University of Groningen Studies on the role of dopamine and serotonin in tumors and their microenvironment Peters, Marloes A.M.

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University of Groningen

Studies on the role of dopamine and serotonin in tumors and their microenvironment Peters, Marloes A.M.

DOI:

10.33612/diss.135597229

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Publication date: 2020

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Peters, M. A. M. (2020). Studies on the role of dopamine and serotonin in tumors and their microenvironment. University of Groningen. https://doi.org/10.33612/diss.135597229

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548127-L-bw-Peters 548127-L-bw-Peters 548127-L-bw-Peters 548127-L-bw-Peters Processed on: 17-9-2020 Processed on: 17-9-2020 Processed on: 17-9-2020

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CHAPTER 1

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BACKGROUND

Tumor progression is not only determined by tumor cell characteristics, but also by the tumor microenvironment and systemic responses. Here, several cell types play a role including immune cells, platelets, and endothelial progenitor cells. These are attracted towards the tumor, contribute to a pro-inflammatory state, provide growth factors, and facilitate blood vessel formation [1,2]. Together, this resembles the process of wound healing. However, in wound healing, this process is self-limiting, whereas it is constitutively active in cancer [3].

The newly formed tumor blood vessels are structurally and functionally abnormal, causing variations in blood flow [4]. This can activate platelets, which then aggregate and release their content [5]. Platelets store and transport pro- and anti-angiogenic factors, of which vascular endothelial growth factor A (VEGF-A) is studied most extensively [6]. VEGF-A predominantly stimulates angiogenesis via activation of VEGF receptor 2 (VEGF-R2). Not only VEGF-A is transported by platelets, but dopamine and serotonin are as well [7,8].

Dopamine and serotonin are biogenic amines which can function as neurotransmitters. They are produced in the central nervous system and the gastro-intestinal tract [9,10]. Dopamine exerts its function via the dopamine receptor D1 (DRD1) and the dopamine receptor D2 (DRD2). These receptors activate opposite intracellular pathways; DRD1 stimulates formation of cyclic adenosine monophosphate (cAMP), whereas DRD2 inhibits formation of cAMP [9]. Serotonin acts via 7 serotonin receptors (5-HTR1 to 5-HTR7). Some of these have subtypes including 5-HTR1A, 5-HTR1B, 5-HTR2A, and 5-HTR2B [10]. The reuptake of dopamine and serotonin from the synaptic cleft and the plasma is regulated by their respective transporters. The dopamine transporter and the serotonin transporter are located at the plasma membrane of neurons and platelets (Figure 1 and 2) [9,10].

Dopamine and serotonin play a role in limb movement control, the reward system in the brain, vascular tone, gastro-intestinal motility, and other physiological processes. Dopamine is involved in Parkinson’s disease, schizophrenia, and mood disorders. Serotonin plays a role in migraine and nausea [9-12]. Both serotonin and dopamine can be produced by neuroendocrine tumors (NET) [13].

Preclinical studies suggest that dopamine and serotonin can also affect tumor angiogenesis and tumor growth [14-18]. Dopamine can inhibit tumor angiogenesis via activation of DRD2 [14,15]. Contradictory results have been published for DRD1, as both inhibition as well as stimulation of tumor growth has been reported upon receptor activation [14,16]. Serotonin can stimulate angiogenesis and tumor cell proliferation. This is regulated via activation of 5-HTR1B and 5-HTR2B [17,18].

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BACKGROUND

Tumor progression is not only determined by tumor cell characteristics, but also by the tumor microenvironment and systemic responses. Here, several cell types play a role including immune cells, platelets, and endothelial progenitor cells. These are attracted towards the tumor, contribute to a pro-inflammatory state, provide growth factors, and facilitate blood vessel formation [1,2]. Together, this resembles the process of wound healing. However, in wound healing, this process is self-limiting, whereas it is constitutively active in cancer [3].

The newly formed tumor blood vessels are structurally and functionally abnormal, causing variations in blood flow [4]. This can activate platelets, which then aggregate and release their content [5]. Platelets store and transport pro- and anti-angiogenic factors, of which vascular endothelial growth factor A (VEGF-A) is studied most extensively [6]. VEGF-A predominantly stimulates angiogenesis via activation of VEGF receptor 2 (VEGF-R2). Not only VEGF-A is transported by platelets, but dopamine and serotonin are as well [7,8].

Dopamine and serotonin are biogenic amines which can function as neurotransmitters. They are produced in the central nervous system and the gastro-intestinal tract [9,10]. Dopamine exerts its function via the dopamine receptor D1 (DRD1) and the dopamine receptor D2 (DRD2). These receptors activate opposite intracellular pathways; DRD1 stimulates formation of cyclic adenosine monophosphate (cAMP), whereas DRD2 inhibits formation of cAMP [9]. Serotonin acts via 7 serotonin receptors (5-HTR1 to 5-HTR7). Some of these have subtypes including 5-HTR1A, 5-HTR1B, 5-HTR2A, and 5-HTR2B [10]. The reuptake of dopamine and serotonin from the synaptic cleft and the plasma is regulated by their respective transporters. The dopamine transporter and the serotonin transporter are located at the plasma membrane of neurons and platelets (Figure 1 and 2) [9,10].

Dopamine and serotonin play a role in limb movement control, the reward system in the brain, vascular tone, gastro-intestinal motility, and other physiological processes. Dopamine is involved in Parkinson’s disease, schizophrenia, and mood disorders. Serotonin plays a role in migraine and nausea [9-12]. Both serotonin and dopamine can be produced by neuroendocrine tumors (NET) [13].

Preclinical studies suggest that dopamine and serotonin can also affect tumor angiogenesis and tumor growth [14-18]. Dopamine can inhibit tumor angiogenesis via activation of DRD2 [14,15]. Contradictory results have been published for DRD1, as both inhibition as well as stimulation of tumor growth has been reported upon receptor activation [14,16]. Serotonin can stimulate angiogenesis and tumor cell proliferation. This is regulated via activation of 5-HTR1B and 5-HTR2B [17,18].

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Fig. 1 Dopamine pathway. (A) Dopamine, norepinephrine (NE) and epinephrine (E) are

produced in chromaffin cells in the medulla of the adrenal gland. Tyrosine (TYR) is converted into dihydroxyphenylalanine (DOPA) and then to dopamine. Dopamine can be further metabolized into NE and E. These catecholamines are stored in chromaffin granules and are released into the circulation upon activation by the splanchnic nerve. Dopamine is taken up by platelets via the dopamine transporter (DAT). (B) Dopamine is also produced in in the central nervous system,

and stored in vesicles in the presynaptic neuron via vesicular monoamine transporter 2 (VMAT2). Dopamine is released in the synaptic cleft, and can bind to various dopamine receptors (DRD1 and DRD2-like receptors) on the postsynaptic neuron. Ligand-receptor binding induces intracellular signaling. Dopamine re-uptake from the synaptic cleft into the presynaptic neuron is mediated by DAT. Dopamine is inactivated by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) and eventually converted into homovanillic acid (HVA) and undergoes renal clearance.

(C) Synthesis of dopamine, NE and E from the amino acid TYR with the involved enzymes is

shown in the yellow panel. Inactivation of dopamine through conversion into HVA is shown in the green panel.

These findings suggest that dopamine and serotonin receptors may be potential targets for cancer treatment. Dopamine receptor agonists are already used in clinical practice for the treatment of patients with hyperprolactinemia and Parkinson’s disease [9], and serotonin receptor agonists respectively antagonists are used for migraine and severe nausea [11,12]. Furthermore, selective serotonin reuptake inhibitors (SSRIs), which the block serotonin transporter, are extensively used to treat depression and anxiety. Since serotonin transporters are also located on platelets, SSRIs might be able to deprive these delivery vehicles from serotonin, and thus potentially affect free plasma serotonin concentrations. However, before clinical trials can be conducted in cancer patients, more information is needed regarding dopamine’s and serotonin’s mechanism of action, expression of their receptors, and the platelet and free plasma concentrations of serotonin and dopamine.

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Fig. 2 Serotonin pathway. (A) The majority of serotonin is produced in enterochromaffin cells

in the gut. Tryptophan (TRP) is converted to hydroxytryptophan (HTP) and then to 5-hydroxytryptamine (5-HT, serotonin). Serotonin is released in the gut lumen, but also enters the circulation where it is taken up by platelets via the serotonin transporter (SERT). Serotonin is stored in dense granules, uptake in granules is mediated by the vesicular monoamine transporter 2 (VMAT2). (B) Serotonin cannot cross the blood-brain barrier and is produced in the central

nervous system, and stored in vesicles in the presynaptic neuron via VMAT2. Serotonin is released in the synaptic cleft, and can bind to various serotonin receptors (5-HTR) on the post synaptic neuron. Ligand-receptor binding induces intracellular signaling. Serotonin re-uptake form the synaptic cleft into the presynaptic neuron is mediated by SERT. Serotonin is inactivated by monoamine oxidase (MAO) and eventually converted into 5-hydroxyindoleacetic acid (5-HIAA) and undergoes renal clearance. (C) Synthesis of serotonin and melatonin from the amino acid TRP

with the involved enzymes is shown in the yellow panel. In the blue panel the alternative kynurenine pathway is shown. Inactivation of serotonin through conversion into 5-HIAA is shown in the green panel.

Therefore, the aim of this thesis is to explore the role of dopamine and serotonin in cancer by targeting DRD2 in an in ovo ovarian cancer model, assessment of serotonin

and dopamine receptor expression in solid tumors, and analysis of platelet and free plasma serotonin concentrations as well as plasma concentrations of its precursors and metabolites.

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Fig. 1 Dopamine pathway. (A) Dopamine, norepinephrine (NE) and epinephrine (E) are

produced in chromaffin cells in the medulla of the adrenal gland. Tyrosine (TYR) is converted into dihydroxyphenylalanine (DOPA) and then to dopamine. Dopamine can be further metabolized into NE and E. These catecholamines are stored in chromaffin granules and are released into the circulation upon activation by the splanchnic nerve. Dopamine is taken up by platelets via the dopamine transporter (DAT). (B) Dopamine is also produced in in the central nervous system,

and stored in vesicles in the presynaptic neuron via vesicular monoamine transporter 2 (VMAT2). Dopamine is released in the synaptic cleft, and can bind to various dopamine receptors (DRD1 and DRD2-like receptors) on the postsynaptic neuron. Ligand-receptor binding induces intracellular signaling. Dopamine re-uptake from the synaptic cleft into the presynaptic neuron is mediated by DAT. Dopamine is inactivated by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) and eventually converted into homovanillic acid (HVA) and undergoes renal clearance.

(C) Synthesis of dopamine, NE and E from the amino acid TYR with the involved enzymes is

shown in the yellow panel. Inactivation of dopamine through conversion into HVA is shown in the green panel.

These findings suggest that dopamine and serotonin receptors may be potential targets for cancer treatment. Dopamine receptor agonists are already used in clinical practice for the treatment of patients with hyperprolactinemia and Parkinson’s disease [9], and serotonin receptor agonists respectively antagonists are used for migraine and severe nausea [11,12]. Furthermore, selective serotonin reuptake inhibitors (SSRIs), which the block serotonin transporter, are extensively used to treat depression and anxiety. Since serotonin transporters are also located on platelets, SSRIs might be able to deprive these delivery vehicles from serotonin, and thus potentially affect free plasma serotonin concentrations. However, before clinical trials can be conducted in cancer patients, more information is needed regarding dopamine’s and serotonin’s mechanism of action, expression of their receptors, and the platelet and free plasma concentrations of serotonin and dopamine.

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Fig. 2 Serotonin pathway. (A) The majority of serotonin is produced in enterochromaffin cells

in the gut. Tryptophan (TRP) is converted to hydroxytryptophan (HTP) and then to 5-hydroxytryptamine (5-HT, serotonin). Serotonin is released in the gut lumen, but also enters the circulation where it is taken up by platelets via the serotonin transporter (SERT). Serotonin is stored in dense granules, uptake in granules is mediated by the vesicular monoamine transporter 2 (VMAT2). (B) Serotonin cannot cross the blood-brain barrier and is produced in the central

nervous system, and stored in vesicles in the presynaptic neuron via VMAT2. Serotonin is released in the synaptic cleft, and can bind to various serotonin receptors (5-HTR) on the post synaptic neuron. Ligand-receptor binding induces intracellular signaling. Serotonin re-uptake form the synaptic cleft into the presynaptic neuron is mediated by SERT. Serotonin is inactivated by monoamine oxidase (MAO) and eventually converted into 5-hydroxyindoleacetic acid (5-HIAA) and undergoes renal clearance. (C) Synthesis of serotonin and melatonin from the amino acid TRP

with the involved enzymes is shown in the yellow panel. In the blue panel the alternative kynurenine pathway is shown. Inactivation of serotonin through conversion into 5-HIAA is shown in the green panel.

Therefore, the aim of this thesis is to explore the role of dopamine and serotonin in cancer by targeting DRD2 in an in ovo ovarian cancer model, assessment of serotonin

and dopamine receptor expression in solid tumors, and analysis of platelet and free plasma serotonin concentrations as well as plasma concentrations of its precursors and metabolites.

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OUTLINE OF THE THESIS

Chapter 2 provides an overview of the literature on dopamine and serotonin in tumor

angiogenesis and tumor growth. Literature search was performed using the search terms “dopamine”, “serotonin”, “5-hydroxytryptamine”, “dopamine receptor”, “serotonin receptor”, “platelets”, “angiogenesis”, “neovascularization”, “neoplasm”, and “cancer”. The direct effects of these biogenic amines on tumor and endothelial cells in vitro, as well as on tumor growth and angiogenesis in vivo are evaluated.

Furthermore, we discuss literature findings regarding the role of dopamine and serotonin in angiogenesis in fields outside oncology, including Parkinson’s disease, ovarian hyperstimulation syndrome, endometriosis, and wound healing.

In preclinical studies, dopamine inhibited tumor angiogenesis and tumor growth via activation of DRD2. In one small clinical study, treatment with dopamine was evaluated in melanoma patients. However, this approach turned out to be not feasible in patients due to the rapid conversion of dopamine to epinephrine and norepinephrine and the associated cardiovascular toxicity [10]. Therefore, direct targeting of DRD2 with DRD2 agonists would be preferable. In chapter 3, we

studied if the DRD2 agonist quinpirole has a direct effect in vitro on proliferation of

human umbilical vein endothelial cells (HUVEC) and human A2780 ovarian tumor cells. Furthermore, we studied if quinpirole could inhibit tumor angiogenesis and reduce A2780 ovarian tumor weight in an in ovo chick chorioallantoic membrane

model. In this model, A2780 ovarian tumors were grown for 14 days, and treated for 4 consecutive days with vehicle, dopamine hydrochloride, or quinpirole hydrochloride. Tumor microvessel density as well as DRD2 expression on avian blood vessels was assessed with immunohistochemistry.

Data regarding serotonin and dopamine receptor expression in human tumors is limited. Therefore, in chapter 4, we aimed to investigate expression of serotonin and

dopamine receptors in solid tumors. We explored predicted mRNA overexpression of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 in a broad panel of 43 tumor types compared to healthy tissue using functional genomic mRNA profiling [19]. Furthermore, we used immunohistochemistry to assess the presence and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 protein in colon cancer, ovarian cancer, breast cancer, renal cell carcinoma (RCC), pancreatic cancer, gastro-intestinal stromal tumors, melanoma, and pheochromocytoma samples. These tumor types were selected based on results of preclinical studies and tumor characteristics.

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RCC and pancreatic NET (pNET) are both highly vascular tumors [20, 21]. We hypothesized that platelet activation in the microenvironment of these tumors [4] results in local release of angiogenic factors, consequently lowering platelet serotonin concentrations. In chapter 5, we report a study that compared platelet serotonin

concentrations in patients with metastatic RCC (n = 20) and patients with metastatic

non-serotonin producing pNET (n = 20) with matched healthy controls (n = 20 per

group) using high performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Potential other reasons for the observed low platelet serotonin concentrations include competition of other platelet-bound angiogenic factors, such as VEGF-A as well as diminished availability of serotonin’s precursor tryptophan or enhanced break-down of serotonin. We therefore measured platelet concentrations of the pro-angiogenic factor VEGF-A with enzyme-linked immunosorbent assay (ELISA) and analyzed a possible correlation with platelet serotonin concentrations. Furthermore, we evaluated plasma concentrations of serotonin’s precursor tryptophan, tryptophan’s metabolites kynurenine and 3-hydroxykynurenine, and serotonin’s metabolite 5-hydroxyindoleacetic acid in plasma using LC-MS/MS.

Platelets are the main circulating reservoir of serotonin [7]. SSRIs are prescribed to patients with mood and anxiety disorders. Their mechanism of action is to enhance availability of serotonin in the synaptic cleft by selective blockage of the serotonin transporter. SSRIs also block these transporters on platelets and prevent uptake of serotonin. As a result, platelet serotonin concentrations are low in patients using SSRIs [22]. Reliable information on free plasma serotonin, which is available for receptor binding, is scarce. Because of the very low free serotonin concentrations in the plasma, analysis of free plasma serotonin requires a sensitive assay and careful handling of samples to prevent platelet activation and thereby contamination with platelet-bound serotonin. In chapter 6, we aimed to assess free plasma serotonin and platelet

serotonin concentrations in patients using SSRIs, both compared with matched healthy individuals. For this analysis, we used careful sample preparation combined with LC-MS/MS [23,24].

Serotonin not only metabolizes to 5-hydroxyindoleacetic acid, but also to melatonin [25]. In previous studies, ELISA was used to demonstrate the presence of melatonin in platelets [26]. Currently, a more specific and sensitive analysis of melatonin is feasible by LC-MS/MS [27]. In chapter 7, we therefore evaluated melatonin

concentrations in platelets and plasma of healthy individuals using LC-MS/MS and compared this with measurements by ELISA.

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