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

Many cancer cells alter their metabolism to provide energy and cellular building blocks required for their rapid proliferation. Amino acids, particularly glutamine and leucine, are essential for cancer cell growth, as they are critical for controlling protein translation and driving cell cycle progression through regulation of the mammalian target of rapamycin complex1 (mTORC1) pathway [1, 2]. This pivotal need for intracellular amino acids is reflected in the increased expression of amino acid transport systems in the majority of cancers, which is regulated by various transcription factors such as c-MYC, hormone receptors, and nutrient starvation responses [3‐5].

Solute Carrier Family 7 Member 8 (SLC7A8) and Member 5 (SLC7A5) are sodium-independent amino acid exchangers (antiports), which transport small and large neutral amino acids, such as alanine, serine, threonine, cysteine, phenylalanine, tyrosine, leucine, and glutamine [6]. Both solute carriers require heterodimerisation with the heavy chain of SLC3A2 for their proper localisation in the plasma membrane [7‐9].

Although the function of SLC7A8 and SLC7A5 are similar, the former displays relatively lower affinity for its substrates, glutamine, and serine. SLC7A5 has been extensively studied in a variety of cancers and it is regulated by the oncogene c-MYC [10‐13]. We have previously described the potential utility of SLC7A5 as a poor prognostic factor for the highly proliferative breast cancer (BC) subtypes [14]. However, there is limited information whether SLC7A8 plays an equal role in BC. Previous studies showed that SLC7A8 is upregulated in ER+BC and it is controlled by oestrogen [4, 15]. Luo et al. also identified SLC7A8 as a novel progesterone target gene in uterine leiomyoma cells [16]. To our knowledge, the prognostic impact of SLC7A8 has not been studied.

The correlations of SLC7A8 mRNA with other relevant genes were investigated using the METABRIC dataset (Table 3). These genes were selected based on previous publications showing a functional association between SLC7A8 and glutamine transport or metabolism [6, 26‐28]. High SLC7A8 mRNA expression was significantly associated with enzymes involved in glutamine metabolism: glutaminase (GLS and GLS2;P < 0.001), which mediate the conversion of glutamine to glutamate. While the correlation with GLS was negative, it was positive with GLS2. There was also a positive association with the enzymes which mediate conversion of glutamine to proline, namely, ALDH4A1 and PRODH (P < 0.001). In contrast, some glutamine transporters were negatively correlated with SLC7A8 expression (P ≤ 0.009), including SLC1A5, SLC7A5, SLC7A6, SLC7A7, and SLC38A3, while others showed a positive association, including SLC7A9, SLC38A1, SLC38A2, and SLC38A7 (P ≤ 0.003). The associations between SLC7A8 and glutamine metabolic enzymes and transporters were primarily observed within luminal A tumours and to lesser extent in luminal B, HER2+ and TN subtypes. High SLC7A8 mRNA expression was associated with tumours which showed wild-type TP53 expression (P < 0.001, Table 3).

SLC7A8 protein was significantly expressed with high GLS and GLS2 enzymes (P < 0.001 and P = 0.006, Table 4), respectively. High PRODH, ALDH18A1, and ALDH4A1 were expressed in breast tumours with high SLC7A8 expression (all P ≤ 0.004, Table 4). High SLC7A8 expression was associated with low levels of SLC7A5 (P < 0.03, Table 4), whereas paradoxical associations were observed with the other transporters, SLC38A2 and SLC7A11 (P ≤ 0.02, Table 4).

BC represents a group of heterogeneous diseases that vary at the histopathological and molecular levels. These subtypes differ in their biology, clinical outcome, and response to therapy [29]. In addition, different BC subtypes showed disparity in their metabolic profiles and nutritional requirements. ER+/luminal tumours are the predominant BC subtype [30, 31] and characterised by having better prognosis and lower mortality rates as well as being targets for endocrine therapy [32]. In clinical practice, however, recognising patients who are likely to exhibit relapse or distant metastasis is challenging. Therefore, understanding the biology of BC is crucial in the pursuit of identifying targets for treatment and/or prognosis of BC patients particularly those with luminal tumours.

Altered metabolic pathways in human cancers are imperative to support cell proliferation and survival. Amino acid metabolism can vary substantially among BC subtypes, where TNBC display increased activity of amino acid consumption and metabolism compared with ER+ tumours [33, 34], suggesting that the latter subtype may allow for expression of lower levels of amino acid metabolic markers or express solute carriers that have lower affinity for their substrates. This study has revealed, for the first time, that SLC7A8 is a key amino acid transporter in the most predominant low proliferative ER+ tumours.

Unlike SLC7A5, SLC7A8 lacks studies that illustrate its prognostic role in human cancer. Data from Oncomine revealed a significant upregulation of SLC7A8 in several cancers, including breast, colorectal, head and neck, leukaemia, lymphoma, and melanoma [35]. However, this only has been validated at the mRNA level in a subset of breast tumours [15] and melanoma cell lines, which showed however more than five times increase in SLC7A5 expression compared to SLC7A8 [36]. Herein, we used large BC cohorts to reveal the significant associations between the high SLC7A8 expression, at mRNA and protein levels, and good prognostic clinicopathological parameters, including small tumour size, low tumour grade, and good NPI.

With respect to BC subtypes, the lowest levels of SLC7A8 mRNA were observed in the ER- and TNBC tumours, while SLC7A8 was higher in the ER+ subtypes which was more prominent in the luminal A tumours. These results were compatible with other studies which showed that SLC7A8 mRNA was expressed in the ER+, MCF7, cell line but not in the ER−, MDA-MB-231 cells [37]. Furthermore, we have shown that SLC7A8 was associated with better patient outcome and longer DMFS in the ER+ low proliferative tumours only and not in the other subtypes. Thakkar et al. also found that upregulation of SLC7A8 alongside GATA3 and MLPH significantly associated with longer relapse free survival in ER+ lymph node positive breast tumours [15]. These results may suggest that ER+ low proliferative tumours settle for the lower affinity transporter, SLC7A8, to satisfy their nutritional needs as they exhibit lower metabolic activity compared to the aggressive forms of BC.

The relationship between hormone receptor positivity and SLC7A8 indicate that hormone receptors have a possible role in stimulating SLC7A8 expression. It has been shown that 17β estradiol, in ER+ BC cells, regulates l-leucine uptake through SLC7A5 and SLC7A8 while no effect was observed in the ER- BC cells [4]. It has been further shown that SLC7A8 has oestrogen-dependent expression, in ER+ BC cells, and the existence of inhibitors of oestrogen signalling pathway (ICI182780 and tamoxifen) eliminates the oestrogen-induced upregulation of SLC7A8 [15]. Another study also revealed that progesterone significantly upregulates SLC7A8 mRNA and SLC7A8 protein expressions, in uterine leiomyoma tissues, and knockdown of SLC7A8 markedly increased leiomyoma cell proliferation [16].

P53 is a well-known tumour suppressor gene that responds to various stress signals through modulating other cellular processes, including cell cycle arrest and apoptosis. P53 also has a role in mediating other cellular mechanisms such as regulating metabolic pathways, including glutamine metabolism by inducing GLS2 expression [38]. Interestingly, this study showed a positive association between wild-type P53 and SLC7A8. This suggests that SLC7A8 may contribute to P53-dependent tumour suppression, which resulted in the presence of favourable prognosis and patient outcome in tumours expressing high SLC7A8. In addition, SLC7A8 was upregulated alongside two tumour suppressor genes, CEACAM1 and BMP2, in human colon cancer cells after exposure to anti-tumour agent [39].

We have previously showed that SLC7A5 is highly expressed in the aggressive BC subtypes and it was predictive of poor prognosis and poor patient outcome [14] while analysing SLC7A8 in the same cohort has resulted in opposite findings. Furthermore, this study showed that SLC7A8 at both mRNA and protein levels were mutually exclusive with SLC7A5 expression. It is noteworthy that although SLC7A5 and SLC7A8 have similar substrate selectivity and function, the latter has narrower tissue expression pattern and exhibits lower affinity to its substrates [6]. There is also evidence which suggests that the role of SLC7A8 is limited to equilibration of amino acids distribution across the cell membrane while SLC7A5 mediates the actual net of amino acid flux [40], which is required for further mTORC1 activation and cellular proliferation.

Furthermore, it seems that both solute carriers have contrasting effect in tumourigenesis and they could be a subject of different regulatory mechanisms, as SLC7A5 expression is induced by c-MYC while hormone receptors appear to control SLC7A8 expression [4, 14, 16].

This study further investigated the association of SLC7A8 expression with other solute carriers which involved in amino acid transport. While the majority of these transporters were negatively associated with SLC7A8, others were positively correlated, including SLC38A7 which showed consistent correlation in all BC subtypes. Positive associations with several glutamine metabolic enzymes were also detected, among these, GLS2 and glutamate dehydrogenase (GLUD1) which are associated with tumours of good prognosis and favourable patient outcome [21, 41]. Some variability in the expression of investigated markers across molecular subtypes was observed. For example, luminal A tumours were the main class which showed association between SLC7A8 and markers required for glutamine transport and metabolism. This could be attributed to the increased SLC7A8 expression and function in this particular BC subtype.

It is noteworthy that different transcriptional pathways such as c-MYC oncogenic transcription, hormone receptors and nutrient starvation responses regulate the expression of amino acid transporters in human cancers [27, 35]. This also applies to SLC7A5 and SLC7A8, as both belong to the system L transport family but their expression is controlled by different pathways. Furthermore, both solute carriers mediate the uptake of large neutral amino acids, with the latter showing decreased transporting capacity. However, SLC7A8 can also accept smaller neutral amino acids, such as glycine, alanine, serine, cysteine, and glutamine [42], which are known substrates of the key system A amino acid transporter, SLC1A5 that is highly expressed in TNBC [6, 43]. These statements indicate that SLC7A8 could be a key transporter in luminal A tumours through determining the actual net flux of not only the large but also the small neutral amino acids.

This study has revealed that the solute carrier SLC7A8 is an independent good prognostic marker in BC. Overexpression of SLC7A8 appears to have tumour suppressive characteristics especially in the low proliferative ER+ subtype, thus it could act as a potential prognostic factor. Functional assessment is necessary to reveal the specific role played by this amino acid transporter in the low proliferative ER+ tumours.