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

Altered metabolic pathways have been readily accepted as part of the revised hallmarks of cancer where cancer cells are able to regulate their metabolism to provide energy and cellular building blocks required for their unremitting proliferation [1]. Many cancer cells are highly reliant on amino acids for their growth, not only because they are a nitrogen donor for the synthesis of nucleotides and other amino acids, but also because they activate mammalian target of rapamycin complex1 (mTORC1) through nutrient signalling pathways which in turn regulates protein translation and cell growth [2, 3]. There is also increasing evidence that oncogenes and/or tumour-suppressor genes can reprogramme tumour cell metabolism including the direct regulation of the amino acid transporter, solute carrier family 7 member 5 (SLC7A5), by the oncogene MYC [4, 5] and the regulation of expression of the glutamine transporter, SLC1A5, by the tumour suppressor retinoblastoma (Rb) [6].

SLC7A5 is a sodium-independent transporter and acts as an amino acid exchanger by transporting large neutral amino acids such as leucine, phenylalanine and tryptophan by exchange with intracellular glutamine [7]. It therefore functions in supplying amino acids to cancer cells and maintaining intra-cellular leucine, which is considered a master regulator of the mTORC1 signalling pathway [8‐10]. For functional expression on the plasma membrane, SLC7A5 must heterodimerise with the heavy chain of SLC3A2 [7, 11].

It has been reported that SLC7A5 is highly expressed in a variety of cancers including oesophageal carcinoma [12], oral cancer [13] and lung adenocarcinoma [14]. SLC7A5 is co-expressed with the glutamine transporter, SLC1A5, in many cancers suggesting a functional coupling of these transporters in supporting tumour progression [15]. In this study, we aimed to assess SLC7A5 gene copy number and mRNA expression, alongside protein expression in large and well-characterised annotated cohorts of BC to determine its biological, clinicopathological and prognostic value in the different BC molecular classes with particular interest in the highly proliferative aggressive subgroups.

Breast cancer is a heterogeneous disease with various subtypes [42] differing in terms of morphology, molecular and biological profiles, response to therapy and clinical behaviour. In addition, different subtypes exhibit disparity in their metabolic pathways and their nutritional needs. The most common form of BC (~ 55–80%) is the ER+/luminal tumour [43, 44], and tumours that belong to this class are also variable in terms of recurrence, mortality rates and disease prognosis [43]. Therefore, understanding the biology of BC and exploring the metabolic pathways could help to identify potential novel therapeutic targets.

Cancer cells must alter their metabolism in order to satisfy the demands of necessary energy and cellular building blocks. It is widely known that amino acid transport systems play a principal role in sustaining the proliferation of cancer cells by supplying the required amino acids for protein synthesis and by activation of nutrient signalling through the mTORC1 complex. This study has revealed for the first time that SLC7A5 is a key amino acid transporter in the more aggressive and highly proliferative ER+ tumours.

SLC7A5 is located in 16q24.2. This study showed that SLC7A5 deletion, but not amplification, was significantly correlated with all the annotated genes located in the same chromosomal region, indicating that the deletion was not locus-specific. Interestingly, E-cadherin (CDH1), which was located in 16q22.1, was also implicated. It is widely known that most lobular tumours harbour loss of heterozygosity (LOH) at chromosome 16 and are missing the wild type CDH1 allele [45]. In this study, approximately 40% of METABRIC cases with SLC7A5 loss were associated with invasive lobular histology. In addition, SLC7A5 protein expression in lobular carcinoma has a relatively lower mean rank value compared to the other histological subtypes, confirming that deletions involve large segments of q16, which can reflect the BC phenotype.

SLC7A5 is widely expressed in many human cancers and various cancer cell lines [46]. The current study included two large discovery and validation cohorts to confirm the significant association between the high SLC7A5 protein expression and the poor prognostic clinico-pathological parameters, including larger tumour size, higher grade and poor NPI. Furthermore, high SLC7A5 expression was significantly associated with higher expression of the proliferative marker (Ki67). This supports the results of previous studies which demonstrated that these two biomarkers are significantly correlated in tongue cancer [47], neuroendocrine carcinoma of the lung [48], thymic carcinoma [49] and breast cancer [50], indicating that SLC7A5 is critical for proliferation in cancer cells.

With respect to the breast cancer ER+ subtypes, SLC7A5 expression was lower in ER+ tumours that have low proliferation (luminal A subtype) compared with the highly proliferative ER+ (luminal B) malignancies, and it was primarily associated with poor patient outcome and shorter DMFS in the latter class only. This is most likely due to their heavier energy and nutrient requirements for cell survival, proliferation and metastasis. This was anticipated, as it has been shown that over expression of SLC7A5 is actively involved in the proliferation of vascular smooth muscle cells [51] and it is co-expressed with vascular endothelial growth factor (VEGF) in the primary and metastatic sites of many cancers [37], which may be implicated for the metastatic process. In this study the most significant positive correlation between mRNA expression of SLC7A5 and VEGFA was identified in the luminal B subtype. In this regard, Bartlett et al. included SLC7A5 as a part of the five-gene Mammostrat® immunohistochemistry panel, where the higher expression is used to predict recurrence-free survival (RFS), DMFS and overall survival (OS) in ER+ breast cancer during endocrine therapy [52]. However, they did not consider the different molecular subtypes of BC.

SLC7A5 mRNA and protein was also highly expressed in TNBC and HER2+ BC, in concordance with Furuya et al. [50]. However, in these subtypes the significant association between SLC7A5 protein expression and patient outcome was only observed in the HER2+ tumours. Among all BC subtypes, SLC7A5 protein expression was an independent predictor of short BCSS in ER+ high-proliferation tumours only. In this regard, the larger sample of ER+ high-proliferation cases might reflect the stronger significance compared with the smaller sample of HER2+ and TNBC tumours. We therefore suggest that further confirmation in larger cohorts of HER2+ and TN tumours is warranted.

Previous studies have shown regulation of SLC7A5 by other proteins including the tumour oncogene Myc, which induces SLC7A5 [4, 5]. In the current study, the relationship between SLC7A5 and other regulatory proteins in both mRNA and protein expression was investigated. A positive relationship was observed between SLC7A5 and Myc in both protein and mRNA levels, and this correlation remained significant only in luminal B subtype, when different subtypes were investigated. ATF4-dependent transcripts also encode for SLC7A5 upon amino acid deprivation [39] and in this study there was positive correlation between ATF4 and SLC7A5 gene expression, in line with expectations. A previous study showed that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5 [38] and the current study confirmed the positive correlation between HIF2α and SLC7A5, which was only observed in luminal B tumours. SLC7A5 functions by importing essential amino acids to cancer cells and research has detailed the role of amino acids, particularly leucine, in activating mTORC1, which in turn controls protein translation and cell proliferation, and prevents apoptosis in malignant cells [2, 3]. This study showed positive correlation between SLC7A5 and mTOR at the mRNA level. However, there were conflicting results in the analysis of protein levels of SLC7A5, whereby high SLC7A5 expression was associated with lower expression of the mTORC1 phosphorylated at ser (2448), which was included in this study. This was unsurprising as Cheng et al. confirmed that phosphorylation of mTORC1 at ser (2448), which is stimulated by growth factors, was mutually exclusive with mTORC1 phosphorylated at thr (2446), which is regulated by amino acids [53]. These observations may explain why SLC7A5 over expression is primarily associated with poor outcome only in the high proliferation ER+ tumours.

This study further investigated the association of SLC7A5 expression with other glutamine transporters, in which some variability in the expression of amino acid transporters across molecular subtypes was observed. For example, the TN subtype was the main class associated with the transporters required for glutamine influx, perhaps because it depends on delivery of glutamine instead of synthesis. In contrast, positive correlation between SLC7A5 and the glutamine synthase enzyme GLUL was observed in luminal A tumours, suggesting that this subtype might rely on glutamine neosynthesis rather than uptake. SLC1A5 functionally couples with SLC7A5 to allow the cellular influx and efflux of glutamine, as SLC1A5 mediates uptake of glutamine, while SLC7A5 uses intracellular glutamine concentrations to adjust the essential amino acid cytoplasmic pool for metabolic demands and signalling to mTORC1 [15]. Here we observed that SLC7A5 and SLC1A5 are positively correlated in all the BC subtypes.

Previous studies have raised awareness and revealed the importance of the proline-glutamine (Pro-Gln) regulatory axis in BC. SLC7A5 appears to have a pivotal role in this regulatory axis, as its expression was highly associated with the enzymes that mediate glutamate degradation to form the amino acid proline, which has been shown to play a role in assisting tumour growth by different mechanisms [54].

Blocking of SLC7A5 using its inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), efficiently decreased colony formation of MDA-MB-231 TNBC cells [55]. Even though the consequences of blocking SLC7A5 in the highly proliferative ER+ tumours remain undetermined, this study suggests that SLC7A5 can potentially be used as a therapeutic target for luminal B BC.

This study revealed and confirmed that the glutamine transporter SLC7A5 is associated with poor prognostic characteristics and poor survival outcome. Over expression of SLC7A5 appears to play a role in the proliferation and progression of the aggressive ER+ subtype of breast cancer, thus it could act as a potential therapeutic target. Functional assessment is necessary to reveal the specific role played by this amino acid transporter in the highly proliferative subclass and HER2+ BC.