A8 were limited to the ER+ low proliferative/luminal A subtype. In multivariate analysis, all aforementioned biomarkers were independent factors for predicting patient survival, excluding SLC1A5, which was significant only in the univariate analysis, and SLC7A11, which did not show any significant associations with patient outcome. A significant association was also observed with other relevant biological markers.The most prominent was the key regulator of tumour cell metabolism, MYC, and a group of glutamine metabolic enzymes, particularly those involved in the glutamine-proline regulatory axis. Clustering analysis of SLC1A5, SLC7A5, and SLC3A2 protein expression resulted in classifying BC patients into three clusters; low SLCs (SLC1A5-/SLC7A5- /SLC3A2-), high SLC1A5 (SLC1A5+/SLC7A5-/SLC3A2-), and high SLCs (SLC1A5+/SLC7A5+/SLC3A2+). Each cluster had distinct correlations to known prognostic factors and patient outcome. Multivariate analysis showed that the SLC clusters were independent risk factors for shorter BC-specific survival only in the ER+ highly proliferative tumours. The high SLCs cluster showed a significant association with different immune cell markers, primarily in the TN subtype. In addition, the co-occurrence of SLCs with immune markers had an additive effect on predicting patient outcome. Targeting SLC7A5 but not SLC1A5 significantly reduced the expression of PDL1 in the TN cell line. In this context, while silencing SLC1A5 or SLC7A5 significantly reduced amino acid uptake in the respective BC cell lines, the impact on cell proliferation and cell invasion was significant only upon silencing SLC7A5. Conclusion: Continued refinement in understanding the biological diversity of BC and linked development of classification strategies suitable for routine clinical use are essential to achieve a personalised approach to BC management. This thesisdemonstrated that the amino acid transport system for glutamine has a crucial prognostic role in BC, which is variable among BC molecular subtypes. Solute carriers that showed the liability of being independent predictors for poor patient outcome can be potential targets for BC treatment, particularly in synergism. Furthermore, this study deduced that enhanced amino acid uptake by cancer cells can influence the composition of immune cell infiltrates, which has an additional effect on patient outcome. Co-targeting the key amino acid transporters and the pro-tumorigenic immune cells may be a novel approach for BC treatment, particularly in the TN subgroup. Further elaborated functional studies may provide new insights into the specific role played by the amino acid transporters in the aggressive BC molecular subtypes

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

The prognostic significance of ALDH1A1 expression in early invasive breast cancer

Aims: Aldehyde dehydrogenase family 1 member A1 (ALDH1A1)is reportedly a key ALDH isozyme linked to the cancer stem cells (CSC) of many solid tumours, where it is involved in self-renewal, differentiation and self-protection. In this study, the prognostic significance of ALDH1A1 expression in early invasive breast cancer (BC) and its role as a BC stem cell (BCSC) were evaluated.Methods: ALDH1A1 expression was assessed, using immunohistochemistry and tissue microarrays, in a large well- characterised BC cohort. ALDH1A1 mRNA expression was also assessed at the transcriptomic levels, utilising data from the Molecular Taxonomy of Breast Cancer International Consortium. The associations of ALDH1A1 with clinicopathological parameters, other stem cell markers and patient outcomes were determined.Results: ALDH1A1 was expressed in 71% of BC cases, at both the protein and mRNA levels. High ALDH1A1 expression was associated with poor prognostic features, including high grade, poor Nottingham Prognostic Index (NPI), lymph node metastasis and highly proliferative ER+ (luminal B) and triple negative (TNBC) subtypes. ALDH1A1 expression was positively correlated with the expression of CD44, CD24, TWIST, SOX9, EPCAM and CD133. The high immunoexpression of ALDH1A1 was significantly associated with poor BC-specific survival [less than] 0.001), and specifically in the luminal B and TNBC subtypes (P=0.042 and P=0.003, respectively). The immunoexpression of ALDH1A1 was an independent predictor of poor prognosis (P=0.015).Conclusions: ALDH1A1, as assessed using IHC, seems to act as a BCSC marker associated not only with other BCSC markers but also with poor prognostic characteristics and poor outcomes, particularly in the luminal B and TNBC subtypes

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

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

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 (

The amino acid transporter SLC7A5 confers a poor prognosis in the highly proliferative breast cancer subtypes and is a key therapeutic target in luminal B tumours

Background: Breast cancer (BC) is a heterogeneous disease characterised by variant biology and patient outcome. The amino acid transporter, SLC7A5, plays a role in BC although its impact on patient outcome in different BC subtypes remains to be validated. This study aimed to determine whether the clinicopathological and prognostic value of SLC7A5 is different within the molecular classes of BC.Methods: SLC7A5 was assessed at the genomic, using METABRIC data (n=1,980), and proteomic, using immunohistochemistry and TMA (n= 2664; 1,110 training and 1,554 validation sets), levels in well-characterised primary BC cohorts. SLC7A5 expression was correlated with clinicopathological and biological parameters, molecular subtypes, and patient outcome.Results: SLC7A5 mRNA and protein expression were strongly correlated with larger tumour size, and higher grade. High expression was observed in triple negative (TN), HER2+, and luminal B subtypes. SLC7A5 mRNA and protein expression was significantly associated with the expression of the key regulator of tumour cell metabolism c-MYC, specifically in Luminal B tumours only (p=0.001). High expression of SLC7A5 mRNA and protein was associated with poor patient outcome (

The amino acid transporter SLC7A11 expression in breast cancer

Breast cancer (BC) is a complex disease with diverse molecular profiles and clinical outcomes, making it challenging to develop effective treatments. Metabolic reprogramming is a hallmark of cancer and SLC7A11, an amino acid transporter, plays a crucial role in this process. This study investigated the role of SLC7A11 in BC using genomic, transcriptomic, and protein analyses.SLC7A11 gene copy number and mRNA expression were evaluated using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n=1,980) and Breast Cancer Gene Expression Miner (n=4,712). SLC7A11 protein was assessed using immunohistochemistry in a large BC cohort (n=1,981). Additionally, The Cancer Genome Atlas (TCGA) dataset was used to explore SLC7A11 DNA methylation patterns using MethSurv (n=782) and association of SLC7A11 mRNA expression with immune infiltrates using TIMER (n=1,100). High SLC7A11 mRNA and SLC7A11 protein expression were significantly associated with high tumor grade (p≤0.02). Interestingly, SLC7A11 copy number gain was observed in HER2+ tumors (p=0.01) whilst SLC7A11 mRNA expression was higher in basal-like/triple-negative (TN) and luminal B tumors (p≤0.02). In contrast, high SLC7A11 protein expression was predominantly observed in Estrogen Receptor (ER)-negative and TN BC. SLC7A11 correlated with other amino acid transporters and glutamine metabolism enzymes and with neutrophil and macrophage infiltration.These findings suggest that SLC7A11 plays a significant role in BC metabolism and may be a potential therapeutic target. Further studies are needed to elucidate its precise mechanisms and explore its therapeutic potential

The Biological and Clinical Significance of Glutaminase in Luminal Breast Cancer

Glutamine metabolism has a key role in the regulation of uncontrolled tumour growth. This study aimed to evaluate the expression and prognostic significance of glutaminase in luminal breast cancer (BC). The glutaminase isoforms (GLS/GLS2) were assessed at genomic/transcriptomic levels, using METABRIC (n=1 398) and GeneMiner datasets (n=4 712), and protein using immunohistochemistry in well characterised cohorts of Oestrogen Receptor-positive/HER2-negative BC patients: ductal carcinoma in situ (DCIS; n=206) and invasive breast cancer (IBC; n=717). Glutaminase expression was associated with clinicopathological features, patient outcome and glutamine-metabolism related genes. In DCIS, GLS alone and GLS+/GLS2- expression was a risk factor for shorter local recurrence-free interval (