Prognostic impact of co-expression of LLCL2 and SLC7A5 in ERα-positive breast cancer patients

doi:10.21203/rs.3.rs-728950/v1

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Prognostic impact of co-expression of LLCL2 and SLC7A5 in ERα-positive breast cancer patients

https://doi.org/10.21203/rs.3.rs-728950/v1

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Background

Lethal giant larvae homolog 2 (LLGL2) functions as a promoter of tumor growth and localizes at cell junctions and membranes with solute carrier family 7 member 5 (SLC7A5) in estrogen receptor α (ERα)-positive breast cancer. LLGL2 and SLC7A5 have been reported to be involved in resistance to endocrine therapy. This study aimed to assess the effects of LLGL2/SLC7A5 co-expression in predicting prognosis and response to endocrine therapy in ERα-positive breast cancer patients.

Methods

The associations of clinicopathological factors with LLGL2 and SLC7A5 expression or LLGL2/SLC7A5 co-expression at the mRNA and protein level were assessed in invasive breast cancer patients with long-term follow-up. The median follow-up period was approximately10 years. Survival curves were analyzed using the Kaplan–Meier method and verified by the log-rank test. A Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values using stepwise linear regression.

Results

We identified a positive association between low mRNA expression of LLGL2 or SLC7A5 alone and longer disease-free survival (DFS) and overall survival (OS) in ERα-positive breast cancer patients, but not in ERα-negative patients. We also identified that low LLGL2/SLC7A5 mRNA co-expression (LLGL2^low/SLC7A5^low) was associated with longer survival compared with other combination groups in all breast cancer patients. In ERα-positive breast cancer patients, LLGL2^low/SLC7A5^low showed longer survival compared with LLGL2^high/SLC7A5^high and a positive trend of longer survival compared with other combination groups. We also observed that LLGL2^low/SLC7A5^low showed longer survival compared with LLGL2^high/SLC7A5^high in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy. Multivariate analysis demonstrated that LLGL2^low/SLC7A5^low was an independent favorable prognostic factor of both DFS and OS in ERα-positive breast cancer patients. High co-expression of LLGL2 and SLC7A5 protein showed a positive trend of shorter survival.

Conclusions

Our study showed that co-expression of LLGL2 and SLC7A5 mRNA is a promising candidate biomarker and suggested that the LLGL2–SLC7A5 axis may be a therapeutic target in early breast cancer patients, especially in those receiving adjuvant tamoxifen therapy.

breast cancer

LLGL2

SLC7A5

Breast cancer remains a leading cause of cancer-related death in women. Approximately 70% of all breast cancer cases express estrogen receptor α (ERα) [1, 2]. In ERα-positive breast cancers, estradiol is a critical regulator of cell proliferation and survival. Estradiol directly regulates genes through binding to ERα or indirectly through activating plasma membrane-associated ERα. Treatment options for ERα-positive breast cancer patients include endocrine therapies that inhibit ERα signaling, either by antagonizing ligand binding to ERα, downregulating ERα, or suppressing estrogen production [3].

Tamoxifen is one of the most common endocrine treatments for breast cancer. Tamoxifen treatment was demonstrated to reduce risk of breast cancer recurrence and death in ERα-positive breast cancer patients [4]. Although endocrine therapy has dramatically improved survival in ERα-positive breast cancer patients, some tumors show de novo or acquired drug resistance to endocrine therapy [5–7]. Resistance to endocrine therapies including tamoxifen remains a major challenge in the treatment of ERα-positive breast cancer patients.

Recently, Saito et al. reported that lethal giant larvae homolog 2 (LLGL2) functions as a promoter of tumor growth in ERα-positive breast cancer [8]. They reported that the intracellular concentration of leucine decreased in MCF-7 ERα-positive breast cancer cells when LLGL2 expression was knocked down. They also reported that the proliferation of MCF-7 cells was suppressed when LLGL2 expression was knocked down, and that excess leucine could rescue the proliferation of LLGL2-knockdown cells [8].

LLGL2 is reported to be localized at cell junctions and membranes with a member of the solute carrier (SLC) family, SLC7A5, which is the primary leucine transporter in cells [8, 9]. SLC7A5 is a sodium-independent amino acid transporter that imports leucine [10]. High SLC7A5 expression was reported to be associated with poor prognosis in various cancers including breast cancer [11–15] .

LLGL2 and SLC7A5 are involved in resistance to tamoxifen treatment [8, 12, 16]. LLGL2 was also reported to function with SLC7A5 at cell junctions and membranes in ERα-positive breast cancer cells, and LLGL2 interacts with SLC7A5 to promote cell proliferation [8]. Therefore, we hypothesized that both LLGL2 and SLC7A5 are required for the efficacy of tamoxifen treatment in ERα-positive breast cancer patients.

In this study, we assessed the effects of LLGL2/SLC7A5 co-expression in predicting prognosis and response to endocrine therapy in ERα-positive breast cancer patients with long-term follow up.

Patients and samples

A total of 626 consecutive invasive breast cancer tissue samples collected between 1992 and 2008 from the archive of the Department of Breast Surgery, Nagoya City University Hospital, Japan, were included in this study to measure LLGL2 and SLC7A5 mRNA expression. Furthermore, 415 consecutive invasive breast cancer tissues collected between 2000 and 2009 as tissue microarrays were included to evaluate LLGL2 and SLC7A5 protein expression. The tissues were fixed in 10% buffered formalin and embedded in paraffin or snap-frozen in liquid nitrogen immediately after resection and stored at − 80ºC until RNA extraction. The histological grade was estimated according to the Bloom and Richardson method proposed by Elston and Ellis [17]. Disease-free survival (DFS) was defined as the interval from the date of primary surgery to the earliest occurrence of one of the following: locoregional recurrence, distant metastasis, or death from any cause. Overall survival (OS) was defined as the interval from the date of primary surgery to death from any cause. The median follow-up period was 10.1 years (range, 0.2–215.1 months) and 9.7 years (range, 0.8–215.1 months) for mRNA and protein expression analyses, respectively. Written informed consent for comprehensive research use was obtained from all patients before surgery. This protocol was approved by the institutional review board of Nagoya City University Graduate School of Medical Sciences and conformed to the guidelines of the Declaration of Helsinki.

RNA extraction and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Tokyo, Japan) in accordance with the manufacturer’s protocol. The quantity of RNA extracted from breast cancer tissues was evaluated using a DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. TaqMan Gene Expression assays (Thermo Fisher Scientific) were used to measure the mRNA expression of LLGL2 and SLC7A5 as well as that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Duplex quantitative RT-PCR assays were performed using the StepOnePlus real-time PCR system (Thermo Fisher Scientific). The reaction was analyzed using a FAM-labeled probe for LLGL2 or SLC7A5 (Thermo Fisher Scientific) and a VIC-labeled probe for GAPDH (Thermo Fisher Scientific) as a single assay for each sample. The amplification reaction mixture comprised 2 µL cDNA, 10 µL TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), 1 µL TaqMan Gene Expression assay for LLGL2 or SLC7A5, 1 µL TaqMan Gene Expression assay for GAPDH, and 6 µL DEPC-treated water (Thermo Fisher Scientific) in a total volume of 20 µL. The PCR conditions used for LLGL2 or SLC7A5 and GAPDH were 50°C for 2 min and 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 1 s and annealing and extending at 60°C for 20 s. Samples were amplified independently in duplicate. Results were converted into relative concentrations using a standard curve. The relative concentration of LLGL2 or SLC7A5 mRNA for each sample was normalized to that of GAPDH mRNA expression as an internal control to account for variations in the amount of input cDNA.

We performed a receiver operating characteristic (ROC) curve analysis to determine the cut-off value of LLGL2 and SLC7A5 mRNA. We used Youden’s index (sensitivity + specificity − 1), which corresponded to a point on the ROC curve with the highest vertical distance from the 45° diagonal line. The area under the curve (AUC) was 0.51 and 0.57 when ROC analysis was performed for DFS according to the level of LLGL2 and SLC7A5 mRNA expression, respectively ( Supplementary Figure S1 and S2). As a result of this analysis, we determined the cut-off values for LLGL2 and SLC7A5 mRNA expression to be 0.815 and 0.54, respectively.

Immunohistochemistry (IHC) of ERα, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2)

One 4-µm-thick section from each paraffin-embedded specimen was first stained with hematoxylin and eosin to ascertain whether an adequate number of invasive ductal carcinoma cells were present and that the quality of fixation was adequate for immunohistochemical analysis. Serial sections (4-µm-thick) were then prepared from suitable tissue blocks and float-mounted on adhesive-coated glass slides for ERα (Dako Envision FLEX-ER, EP1; Agilent Technologies, Santa Clara, CA, United States), PgR (Dako Envision FLEX-ER, PgR636; Agilent Technologies), and HER2 (HercepTest II; Agilent Technologies) staining. Staining of these hormone receptors was performed using the Autostainer Link 48 (Agilent Technologies). Immunostained specimens were scored after the entire section had been evaluated by light microscopy. ERα and PgR expression was evaluated by the percentage of cells with positive nuclear staining. A positive nuclear staining ratio of ≥ 1/100 was considered positive. Scoring of HER2 expression was based on the membrane staining pattern and was scored on a scale of 0–3+. Tumors with scores of 0 or 1 were considered negative for HER2 overexpression, and those with a score of 3 were considered positive. Tumors with a score of 2 + were tested for gene amplification by fluorescence in situ hybridization (FISH) using the PathVysion assay (Vysis; Abbott Laboratories, Abbott Park, IL, USA) in accordance with the manufacturer’s protocol. A ratio of > 2.0 for HER22 gene/chromosome 17 was considered positive. Tumors were considered HER2-positive if immunohistochemical staining was 3 + or positive by FISH.

Ihc Of Llgl2 And Slc7a5

For the immunohistochemical analysis of LLGL2 and SLC7A5, tissue microarrays on 2-mm-diameter slides were prepared after confirming whether an appropriate number of invasive ductal carcinoma cells were present and whether the fixation quality was suitable for immunohistochemical analysis. The primary antibodies for LLGL2 and SLC7A5 protein were rabbit monoclonal anti-LLGL2 antibody (Santa Cruz Biotechnology, CA, USA) and rabbit monoclonal anti-SLC7A5/LAT1 antibody (Abcam, Cambridge, UK) at 1:300 and 1:100 dilution, respectively. Immunostaining was performed using the Leica Bond-Max automated system and Leica Refine detection kits (Leica Biosystems).

Tissue microarray slides were scanned at ×20 magnification using an Aperio scanscopeCS2 (Leica Biosystems, San Diego, CA, USA). At least 1,000 tumor cells were evaluated in each tissue core. The protein expression level of LLGL2 was evaluated according to H-score [18] using the eSlide manager application, a digital pathological system (Leica Biosystems). H-score was calculated by classifying the immunostaining intensity into three categories (weak staining, 1; moderate, 2; and strong, 3) and adding the evaluation of the proportion of each staining. The threshold optical density for each staining intensity was defined as 210, 180, and 150, respectively. H-score was assigned using the following formula: [1× (% cells with category 1) + 2 × (% cells with category 2) + 3 × (% cells with category 3)] [18].

SLC7A5 protein expression was localized to the cell membrane, and therefore SLC7A5 immunostaining was evaluated in accordance with the same method employed by the HercepTest (Dako) for HER2 immunostaining, as described above. Scoring of SLC7A5 expression was based on the membrane staining pattern and scored on a scale of 0–3+ [19, 20].

Statistical analysis

The associations of LLGL2 and SLC7A5 mRNA expression with clinicopathological factors were assessed by χ² and Fisher’s exact probability tests. Survival curves were analyzed using the Kaplan–Meier method and verified by the log-rank test. DFS was censored at the date of last follow up if patients were still relapse-free and alive, and OS was censored at the time when patients were alive. A Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values using the stepwise variable selection method. The level of statistical significance was set at a P-value of less than 5%. Multiple survival curves were compared by the log-rank test with Bonferroni adjustment. In this study, there were three comparisons: LLGL2^high/SLC7A5^high vs LLGL2^low/SLC7A5^low; LLGL2^high/SLC7A5^low vs LLGL2^low/SLC7A5^low; and LLGL2^low/SLC7A5^high vs LLGL2^low/SLC7A5^low. Therefore, a two-sided P-value of 0.017 (0.05/3) was taken to indicate statistical significance. Missing data points were excluded from the analysis. Statistical calculations were performed with JMP12.2 software (SAS Institute, Inc., Cary, NC, USA).

LLGL2 mRNA expression and prognosis of breast cancer patients

We first investigated the association between LLGL2 mRNA expression level and prognosis of breast cancer patients with long-term follow up. A total of 624 breast cancer tissue samples were subjected to LLGL2 mRNA expression analysis. The associations between LLGL2 mRNA expression and clinicopathological characteristics are shown in Supplementary Table S1. Low LLGL2 mRNA levels were positively associated with larger tumor size (P = 0.047) and lymph node-negativity (P = 0.031). Low LLGL2 mRNA expression was positively associated with longer DFS in all breast cancer patients analyzed in this study (P = 0.023; Supplementary Figure S3). Furthermore, patients with tumors showing low LLGL2 mRNA expression showed a tendency towards longer OS (P = 0.072; Supplementary Figure S3).

LLGL2 was reported to be involved in prognosis only in ERα-positive breast cancer patients [8]. Therefore, we next investigated the association of LLGL2 mRNA expression with prognosis according to ERα status. As shown in Fig. 1A and B, positive associations were found between low LLGL2 mRNA expression and longer DFS and OS in ERα-positive breast cancer patients (n = 491; P = 0.0009 and P = 0.005, respectively). However, no association was observed between LLGL2 mRNA expression and prognosis in ERα-negative breast cancer patients (n = 132; Supplementary Figure S3). The clinicopathological characteristics of ERα-positive breast cancer patients are shown in Table 1. Low LLGL2 mRNA expression level was positively associated with lymph node negativity (P = 0.007).

LLGL2 was reported to be involved in resistance to tamoxifen in ERα-positive breast cancer patients [8]. Therefore, we investigated the association of LLGL2 mRNA expression with prognosis in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy (n = 272). As shown in Fig. 1C and D, positive associations were found between low LLGL2 mRNA expression and longer DFS and OS in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy (P = 0.016 and P = 0.018, respectively). Interestingly, no associations were identified between LLGL2 mRNA expression level and prognosis in ERα-positive breast cancer patients without adjuvant tamoxifen therapy (Supplementary Figure S3).

We next performed univariate and multivariate Cox regression analyses of clinicopathological factors associated with prognosis using stepwise linear regression in all breast cancer patients (Supplementary Table S2) and in ERα-positive breast cancer patients analyzed in this study (Table 1). Although low LLGL2 mRNA expression was not an independent favorable prognostic factor in all breast cancer patients, we showed that low LLGL2 was an independent favorable prognostic factor for both DFS and OS in ERα-positive breast cancer patients, as well as nodal status (P = 0.012 and P = 0.011, respectively; Table 2).

SLC7A5 mRNA expression and prognosis of breast cancer patients

Next, we investigated the association between SLC7A5 mRNA expression and prognosis of breast cancer patients. The characteristics of ERα-positive breast cancer patients according to SLC7A5 mRNA expression are shown in Supplementary Table S3. Low SLC7A5 mRNA expression was positively associated with favorable prognosis in both DFS and OS in all breast cancer patients analyzed (P = 0.002 and P = 0.0005, respectively). Low SLC7A5 mRNA expression was also positively associated with favorable prognosis in both DFS and OS in ERα-positive breast cancer patients (P = 0.004 and P = 0.004, respectively; Fig. 2A and B). However, no association was observed in ERα-negative breast cancer patients (Supplementary Figure S4). As shown in Fig. 2C, positive associations were identified between low SLC7A5 mRNA expression and longer DFS in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy (P = 0.014).

Combination of LLGL2 and SLC7A5 mRNA expression and prognosis of breast cancer patients

We then investigated the prognostic impact of the combination of LLGL2 and SLC7A5 mRNA expression. Supplementary Table S4 shows the characteristics of breast cancer patients classified by the combination of LLGL2 and SLC7A5 mRNA expression. Low LLGL2/SLC7A5 mRNA co-expression (LLGL2^low/SLC7A5^low) was positively associated with lower tumor grade, lymph node negativity, and ERα positivity. As shown in Fig. 3A and B, LLGL2^low/SLC7A5^low was associated with longer survival compared with other combination groups in all breast cancer patients analyzed in this study. The characteristics of ERα-positive breast cancer patients according to LLGL2/SLC7A5 mRNA co-expression are shown in Table 3. LLGL2^low/SLC7A5^low was associated with lower grade and lymph node negativity in ERα-positive breast cancer patients. As shown in Fig. 3C and D, LLGL2^low/SLC7A5^low showed longer survival compared with high LLGL2/SLC7A5 mRNA co-expression (LLGL2^high/SLC7A5^high) and a positive trend of longer survival compared with other combination groups in ERα-positive breast cancer patients. Then, we investigated the association of prognosis with the combination of LLGL2 and SLC7A5 mRNA expression in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy. As shown in Fig. 3E and F, LLGL2^low/SLC7A5^low showed longer survival than LLGL2^high/SLC7A5^high and a positive trend of longer survival compared with other combination groups in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy. However, no significant difference was observed between these four combination groups in ERα-positive breast cancer patients who had not received adjuvant tamoxifen therapy (Supplementary Figure S5).

We performed univariate and multivariate Cox regression analyses of clinicopathological factors associated with prognosis using stepwise linear regression in each group of breast cancer patients. Multivariate analyses demonstrated that LLGL2^low/SLC7A5^low was an independent favorable prognostic factor for DFS as well as lymph node negativity and ERα positivity in all breast cancer patients analyzed (Supplementary Table S5). Then, we performed univariate and multivariate analyses in ERα-positive breast cancer patients, which identified LLGL2^low/SLC7A5^low as an independent favorable prognostic factor for both DFS and OS, as well as lymph node negativity (Table 4).

Protein expression of LLGL2 and SLC7A5 in breast cancer patients

The expression levels of LLGL2 protein in breast cancer tissue samples were examined by IHC. LLGL2 protein expression was observed in the cytoplasm. Representative images of LLGL2 and SLC7A5 are as shown in Fig. 4A. Immunostaining results were evaluated using the Aperio scanscopeCS2 and eSlide manager application, and H-scores were calculated by this digital pathological system. In this study, a total of 285 consecutive breast cancer tissue samples for which mRNA expression data were available were analyzed for LLGL2 protein expression.

We also evaluated the expression levels of SLC7A5 protein in breast cancer tissue samples. The analysis was performed using the same tissue microarray as the protein expression analysis of LLGL2. SLC7A5 protein expression was observed in the cell membrane (Fig. 4A). Furthermore, SLC7A5 protein expression was observed in 10% of breast cancer tissues. We investigated the association between prognosis and the combination of LLGL2 and SLC7A5 protein expression. The LLGL2^high/SLC7A5^pos group seemed to show the worst prognosis among the four groups, although there was no statistically significant difference between them (Fig. 4B). The median H-score was used as the cutoff value for LLGL2, and SLC7A5 was divided into two groups based on the presence or absence of staining.

In this study, we evaluated the prognostic value of co-expression of LLGL2 and SLC7A5 in primary breast cancer patients with long-term follow up. First, we showed that low LLGL2 or low SLC7A5 mRNA expression was an independent favorable prognostic factor in ERα-positive breast cancer patients. Second, we showed that low LLGL2/SLC7A5 mRNA co-expression (LLGL2^low/SLC7A5^low) was also an independent favorable prognostic factor both in all breast cancer patients and in ERα-positive breast cancer patients. We also observed that LLGL2^low/SLC7A5^low showed longer survival compared with LLGL2^high/SLC7A5^high and a positive trend of longer survival compared with other combination groups in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy.

Saito et al. recently reported that LLGL2 promoted leucine uptake and conferred tumor growth and resistance to tamoxifen treatment by increasing the cell surface level of SLC7A5 in ERα-positive breast cancer. They also reported that low LLGL2 mRNA expression was a favorable prognostic factor in ERα-positive breast cancer [8]. In this study, our data supported the report by Saito et al [8]. However, LLGL2 was originally discovered as a tumor suppressor protein in Drosophila. In colorectal cancer and ERα-negative breast cancer, downregulation of LLGL2 was shown to be involved in cancer progression [21–23]. LLGL2 was reported to act as a suppressor for epithelial–mesenchymal transition (EMT), and to interact with the EMT-related transcription factors such as SNAIL and ZEB1 [21, 22]. EMT is generally a characteristic of ERα-negative breast cancer [24]. In our study, ERα-negative breast cancer patients with tumors showing high LLGL2 mRNA expression appeared to show a better prognosis compared with those with tumors showing low LLGL2 mRNA expression. Therefore, our findings were consistent with previous reports of colorectal cancer and ERα-negative breast cancer [21, 23]. Thus, previous reports and our data suggested that the functions of LLGL2 could be completely different between ERα-positive tumors and ERα-negative tumors.

In this study, we showed that low SLC7A5 mRNA expression was positively associated with favorable prognosis in ERα-positive breast cancer patients but not in ERα-negative breast cancer patients. Our data in this study supported the previous report by Ansari et al. [11]. Although SLC7A5 is a systemic L amino acid transporter that carries branch-chain amino acids including leucin, and bulky amino acids including glutamine, which are considered master regulators of the mTORC1 signaling pathway [25, 26], the mechanism by which SLC7A5 affects the prognosis of ERα-positive breast cancer patients is not yet fully understood. Recently, Ansari et al. reported that enhanced glutamine uptake by SLC family members including SLC7A5 affects the composition of immune cell infiltrates, and might be involved in breast cancer progression [27, 28].

In this study, we demonstrated that LLGL2^low/SLC7A5^low was an independent favorable prognostic factor not only in all breast cancer patients, but also in ERα-positive breast cancer patients. In ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy, we observed that LLGL2^low/SLC7A5^low showed longer survival compared with LLGL2^high/SLC7A5^high and a positive trend of longer survival compared with other combination groups. Saito et al. reported a novel mechanism by which LLGL2 interacts with its cargo, SLC7A5, in the cytoplasm and transports it to the membrane, increasing SLC7A5 levels on the cell surface in ERα-positive breast cancer. They also reported that knockdown of LLGL2 decreased cell surface levels of SLC7A5, and that knockdown of LLGL2 or SLC7A5 was sufficient to restore tamoxifen sensitivity to tamoxifen-resistant ERα-positive breast cancer cells under low leucine concentration. Our data and that of other groups suggested that co-expression of LLGL2 and SLC7A5 is involved in tamoxifen resistance in ERα-positive breast cancer patients, and that the LLGL2–SLC7A5 axis may be an important determinant of the therapeutic effect of tamoxifen in ER-positive breast cancer.

This study had some limitations. First, this was a retrospective analysis at a single institute using archived materials. For the mRNA analysis, we used samples from surgical specimens that were macro-dissected and cryopreserved immediately after resection. However, we did not confirm the amount of cancer tissue in the cryopreserved samples; therefore, the percentage of cancer cells was likely to vary. A total of 626 consecutive invasive breast cancer tissue samples collected between 1992 and 2008 from the archive of our institute were included in this study, and adjuvant therapies for breast cancer have progressed during in this period. Therefore, we could not eliminate the effects of different adjuvant therapies in this study. Second, we determined the mRNA cutoff values of LLGL2 and SLC7A5 by ROC curve analysis. As results of these analyses, we determined 0.51 and 0.57 as the cut-off levels of relative LLGL2 and SLC7A5 mRNA expression, respectively. Because AUC values of 0.51 and 0.57 do not indicate good discriminatory power, the cut-off value for both mRNAs should be re-evaluated using a different dataset in the future. Third, we did not find a positive association between LLGL2/SLC7A5 protein expression and prognosis in this study. The positive rate of SLC7A5 was only 10% in this study. Because long-term follow-up tissues were used in this study, the positive rate of staining might have decreased due to tissue deterioration over time, but the prognosis of cases with staining was poor, which was consistent with the mRNA results of this study.

In this study, we showed that low LLGL2/SLC7A5 mRNA co-expression (LLGL2^low/SLC7A5^low) was an independent favorable prognostic factor in ERα-positive breast cancer patients, as well as low LLGL2 or low SLC7A5 mRNA expression. We also observed that LLGL2^low/SLC7A5^low showed longer survival compared with LLGL2^high/SLC7A5^high and a positive trend of longer survival compared with other combination groups in ERα-positive breast cancer patients receiving adjuvant tamoxifen therapy. Thus, our study showed that the co-expression of LLGL2 and SLC7A5 mRNA is a promising candidate biomarker and suggested that the LLGL2–SLC7A5 axis might be a therapeutic target in early breast cancer patients, especially in those receiving adjuvant tamoxifen therapy.

ERα, estrogen receptor α; LLGL2, lethal giant larvae homolog 2; SLC7A5, solute carrier family 7 member 5; DFS, disease-free survival; OS, overall survival; ROC, receiver operating characteristic; IHC, immunohistochemistry; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; OD, optical density.

Acknowledgements

We thank Mrs. Makino for the excellent technical assistance. We also thank H. Nikki March, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Author’s contributions

TH and TT contributed to the study conception and design. NK, YW-E, TA, HS, YK, MT, AK, KO, and TT contributed to the acquisition of patient data. TH, YU, and SN performed the mRNA analyses and TH analyzed patient data. TH, MK, and HK evaluated the immunohistological analysis and ST supervised the pathological evaluation. SO performed the statistical analysis and SM supervised these analyses. TH drafted the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (KAKENHI grant number: 19K18065).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the institutional review board of Nagoya City University Graduate School of Medical Sciences. All tissue samples were provided from a biobank that is maintained by the Department of Breast Surgery, Nagoya City University Graduate School of Medical Sciences and conformed to the guidelines of the Declaration of Helsinki.

Written informed consent for comprehensive research use was obtained from all patients involved in the study.

Consent for publication

All authors have given consent for publication.

Competing interests

The authors declare that they have no competing interests.

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Supplementary Figure S1. ROC analysis to determine the cut-off value of LLGL2 mRNA expression.
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Supplementary Figure S2. ROC analysis to determine the cut-off value of SLC7A5 mRNA expression.
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Supplementary Table S1. Association between LLGL2 mRNA expression and clinicopathological characteristics in all breast cancer patients.
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Supplementary Figure S3. Association between LLGL2 mRNA expression and clinicopathological characteristics in all breast cancer patients. Graphs show DFS and OS curves for all breast cancer patients (A, B), ERα-negative breast cancer patients (C, D), and ERα-positive breast cancer patients without adjuvant tamoxifen therapy (E, F).
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Supplementary Table S2. Univariate and multivariate analysis of factors associated with prognosis in all breast cancer patients.
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Supplementary Table S3. Association between SLC7A5 mRNA expression and clinicopathological characteristics in ERα-positive breast cancer patients.
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Supplementary Figure S4. Kaplan–Meier survival curves according to SLC7A5 mRNA expression levels. Graphs show DFS and OS curves for ERα-negative breast cancer patients (C, D).
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Supplementary Table S4. Univariate and multivariate analysis of factors associated with prognosis in all breast cancer patients.
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Supplementary Figure S5. Kaplan–Meier survival curves according to the combination of LLGL2 and SLC7A5 mRNA expression. Graphs show DFS and OS curves for ERα-positive breast cancer patients who had not received adjuvant tamoxifen therapy (A, B).
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Supplementary Table S5. Univariate and multivariate analysis of factors associated with prognosis including LLGL2/SLC7A5 in all patients.

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Prognostic impact of co-expression of LLCL2 and SLC7A5 in ERα-positive breast cancer patients

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Abstract

Background

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Background

Methods

Patients and samples

Ihc Of Llgl2 And Slc7a5

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

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References

Supplementary Files

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