International Journal of Gynecological Pathology 00:1–8, Lippincott Williams & Wilkins, Baltimore Copyright © 2018 by the International Society of Gynecological Pathologists
Original Article
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Serum Prolactin Contributes to Enhancing Prolactin Receptor and pJAK2 in Type I Endometrial Cancer Cells in Young Women Without Insulin Resistance Chimeddulam Erdenebaatar, B.Sc., Munekage Yamaguchi, M.D., Ph.D., Mahina Monsur, M.D., Fumitaka Saito, M.D., Ph.D., Ritsuo Honda, M.D., Ph.D., Hironori Tashiro, M.D., Ph.D., Takashi Ohba, M.D., Ph.D., Ken-ichi Iyama, M.D., Ph.D., and Hidetaka Katabuchi, M.D., Ph.D.
Summary: Elevated levels of serum prolactin and a high expression of prolactin receptor (PRLR) in cancer cells was recently identified in patients with endometrial cancer (EC). However, the impact of prolactin on EC remains unknown. The aim of this study was to elucidate the clinical and immunohistochemical characteristics of hyperprolactinemic patients with EC according to the pathogenetic types, type I and type II. EC patients were retrospectively divided into a high prolactin (HP) group and a low prolactin (LP) group by a serum prolactin level of 20 ng/mL and were compared between 2 groups. The expression of PRLR, phosphorylated Janus-kinase 2 (pJAK2), estrogen receptor-α, progesterone receptor, and PTEN in cancer tissue were evaluated by immunohistochemistry. Ninety-nine patients were identified. In the type I group, HP group was significantly younger (45.2 vs. 52.2, P = 0.028) and their insulin resistance was significantly lower (1.6 vs. 2.5, P = 0.033) than those in LP group, and the expression of PRLR and pJAK2 in the HP group was significantly higher than that in the LP group (immunoreactive score: 6.8 vs. 3.9, P = 0.003; 5.7 vs. 2.6, Po0.001, respectively). In the type 2 group, there were no differences between all the term. In the type I group, the rate of loss of PTEN in the HP group was significantly lower than the LP group (25.0% vs. 60.7%, P = 0.024). Prolactin-PRLR signaling may play a crucial role for the progression of type I EC without involving the PTEN mutation in young hyperprolactinemic women without insulin resistance. Key Words: Endometrial cancer—Prolactin—Prolactin receptor—PTEN—Insulin resistance.
Prolactin, a polypeptide hormone that is a member of the growth hormone family, is primarily synthesized and
secreted from the lactotrope cells of the anterior pituitary gland. In addition to the pituitary, prolactin is produced at many extrapituitary sites that include the endometrium, myometrium, decidua, immune cells, brain, breast, prostate, skin, and adipose tissue (1). Prolactin is involved in various physiological functions, including the development of the mammary gland, lactation, implantation and pregnancy, angiogenesis, and the regulation of immune function (2). However, the roles of prolactin in the human physiology remains to be completely elucidated. The actions of prolactin are mediated by the prolactin receptor (PRLR). The PRLR is a transmembrane protein that belongs to the cytokine receptor superfamily and is
From the Department of Obstetrics and Gynecology (C.E., M.Y., M.M., F.S., R.H., T.O., H.K.); Department of Mother-Child Nursing (H.T.), Faculty of Life Sciences, Kumamoto University; and Department of Surgical Pathology, Kumamoto General Hospital, Japan Community Health Care Organization (K.-I.I.), Kumamoto, Japan. The authors declare no conflict of interest. Address correspondence and reprint requests to Munekage Yamaguchi, MD, PhD, Department of Obstetrics and Gynecology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, ChuoKu, Kumamoto-City, Kumamoto 860-8556, Japan. E-mail:
[email protected].
1
DOI: 10.1097/PGP.0000000000000527
Copyright r 2018 International Society of Gynecological Pathologists.
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C. ERDENEBAATAR ET AL.
expressed in a variety of tissues (3). The most well characterized mediator of prolactin signaling is the nonreceptor tyrosine kinase Janus-kinase 2 (JAK2) (4,5). JAK2 is activated rapidly following PRLR stimulation and induces the phosphorylation of the receptor itself, the associated kinases, and the Stat proteins, suggesting that JAK2 fulfills a critical role within the PRLR signaling pathway (6,7). Elevated levels of serum prolactin were recently identified in patients with various types of cancer (8–14). In addition, the expression of PRLR was higher in cancer cells of various types of cancer in comparison to respective normal tissues (13,15–17). A high expression of pJAK2 in cancer cells with a high expression of PRLR was associated with poor prognosis in patients with hepatocellular carcinoma (14). The highest levels of serum prolactin were identified in patients with endometrial cancer (EC) and ovarian cancer, and the expression of PRLR in EC and ovarian cancer was also significantly higher than that of the respective normal tissues (13). However, the expression of PRLR in cancer cells has not been examined according to serum prolactin levels. EC is categorized into 2 pathogenetic types that are primarily based on clinical, metabolic, and endocrine characteristics, including type I and type II. Type I EC is relevant to persistent hyperestrogenism and progesterone insufficiency, and the risk factors for type I include anovulation and infertility (18). Hyperprolactinemia is well known as a cause of infertility or anovulation, which strongly overlaps the risk factors for type I. In our clinical practice, EC patients who were treated with fertility-sparing therapy have been concomitantly examined for factors related to infertility including serum prolactin because their goal was to become pregnant and experience parturition. Of these patients, a population in which EC patients had high levels of serum prolactin was identified after endocrine screening. Our retrospective observational study revealed that cabergoline which is a selective and long-acting dopamine agonist contributed to preserving the uterus in young hyperprolactinemic patients with atypical endometrial hyperplasia (AEH) or well-differentiated EC treated with medroxyprogesterone acetate by reducing the serum prolactin levels (19). Prolactin may be expected to be associated particularly with type I EC. However, serum prolactin levels and the expression of PRLR also were not investigated according to the pathogenetic type of EC. The aim of this study was to elucidate the clinical and immunohistochemical characteristics of hyperprolactinemic patients with EC according to the pathogenetic type.
PATIENTS AND METHODS Patients Hospital records were used to identify a cohort of patients with EC who were treated at Kumamoto University Hospital between January 2010 and December 2012. A retrospective observational study was conducted. The clinical characteristics, serum prolactin level, and pathologic diagnoses were retrospectively assessed by reviewing the medical records. Patients were excluded if their preoperative serum prolactin levels were not examined or their formalin-fixed postoperative tissues were not suitable for the evaluation. Insulin resistance was evaluated using the homeostasis model assessment (HOMA), which was calculated as the fasting insulin concentration (μU/mL)×fasting glucose concentration (mg/dL)/405. Serum prolactin levels were measured during the morning fasting state, and the number of examinations was variable according to their primary physician. The mean prolactin level of the individual patients was used to divide all the participants into 2 groups using a cut-off level of 20 ng/mL, including a high prolactin (HP) group and a low prolactin (LP) group. For the classification of pathogenetic types, endometrioid carcinoma grade 1 (G1) and endometrioid carcinoma grade 2 (G2) were classified into “type I” and endometrioid carcinoma grade 3 (G3), serous carcinoma, and clear cell carcinoma were classified into “type II” in reference to previous articles (20,21). For each type, the characteristics of the patients and the pattern of the protein expression in the tissues using immunohistochemistry were compared between the HP group and LP group. This study protocol was approved by the Institutional Review Board of Kumamoto University. Immunohistochemistry Immunohistochemistry was performed on 3 μm sections of formalin-fixed, paraffin-embedded tissues. Each section was deparaffinized and hydrated by passing it through xylene and a graded series of alcohol. Antigen retrieval was performed for 15 min in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven. The slides were washed 3 times in phosphate-buffered saline after cooling. The endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol for 40 min. The slides were incubated with anti-PRLR (B6.2) antibody (mouse monoclonal, ab74608, 1:3; Abcam, Tokyo, Japan), anti-estrogen receptor-α (ER-α) antibody (mouse monoclonal, clone 1D5, 1:2; Dako, Denmark, A/ S), and anti-progesterone receptor (PgR) antibody (mouse monoclonal, clone PgR 1294, 1:6; Dako Denmark A/S) overnight at 4°C after blocking for 30 min in 5% normal
Int J Gynecol Pathol Vol. 00, No. 00, ’’ 2018
Copyright r 2018 International Society of Gynecological Pathologists.
PROLACTIN AND PROLACTIN RECEPTOR IN ENDOMETRIAL CANCER goat serum. The slides were incubated in with a Dako Envision+Dual Link System-HRP (K4063; Dako Denmark A/S) for 30 min. The immune complexes with anti-pJAK2 (Tyr 1007/Tyr 1008) antibody (goat polyclonal, sc-21870, 1:100; Santa Cruz Biotechnology, CA) and antiphosphatase and tensin homolog deleted on chromosome 10 (PTEN) antibody (rabbit monoclonal, clone 138G6, 1:200; Cell Signaling Technology, Danvers, MA) were detected using the avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA). Each slide reacted with all 5 antibodies was stained with diaminobenzidine substrate (Vector Laboratories). The slides were evaluated under light microscopy after counterstaining with hematoxylin and mounting. Postoperative tissue was used for the evaluation of PRLR, pJAK2, ER-α, and PgR. Preoperative biopsy tissues were also collected for immunohistochemistry with anti-PTEN antibody, because endometrial biopsy tissue is more appropriate for evaluating the expression of PTEN than matched hysterectomy tissue, due to possible delayed fixation and overfixation (22). Immunohistochemical Evaluation The immunoreactive score was calculated by multiplying the optical staining intensity (graded as 0, no; 1, weak; 2, moderate; and 3, strong staining) and the percentage of positive stained cells (0, no staining; 1 ≤ 10% of the cells; 2, 11%–50% of the cells; 3,
51%–80% of the cells; and 4 ≥ 81% of the cells) for an evaluation of the expression of PRLR, pJAK2, ER-α, and PgR as previously described (23) (Fig. 1). The expression of PTEN in the cancer cells in the preoperative biopsy tissues was classified as positive, negative, and heterogenous (Fig. 2). The positive expression in the stromal cells and blood vessels was used as an internal positive control. All the specimens were divided into the 2 groups, including “positive,” which includes positive tissues and “loss of PTEN,” which includes negative tissues and heterogenous tissues for comparison of the expression of PTEN. The immunostained slides were evaluated independently by 2 of the authors who were not informed about the clinical outcomes. An average score of 2 authors was used for comparison of PRLR, pJAK2, ER-α, and PgR. The expression of PTEN was finally classified as positive, negative, and heterogenous after the joint review using a multiheaded microscope when each classification by 2 authors were different. Statistical Analysis Descriptive statistics are given as the mean (SD) or percentages. The mean values were compared between the 2 groups using Student t tests or Mann-Whitney tests. The frequency distributions were compared using the χ2 test except if the expected frequency was
