Down-Regulation of 5-HT1B and 5-HT1D Receptors Inhibits Proliferation, Clonogenicity and Invasion of Human Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B– and 5-HT1D–mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.


Introduction
Pancreatic cancer (PaCa), which has a strong invasive capacity with frequent metastasis and recurrence, is known to be one of the most lethal human cancers with ,5% 5-year survival rate [1]. Although it only ranks tenth in incidence among the most common human cancers, PaCa is the fourth leading cause of cancer deaths in Western countries and its death rate has not decreased over the past few decades [2,3]. Overall, PaCa has about 100% mortality because it is generally detected at advance stages since it typically does not cause any symptoms at earlier stages [4]. PaCa is intrinsically resistant to apoptosis and poorly responds to existing therapeutics, including combination chemotherapeutic regimens [5]. To overcome this global health problem, the investigations are focused on the identification of novel molecular targets to develop new treatment strategies.
The mitogenic neurotransmitter, serotonin (5-HT) was previously known to acts as a growth factor [6] for several types of nontumoral cells (e.g. vascular smooth muscle cells, lung fibroblasts and renal mesangial cells) [7,8], and tumor cells (e.g. pancreatic carcinoid cells, small cell lung carcinoma cells and colorectal carcinoma) [9,10,11]. Recently, 5-HT has emerged as an important regulator of cell proliferation and tumor growth in a variety of cancer types [12,13,14,15]. During tumor progression, tyrosine hydroxylase, the rate-limiting enzyme in the serotonin biosynthesis pathway, is often up-regulated [16]. Importantly, different 5-HT receptors have been identified (5-HT-1-7) based on their structural, functional and pharmacological characteristics [17,18]. Six of the families of 5-HT receptors are G-proteincoupled, including Gi: 5-HT-1, Gs: 5-HT-4,6,7, and Gq/11: 5-HT-2,5. Only 5-HT-3 is uniquely a ligand-gated cation channel, related to the nicotinic acetylcholine receptor [16]. 5-HT receptors are further divided into different subtypes, e.g. 5-HT-1 family has five subtypes [18], comprising the 5-HT-1A, -1B, -1D, -1E and -1F receptors and couples preferentially to Gi/o to inhibit cAMP formation [19,20]. In particular, the human 5-HT 1B and 5-HT 1D receptors are especially similar in sequence despite being encoded by two distinct genes. The precise function of these receptors remains undefined, and progress toward this has been hampered by the lack of selective ligands [21]. It was previously indicated that the 5-HT-1 receptors are extensively expressed in the human breast cancer [22], prostate cancer [23] and bladder cancer cells [18], which could explain the mitogenic effects of the agonists of these receptor in such cancers. Pancreatic cancer research has mostly focused on the study of gene mutations and signal transduction pathways in pancreatic ductal adenocarcinoma (PDAC) cells, whereas the potential role of neurotransmitter receptors in the development and progression of this deadly neoplastic disease has been largely ignored [4]. Given the potential involvement of 5-HT-1 receptors signaling to the proliferation of several types of cancers, with unknown implications of these receptors on PaCa progression, we investigated here the role of 5-HT 1B and 5-HT 1D receptors in the proliferation and the invasive phenotype of PaCa.
Local invasion can be considered as an initial and essential step in the malignancy of carcinomas, leading to the generation of usually fatal distant metastasis. For the cancer cells to invade distant tissues, they have to penetrate surrounding extracellular matrices. Such cancer cell/ECM interactions are facilitated by the integrin family of cell adhesion molecules including tyrosinephosphorylated substrates (the tyrosine kinase Src and focal adhesion kinase) [24]. Integrins are trans-membranous a/b heterodimeric receptors that mediate cell-cell interactions and cell attachment to extracellular matrix (ECM) [25], and they serve as receptors for some ECM proteins (e.g., Fibronectin, Vitronectin, Laminin and Collagen) [26]. Altered integrin activity or substrate affinity can contribute to the neoplastic phenotype. Normally, cellular Src is held in an inactive state, but in several cancer types, abnormal events lead to elevated kinase activity of the protein and cause pleiotropic cellular responses inducing transformation and metastasis [27].
As carcinomas progress, the tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial to mesenchymal transition (EMT), a critical process similar to the process of embryonic development, is thought to be an important mechanism for promoting cancer invasion and metastasis [28]. In recent years, the ZEB family of zinc finger transcription factors has been documented as essential players of EMT [29]. The epithelial adhesion protein, E-cadherin, is an active suppressor of invasion and growth of many epithelial cancers, and its down-regulation is considered a hallmark of EMT [30,31]. E-cadherin is a major target gene of the ZEB family transcriptional repressors. EMTinducing mediators, such as TCF8, trigger epithelial dedifferentiation by impairing the expression/function of E-cadherin [32]. The E-cadherin repressors may regulate developmental transcriptional programs of EMT in tumor cells predisposing them to invasion and metastasis [33]. Thus, mutations in ZEB encoding genes link these factors to malignant tumor progression [29].
The current study focused on investigating the role of 5-HT 1B and 5-HT 1D receptors on PaCa proliferation and invasion. Our data demonstrates significant reductions in PaCa cells growth, invasion and correlated downstream signaling in response to down-regulation of these serotonin receptors expressions, suggesting the significant involvement of these receptors in promoting pancreatic cancer.

Cell lines, culture conditions and reagents
The human pancreatic cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). PANC-1 and MIAPaCa-2 cells were cultured in DMEM/F12 supplemented with 10% FBS. All media contain penicillin and streptomycin (100 units/ml). Cells were maintained at 37uC in a humidified atmosphere containing 5% CO 2 /95% air, and were used between passages 4 and 15. Human pancreatic duct epithelial (HPDE) cells were kindly provided by Dr. Kapil Mehta, Department of experimental Therapeutic, M.D. Anderson Cancer Center, as a generous gift. HPDE cells were maintained in keratinocyte serumfree medium (Keratinocyte-SFM, 1X) containing L-glutamine, and supplemented with prequalified human recombinant Epidermal Growth Factor 1-53 (EGF 1-53) and 25 mg/500 ml Bovine Pituitary Extract (BPE) (Invitrogen/Life Technologies, Carlsbad, CA). NF-kB activation inhibitor II, JSH-23 (4-Methyl-N 1 -(3phenylpropyl)benzene-1,2-diamine) (EMD Millipore Billerica, MA), was dissolved in DMSO at a final stock concentration of 10 mM, and directly added to cell cultures at 25 and 50 mM concentrations, which selectively blocks nuclear translocation of NF-kB p-65 and its transcription activity.

Cell viability and proliferation/growth assays
The viability and/or proliferation of cells were detected by MTS assay (Promega, Madison, WI, USA), after cells treatment, to measure cell growth. Cells were counted using a hemocytometer and viable cells were identified by trypan blue exclusion. Viable cells were seeded in 96-well plates (1.5610 3 cells/well), and transfected with indicated siRNAs. After 72 h of treatment, a solution containing MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and PMS (phenazine methosulfate) (20:1 v/v) was added to the cells. After 2-3 h of incubation at 37uC, the viable growing cells were estimated by monitoring the absorption of the product at 490 nm, based on the generation of formazan via live cells. All experiments were performed in triplicate and the results were reported as mean of absorption 6 standard deviation.

Clonogenic survival assay
PaCa cells were seeded in 6-wells plates (1.5610 3 cells/well), transfected with non-silencing control siRNA, or siRNA against 5-HT 1B or 5-HT 1D (once/week), and grown for 2 weeks. The formed-colonies were stained with crystal violet and the coloniesarea distribution regions were measured densitometrically [36].
Each experiment was performed in triplicate and the results were reported as mean of absorption 6 standard deviation.

Matrigel invasion assay
PaCa cells were transfected with 50 nM of indicated siRNAs, and 72 h later, equal number of the treated viable cells (4610 4 cells), were seeded onto Matrigel-coated Transwells (with 8mm pore size filters) in Matrigel invasion chambers (BD Biosciences, San Jose, CA). The number of cells that invaded the lower side of the membrane after 24 h was determined by counting cells in a minimum of four randomly selected areas. The experiments were performed in triplicate and the results were reported as mean of percentages of invasion 6 standard deviation.

Migration Assay
In-vitro wound-healing assay was used to assess cell motility and the ability to migrate. PANC-1 cells were plated in 6-well plates (5610 5 cells/well), and cultured in medium containing 10% FBS to achieve a nearly confluent cell monolayer. A scratch was then carefully made on the cell layer using a 10 mL sterile micropipette tip, and any cellular debris was removed by washing with PBS to remove floating cells. The wounded monolayers were then transfected with 50 nM of indicated siRNAs. Immediately after the treatments, the cells were photographed using a phase-contrast microscope (Nikon), to determine the wound width at time 0. The cultures were continued, and the cells were photographed again after 12 h and after 24 h of wounding the cell layer. The Wound healing was visualized by comparing photographs taken at 0 h with those taken at 12 h and 24 h later, and analyzed for the distance migrated by the leading edge of the wound at each time point. The distance traveled by the cells was determined by measuring the wound width at time 12 h and 24 h, and subtracting it from the wound width at time 0. The values obtained were then expressed as % migration, setting the gap width at 0 h as 100%. Three experiments were done in triplicate.

Western blot analysis
Cells were seeded in 25-cm 2 culture flasks (0.5610 6 cells/flask). Following treatments, the cells were collected, centrifuged, washed twice in ice cold PBS and whole-cell lysates were obtained by suspending the cells in a lysis buffer at 4uC. Lysates were centrifuged at 13,000 g for 10 min at 4uC, and the supernatant fractions were collected. Total protein concentration for each sample was determined by a detergent compatible protein assay kit (Bio-Rad, Hercules, CA), and Western blotting was performed as 40 mg protein/lane on 4-15% SDS-PAGE gel. The proteins were electro-transferred to PVDF membranes and were first incubated with the following primary antibodies; p-Src (Tyr-416), Src, b1 integrin, uPAR, MMP-2, Snail, TCF8/ZEB1, NF-kB (p-105/ p-50), and Claudin-1 (Cell Signaling Technology, Danvers, MA); p-FAK (Tyr-397), FAK (BD Biosciences, Franklin Lakes, NJ); 5-HT 1B (Sigma Chemical, St. Louis, MO); TG2 and a-SMA (Abcam; Cambridge, MA); Fibronectin and 5-HT 1D (Santa Cruz Biotechnology, Santa Cruz, CA), and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA). b-actin (Sigma Chemical, St. Louis, MO), or a-b-Tubulin (Cell Signaling Technology, Danvers, MA) were used as loading controls. All antibodies were diluted in TBS-Tween 20 containing 5% dry milk. Chemiluminescent detection was performed with Chemi-glow detection reagents (Alpha Innotech, San Leandro, CA), and the blots were visualized with a FluorChem 8900 imager, and quantified by a densitometer using the image analysis program (ImageJ 1.48s processing software, National Institutes of Health, Bethesda, MD, USA). All experiments were independently repeated at least twice.

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from the collected cells with TRIzol Reagent (Invitrogen/Life Technologies, Carlsbad, CA), and cDNA was obtained from 1 mg of total RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The cDNA for 5-HT 1B , 5-HT 1D , b1 integrin, TG2 and GAPDH were amplified using Platinum Taq DNA Polymerase kit (Invitrogen/ Life Technologies), with specific primers. Briefly, 2 mL of the total 20 mL of reverse-transcribed product were used for PCR in 16 PCR buffer containing 1. The sequences of the sense and anti-sense TG2 primers are 59-TAAGAGATGCTGTGGAGGAG-39; and 59-CGAGCCCTG-GTAGATAAA-39, respectively. The cDNA samples were incubated at 94uC (2-5 min.) to denature the template and activate the enzyme. This step was followed by 35 cycles of PCR amplification (as 94uC for 30 s, 55uC for 30 s and 72uC for 60 s with 5-HT 1B primer; 94uC for 30 s, 60uC for 30 s and 72uC for 60 s with TG2 primer; 94uC for 30 s, 58uC for 45 s and 72uC for 60 s with 5-HT 1D and b1 integrin primers, in each cycle). The PCR reaction was terminated with a final extension step of 5 min. at 72uC. The amplified reaction products were analyzed on a 1.2% agarose gel containing ethidium bromide. The cDNA synthesis was verified by detection of the GAPDH transcript, which was used as an internal control.

Statistical analysis
The data were expressed as the mean 6 SD of three independent experiments, and statistical analysis was performed using the Student's t-test, to determine statistical significance. P values less than 0.05 were considered statistically significant and are indicated by an asterisk.

5-HT 1B and 5-HT 1D receptors are overexpressed in pancreatic cancer cells
Increased 5-hydroxytryptamine biosynthetic capacity as well as severe alterations in the expression patterns of the 5-HT receptors, has been previously reported during progression of breast cancer [16]. Here, we evaluated the expression of 5-HT 1B and 5-HT 1D receptors in different PaCa cells as well as in normal human pancreatic duct epithelial (HPDE) cells. We found that these receptors are up-regulated in all PaCa cells tested, comparing with its low expression in normal pancreatic epithelium (Fig. 1A), suggesting that the dys-regulation of these receptors might promote signaling favor tumor progression in PaCa cells.

Knockdown of 5-HT 1B and 5-HT 1D receptors expression inhibits proliferation/viability of PaCa cells
We aimed to investigate the involvement of these receptors in the proliferation and growth of PaCa cells. Toward this end, we knocked down the expression of each receptor subtype in PANC-1 and MIAPaCa-2 cells, using specific small interfering RNA (siRNA). Despite being encoded by two distinct genes, the human 5-HT 1B and 5-HT 1D receptors are especially similar in sequence [21]. Therefore, as shown in Figure 1B and C, 5-HT 1B siRNA treatments led to a relatively lower expression of 5-HT 1D protein level, with a similar lower 5-HT 1B protein levels induced by 5-HT 1D siRNA. This could be attributed to the fact that both 5-HT 1B and 5-HT 1D receptors subtypes share a high amino acid sequence identity (,68% amino acid sequence homology), have similar ligand binding properties and are almost indistinguishable pharmacologically [37]. Such similarities-induced interactions between the two receptor types at the protein level could be occurred after gene expression (e.g, during protein maturation or folding). Therefore, the detecting of the receptors protein expression by Western blotting was not completely sufficient in distinguishing these closely-related receptor subtypes to establish their respective physiological relevance. To overcome such problem in order to investigate the biological effects of targeting each subtype individually, we used RT-PCR analysis and examined the effect of siRNA treatments on the transcription levels of the corresponding gene subtype. Our results clearly show that 5-HT 1B specific siRNA and 5-HT 1D specific siRNA were confirmed to inhibit the expression of the corresponding mRNA without any significant effect on the other cross analogue subtype in both PANC-1 and MIAPaCa-2 cell lines ( Fig. 1D and E). We next analyzed the proliferation after 72 h of siRNA treatment by MTS assay. As shown in Figure 1F and G, our results demonstrated that knockdown of 5-HT 1B and 5-HT 1D expression significantly inhibited the proliferation of both PANC-1 and MIAPaCa-2 cells. The combined down-regulation of both 5-HT 1B and 5-HT 1D subtypes impairs proliferation more than downregulation of either receptor alone ( Figure S1), suggesting the biological benefits provided from simultaneous targeting both receptors.

Targeting 5-HT 1B and 5-HT 1D receptors inhibits cell clonogenicity of PaCa cells
To further verify the role of 5-HT-1 serotonergic-receptors in PaCa cell proliferation and colony formation, we evaluated the clonogenic capacity of PaCa cells following knock-down of 5-HT 1B and 5-HT 1D receptors expression. This assay is an in-vitro cell survival assay based on the ability of a single cell to grow and form foci into a colony [36]. Knockdown of 5-HT 1B and 5-HT 1D receptors, using their specific siRNAs, markedly inhibits the ability of PANC-1 and MIAPaCa-2 cells to form colonies ( Fig. 2A and B, respectively). Overall, these findings suggest that 5-HT 1B -and 5-HT 1D -mediated signaling is involved in the proliferation and clonogenic capability of PaCa cells.

Targeting 5-HT 1B and 5-HT 1D receptors impairs cell invasion/migration of PaCa cells
Pancreatic ductal adenocarcinoma is characterized by highly invasive phenotype and strong metastatic capacity [38]. Because the expression of 5-HT 1B and 5-HT 1D receptors is elevated in PaCa cells, we assessed whether these receptors are involved in promoting such invasive phenotype. Therefore, we knocked down these receptors in PANC-1 and MIAPaCa-2 cells by siRNAs and evaluated the changes in their invasive capability by in-vitro invasion assay using Matrigel-coated Boyden chambers. This assay mimics the in-vivo invasion process and measures the number of cancer cells passing through a basement membrane matrix towards media containing chemo-attractants [39]. The most striking finding was that knockdown of 5-HT 1B and 5-HT 1D receptors significantly reduced the invasion of PANC-1 cells by about 76% and 66%, respectively (Fig. 3A), and reduced the invasion of MIAPaCa-2 cells by about 75% and 71%, respectively (Fig. 3B). We next examined the involvement of 5-HT 1B and 5-HT 1D receptors in mediating PANC-1 cell motility using the scratch assay at 12 h and 24 h time points. The analysis revealed that the distance covered by migrating cells was significantly decreased when the cells transfected with 5-HT 1B or 5-HT 1D receptors siRNAs compared to cells exposed to the non-silencing control siRNA (Fig. 3C). These results demonstrate a correlation between PaCa cell motile behavior and 5-HT 1B/1D expression. Overall, this data indicates that 5-HT 1B and 5-HT 1D receptors play a role in mediating PaCa cells migration and invasion.

5-HT 1B and 5-HT 1D receptors are involved in regulation of b1 integrin expression in PaCa Cells
After finding that 5-HT 1B and 5-HT 1D receptors are overexpressed in PaCa cells, suggestive of significant alterations in growth-promoting downstream signaling, we next investigated some downstream molecular effects of knockdown of these receptors. The integrin family of trans-membrane receptors links the extracellular matrix (ECM) to the intracellular actin cytoskeleton at focal adhesions interaction points. In addition to this structural role, integrin clustering can initiate intracellular signaling events that promote cell proliferation, survival and migration in both normal and tumorigenic cell contexts [40]. From integrin family, b1-subtype is known to induce Src and FAK activity through the recruitment and activation of Src/FAK dual kinase complex [41]. Because, down-regulation of 5-HT 1B/1D receptors suppresses PaCa cells migration/invasion (Fig. 3), we examined whether these receptors regulate the expression of b1 integrin. We employed Western blot and RT-PCR analysis to determine the expression of b1 integrin protein and mRNA levels, respectively, after silencing these receptors. We found that 5-HT 1B and 5-HT 1D receptors knockdown significantly induce downregulation of b1 integrin expression at both protein and mRNA level in both PANC-1 and MIAPaCa-2 cells (Fig. 4A and B).

5-HT 1B and 5-HT 1D receptors promote activation of Src-FAK signaling
The tyrosine-phosphorylated substrates (e.g, the non-receptor protein tyrosine kinase Src and focal adhesion kinase) function in integrin signaling [24]. FAK is associated with Src, and they have been shown competent in recruiting/engaging as a major component of an integrin signaling pathway [24,27]. Integrinmediated activation of Src/FAK linkage contributes to cancer progression and metastasis in multiple ways. Thus, the disruption of integrin/Src/FAK signaling may contribute to increased cancer cell death and metastatic phenotype [41]. Importantly, Src kinases are overexpressed in a variety of human tumors, and increased Src activity often correlates with malignant potential and metastasis of pancreatic cancer and many other tumors, as it plays an integral part of cellular migration, proliferation, adhesion, and angiogenesis. Therefore, Src kinases have been proposed as a molecular target for therapy in such cancers [42,43]. Src is activated by the phosphorylation of Tyr-416 and dephosphorylation at the negative regulatory site Tyr-527 [44]. Activation of FAK, by integrin clustering, leads to auto-phosphorylation at Tyr-397, the binding site for the Src family kinases [24]. Therefore, we investigated the activity of Src (p-Tyr-416) and FAK (p-Tyr-397) after targeting 5-HT 1B and 5-HT 1D receptors. In correlation with b1 integrin reduction, silencing the expression of 5-HT 1B and 5-HT 1D receptors, lead to profound reductions of active Src and active FAK in both PANC-1 cells (Fig. 4A) and MIAPaCa-2 cells (Fig. 4B). This data suggests that 5-HT 1B -and 5-HT 1D -mediated signaling is involved in activation b1 integrin/Fak/Src complex. Inhibition of this engagement cascade may be one of the underlying mechanisms that mediate inhibition of PaCa cell invasion and proliferation evolved after targeting 5-HT 1B and 5-HT 1D receptors.

5-HT 1B and 5-HT 1D receptors regulate ECM/uPAR/MMP-2 signaling
A prerequisite of the ability of a cancer cell to undergo metastasis into distant tissues is to penetrate surrounding extracellular matrices (ECM) [27]. Plasma membrane urokinase-type plasminogen activator receptor (uPAR) is a protein that binds with high-affinity and activates the serine protease uPA, thus regulating proteolytic activity at the cell surface [45]. Plasmin activated by uPA can break down ECM directly or degrade the ECM indirectly through activation of pro-matrix metalloproteinases (MMPs) [46]. Although plasmin has been shown to principally activate MMP-1, -3, and -9, increasing evidence proves that uPA/plasmin can activate pro-MMP-2 and thereby promoting tumor invasion and metastasis [46,47]. Thus, we examined whether 5-HT 1B and 5-HT 1D receptors regulate the expression of uPAR/MMP-2, as important markers for cell invasion [45]. Knockdown of such receptors results in significant reductions in the expression level of both ECM proteins in both PANC-1 and MIAPaCa-2 cells (Fig. 4A and B respectively).

5-HT 1B and 5-HT 1D receptors regulate the zinc finger transcriptional factors of epithelial mesenchymal transition (EMT)
Epithelial-to-mesenchymal transition (EMT) is implicated in the progression of primary tumors towards metastasis, and is likely caused by a pathological activation of transcription factors regulating EMT [48]. This critical process requires a loss of cellcell adhesion, as well as the acquisition of a fibroblastoid motile phenotype [49]. To explore the role of 5-HT 1B and 5-HT 1D receptors in regulating EMT-related regulators, we investigated the expression of the zinc finger ZEB1 transcriptional factors that repress the cell-cell junctions [50], after knock-down of these receptors, using Western blot analysis. We investigated the expression of TCF8, as one of the EMT-inducing transcription factors belongs to ZEB1 family, which triggers epithelial dedifferentiation by impairing the expression of E-cadherin, the epithelial adhesion protein and the key mediator of cell-cell junctions [48]. The EMT tight junction proteins, claudins, were identified as potent inhibitors of the invasiveness and metastatic phenotype of pancreatic cancer cells [51]. These tight junctions are the most apical components of intercellular junctional complexes in epithelial and endothelial cells. They separate the apical and baso-lateral cell surface domains, maintaining cell polarity [38]. The loss of tight junction function leads, in a part, to invasion of cancer cells [52], and enhances tumor cell proliferation [48]. In particular, claudin-1 plays crucial roles in epithelial cell polarity during EMT [38]. Thus, we also examined the levels of Snail, the important transcriptional repressor of both E-cadherin [50], and claudin-1 [38,53]. Our results demonstrated that down-regulation of 5-HT 1B and 5-HT 1D receptors significantly reduce the expression of TCF8/ZEB1 and Snail, indicating the effective inhibition of EMT. The basal level of E-cadherin expression in untreated cells was un-detectable by Western blotting, probably due to the high basal expression of TCF8/ZEB1, which impairs the E-cadherin expression. Because Snail acts as transcription repressor of the genes of claudin/occludin, EMT tight junction proteins [53], we further investigated the expression of claudin-1, which is known to be expressed in various types of epithelial cells, playing an important role in epithelial cell polarity, cancer invasion and metastasis [54,55]. Parallel to the reduction of the Snail and TCF8/ZEB1 levels, down-regulation of 5-HT 1B and 5-HT 1D receptors was accompanied with obvious up-regulation of claudin-1 expression in both PANC-1 and MIAPaCa-2 cells (Fig. 4C and D). Furthermore, as shown by our RPPA data (Fig. 4E), the inhibition of the expression of the 5-HT 1B and 5-HT 1D receptors was associated with up-regulation of E-cadherin, as well as decrease in its transcriptional repressor, TWIST, one of the transcription factors that modulate EMT, along with Snail1, Slug, ZEB1, ZEB2, E12, E47 [50]. In conclusion, targeting 5-HT 1B and 5-HT 1D receptors mediate inhibition of EMT attainment and induce epithelial molecular characteristics.

TG2/NF-kB signaling underlies 5-HT 1B/1D receptorsmediated pathways promoting proliferation and invasion
Tissue transglutaminase (TG2) is implicated in regulation of cell attachment, interactions of the cells with the surrounding ECM, motility, invasion and is considered as a bad prognostic factor in different cancers, including PaCa [50,56,57,58]. Importantly, TG2 can exist in complex with integrins (b1, b4 and b5) in cancer cell membranes [26]. It also stabilizes the b1-integrin/ Fibronectin complex at the cell surface to support cell adhesion, motility and invasion [57]. Beside modifying ECM proteins  interactions, extracellular TG2 was found to activate nuclear factor-kappa B (NF-kB) signaling, leading to CD44 up-regulation and EMT activation, contributing to increased cancer cell invasiveness and peritoneal dissemination [59]. On the other hand, intracellular TG2 is known to modulate intracellular signaling including FAK, Akt [60], and NF-kB signaling [61], to promote proliferation and cell adhesion. Interestingly, constitutive NF-kB activation is known to play a key role in the aggressive behavior of pancreatic cancer [62]. The tight correlation between TG2 expression/activation and constitutive activation of NF-kB in PaCa cells and many other cancers [61], warranted our attention to address the question whether 5-HT 1B/1D receptors-activated proliferation/invasion signaling pathways is mediated through TG2 and/or NF-kB. Thus, we first examine if 5-HT 1B and 5-HT 1D receptors regulate the TG2/NF-kB expression. Our results show that down-regulation of 5-HT 1B and 5-HT 1D receptors significantly reduce the expression of both TG2 and NF-kB (Fig 5A), suggesting that 5-HT 1B/1D receptors lie as an upstream of TG2/NF-kB complex. We next knocked TG2 down and examined the same molecular targets that are regulated by 5-HT 1B/1D receptors and involved in proliferation, invasion and EMT process. Consistent with the events that produced after down-regulation of HT 1B/1D receptors, knockdown of TG2 induces similar trend (Fig. 4B), further supporting the role of TG2 in regulation proliferation and invasion pathways in PaCa cells. In fact, there is a direct correlation between TG2 expression and NF-kB activation in various cancer cell lines [61], and NF-kB activation is a known inducer of EMT in TG2-expressing cells [50,63]. TG2 and NF-kB form a recruitment complex at to the promoter sequence of Snail leading to its transcriptional regulation, facilitating EMT attainment [50]. Because of this positive cross-linking and regulatory loop, we next examined the effect of specific inhibition of constitutive NF-kB activation in PaCa cells, using a specific NF-kB activation inhibitor, JSH-23. Treating the cells with JSH-23 led to a concentration-dependent decrease in TG2 expression, suppression of b1 integrin/Src signaling along with inhibition of EMT markers a-SMA and Fibronectin. Parallel to our results, NF-kB was recently found to regulate b1 integrin expression in breast cancer cells [64]. Taken together, this data suggest a close link between TG2/NF-kB, and 5-HT 1B/1D receptors in mediating pro-tumorigenic pathways and in contributing to PaCa cells proliferation and invasion. It also suggest a rationale for inhibiting endogenous 5-HT 1B/1D receptors to inhibit TG2/NF-kB axis, leading to suppression of the potential cellular tumorigenic downstream signaling. Figure 6 depicts a summary of the suggested molecular mechanisms of the down-regulation of 5-HT 1B -and 5-HT 1Dmediated inhibition of proliferation and invasion of PaCa cells.

Discussion
The essential phenotype of epithelial cancers (e.g., pancreas, breast, prostate) includes: self-sufficiency for growth signals, sustained tissue invasion and insensitivity to growth inhibitors [16,65]. Pancreatic cancer is one of the most deadly neoplastic diseases because it typically diagnosed at an advanced stage. At the time of diagnosis, most pancreatic cancers are, therefore, inoperable and have metastasized to distant organs. In addition, this malignancy is generally unresponsive to conventional radioand chemotherapy, resulting in a mortality rate near 100% within 6 months of diagnosis [4]. In the current study, we demonstrated the over-expression of 5-HT 1B and 5-HT 1D receptors subtypes in PaCa cells, determined their involvement in PaCa cells progression, and investigated the effect of down-regulation of these receptors on PaCa cells growth and invasion. We showed that 5-HT 1B and 5-HT 1D receptors promote the key survival factors involved in supporting PaCa cells proliferation and invasion and thus they may be considered as one of the novel potential therapeutic targets in PaCa treatment.
The biogenic amines (e.g; serotonin, 5-HT) have been reported to be released as growth factors in different malignancies [16]. 5-HT can exert its mitogenic effect in interaction with other hormones or growth factors [22,66]. It has been previously shown that different 5-HT receptors can trigger the proliferative properties of 5-HT and mediate cascades of mitogenic signaling pathways [67]. The stimulation of 5-HT receptors leads to transcription factors phosphorylation and cell division activation, which is a common reaction to many extracellular stimuli, including growth factors, hormones and neurotransmitters in mammalian cells, such as human pancreatic carcinoid, and mouse fibroblast cells [68,69]. In prostate cancer, these growth factors act on the adjacent (exocrine) tumor cells to enhance tumor growth, differentiation and angiogenesis, the events that lead to a poor prognosis [18,70]. Recently, 5-HT 1B receptors were associated with an increased proliferation index, correlated with the size of the tumor in hepatocellular cancer patients [71], and were shown to play a potential role in colorectal cancer [69]. Since we showed here that the 5-HT 1B and 5-HT 1D receptors are highly overexpressed in PaCa cells, we tried to explore the etiological associations between its expression and PaCa progression.
Mechanisms responsible for elaboration of growth-and invasion-stimulating signals and molecular events responsible for their sensing are deregulated in cancer cells. To get insight into the role of these 5-HT-1 receptors in PaCa progression, we investigated some proliferation and invasion biomarkers, in order to speculate some of the contributing transduction signaling mediated by these receptors in PaCa cells. uPAR was proposed as an important regulator of the invasive properties of cancer cells. It is highly expressed in virtually all human cancers, and such overexpression is mediated by some extracellular matrix proteins [45,72]. The over-expression of the uPA/uPAR-system components correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of the cancer [45]. Invasive tumor cells have a marked ability to degrade extracellular matrix via activation of matrix metalloproteases (MMPs). MMP-2 rather than MMP-9 was activated in the metastatic pancreatic cancer, and it is secreted as an inactive zymogen and requires distinct activation processes [46]. MMP-2 can be activated through plasminogen activator/plasmin system, in which pro-uPA binds to its receptor, uPAR [73], resulting in uPA activation, acceleration of the conversion of plasminogen to plasmin on the cell surface, and localization these enzymes to focal contact sites [46,74]. Our data shows the effective down-regulation of uPAR and MMP-2 following the silencing of 5-HT 1B and 5-HT 1D receptors, supporting the notion of the distinct correlation between the expression of these receptors and the expression of uPAR/ MMP-2. Although the involvement of uPA cascade in MMP-2 activation was previously reported in metastatic pancreatic cancer BxPc3 cells [46], we showed for the first time that the expression pattern of these proteins is regulated by 5-HT 1B/1D receptors, in the metastatic PANC-1 and MIAPaCa-2 cells.
On the other hand, the uPAR-mediated activity requires integrin-dependent signaling [75,76]. The integrin family of cell adhesion molecules facilitates the penetration and invasion of the cancer cell to the surrounding extracellular matrices [27]. In pancreatic BxPC3 cells, the blocking of the over-expressed b1 integrin was found to decrease the activation of uPA/MMP-2 with subsequent decrease in metastasis [46]. b1 integrin is proposed as an emerging target that limits the metastasis of the tumors in-vivo [77], since it plays a profound role in cancer initiation, tumor growth progression and invasion/metastasis, through cell binding to ECM [78,79]. Moreover, b1 integrin expression is known to induce Src activity, which is associated with shorter patient survivals, making both b1 integrin and Src appealing targets for cancer therapy [80]. Src is one of the tyrosine kinases that plays a critical role in signal transduction associated with cell-extracellular matrix interactions, migration and adhesion [81]. Interestingly, Src inhibitors have shown a significant inhibition of the tumor growth in a subset of human pancreatic tumor xenografts [82]. In addition, recruitment of Focal adhesion kinase (FAK)/Src complex mediates and regulates the signaling events downstream of integrin-dependent pathway [27]. The cytoplasmic protein tyrosine kinase, FAK, is involved in integrin-mediated signal transduction and plays an important role in the control of cell spreading, migration, and survival [83]. Src/FAK mutually regulates the activity of each other and promotes normal and cancer cell migration by regulating focal adhesion formation and turnover through multiple signaling connections [41]. Enhanced FAK signaling was also connected to elevated uPA expression, directly contributing to the proliferation, invasion and metastatic phenotype [84]. It was documented that the cells expressing an activated FAK-Src signaling alter the surrounding stromal environment, facilitating breakdown of cell-cell adhesion, increased cell-matrix-and focal-adhesions and tissue invasion. Accordingly, inhibition of Src/FAK activity leads to restoration of cell-cell adhesion and inhibits cell migration and invasion [41]. In the current study, we demonstrated the potential inhibition of b1 integrin protein and gene expression and the inhibition of Src/ FAK activity after silencing of 5-HT 1B and 5-HT 1D receptors in PANC-1 and MIAPaCa-2 cells (Fig. 4 A-B). Furthermore, Integrins serve as receptors for some ECM proteins (e.g., Fibronectin, Vitronectin, Laminin and Collagen) [26]. The cell attachment/motility regulatory protein, tissue transglutaminase (TG2), was found to play a role in stabilizing the b1-integrin/ Fibronectin complex [84,85]. We showed here that molecular downstream signaling mediated through TG2 is similar to that regulated by 5-HT 1B and 5-HT 1D receptors, and that the inhibition of 5-HT 1B and 5-HT 1D receptors suppressed the expression of TG2 and NF-kB ( Fig. 5A and B). In many types of tumor cells including PaCa, TG2 contributes to constitutive activation of NF-kB, which, in turn, activates gene transcription [61]. Intracellular TG2 promotes degradation of IkBa resulting in constitutive activation of NF-kB through a canonical pathway, via the formation of the p52/RelB complex and its translocation to the nucleus [59]. Alternatively, association of TG2 with p65/p50 complex could mitigate the binding of IkBa to NF-kB complex, resulting in NF-kB constitutive activation. TG2-mediated NF-kB activation/nuclear translocation can result in constitutive activation of transcription of various target genes, including TG2 [61]. In addition, NF-kB was recently found to bind at the b1 integrin promoter region in breast cancer cells, leading to significant increase in b1 integrin expression [64]. This indicates a regulatory role for NF-kB to b1 integrin and subsequent downstream signaling (eg., Src, FAK, etc) as well as other recruited proteins involved in cell attachment and motility. Parallel with these results, our current data demonstrates that specific inhibition of NF-kB led to significant inhibition of b1 integrin/Src activation signaling and other b1-integrin-associated ECM proteins (e.g. TG2 and Fibronectin) (Fig. 5C).
Tumor invasion appears to be also controlled by other coordinated series of complex cellular and molecular processes that enable tumor cells to dissociate and migrate from the primary tumor [86]. The changes in cell adhesion and migration during tumor invasion are reminiscent of EMT; a process that reorganizes epithelial cells to become migratory mesenchymal cells. EMT is a crucial process in tumor progression providing tumor cells with the ability to escape from the primary tumor, to migrate to distant regions and to invade tissues, promoting oncogenic progression and metastasis [28]. Alterations of EMT transcription factors define consequences for tumorigenesis [87]. The zinc finger ZEB1 EMT-inducing factor, TCF8, has been identified as a potent transcriptional repressor of E-cadherin, the protein that rescues epithelial architecture [48]. Snail is another central EMT inducer, by its inhibitory effect on both E-cadherin and other EMT-tight junction proteins [50,53]. Snail directly binds to the promoters of claudin/occludin genes, resulting in repression of the promoter activities and loss of epithelial cell polarity [53]. These EMTmolecular triggers repress genes encoding cadherins, claudins, cytokines, integrins, mucins and occludin proteins, thereby promoting EMT [56]. In addition, Snail confers resistance to cell death [88]. In pancreatic cancer, expression of claudin-4 was associated with significant reduction of in-vitro invasive potential, inhibition of colony formation, as well as reduction of in-vivo metastases [51]. Thus, it is thought that the loss of tight junction function leads, in a part, to invasion and metastasis of cancer cells [52]. The functional loss of epithelial cell polarity also enhances tumor cell proliferation [48]. Here, we show that targeting 5-HT 1B and 5-HT 1D receptors was associated with up-regulation of Ecadherin (the fundamental key adhesion and epithelial marker), and claudin-1 expression, along with concomitant decreases in the Figure 6. The postulated molecular regulation of 5-HT 1B and 5-HT 1D receptors to b1 integrin/Src/FAK complex, ECM/uPAR/MMP-2 signaling and the zinc finger transcriptional regulators of EMT in PaCa cells. These receptors-regulated signaling might be mediated through TG2/NF-kB axis. 5-HT 1B and 5-HT 1D receptors mediate b1-integrin activity to recruit a Src-FAK complex promoting the cell proliferation and migration. Upon different extracellular mitogenic stimuli (e.g; growth factors, hormones and neurotransmitters), the over-expressed 5-HT 1B/1D receptors promote the activation of urokinase plasminogen activator receptor (uPAR), and matrix metalloproteinase (MMP-2), facilitating extracellular matrix degradation and enhancement of invasion process. Also, 5-HT 1B/1D receptors stimulate the expression of zinc finger transcriptional factors (Snail and TCF8/ZEB1). These mesenchyme markers, in turn, repress the gene transcription of epithelial markers (claudin-1 and E-cadherin) leading to stimulation of epithelial mesenchymal transition (EMT), further supporting the proliferation and invasiveness of PaCa cells. doi:10.1371/journal.pone.0105245.g006 expression levels of the E-cadherin-repressors, TCF8/ZEB1, Snail and TWIST (Fig. 4C-E).
In conclusion, the in-vitro data pioneered by our study suggests the involvement of 5-HT 1B and 5-HT 1D receptors in the activation of the b1 integrin-mediated proliferation/invasion promoting signaling and EMT process, representing these receptors as novel important regulators of pro-tumorigenic signals. To have generally accepted view, we are currently investigating the value of in-vivo targeting 5-HT 1B and 5-HT 1D receptors in nude mice-bearing PANC-1 tumor xenografts using siRNA-nanoparticles, to demonstrate the role of these receptors in pancreatic tumor progression, in order to establish a novel and efficient molecularly targeted therapy for pancreatic cancer. Figure S1 Effects of dual down-regulation of 5-HT 1B and 5-HT 1D receptors on PaCa cell proliferation. PANC-1 cells were transfected with control, 5-HT 1B or 5-HT 1D siRNAs, or transfected with both 5-HT 1B and 5-HT 1D simultaneously. After 72 h, proliferation was evaluated by an MTS assay. Data are represented as mean 6 SD of three independent experiments. * P,0.05 vs. control cells. # represents significant difference between indicated groups (P,0.05). (TIF)

Author Contributions
Conceived and designed the experiments: NG AAA BO. Performed the experiments: AAA NG. Analyzed the data: AAA NG SNA BO. Contributed reagents/materials/analysis tools: NG AAA SNA BO. Contributed to the writing of the manuscript: AAA BO NG. Carried out the statistical analysis: AAA. The study conception, coordination, and results evaluation: AAA NG SNA BO. Contributed equally to this work and share the first authorship: AAA NG.