Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival

Primary human mammary epithelial cells (pHMEC), obtained from reduction mammoplasty under Institutional Review Board approval, were a generous gift from Eric R. Hugo at The University of Cincinnati; cell lines and anonymous tissue microarray specimens purchased from the National Cancer Institute were considered to be exempt. The research carried out in this article is in compliance with the Declaration of Helsinki.

Primary human mammary epithelial cells (pHMEC) were prepared using a modification of previously described protocol [30]. Briefly, excised human mammary tissue was finely minced, transferred to conical tubes, and digested overnight at 37°C in M199 media containing 2.5 mg/ml BSA (Sigma-Aldrich, St. Louis, MO, USA), 0.1% collagenase type III (Worthington Biochemical Corporation, Lakewood, NJ, USA) and antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA). Digested tissue was pelleted by centrifugation, washed in phosphate buffered saline (PBS), and either plated in the pHMEC media (see below) or frozen back for later use.

All the cell lines used in the aforementioned studies were procured from the American Type Culture Collection (ATCC), and used within 10 passages after acquisition. MCF10A media consisted of the following: DMEM-F12 50:50 (Invitrogen) supplemented with 5% horse serum, 2 mM L-glutamine, 10 μg/ml insulin (Sigma-Aldrich), 20 ng/ml EGF (Upstate Biotechnology, Waltham, MA, USA) 0.5 μg/ml hydrocortisone and antibiotic/antimycotic (Invitrogen). pHMEC media contained DMEM-F12 50:50, 5% FBS (Hyclone, Logan, UT, USA), insulin (Sigma-Aldrich), hydrocortisone (Sigma-Aldrich), EGF (Upstate), 1 ng/ml cholera toxin (Sigma-Aldrich) (except where noted) and antibiotic-antimycotic (Invitrogen). MDA-MB-231 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS, L-glutamine and antibiotic-antimycotic (Invitrogen). T47D cells were grown in same media as MDA-MB-231 with the addition of 5 μg/ml of insulin. MCF7 cells were grown in media consisting of DMEM-F12 50:50 (Invitrogen) supplemented with 10% FBS, L-glutamine, 1 mM sodium pyruvate (Invitrogen), 1× concentration of non-essential amino acids (Invitrogen), and antibiotic-antimycotic (Invitrogen).

Paraformaldehyde fixed paraffin embedded mouse mammary gland sections and tissue microarray sections were deparaffinzed in xylene and rehydrated in decreasing concentrations of ethanol from 100% to 50%. Endogenous peroxidases were blocked by incubation in 3% H₂O₂ at room temperature for 30 min. Antigen retrieval was performed using borate buffer pH = 8.5 (80 mM boric acid, 20 mM sodium borate) in a microwave (60% power) twice for 5 min. Sections were then permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 30 min followed by normal serum blocking for 1 h at room temperature and incubation in primary antibody (sheep anti-TPH 1:100 Abcam, Cambridge, MA, USA) overnight at 4°C in a humid chamber. The immune reaction was visualized using HRP-conjugated secondary antibody (Sigma-Aldrich) and ABC-DAB system (Zymed, S. San Francisco, CA, USA; and Vector Labs, Burlingame, CA, USA, respectively).

Cells grown on coverslips were fixed in 4% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100, incubated in borate buffer overnight at 75°C and incubated in primary antibodies overnight at 4°C. Images were collected using a Zeiss LSM510 Confocal Microscope, Göttingen, Germany) using the Zeiss LSM Image Software version 3.5, Munich, Germany)

Total cellular RNA was extracted using TRI-REAGENT (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer's instructions. Two μg of RNA was subjected to reverse transcription by standard methods. One μl cDNA was used for 25 μl PCR reactions. For primer information, please see Table S1 in Additional data file 1.

Cellular protein extracts were prepared using the Cell Lysis kit (Cell Signaling Technology, Boston, MA, USA) as per the manufacturer's instructions. Proteins were quantified using Lowry assay and equal amounts were separated on SDS-PAGE gel. After transferring the proteins to nitrocellulose membrane, the specific proteins were visualized using specific antibodies and detected using HRP tagged secondary antibodies.

After experimental treatments, cell proliferation was measured by colorimetric assay based on cleavage of a tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by mitochondrial dehydrogenase enzyme present in proliferating cells (Cell titer 96 - Promega, Madison, WI, USA). For trypan blue experimental procedures, the cells were gently trypsinized and re-suspended in 0.2% trypan blue solution and counted using a hemocytometer.

Human breast cancer tissue microarrays were purchased from The National Cancer Institute (Bethesda, MD, USA [31]. The array has 288 cores in quadruplicate with tissue matched controls. A modified histochemical-score (H-score) [32, 33] system was used to evaluate the breast cancer tissue microarrays. An H-score comprises of a semi-quantitative assessment of both the intensity of staining and percentage of positive mammary epithelial cells. For intensity, a score of 0 to 3, corresponding to negative, weak, positive and strong positive was recorded blindly by two independent observers with final scores resulting in an average. It was not necessary to correct the staining intensities to account for the percentage of positive cells because of uniform epithelial staining within given specimens.

TPH1 catalyzes the first and the rate-limiting step in 5-HT biosynthesis. We have previously shown that TPH1 is expressed in the mammary gland and TPH1 expression directly correlates with 5-HT production [5, 6]. We therefore addressed the question of whether there were any changes in 5-HT synthetic capacity of breast cancer cells. Toward this end, we analyzed TPH1 mRNA levels by reverse-transcriptase-couple polymerase chain reaction (RT-PCR) in different human breast cancer cells (MCF7, MDA-MB-231 and T47D), and compared them to that of non-transformed human mammary epithelial cells (MCF10A). The mRNA levels of TPH1 were elevated more than two-fold in MDA-MB-231 and T47D cells compared to that of MCF10A cells (Figure 1A). The TPH1 protein was analyzed by western blot in extracts of these cells and was significantly elevated not only in MDA-MB-231 and T47D, but also in MCF7 cells, demonstrating that 5-HT biosynthetic capacity was increased in all breast cancer cell lines tested, with the highest level of expression occurring in MDA-MB-231 cells (Figure 1B). This was contrary to what occurred in MCF10A cells, where TPH1 protein was near the lower limit of detection by western blotting (Figure 1B). An additional factor that regulates cellular exposure to 5-HT is SERT, which pumps 5-HT back into the cells contributing to the recycling and controlling the extracellular concentration of 5-HT. We assessed SERT protein levels and found them to be similar among these cell lines [see Figure S1 in Additional data file 1].

To gain insight into the possible association between altered 5-HT synthesis and breast cancer progression in actual human tumors, we analyzed TPH1 in histological specimens. The specimens used were human breast cancer tissue microarrays acquired from The National Cancer Institute Cooperative Breast Cancer Tissue Resource [31]. Quadruplicate samples of 288 cores comprised the array, which included both tissue-matched non-cancer and non-diseased controls, along with cell line controls. Figure 2A shows a representative section of normal human breast tissue from an array at two different magnifications, stained for TPH1. In normal breast tissue the epithelium was dispersed in an orderly fashion within the stroma, and the epithelium stained distinctly for TPH1. Scattered blood vessel-associated cells were also positive for TPH1 within the stroma. A modified H-score [34, 35] was used by blinded examiners to score the stained tissues. Sections were scored on a scale of 0 to 3 with respect to TPH1 staining separately within epithelia and stroma, using a key generated from among the array sections [see Figure S2 in Additional data file 1]. Cores of cell lines on the array (MCF7 and T47D) validated the scoring for TPH1. Similar to the expression pattern we had seen previously in the cell lines (Figure 1), TPH1 staining intensity was significantly elevated in the cancer cell lines, compared with normal breast tissue (Figure 2B).

The tissue specimens on the array were all from primary tumor sites, and were scored based on a variety of pathological criteria. An obvious and clinically meaningful characterization is reproductive steroid receptor (estrogen receptor (ER) and progesterone receptor (PR)) status. Levels of TPH1 were lower in ER negative cases, compared with ER positive cases. There was no difference in TPH1 levels associated with PR status of the breast cancers (Figure 2C and 2D, respectively).

Segregating the cases crudely according to tumor size and according to T-stage (another size-based classification) showed an inverse relationship between tumor size (>20 mm) and TPH1 staining [see Figure S3 in Additional data file 1]. When the breast cancer cases were sorted according to invasion and progression criteria, TPH1 staining showed a set of nonlinear relationships (Figure 3). Staining for TPH1 was lower in locally-invasive (IN+) cases compared with non-invasive samples (NI), which included both normal tissue and ductal carcinoma in situ (DCIS). However, TPH1 was high in cases of tumors with distant metastases (IN-Mets) (Figure 3A). The relationship of TPH1 staining to progression was clearest among samples sorted according to N-stage (extent of lymph node involvement) (Figure 3B). Staining for TPH1 was significantly decreased in the N1 staged tumors (one to three ipsilateral nodes). However, with progression to N1a/b stage (micrometastases to four or more nodes, including extension beyond the node capsule) and higher stages (N2/3), the TPH1 staining was elevated. Representative core sections from each sorted category are depicted in Figure 3C.

To determine the expression of 5-HT receptor mRNA profiles, we performed a comprehensive analysis by RT-PCR (Figure 4). In addition to HTR7, which has been studied in detail in the breast [6, 7], we report for the first time the expression of HTR1D, 2B and 3A in an untransformed cell line and primary human mammary epithelial cells (MCF10A and pHMEC) (Figure 4). The HTR1D and 2B were also present in all breast cancer cells tested and, using human hypothalamus as the reference level, expression in the cancer cells was elevated compared to the untransformed cells (Figure 4Bi and 4Biv).

Among the receptor isoforms that were expressed differentially in breast cancer cell lines, HTR3A was markedly downregulated in all of the cancer lines (Figure 4A and 4Bvi). Three receptor types were upregulated in MCF7 to levels that were at least 40% of the brain (1E, 1F, 2C) (Figure 4A, Bii, iii and 4Bv). MDA-MB-231 expressed 1F, and T-47D expressed both 1F and 2C at modest levels relative to the brain (Figure 4B). No detectable transcripts for HTR1A, 1B, 2A, 4, 5A and 6 were found in the untransformed (MCF10A and pHMECs) or breast cancer cells (MCF7, T47D and MDA-MB-231) (Figure 4A) and [see Figure S4 in Additional data file 1]. HTR7 was expressed in MCF10A [6, 7], pHMEC and MDA-MB-231 cells. In contrast, MCF7 and T47D, both of which are ER positive cancer cells, lacked any detectable HTR7 transcript (Figure 4A).

To address the question of 5-HT receptor profiles in breast cancers, we examined expression using the Oncomine database [36, 37] to search preliminarily for patterns of 5-HT receptor gene expression (Table 1). Among all of the receptor isoforms, HTR1B, 1D, 1F, 2A, 2B, 2C, 3, 4, 5A, 7 were found to be expressed in breast tissues (cancer and other). Of these receptors, the expression levels of HTR1D, 2A and 3 were unchanged in the archived studies (data not shown). The HTR1D pattern was in accord with the breast cancer cell lines (Table 1). HTR1B and 2A were not expressed in either pHMEC or established breast cell lines (Figure 4), which implies that expression in tumor specimens represents the presence of stromal or vascular elements, which typically express HTR1B and 2A in smooth muscle cells [38, 39].

A significant subset of 5-HT receptor mRNAs (HTR1F, 2B, 4 and 7), were significantly decreased in ER negative tumors, relative to ER positive tumors (Table 1). These observations were confirmed by multiple independent studies. HTR2B expression was also lower in basal tumors (ER negative), compared with luminal tumors, which are most commonly ER positive (Table 1).

Expression levels for HTR1F, 2B, 2C, 5A and 7 showed overall increases in breast cancers. HTR2B, which is expressed in untransformed human mammary epithelium (Figure 4), was elevated in carcinomas and was found to increase with tumor stage, and concomitantly was higher in lymph node-positive tumors as compared to node-negative tumors (Table 1). This observation was supported by a study of HMECs, showing c-Myc transformation induced an increase in HTR2B expression (Table 1). Similar to HTR2B, HTR2C showed an increase in expression with tumor stage. The HTR2C pattern was in accord with the increased expression observed in human breast cancer cell lines (Figure 4). Analogously, HTR2C expression was found to be higher in metastatic and Her2/neu-overexpressing tumors. The expression of HTR7 increased with tumor grade, and was higher in p53-mutated tumors (Table 1). This observation also was supported by a study of HMECs, showing H-Ras transformation induced increases in HTR7 expression. Other 5-HT receptor expression changes in breast cancers included HTR1F (higher in recurring tumors) and HTR5A (higher in p53-mutant) (Table 1).

The variety of examples showing aberrant expression of 5-HT receptors in breast cancers suggests that multiple modifications of 5-HT signaling can contribute to the loss of tissue homeostasis during tumor progression.

Our previous studies on 5-HT physiology in mammary gland cells revealed the critical roles of 5-HT in regulating epithelial homeostasis during involution, which is characterized by epithelial tissue regression [5‐7, 40, 41]. One expected effect of elevated 5-HT activity in the normal breast is widespread apoptotic cell death. Hence, we tested the effects of 5-HT on apoptosis in mammary epithelial cells. The pHMEC and MCF10A cells, as expected, showed significant increases in active caspase 3 staining when treated with 5-HT, as compared to untreated controls (Figure 5A). In contrast, all of the breast cancer cell lines were highly resistant to 5-HT-induced apoptosis under similar experimental conditions.

In response to 5-HT, MDA-MB-231 cells underwent dramatic changes in their morphological phenotype, assuming exaggerated fibroblastic spindle morphologies (Figure 5B). In the presence of 5-HT, the MDA-MB-231 cells projected long axial appendages, which inconsistently contacted neighboring cells, sometimes crossing cells without apparent interactions. Previously, similar changes in morphology of MDA-MB-231 cells have been associated with exaggerated motility and invasiveness [42, 43]. No similarly obvious phenotypic effects of 5-HT treatment were observed in MCF10A, pHMEC, T47D or MCF7 cells, suggesting that this response to 5-HT may be limited to the most aggressively-transformed breast cancer cells, which have undergone an epithelial-mesenchymal transition (Figure 5B).

Because breast cancer cells were resistant to 5-HT-induced apoptosis, we decided to explore its impact on proliferation in these cells. Treatment with 5-HT for 36 h (in serum-containing medium) resulted in significant inhibition of proliferation in both pHMECs (approximately 55%) and MCF10A (approximately 37%), compared to untreated controls (Figure 6Ai and 6ii). This observation was verified by a standard trypan blue assay under similar experimental conditions [see Figure S5 in Additional data file 1]. Methysergide (MS), a broad spectrum 5-HT receptor antagonist, attenuated growth inhibition by 5-HT in pHMECs and MCF10A (Figure 6Ai and 6ii). Our previous study showed that mammary epithelial cells express the 5-HT₇ receptor [6] and stimulation of this receptor results in cyclic AMP (cAMP)-mediated activation of both protein kinase A (PKA) and p38 mitogen activated protein kinase (p38 MAPK) [7]. Treatment of MCF10A cells with a 5-HT₇ antagonist (SB269970 [SB]) resulted in near complete extinction of 5-HT inhibition of proliferation (Figure 6Aiii). In addition, specific inhibition of p38 MAPK blocked the inhibition of proliferation (Figure 6Aiv), however PKA inhibition had no significant effect on proliferation. These data suggest that 5-HT₇-mediated p38 MAPK activation may, in part, be responsible for growth inhibitory actions of 5-HT in mammary epithelial cells.

It seemed paradoxical that breast cancer cell lines had an increased capacity for 5-HT biosynthesis (Figure 1), while 5-HT acted as a growth-inhibitor in mammary epithelial cells (Figure 6A). The juxtaposition of these findings suggested that the breast cancer cells might be resistant to growth inhibition by 5-HT. Hence, we tested the effect of 5-HT on proliferation of breast cancer cells. Confirming the previous result, 5-HT, in a concentration dependent manner, inhibited proliferation of mammary epithelial cells (pHMECs and MCF10A) (Figure 6B). However, breast cancer cells (T47D and MDA-MB-231) responded differently to 5-HT. The T47D cells were resistant to 5-HT, which required five- to ten-fold higher concentrations to show growth inhibition (Figure 6Bi). However, MDA-MB-231 cells were not only resistant to growth inhibition, but also, were significantly stimulated by 5-HT at low concentrations (Figure 6Aii). The altered response of MDA-MB-231 cells to 5-HT was seen in spite of the presence of strong expression of the HTR7 (Figure 4A and 4Bvi). To determine whether differences in cell death could account for the effects of 5-HT on proliferation, we measured lactate dehydrogenase (LDH) levels in the media, which is indicative of lysed/dead cells. In both the MCF10A and MDA-MB-231 cells, the LDH levels in the media remained unchanged by 36 h after 5-HT treatment [see Figure S6 in Additional data file 1]. At later time points (72 h, see Figure 5), there was substantial apoptosis in the untransformed (i.e., MCF10A) cells and pHMECs, which implied that these cells underwent growth arrest early, followed by apoptosis if 5-HT signaling was sustained.

Given that growth inhibition by 5-HT occurs through the 5-HT₇ receptor, resistance to growth inhibition in breast cancer cells could be explained by: 1] a loss of 5-HT₇ receptor expression (Figure 4A and 4Bvi), 2] expression of 5-HT receptors that counteract 5-HT₇ (that is, G_i-coupled), (Figure 4A, Bii and 6Biii) or 3] changes in signaling downstream of 5-HT₇; and these possibilities are not mutually exclusive. Expression of HTR7 in MDA-MB-231 cells was greater that in pHMEC and MCF10A, yet MDA-MB-231 cells showed significant differences in their response to 5-HT (Figure 6Bii). Therefore, we sought to determine whether G_s-coupled signaling, which is downstream of 5-HT₇, was altered in MDA-MB-231. As previously described [6], MCF10A cells showed a 5-HT concentration-dependent increase in cAMP accumulation (Figure 6Biii). In MDA-MB-231 cells, the maximum cAMP activation was observed at low concentrations of 5-HT, followed by a progressive decline in cAMP to levels below that of controls. Peak cAMP accumulation was identical in both cells (Figure 6Biii). These data imply altered signaling events associated with 5-HT₇.