Association of L-type amino acid transporter 1 (LAT1) with the immune system and prognosis in invasive breast cancer

L-type amino acid transporter 1 (LAT1), also referred to as SLC7A5, is believed to regulate tumor metabolism and be associated with tumor proliferation. In invasive breast cancer, we clinicopathologically investigated the utility of LAT1 expression. LAT1 expression was evaluated via immunohistochemistry analyses in 250 breast cancer patients undergoing long-term follow-up. We assessed the relationships between LAT1 expression and patient outcomes and clinicopathological factors. Breast cancer-specific survival stratified by LAT1 expression was assessed. Human epidermal growth factor receptor 2 (HER2)-positive patients with metastasis received trastuzumab therapy. The density of tumor-infiltrating lymphocytes (TILs) was evaluated according to the International Working Group guidelines. In the current study, high LAT1 expression was significantly correlated with estrogen receptor (ER) negativity, progesterone receptor negativity, high histological grade, increased TILs, and programmed death ligand 1 positivity. Among the ER-positive and HER2-negative patients, high LAT1 was an independent indicator of poor outcomes (hazard ratio (HR) = 2.97; 95% confidence interval (CI), 1.16–7.62; p = 0.023). Moreover, high LAT1 expression was an independent poor prognostic factor in luminal B-like breast cancer with aggressive features (HR = 3.39; 95% CI 1.35–8.52; p = 0.0094). In conclusion, high LAT1 expression could be used to identify a subgroup of invasive breast cancer characterized by aggressive behavior and high tumor immunoreaction. Our findings suggest that LAT1 might be a candidate therapeutic target for breast cancer patients, particularly those with luminal B-like type breast cancer.

www.nature.com/scientificreports/ cell proliferation via the mTOR pathway 3 . The L-type amino acid transporter (LAT) enables the transport of large neutral essential amino acids into cells 4,5 . Interestingly, LAT1, encoded by the SLC7A5 gene, is generally overexpressed in malignant cells [6][7][8][9][10][11][12][13][14][15] . High LAT1 expression is closely related to the proliferation of tumor cells and angiogenesis in various types of cancer, such as melanoma 16 , lung cancer 17 , pancreatic cancer 18 , gastrointestinal cancer 19,20 , and triple-negative BCa (TNBC) 21 . LAT1 overexpression is also associated with lymphovascular invasion, lymphatic metastasis, and advanced stages of cancer 22 and contributes to the development of therapeutic resistance in cancer cells. A previous large BCa study by El Ansari et al. 23 reported that a high LAT1 expression level was associated with high proliferation potential, as indicated by the high Nottingham prognostic index and Ki67 labeling index; moreover, high LAT1 expression level was a poor prognostic factor in luminal B-like type breast tumors.
The immune system affects all phases of tumor growth from initiation to progression and dissemination. In addition, previous studies confirmed that tumor immunity-associated biomarkers, such as tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1), are related to the treatment response and prognosis of BCa [24][25][26][27] . Recently, El Ansari et al. 28 reported that LAT1 expression was associated with the expressions of PD-L1, PD1, FOXP3, CD68 and CD20 in the breast tumor microenvironment.
In this study, we attempted to validate the relationship between LAT1 expression and patient outcome. We also confirmed the correlation between LAT1 protein expression and key clinicopathological factors, including TILs and PD-L1, in BCa patients.
Prognostic analysis of SLC7A5 mRNA based on ER status. To obtain insight into how LAT1 might be linked to survival, we evaluated the prognostic utility of SLC7A5 mRNA expression according to ER status using the TCGA database. Among patients with ER-positive cancer, those with high SLC7A5-expressing tumors had a significantly lower BCSS than those with low SLC7A5-expressing tumors (HR = 2.13; 95% CI 1.22-3.71; p = 0.0075). In contrast, SLC7A5 expression was not a prognostic factor among patients with ER-negative cancer ( Supplementary Fig. 2). At the protein level, high LAT1 expression was a significant prognostic marker in the ER-positive type (HR = 2.22; 95% CI 1.07-4.59; p = 0.032) but not in the ER-negative type ( Supplementary  Fig. 2). Moreover, LAT1 expression was not a significant prognostic marker in the HER2-positive type (Supplementary Fig. 3).

Prognostic utility of LAT1 expression in ER-positive and HER2-negative BCa.
In the 142 ERpositive and HER2-negative tumors, we investigated which factors were associated with LAT1 staining. The frequency of high LAT1 expression was 39.4% in luminal-like tumors. Among luminal-like tumors, high LAT1 expression was significantly correlated with tumor size (p = 0.018), high Ki67 labeling index (p = 0.0017), and high histological grade (p = 0.018) ( Table 2). In patients with luminal-like cancer, those with high LAT1-expressing tumors had a significantly lower BCSS than those with low LAT1-expressing tumors (HR = 2.86; 95% CI 1.26-6.48; p = 0.012; Fig. 2a).

Discussion
The current study demonstrates that high LAT1 expression can be used to identify a subgroup of invasive BCa with aggressive behavior and high tumor immune reaction (PD-L1 positivity and TIL upregulation). The present study indicated that high LAT1 expression was an independent poor prognostic factor in luminal B-like BCa with long term follow-up. The indications for chemotherapy in ER-positive/HER2-negative breast cancer patients have been debated for many years [29][30][31] . At the St. Gallen consensus conference 32 , Ki67 and PgR expression levels were suggested to be important factors to consider for postoperative chemotherapy. The St. Gallen consensus meeting recommended that hormone receptor-positive/HER2-negative breast cancer should be divided into the luminal A-like type (high ER/PgR and clearly low Ki67) and the luminal B-like type (low ER/PgR and clearly high Ki67) 32 . Adjuvant chemotherapy combined with endocrine treatment was recommended for luminal B-like type tumors. Recently, cyclin dependent kinase inhibitors have been suggested to be effective in ER-positive/HER2-negative BCa with a high risk of recurrence 33 . However, effective molecular targets for luminal B-like type tumors have not yet been fully identified as alternatives to chemotherapy. In this study, LAT1 expression was related to the expression of Myc and the VEGF family. VEGF and Myc may be related to proliferation in luminal B-like BCa 34 . LAT1 contributes to angiogenesis in cancer in the presence of VEGF 35 . Moreover, Myc asserts its oncogenic functions  www.nature.com/scientificreports/ partially through its control of LAT1 expression 36 . The results of this study suggest that the molecular pathways involving LAT1 are related to the proliferation or metastasis abilities of luminal B-like BCa. It may be possible to support the development of new drugs targeted against luminal B-like BCa by conducting a more detailed functional analysis of the genes related to this molecular pathway. El Ansari et al. 28 clarified that glutamine transporters, including LAT1, are associated with the expression of CD68-positive macrophages and PD1-positive lymphocytes in tumors. Moreover, they used TNBC cell lines to demonstrate that the inhibition of LAT1 reduced the expression of PD-L1. HIF1α is known to activate tumorassociated CD68-positive macrophages 37 . Thus, LAT1 may be involved in the function of tumor-associated macrophages because it enhances the function of mTORC1 by controlling the HIF pathway 38 . The molecular pathway related to mTOR plays an essential role in the progression of BCa 39 . Although an mTOR inhibitor is used to treat metastatic ER-positive BCa in clinical practice 40 , its effectiveness in early-stage BCa remains to be elucidated. Recent investigations have observed an antitumor effect of using a LAT1 inhibitor because it suppresses the phosphoric acid of mTOR in tumor cells, inhibits its downstream cell proliferation signals, and elicits G1 arrest and apoptosis 41 . The mTOR pathway suppresses Treg cells and promotes the differentiation of CD8positive T cells 42,43 . There are ongoing clinical trials for the combination of PI3K and PD-L1 inhibitors in TNBC 44 .
To determine how LAT1 works in antitumor immune reactions, additional functional studies are necessary.
The present study revealed that high LAT1 expression was significantly associated with PD-L1 positivity at the protein level but not at the mRNA level. In recent years, with the rapid development and widespread use of comprehensive genomic analysis methods using microarrays and next-generation high-speed sequencing, candidate factors that may play a role in the prognosis and drug efficacy of BCa have been discovered from the analysis of vast amounts of genomic and transcriptomic information. However, although genomic and transcriptomic information is excellent for assessing the expression intensity of targets as continuous variables, it is difficult to determine how the factors are localized in the cells. The expression of PD-L1 in tumor cells is heterogeneous. It may bind not only to PD-L1-positive cancer cells but also to PD-L1-positive lymphocytes. In a recent study, PD-L1 expression on tumor cells was associated with high-risk clinicopathological parameters and poor prognosis, while PD-L1 expression on TILs was associated with favorable survival outcomes 45 . Further studies are needed to clarify the relationship between PD-L1 expression in tumor cells and lymphocytes and LAT1 expression in tumor cells.
In conclusion, LAT1 expression was associated with immune-related biomarkers, such as TILs and PD-L1, and was strongly correlated with poorly differentiated tumors. We evaluated TILs grade based on the guidelines of the International Working Group. Moreover, we examined the relationship of TILs and PD-L1 with LAT1 using the recent clinical breast cancer samples. These findings indicate that LAT1 may play important roles in antitumor immunity and may promote BCa progression and metastasis, particularly in ER-positive and HER2-negative BCa. This study has several limitations. First, the number of enrolled patients was small. Second, this study was a retrospective trial. Further biological research regarding the ability of this new agent to inhibit LAT1 expression is warranted 9 . Moreover, concomitant treatment using LAT1 inhibitors and immune checkpoint inhibitors 46 is expected to become an innovative therapeutic target in the luminal B-like type of BCa. Table 3. Survival analysis based on clinicopathological factors, including LAT1 protein expression, in ER-positive/HER2-negative patients. LAT1 L-type amino acid transporter 1, ER estrogen receptor, PgR progesterone receptor, HER2 human epidermal growth factor receptor 2.  29,31 . The ER positivity (≥ 1%) rate was 67.6%; 58.0% of the patients were PgR-positive (≥ 20%), and 17.2% were HER2-positive. The cohort was classified according to the intrinsic molecular subtypes (luminal A-like, luminal B-like, HER2positive, and triple-negative types). The luminal A-like type was defined as patients who are PgR-positive and display low Ki67 staining (labeling index of ≤ 10%) in ER-positive and HER2-negative breast cancer, whereas other ER-positive and HER2-negative tumors were classified as the luminal B-like type.
The density of stromal TILs was evaluated according to the International Working Group guidelines 24,47 . Cytoplasmic and/or membranous PD-L1 expression was assessed by immunohistochemistry (SP142; Roche, Switzerland; diluted 1:50), and the PD-L1 positivity cutoff value was determined to be 1%. Detailed assessments of these biomarkers have been described in our previous paper 25 . LAT1 immunohistochemistry. LAT1 expression was assessed by immunohistochemistry using an affinity-purified polyclonal rabbit anti-human LAT1 antibody 6 diluted to 1:5000. LAT1 protein expression was evaluated for cytoplasmic and membrane-associated staining in full-face slides obtained from 250 patients. For clinicopathological and prognostic analyses, the 250 samples were stratified into high-and low-LAT1 expression groups based on staining intensity.
The staining intensity of LAT1 expression on cancer cells was scored as follows: 0 (no staining or staining of < 10% of tumor cells), 1 (weak staining of ≥ 10% of tumor cells), 2 (moderate staining of ≥ 10% of tumor cells), and 3 (strong staining of ≥ 10% of tumor cells). In addition, tumors with a score of 2 or 3 were assigned to the high LAT1 expression group, whereas tumors with a score of 0 or 1 were assigned to the low LAT1 expression group (Fig. 3a-d: intensity score 0-3).
Statistical analysis. Statistical analyses were performed using SPSS statistical software v24.0 (IBM, Armonk, NY, USA). The relationships between LAT1 expression and several clinicopathological factors were evaluated using the chi-square test and Fisher's exact test. BCSS was used to evaluate the prognostic utility of www.nature.com/scientificreports/ LAT1 expression. For the univariable and multivariable prognostic assessments of several clinicopathologically important parameters (LAT1, tumor size, nodal status, histological grade, Ki67 labeling index, ER, PgR, HER2, and TILs), the Cox proportional hazards regression model was used to calculate HRs and 95% CIs. The prognostic value of LAT1 mRNA (SLC7A5) expression was further evaluated using the TCGA BRCA dataset as an external validation cohort. In brief, the datasets of mRNA expression from RNA-Seq V2 were accessed along with deidentified clinical information for several clinicopathological factors and outcomes 48,49 . The median value of SLC7A5 expression was defined as the cutoff point.
Ethical approval and informed consent. This study was approved by the Saitama Cancer Center Institutional Review Board (reference number 738). All procedures performed in studies involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from the participants included in this study.

Data availability
The datasets generated and/or analyzed during the current study are not publicly available due to the regulation of the Institutional Review Board of the Saitama Cancer Center but are available from the corresponding author on reasonable request.