Objectives: The aims of this study were to determine whether the altered L-type amino acid transporter 1 (LAT1) expression is related to clinicopathologic factors, expressions of Ki-67, p53, estrogen receptor, and progesterone receptor and clarify the significance of LAT1 as a prognostic factor and the novel possibility of using it to treat endometrial endometrioid adenocarcinoma.
Methods: The LAT1 expression was analyzed immunohistochemically in atrophic (6 cases), secretory phase (6 cases), proliferative phase endometria (6 cases), atypical hyperplasia (6 cases), and endometrioid adenocarcinoma (26 well-differentiated [G1], 17 moderately differentiated, and 11 poorly differentiated [G3] adenocarcinoma patients).
Results: The LAT1 expression was observed in the cell membrane. Its expression increased in the atrophic, secretory, and proliferative phases of the endometrium in that order. There was no difference between the proliferative phase endometrium, atypical hyperplasia, and G1 adenocarcinoma. The LAT1 expression in G1 adenocarcinoma was significantly higher than that in G3 adenocarcinoma. The LAT1 expression was inversely correlated with p53 expression but not with those of Ki-67, estrogen receptor, or progesterone receptor.
Conclusions: It is suggested that the significance of LAT1 as a prognostic factor is low because LAT expression was low in G3 adenocarcinoma, not correlated with the International Federation of Gynecology and Obstetrics stage and proliferative activity and inversely correlated with p53. The LAT1 inhibitors can be used as anticancer drugs for G1 and moderately differentiated adenocarcinoma that express high LAT1.
*Division of Medical Life Sciences, Department of Pathologic Analysis, Graduate School of Health Sciences, and †Department of Obstetrics and Gynecology, Graduate School of Medicine, Hirosaki University, Hirosaki; ‡Department of Pathology, Kitasato University School of Medicine, Sagamihara; and §J-Pharma Co, Ltd; and ∥Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan.
Address correspondence and reprint requests to Jun Watanabe, MD, PhD, Division of Medical Life Sciences, Department of Pathologic Analysis, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-Cho, Hirosaki 036-8564, Japan. E-mail: email@example.com.
This study was supported by a grant for Hirosaki University Institutional Research (2013).
The authors declare no conflicts of interest.
Received January 23, 2014
Accepted February 16, 2014
The incidence of endometrial cancers has recently increased in Japan because of change to a Western lifestyle. A total of 7273 cases of uterine corpus cancer were registered by the Gynecologic Oncology Committee of Japan in 2012.1 The number of young patients with endometrial cancer has also increased, so a new conservative therapy that safeguards fertility is needed. Furthermore, the survival rate is more than 90% at 5 years for International Federation of Gynecology and Obstetrics (FIGO) stage I, but it is almost 70% for stage III and 20% for stage IV; the prognosis worsens with an increasing stage.
Endometrial endometrioid adenocarcinoma (endometrioid adenocarcinoma) is derived from endometrial hyperplasia caused by continuous hyperestrogenic proliferative effects. An autocrine function regulated by the estrogen-induced expression of TGF-α and its receptor is activated in endometrial cancer cells (Ishikawa cells).2 We previously reported that the stimulatory effect of estrogen on cell proliferation may be up-regulated by cyclin D1 and cyclin A, and down-regulated by p53, p21, and p27.3 Furthermore, we analyzed whether gene variation and the expression of various factors, including those mentioned previously, were correlated with clinicopathologic factors and the prognosis using clinical materials. As a result, a prognostic algorithm for endometrioid adenocarcinoma patients was tentatively proposed by clarifying that mutation and overexpression of the p53 gene,4 expression of some cell cycle regulators,5 and the loss of progesterone receptor-B expression are useful factors indicating a poor prognosis.6,7 Moreover, it is necessary to clarify new biomarkers for more useful prognosis and prediction to develop new therapies. Therefore, we must clarify prognostic factors and treatment predictors by elucidating the mechanism of carcinogenesis of endometrioid adenocarcinoma.
The L-amino acid transporter (LAT) system responsible for the transport of large neutral amino acids, such as leucine, largely depends on the L-type amino acid transporter 1 (LAT1), originally cloned by Kanai et al.8 The LAT1 is expressed in some normal human cells and carcinomas such as prostatic, breast, esophageal, pulmonary, gastric, and pancreatic carcinomas.9–15 Previously, we demonstrated that LAT1 expression could be a reliable prognostic marker in prostatic, nonscirrhous gastric, and pancreatic ductal carcinomas.10,14,15 Therefore, LAT1 overexpression was strongly suggested to be associated with the aggressive phenotype of a malignant tumor. Its potentiality as a therapeutic target in endometrial lesions has not been reported yet.
The aims of this study were to determine whether altered LAT1 expression is related to clinicopathologic factors, Ki-67 expression indicated as cellular proliferation activity, expressions of p53, estrogen receptor (ER), and progesterone receptor (PR) and to clarify the significance of LAT1 as a prognostic factor and investigate the possibility treating endometrioid adenocarcinoma with anti-LAT1 drugs.
PATIENTS AND METHODS
Tissue samples of 18 normal endometria (6 in atrophy, 6 in secretory, and 6 in proliferative phases), 6 atypical hyperplasias, and 54 endometrioid adenocarcinomas (26 well-differentiated [G1], 17 moderately differentiated [G2], and 11 poorly differentiated [G3] adenocarcinomas) were surgically obtained between 2006 and 2013 at Hirosaki University Hospital with informed consent. The study was approved by the Hirosaki University School of Medicine and Hospital Ethics Committee (2013-232). The mean age of the patients was 56.6 years old (range, 22–88 years). Myometrial invasion of more than half, cervical involvement, adnexal involvement, vascular space involvement, and lymph nodes metastasis were confirmed in 17 (31.5%), 7 (13.0%), 6 (11.1%), 11 (20.4%), and 6 (11.1%), respectively. No patients had received either neoadjuvant chemotherapy or radiotherapy before surgery. The patients generally underwent a modified radical hysterectomy, bilateral salpingo-oophorectomy, and a systematic retroperitoneal lymph node dissection, and they received a combination chemotherapy consisting of paclitaxel and carboplatin if they had risk factors such as deep myometrial invasion, cervical involvement, adnexal involvement, vascular space involvement, or retroperitoneal lymph node involvement. In this study, simple total hysterectomy, modified radical hysterectomy, and radical hysterectomy were performed in 5, 38, and 11 cases of endometrial adenocarcinoma, respectively. Surgical staging and the histological diagnosis were based on criteria established by the FIGO.16
Immunohistochemical staining of LAT1 using 4-µm-thick formalin-fixed paraffin-embedded sections was performed according to the methods previously described.10 In brief, tissue sections were deparaffinized in xylene and rehydrated and then treated with 1% hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity. After antigen retrieval, the specimens were incubated with LAT1 (2 µg/mL, J-Pharma, Tokyo, Japan) at 4°C overnight. The slides were then incubated with a peroxidase-labeled polymer (EnVision kit, Dako, Kyoto, Japan) for 30 minutes. The peroxidase activity was reacted with 3,3′-diaminobenzidine. Finally, nuclei were counterstained with 0.3% methyl green, and the sections were dehydrated, cleared, and mounted. Immunohistochemical staining of Ki-67, p53, ER, and PR was performed according to the methods previously described.6,7
Evaluation of Immunohistochemistry
The expression of LAT1 was evaluated immunohistochemically based on a combination of intensity and distribution with a slightly modified revision of the method of Sinicrope et al.17 Intensity was scored 0 to 3 depending on whether the staining was 0, absent; 1, weak; 2, moderate; and 3, intense and complete membrane staining. Distribution was scored 0 to 3 depending on the proportion of positive cells as follows: 0, 0%; 1, 1% to 10%; 2, 11% to 30%; 3, 30 or less than. Immunoreactivity scores for each case were obtained by multiplying the values of 2 parameters. Expressions of Ki-67, p53, ER, and PR were evaluated with a nuclear labeling index (LI; in percent) by counting at least 1000 cells.
Statistical significance was analyzed by the Mann-Whitney U test and the Spearman rank correlation, using SPSS 16.0 J for Windows. P < 0.05 was considered statistically significant.
The LAT1 expression was observed in the membranes of cells in normal endometria, atypical hyperplasias, and endometrioid adenocarcinoma (Fig. 1). Scores of LAT1 expression are shown in Table 1. The LAT1 scores (mean [SD]) of atrophic, secretory, and proliferative phases of the normal endometrium and atypical hyperplasia were 0.3 (0.5), 2.2 (0.1), 5.0 (1.1), and 5.0 (1.1), respectively. The LAT1 expression in atrophy was significantly the lowest (P < 0.01), and its expression in the secretory phase was significantly lower than those in the proliferative phase and atypical hyperplasia. There was no difference between the proliferative phase and atypical hyperplasia. The LAT1 scores of G1, G2, and G3 adenocarcinoma were 6.4 (2.0), 6.1 (2.2), and 3.8 (2.2), respectively. The LAT expression in G1 and G2 adenocarcinoma was higher than that in G3 adenocarcinoma (G1 vs G3, P < 0.01; G2 vs G3, not significant; G1 vs G2, not significant). The LAT1 scores showed no significant difference between atypical hyperplasia and G1 adenocarcinoma.
Correlation of LAT1 Expression With FIGO Stage
The correlation between the LAT1 score and FIGO stage in endometrioid adenocarcinoma is shown in Table 2. There was no significant correlation of LAT1 expression with the FIGO stage.
Correlation of LAT1 Expression With Ki-67, p53, ER, and PR
The correlation of the LAT1 score with LIs of Ki-67, p53, ER, and PR is shown in Table 3. The LAT1 expression was inversely correlated with p53 LI (rs = 0.39, P < 0.01) but not with those of Ki-67, ER, and PR.
The LAT1 was expressed in the cell membranes of the normal endometria, the atypical hyperplasias, and endometrioid adenocarcinomas. The LAT1 has been reported to be expressed in cell membranes of gonad tissues and several kinds of cells having special functions, as well as being discovered as a component of oncofetal proteins,9 and many kinds of cancer cell.10–15
In endometrial tissues, LAT1 showed significantly higher expression in the order of atrophic, secretory, and proliferative phases. There was no difference among the proliferative phase, atypical hyperplasia, G1, and G2 endometrioid adenocarcinoma. In the stomach, LAT1 expression is higher in the order of the normal epithelia, adenomas, and adenocarcinomas.14 Surprisingly, G3 endometrioid adenocarcinoma was significantly lower than G1 adenocarcinoma. There was no significant difference between the differentiated and undifferentiated types in gastric carcinoma. However, gastric scirrhous carcinoma that is supposed to be undifferentiated shows a lower LAT1 score than nonscirrhous carcinoma, in line with our results.14 On the contrary, high LAT1 intensity cases are relatively correlated with high Gleason score groups in prostate cancer.10 Decreased androgen signaling results from hormone ablation therapy leading to activating transcription factor 4 transcription, which initiates the transcription of LAT1. Androgen receptor signaling activates the transcription of LAT3, an isoform of LATs.18 The LAT1 may be an important system in hormone-independent tumors, but LAT3 expression is detected at all stages of prostate cancer, with no differences observed between Gleason grades.19 It was reported that LAT3 increased in the liver and skeletal muscle of starved mice compared with LAT1.20 It is suggested that LAT3, other than LAT1, might maintain the growth of those cells and G3 cancer such as endometrioid and gastric scirrhous cancer.
In endometrioid adenocarcinoma, there were no significant correlations between LAT1 expression and the FIGO stage. A correlation is also not observed between the LAT1 score and lymph node metastasis in breast cancer.11 However, LAT1 expression has been reported to be a reliable prognostic marker in some carcinomas.10,14,15
In our study, LAT1 expression was inversely correlated with p53 expression but not with Ki-67, ER, and PR. A positive correlation is observed between p53 expression and LAT1 in gastric carcinoma.14 In endometrioid adenocarcinoma, p53 expression is inversely correlated with tumor differentiation, and there was a quantitative correlation reported between p53 overexpression and p53 gene mutation.4 The mechanism of the association between LAT1 and p53 is not clear. Further study is necessary to clarify it.
There is no correlation between LAT1 expression and the Ki-67 LI in pancreatic ductal adenocarcinoma based on double immunostaining of LAT1 and Ki-67.15 In contrast, a significant correlation between those factors in lung and gastric cancers was reported.13,14 It is generally thought that the proliferation of cells such as carcinoma requires many amino acids. However, because Ki-67 LI is higher in G3 than in G1 of endometrioid adenocarcinoma,21 there may be no direct correlation between LAT1 expression and the proliferative activity. Other LAT systems such as LAT3 might act on cell proliferation in endometrioid adenocarcinoma.
The LAT1 expression is significantly correlated with the negative expression of ER and PR in breast cancer.11 In our study, LAT1 expression was lower in G3 than in G1 adenocarcinoma, so ER and PR were expected to correlate inversely with LAT1. However, there was no correlation between them. The LAT1 expression may not be regulated by estrogen and progesterone in endometrioid adenocarcinoma.
Recently, KYT-0353, 1 of the LAT1 inhibitors, showed inhibition of the growth of colon cancer cells in vivo and in vitro.22 Inhibition of LAT1 by melphalan reduced cell growth in breast cancer cells.23 Therefore, LAT1 inhibitors might be useful as a new anticancer drug, especially against high LAT1 expression such as in endometrioid adenocarcinoma, G1, and G2.
In conclusion, the significance of LAT1 as a prognostic factor is low because LAT expression was low in G3 adenocarcinoma, not correlated with the FIGO stage and proliferative activity, and inversely correlated with p53 expression. The LAT1 inhibitors might be new anticancer drugs for G1 and G2 adenocarcinoma that express high LAT1. It is necessary to investigate the expression of other factors such as LAT3 in G3 adenocarcinoma that expresses low LAT1.
1. A report of gynecologic oncology committee of Japan. Acta Obstet Gynecol Jpn. 2012; 64: 2354–2365.
2. Hata H, Hamano M, Watanabe J, et al. Role of estrogen and estrogen-related growth factor in the mechanism of hormone dependency of endometrial carcinoma cells. Oncology. 1998; 55: 35–44.
3. Watanabe J, Kamata Y, Seo N, et al. Stimulatory effect of estrogen on the growth of endometrial cancer cells is regulated by cell-cycle regulators. J Steroid Biochem Mol Biol. 2007; 107: 163–171.
4. Kamata Y, Watanabe J, Hata H, et al. Quantitative study on the correlation between p53
gene mutation and its expression in endometrial carcinoma cell lines. Eur J Gynaecol Oncol. 2003; 25: 55–60.
5. Watanabe J, Kamata Y, Kanai T, et al. Expression of cell cycle regulators in endometrial adenocarcinoma. In: Kuramoto H, Nishida M, eds. Cell and Molecular Biology of Endometrial Carcinoma. Tokyo, Japan: Springer; 2003: 93–106.
6. Miyamoto T, Watanabe J, Hata H, et al. Significance of progesterone receptor-A and -B expressions in endometrial adenocarcinoma. J Seroid Biochem Mol Biol. 2004; 92: 111–118.
7. Watanabe J, Nishimura Y, Tsunoda S, et al. Liquid-based preparation for endometrial cytology—usefulness for predicting the prognosis of endometrial carcinoma preoperatively. Cancer. 2009; 117: 254–263.
8. Kanai Y, Segawa H, Miyamoto K, et al. Expression cloning and characterization of a transporter large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem. 1998; 273: 23629–23632.
9. Nakata N, Mikami T, Hana K, et al. Unique and selective expression of L-amino acid transporter inhuman tissues as well as being an aspect of oncofetal protein. Histol Histopathol. 2013; 29: 217–227.
10. Sakata T, Ferdous G, Tsuruta T, et al. L-type amino acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer. Pathol Int. 2009; 59: 7–18.
11. Furuya M, Horiguchi J, Nakajima H, et al. Correlation of L-type amino acid transporter 1 and CD98 expression with triple negative breast cancer prognosis. Cancer Sci. 2012; 103: 382–389.
12. Kobayashi H, Ishii Y, Takayama T. Expression of L-type amino acid transporter 1 (LAT1) in esophageal carcinoma. J Surg Oncol. 2005; 90: 233–238.
13. Kaira K, Oriuchi N, Imai H, et al. Prognostic significance of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in stage 1 pulmonary adenocarcinoma. Lung Cancer. 2009; 66: 120–126.
14. Ichinoe M, Mikami T, Yoshida T, et al. High expression of L-type amino acid transporter 1 (LAT1) in gastric carcinomas: comparison with non-cancerous lesions. Pathol Int. 2011; 61: 281–289.
15. Yanagisawa N, Ichinoe M, Mikami T, et al. High expression of L-type amino acid transporter 1 (LAT1) predicts poor prognosis in pancreatic ductal adenocarcinoma. J Clin Pathol. 2012; 0: 1–5.
16. FIGO Committee on Gynecologic Oncology. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynaecol Obstet. 2009; 105: 103–104
17. Sinicrope FA, Ruan SB, Cleary KR, et al. Bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res. 1995; 55: 237–241.
18. Wang Q, Bailey CG, Ng C, et al. Androgen receptor and nutrient signaling pathways coordinate the demand for increased amino acid transport during prostate cancer progression. Cancer Res. 2011; 71: 7525–7536.
19. Wang Q, Tiffen J, Baily CG, et al. Targeting amino acid transport in metastatic castration-resistant prostate cancer: effects on cell cycle, cell growth, and tumor development. J Natl Cancer Inst. 2013; 105: 1463–1473.
20. Fukuhara D, Kanai Y, Chairoungdua A, et al. Protein characterization of Na-independent system L amino acid transporter 3 in mice. A potential role in supply of branched-chain amino acids under nutrient starvation. Am J Pathol. 2007; 170: 888–898.
21. Salvesen HB, Iversen OE, Akslen LA. Prognostic significance of angiogenesis and Ki-67, p53, and p21 expression: a population-based endometrial carcinoma study. J Clin Oncol. 1999; 17: 1382–1390.
22. Oda K, Hosoda N, Endo H, et al. L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer sci. 2009; 101: 173–179.
23. Shennan DB, Thomson J. Inhibition of system L (LAT1/CD98hc) reduces the growth of cultured human breast cancer cells. Oncol Rep. 2008; 20: 885–889.
Keywords:© 2014 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.
Endometrial endometrioid adenocarcinoma; LAT1; Histological grade; p53