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Year : 2017  |  Volume : 60  |  Issue : 1  |  Page : 33-37
Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer

Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan

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Date of Web Publication14-Feb-2017


Background: Estrogens are considered to potentially play some roles in the development and progression of prostate cancer through estrogen receptor beta (ERβ). However, additional factors which could influence the clinical outcome of the patients through modulating these steroid signalings have also been proposed. Among these, increased expression of serotonin receptor especially that of 5-hydroxytryptamine receptor Type 4 (5-HTR4) has been recently proposed to be involved in autocrine/paracrine mechanisms of castration-resistant prostate cancer, but the presence and clinical significance of 5-HTR4 in hormone-naive prostate cancer (HNPC) and its interaction with hormonal signaling pathways have remained virtually unknown. Materials and Methods: We evaluated the status of 5-HTR4 in 112 human HNPC cases (acinar adenocarcinoma) using immunohistochemistry and correlated the findings with clinicopathological features of individual patients and the status of androgen receptor (AR) and ERβ. To further elucidate its underlying mechanisms, androgen-dependent human prostate carcinoma cell line, LNCaP, expressing 5-HTR4, was treated by 5-HTR4 agonist. Results: 5-HTR4 immunoreactivity was detected in 34% of prostate cancer cases examined (38/112) and was significantly correlated with the status of ERβ but not with that of AR and other clinicopathological factors of the patients. Results of in vitro studies demonstrated that 24 h incubation with 5-HTR4 agonist (10 nM) increased the expression level of ERβ messenger RNA compared to controls. 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration (P < 0.05). Conclusion: Results of our present study indicated that 5-HTR4 signaling upregulated ERβ expression in HNPCs and could impact on biological processes in HNPC.

Keywords: 5-hydroxytryptamine receptor Type 4, cell migration, estrogen receptor beta, prostate cancer

How to cite this article:
Nakamura Y, Ise K, Yamazaki Y, Fujishima F, McNamara KM, Sasano H. Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer. Indian J Pathol Microbiol 2017;60:33-7

How to cite this URL:
Nakamura Y, Ise K, Yamazaki Y, Fujishima F, McNamara KM, Sasano H. Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Aug 7];60:33-7. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/33/200022

   Introduction Top

Prostate cancer is associated with two distinct phases of disease development. Initially, prostate cancer presents as a highly androgen or hormone-dependent disease whose growth is strongly influenced by androgens acting through the androgen receptor (AR) and estrogens acting through estrogen receptor beta (ERβ). This initial stage is termed hormone-naive prostate cancer (HNPC) and the standard of care is manipulation of the hormonal environment to suppress the growth signals through these receptors above. However, the great majority of the patients will eventually develop castration-resistant prostate cancer (CRPC). This progression is generally associated with aggressive biological behavior and has emerged as one of the most serious clinical problems in the long-term management of prostate cancer patients.

Neuroendocrine differentiation has been proposed to be associated with tumor progression, poor prognosis, and CRPC [1],[2],[3],[4],[5],[6],[7],[8] with serotonin (5-hydroxytryptamine [5-HT]), a neuroendocrine biomarker, being involved in tumor aggression through modulation of growth factor signaling.[9] Serum serotonin levels have also been reported to be correlated with the status of pT4, grading, Gleason score, prostate-specific antigen (PSA) values >100 ng/ml, and the presence of distant metastases in prostate cancer patients.[10] 5-HT receptor Type 4 (5-HTR4) was also reported to be predominantly expressed in high-grade hormone refractory prostate cancers, and its antagonist inhibits cell proliferation in both hormone responsive and nonresponsive prostate carcinoma cell lines.[11]

HNPC is a highly hormone-dependent disease and as such modulation of androgens or estrogen signaling has been reported to play an important role in the development of these cancers and their subsequent clinical outcome.[12],[13],[14],[15],[16] In light of these studies mentioned above, it is interesting that in other tissue types sex steroids and/or these receptors have been reported to interact with 5-HT resulting in enhancement of each signaling pathway and the expression level of each receptor.[17],[18],[19] However, the potential interactions between these hormonal pathways and serotonin signaling pathways in HNPC have remained virtually unexplored. Therefore, in this study, we first examined the status and relative abundance of 5-HTR4 in HNPC, and correlated the findings with the status of AR, ERβ, and other clinicopathological findings of the patients. Second, we evaluated the effects of 5-HTR4 agonists on HNPC cells in vitro to evaluate the possible biological significance of this unique transcription factor.

   Materials and Methods Top

Tissue preparation

The surgical pathology specimens of HNPC (acinar adenocarcinoma) (n = 112) were retrieved from surgical pathology files of Tohoku University Hospital (Sendai, Japan). None of these patients examined in this study received radiation, chemotherapy, or hormone therapy before surgery. The Ethics Committee at Tohoku University School of Medicine approved the research protocol for this study.

Antibodies and immunohistochemistry

Rabbit polyclonal antibody for 5-HTR4 was commercially obtained from MBL International (Woburn, MA, USA). The antibodies against AR, Ki-67, and ERβ were purchased from DAKO Corporation (Carpinteria, CA, USA), Immunotech (Marseilles, France), Gene Tex, Inc., (San Antonio, TX, USA) and Abcam (Cambridge, MA, USA), respectively. Immunohistochemical analysis was performed employing the streptavidin–biotin amplification method using a Histofine Kit (Nichirei, Tokyo, Japan).

Scoring of immunoreactivity and the number of mast cells

For evaluation of 5-HTR4 immunoreactivity, all the cases were tentatively dichotomized into the following two groups: Positive (more than 10% positive cells); negative (fewer than 10% positive cells). For evaluation of AR, ERβ, and Ki-67 immunoreactivity, a total of more than 500 carcinoma cells from three different representative fields were counted in all the cases examined, and the percentage of immunoreactivity (i.e., labeling index [LI]) was determined.

Cell lines

The human prostate carcinoma cell line, LNCaP (HNPC), was purchased from ATCC (Manassas, VA, USA) and cultured up to subconfluence (70%–80%) in a well with RPM-1640 medium (Sigma Co., St. Louis, MO, USA). A previous study demonstrated abundant expression of 5-HTR4 protein in LNCaP cells.[11]

Cell culture and treatment of LNCaP cell and quantitative real-time-polymerase chain reaction analysis

To test the effects of 5-HTR4 in the LNCaP cell line, the standard RPM-1640 medium was then replaced with phenol red-free RPM-1640 medium (Sigma Co., St. Louis, MO, USA) for 24 h. Following this, the LNCaP cells were treated with 5-HTR4 agonist (10 nM) (cisapride monohydrate) (Sigma) or vehicle (0.1% ethanol) in phenol red-free medium containing 10% dextran-coated charcoal-stripped fetal bovine serum for a duration of 24 h. Cells were subsequently collected and RNA extracted.

Quantitative polymerase chain reaction (qPCR) analysis was carried out using the LightCycler System (Roche Diagnostics GmbH, Mannheim, Germany). The primer sequences used in this study were as follows –

RPL13A (housekeeping gene):






ERβ messenger RNA (mRNA) levels in each case were represented as a ratio of RPL13A and evaluated as a percentage ratio.

Cell migration assay

Cell migration assay was performed using a 24-well tissue culture plate (Becton Dickinson, Franklin Lakes, NJ, USA) and Chemotaxicell (8-µm pore size) (Kurabo, Osaka, Japan) according to a previous report.[20] After incubation for 24 h at 37°C, cells treated by 5-HTR4 agonist (100 nM) (Cisapride monohydrate) (Sigma) or vehicle (0.1% ethanol) on the upper surface of membrane were removed by wiping with a cotton swab, and the migration ability was evaluated as a total number of the cells on the lower surface of membrane, which was counted under light microscopy.

Statistical analysis

Values for patient age, serum PSA levels, and LI for AR, ERβ, and Ki-67 were presented as the mean ± standard error of the mean (SEM), and differences between positive and negative 5-HTR4 cases (divided based on the criteria described above) were evaluated using the unpaired t-test. Statistical differences between immunoreactivity for 5-HTR4 and stage, lymph node status, and histological grade were evaluated in a cross-table using the Chi-square test. Statistical analyses for qPCR and migration assay were done by unpaired t-test. P < 0.05 was considered statistically significant.

   Results Top

Immunohistochemical analysis

5-HTR4 immunoreactivity was detected in the cell membrane and cytoplasm of prostate carcinoma cells, while that of ERβ, AR, and Ki-67 was detected in the nuclei [Figure 1], and 38 cases (34%) were determined as positive for 5-HTR4 immunoreactivity according to the criteria above. There was a significant positive correlation between 5-HTR4 immunoreactivity and ERβ expression (P < 0.034) [Table 1]. The status of 5-HTR4 immunoreactivity was not significantly correlated with other clinicopathological parameters of the patients including patients' age, concentration of serum PSA levels, Gleason score, pT stage, lymph node status, or the LI of Ki-67 [Table 1]. There was no significant correlation between 5-HTR4 and AR immunoreactivity [Table 1]. In addition, the status of 5-HTR4 immunoreactivity was not significantly correlated with the recurrence or survival of the patients (data not shown).
Figure 1: Immunoreactivity of 5-hydroxytryptamine receptor Type 4 (a), estrogen receptor beta (b), androgen receptor, (c) and Ki-67, (d) in human prostate acinar adenocarcinoma

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Table 1: Correlation between 5-hydroxytryptamine receptor Type 4 immunoreactivity and clinicopathological parameters in human prostate cancer. *P<0.05

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Quantitative polymerase chain reaction analysis and cell migration assay in LNCaP cell line

Compared to the negative control, 5-HTR4 agonist (10 nM) increased the expression level of ERβ mRNA in LNCaP prostate carcinoma cells incubated for 24 h (P < 0.05) [Figure 2]. In addition, compared to negative control, 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration incubated for 24 h (*P < 0.05) [Figure 3].
Figure 2: Quantification of estrogen receptor beta messenger RNA levels in LNCaP cells treated by 5-hydroxytryptamine receptor Type 4 agonist (10 nM) or vehicle (0.1% ethanol) for 24 h. Data are presented as mean ± standard error of the mean of values from three independent experiments run and expressed as a percentage of negative control (basal; *P < 0.05)

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Figure 3: Quantification of migration of LNCaP cells treated by 5-hydroxytryptamine receptor Type 4 agonist (10 nM) or vehicle (0.1% ethanol) for 24 h. Data are presented as mean ± standard error of the mean of values from six independent experiments run and expressed as a percentage of negative control (basal; *P < 0.05)

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   Discussion Top

Dizeyi et al. reported that 5-HTR4 immunoreactivity was detected in human prostate cancer tissues of CRPC cases but not in a preinvasive and thus presumably hormonally dependent form prostatic intraepithelial neoplasia (PIN).[11] They hypothesized that increased 5-HTR4 expression in CRPC tissues could represent an escape mechanism from the growth induced by androgen deprivation and could play roles in the process of tumor progression via 5-HT-induced increase in hormone or growth factor levels, resulting in the cell growth in an androgen-depleted environment.[11] In this study, we demonstrated that 5-HTR4 immunoreactivity was detected in HNPC cases (approximately 34%). In addition, we demonstrated the presence of 5-HTR4 in a HNPC cell line and functional consequences of its activation. Dizeyi et al. also examined the same HNPC cell line and compared the findings with those of CRPC cell line models.[11] These studies revealed that the hormone-independent cell line DU145 exhibited reduced proliferation in response to a 5-HTR4 antagonist whereas the androgen-dependent LNCaP cells do not.[11] However, we demonstrated that 5-HTR4 inhibited migration of the androgen-dependent LNCaP cells. Therefore, it awaits further investigations to clarify the extent to which 5-HTR4 may regulate biology in HNPC cases.

One very interesting finding from our present study was the interaction of hormonal pathways and serotonin signaling in both HNPC specimens and cell lines. The most significant correlation in our present study is that between the status of 5-HTR4 and ERβ, with the mechanistic nature of this interaction confirmed by the upregulation of ERβ observed in LNCaP cell lines upon treatment with a 5-HTR4 agonist. The clinicopathological significance of ERβ in prostate cancer has remained controversial. In general, ERβ is considered to play an anti-proliferative role in prostate cancer,[12],[13],[14] while the loss of ERβ expression possibly related to progression to CRPC and/or the development of high-grade prostate cancer.[12],[15] However, contrary to this idea, ERβ expression has also been reported to correlate with increased grade and proliferation in androgen-naive prostate cancer and mechanistically this is suggested to occur at least partially through its regulation of cell cycle proteins such as cyclin D1.[16] Results from this present study hint at the possibility that ERβ-serotonin pathways may also be involved in adverse effects of ERβ signaling. Studies in central nervous system tissues demonstrated cross-talk between estrogens and serotonin pathways with estrogens and/or ERβ involved in synthesis of 5-HT, expression and function of 5-HTRs in the central nervous tissues [17],[18],[19] and 5-HT signaling regulating the expression of sex steroid receptors [21],[22],[23] and similar correlations of 5-HTR4 with ER alpha (ERα) and progesterone receptor in the breast have been demonstrated.[24] These results indicated that the correlation 5-HTR4 with ERβ could be involved in pathogenesis of HNPC in similar manner of 5-HTR4 in breast cancer.

In summary, the data from our histological and cell-based studies indicated that 5-HTR4 could increase expression levels of ERβ and contribute to the progression and pathogenesis of prostate cancer. In addition, results regarding their correlation and interaction could open up the possibility of novel target therapy of prostate cancer in near future.

   Conclusion Top

5-HTR4 is involved in ERβ expression and cell migration in hormone-naive prostate cancer.


This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research [C] (23590387)). A part of the presentation has been presented in the abstract forms in AACR Special Conference on Advances in Prostate Cancer Research (February 6–9, 2012; Orlando, FL) and The Endocrine Society's 95th Annual Meeting and Expo (June 15–18, 2013 - San Francisco).

Financial support and sponsorship

This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research [C] (23590387)).

Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Dr. Yasuhiro Nakamura
Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.200022

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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