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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CHAPTER 8
Summary, discussion and future perspectives
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SUMMARY
The tumor microenvironment is an important contributor to tumor progression. Tumor microenvironment associated blood vessels transport oxygen, proteins, hormones, and tumor-promoting cells towards the tumor, resulting in alleviation of hypoxia and stimulation of tumor growth and metastasis [1,2]. Tumor blood vessels have functional and structural abnormalities, which cause variations in blood flow [3]. This aberrant blood flow easily results in activation of platelets that release their content [4]. Platelets function as a circulating reservoir for pro- and anti-angiogenic factors. Among these factors are dopamine and serotonin, which are stored in the dense granules of platelets [5]. In humans, dopamine and serotonin are involved in a range of physiological and pathological processes, such as mood regulation, gastrointestinal motility, and Parkinson’s disease [6,7]. Preclinical studies show that dopamine and serotonin can also influence tumor behavior.
Therefore, theaim of the research described in this thesis is to explore the role of dopamine and serotonin in cancer, by targeting dopamine receptor D2 (DRD2) in an in ovo ovarian cancer model, by assessing serotonin and dopamine receptor
expression in solid tumors, and by analysis of platelet and free plasma serotonin concentrations as well as plasma concentrations of its precursors and metabolites. In chapter 1 a general introduction of the topic and outline of the thesis is provided.
A literature review, presented in chapter 2, focuses on the ability of dopamine and
serotonin to influence tumor behavior by affecting angiogenesis and tumor cell proliferation. We searched the literature, using the search terms “dopamine”, “serotonin”, “5-hydroxytryptamine”, “dopamine receptor”, “serotonin receptor”, “platelets”, “angiogenesis”, “neovascularization”, “neoplasm”, and “cancer”. In vitro,
vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation was inhibited by dopamine and DRD2 agonists. Dopamine treatment of tumor bearing mice reduced tumor growth, lowered tumor microvessel density and reduced mobilization of endothelial progenitor cells into the circulation. DRD2 knockout mice developed larger tumors with higher microvessel density compared to wild-type mice. Furthermore, mice treated with DRD2 agonists had a relatively low tumor microvessel density. Based on literature, dopamine seems to affect tumor behavior preclinically via DRD2. The role of serotonin receptor 1 (5-HTR1), serotonin receptor 2 (5-HTR2), and serotonin receptor 3 (5-HTR3) was predominantly studied in vitro. Activation of
these receptors stimulated proliferation of endothelial cells and tumor cells of various tumor types. Tumor growth was slower in serotonin-depleted mice, which could be reversed by serotonin replenishment. Treatment with a 5-HTR2B antagonist reduced tumor growth in tumor bearing mice. Based on this literature review, in particular
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SUMMARY
The tumor microenvironment is an important contributor to tumor progression. Tumor microenvironment associated blood vessels transport oxygen, proteins, hormones, and tumor-promoting cells towards the tumor, resulting in alleviation of hypoxia and stimulation of tumor growth and metastasis [1,2]. Tumor blood vessels have functional and structural abnormalities, which cause variations in blood flow [3]. This aberrant blood flow easily results in activation of platelets that release their content [4]. Platelets function as a circulating reservoir for pro- and anti-angiogenic factors. Among these factors are dopamine and serotonin, which are stored in the dense granules of platelets [5]. In humans, dopamine and serotonin are involved in a range of physiological and pathological processes, such as mood regulation, gastrointestinal motility, and Parkinson’s disease [6,7]. Preclinical studies show that dopamine and serotonin can also influence tumor behavior.
Therefore, theaim of the research described in this thesis is to explore the role of dopamine and serotonin in cancer, by targeting dopamine receptor D2 (DRD2) in an in ovo ovarian cancer model, by assessing serotonin and dopamine receptor
expression in solid tumors, and by analysis of platelet and free plasma serotonin concentrations as well as plasma concentrations of its precursors and metabolites. In chapter 1 a general introduction of the topic and outline of the thesis is provided.
A literature review, presented in chapter 2, focuses on the ability of dopamine and
serotonin to influence tumor behavior by affecting angiogenesis and tumor cell proliferation. We searched the literature, using the search terms “dopamine”, “serotonin”, “5-hydroxytryptamine”, “dopamine receptor”, “serotonin receptor”, “platelets”, “angiogenesis”, “neovascularization”, “neoplasm”, and “cancer”. In vitro,
vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation was inhibited by dopamine and DRD2 agonists. Dopamine treatment of tumor bearing mice reduced tumor growth, lowered tumor microvessel density and reduced mobilization of endothelial progenitor cells into the circulation. DRD2 knockout mice developed larger tumors with higher microvessel density compared to wild-type mice. Furthermore, mice treated with DRD2 agonists had a relatively low tumor microvessel density. Based on literature, dopamine seems to affect tumor behavior preclinically via DRD2. The role of serotonin receptor 1 (5-HTR1), serotonin receptor 2 (5-HTR2), and serotonin receptor 3 (5-HTR3) was predominantly studied in vitro. Activation of
these receptors stimulated proliferation of endothelial cells and tumor cells of various tumor types. Tumor growth was slower in serotonin-depleted mice, which could be reversed by serotonin replenishment. Treatment with a 5-HTR2B antagonist reduced tumor growth in tumor bearing mice. Based on this literature review, in particular
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DRD2 and 5-HTR2B were considered of interest for future studies.
In chapter 3, we describe the effects of exposure to dopamine and the DRD2 agonist
quinpirole in vitro, on endothelial cell survival and ovarian tumor cell survival as well as in ovo on tumor angiogenesis and tumor weight in an ovarian tumor model. In vitro,
50% survival inhibition was seen after 96 hours continuous exposure to 67 µM dopamine hydrochloride in human umbilical vein endothelial cells and to 42 µM dopamine hydrochloride in A2780 ovarian tumor cells. Quinpirole did not have an effect in vitro on these cell types. Next, fertilized chicken eggs were grown in a rotary
incubator for 14 days. At day 6, A2780 ovarian tumor cells were added to the chorioallantoic membrane. Vehicle, dopamine hydrochloride (500 µM or 1000 µM), or quinpirole hydrochloride (10 µM, 100 µM, 500 µM or 1000 µM) were daily administered on days 10-13. The tumors, harvested day 14, had a lower microvessel density after treatment with 10-500 µM quinpirole and lower tumor weight when treated with 100 µM quinpirole. Contrary to the in vitro results, treatment with
dopamine did not have an anti-angiogenic or anti-tumor effect in ovo. In conclusion,
dopamine inhibited endothelial and ovarian tumor cell survival in vitro but did not
affect tumor growth and microvessel density in ovo, while quinpirole had no effect in vitro but inhibited tumor angiogenesis and tumor growth in ovo. This suggests that the
anti-tumor effect of quinpirole may be based on interactions with the tumor microenvironment, but more research is required before such conclusions can be drawn.
In chapter 4, we describe the results of screening for serotonin and dopamine
receptor expression in solid tumors. We used functional genomic mRNA profiling to evaluate mRNA overexpression of 5-HTR1B, 5-HTR2B, dopamine receptor D1 (DRD1), and DRD2 in 11,756 tumor samples representing 43 solid tumor types compared to healthy tissue. In addition, we determined the expression and location of these receptors with immunohistochemistry in tumor tissue of eight tumor types: colon cancer, ovarian cancer, breast cancer, renal cell carcinoma (RCC), pancreatic cancer, gastro-intestinal stromal tumors (GIST), melanoma, and pheochromocytoma. Functional genomic mRNA profiling demonstrated that 5-HTR2B mRNA was more frequently overexpressed compared to 5-HTR1B, DRD1, and DRD2. Interestingly, overexpression of the latter receptors was also observed in rare tumor types with limited treatment options. Examples are 5-HTR1B overexpression in 17% of nasopharyngeal carcinomas, DRD1 overexpression in 30% of ependymomas and 21% of synovial sarcomas, and DRD2 overexpression in 13% of astrocytomas. Immunohistochemical staining demonstrated that 5-HTR2B is highly expressed on endothelial cells of colon, ovarian, breast, renal, and pancreatic tumors and tumor
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cells of melanomas and GIST. 5-HTR1B expression was predominantly low. High DRD1 protein expression was observed in subsets of all cancer types studied, ranging from 14% in melanoma up to 57% in RCC. DRD2 protein expression was high in approximately 50% of pheochromocytomas, pancreatic cancers and ovarian cancers studied. Based on functional genomic mRNA profiling as well as on immunohistochemistry, 5-HTR2B is the most interesting receptor for further research as potential treatment target.
Upon platelet activation in the tumor microenvironment, degranulation of platelets takes place and causes local release of serotonin. Therefore, we hypothesized that platelet serotonin concentrations are lower in cancer patients than in matched healthy controls. In chapter 5, we evaluated platelet serotonin concentrations in 20 patients
with metastatic RCC and 20 patients with metastatic pancreatic neuroendocrine tumors (pNET) compared with 20 healthy individuals per group. These tumor types were chosen because of abundant tumor vascularity. We used high performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Platelet serotonin concentrations were approximately 2-fold decreased in cancer patients compared with healthy individuals. We subsequently checked for possible alternative causes of low platelet serotonin concentrations. First, we measured platelet VEGF-A concentrations with enzyme-linked immunosorbent assay (ELISA) as VEGF-A is also present in platelets and could potentially compete for storage capacity. No relation was observed between platelet serotonin and VEGF-A concentrations. Serotonin is produced from its precursor tryptophan and mainly degraded to 5-hydroxyindoleacetic acid (5-HIAA). However, tryptophan can also be converted to kynurenine by indoleamine 2,3-dioxygenase (IDO); an enzyme which is frequently active in cancer patients. We observed that plasma kynurenine/tryptophan ratios were 1.5-fold higher in RCC and pNET patients compared to healthy individuals. This may indicate a shift of tryptophan consumption in the tumor microenvironment towards the kynurenine pathway rather than the serotonin pathway. Therefore, although we did indeed demonstrate a decreased platelet serotonin concentration in RCC and pNET patients, this may not only be due to release in the tumor microenvironment but also to activation of the kynurenine pathway.
The serotonin transporter on neurons is blocked by selective serotonin reuptake inhibitors (SSRIs). The transporter is not only blocked on neurons but also on platelets, causing a decreased platelet serotonin concentration in SSRI users. Data on how SSRIs affect free plasma serotonin, which is available for receptor binding, is scarce. Therefore, in chapter 6, we aimed to analyze free plasma and platelet
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DRD2 and 5-HTR2B were considered of interest for future studies.
In chapter 3, we describe the effects of exposure to dopamine and the DRD2 agonist
quinpirole in vitro, on endothelial cell survival and ovarian tumor cell survival as well as in ovo on tumor angiogenesis and tumor weight in an ovarian tumor model. In vitro,
50% survival inhibition was seen after 96 hours continuous exposure to 67 µM dopamine hydrochloride in human umbilical vein endothelial cells and to 42 µM dopamine hydrochloride in A2780 ovarian tumor cells. Quinpirole did not have an effect in vitro on these cell types. Next, fertilized chicken eggs were grown in a rotary
incubator for 14 days. At day 6, A2780 ovarian tumor cells were added to the chorioallantoic membrane. Vehicle, dopamine hydrochloride (500 µM or 1000 µM), or quinpirole hydrochloride (10 µM, 100 µM, 500 µM or 1000 µM) were daily administered on days 10-13. The tumors, harvested day 14, had a lower microvessel density after treatment with 10-500 µM quinpirole and lower tumor weight when treated with 100 µM quinpirole. Contrary to the in vitro results, treatment with
dopamine did not have an anti-angiogenic or anti-tumor effect in ovo. In conclusion,
dopamine inhibited endothelial and ovarian tumor cell survival in vitro but did not
affect tumor growth and microvessel density in ovo, while quinpirole had no effect in vitro but inhibited tumor angiogenesis and tumor growth in ovo. This suggests that the
anti-tumor effect of quinpirole may be based on interactions with the tumor microenvironment, but more research is required before such conclusions can be drawn.
In chapter 4, we describe the results of screening for serotonin and dopamine
receptor expression in solid tumors. We used functional genomic mRNA profiling to evaluate mRNA overexpression of 5-HTR1B, 5-HTR2B, dopamine receptor D1 (DRD1), and DRD2 in 11,756 tumor samples representing 43 solid tumor types compared to healthy tissue. In addition, we determined the expression and location of these receptors with immunohistochemistry in tumor tissue of eight tumor types: colon cancer, ovarian cancer, breast cancer, renal cell carcinoma (RCC), pancreatic cancer, gastro-intestinal stromal tumors (GIST), melanoma, and pheochromocytoma. Functional genomic mRNA profiling demonstrated that 5-HTR2B mRNA was more frequently overexpressed compared to 5-HTR1B, DRD1, and DRD2. Interestingly, overexpression of the latter receptors was also observed in rare tumor types with limited treatment options. Examples are 5-HTR1B overexpression in 17% of nasopharyngeal carcinomas, DRD1 overexpression in 30% of ependymomas and 21% of synovial sarcomas, and DRD2 overexpression in 13% of astrocytomas. Immunohistochemical staining demonstrated that 5-HTR2B is highly expressed on endothelial cells of colon, ovarian, breast, renal, and pancreatic tumors and tumor
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cells of melanomas and GIST. 5-HTR1B expression was predominantly low. High DRD1 protein expression was observed in subsets of all cancer types studied, ranging from 14% in melanoma up to 57% in RCC. DRD2 protein expression was high in approximately 50% of pheochromocytomas, pancreatic cancers and ovarian cancers studied. Based on functional genomic mRNA profiling as well as on immunohistochemistry, 5-HTR2B is the most interesting receptor for further research as potential treatment target.
Upon platelet activation in the tumor microenvironment, degranulation of platelets takes place and causes local release of serotonin. Therefore, we hypothesized that platelet serotonin concentrations are lower in cancer patients than in matched healthy controls. In chapter 5, we evaluated platelet serotonin concentrations in 20 patients
with metastatic RCC and 20 patients with metastatic pancreatic neuroendocrine tumors (pNET) compared with 20 healthy individuals per group. These tumor types were chosen because of abundant tumor vascularity. We used high performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Platelet serotonin concentrations were approximately 2-fold decreased in cancer patients compared with healthy individuals. We subsequently checked for possible alternative causes of low platelet serotonin concentrations. First, we measured platelet VEGF-A concentrations with enzyme-linked immunosorbent assay (ELISA) as VEGF-A is also present in platelets and could potentially compete for storage capacity. No relation was observed between platelet serotonin and VEGF-A concentrations. Serotonin is produced from its precursor tryptophan and mainly degraded to 5-hydroxyindoleacetic acid (5-HIAA). However, tryptophan can also be converted to kynurenine by indoleamine 2,3-dioxygenase (IDO); an enzyme which is frequently active in cancer patients. We observed that plasma kynurenine/tryptophan ratios were 1.5-fold higher in RCC and pNET patients compared to healthy individuals. This may indicate a shift of tryptophan consumption in the tumor microenvironment towards the kynurenine pathway rather than the serotonin pathway. Therefore, although we did indeed demonstrate a decreased platelet serotonin concentration in RCC and pNET patients, this may not only be due to release in the tumor microenvironment but also to activation of the kynurenine pathway.
The serotonin transporter on neurons is blocked by selective serotonin reuptake inhibitors (SSRIs). The transporter is not only blocked on neurons but also on platelets, causing a decreased platelet serotonin concentration in SSRI users. Data on how SSRIs affect free plasma serotonin, which is available for receptor binding, is scarce. Therefore, in chapter 6, we aimed to analyze free plasma and platelet
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contamination by careful blood collection, rapid sample handling, and drug plus diet restrictions. Free plasma and platelet serotonin concentrations were analyzed with LC-MS/MS. Compared to matched healthy individuals, the free plasma serotonin concentration was 10-fold lower and platelet serotonin concentration was 14-fold lower in 64 SSRI users. Free plasma and platelet serotonin concentrations were lower if the patient used a higher SSRI dose or a SSRI with a higher binding affinity for the serotonin transporter. Concentrations of serotonin’s metabolite 5-HIAA was only slightly elevated in plasma of SSRI users compared with healthy individuals, suggesting that an increased break-down of serotonin to 5-HIAA did not cause the low serotonin concentrations observed.
Serotonin is not only converted to 5-HIAA, but also to melatonin. Previous studies used ELISA to demonstrate that melatonin is present in platelets. We hypothesized that analysis of melatonin with LC-MS/MS, which is a more refined method, would confirm this interesting observation. Therefore, in chapter 7, we compared melatonin
concentrations in platelets measured with LC-MS/MS and ELISA. In 20 healthy volunteers, blood was drawn at 8:00 AM as melatonin secretion displays a circadian rhythm. Melatonin concentrations were analyzed in platelet rich plasma (PRP) and platelet poor plasma (PPP) as a proxy for melatonin concentrations in platelets, because measurement in isolated platelets is prone to artifacts. Contrary to previous results, we did not detect melatonin in platelets as melatonin concentrations did not differ between PRP and PPP. This observation emphasizes the importance of well validated assays, which in this study changed the view on the presence of melatonin in platelets.
DISCUSSION AND FUTURE PERSPECTIVES
Dopamine agonists as anti-angiogenic agents
Various preclinical studies described an inhibitory effect of DRD2 agonists on tumor angiogenesis and tumor growth. In one of our studies, we evaluated the effect of the DRD2 agonist quinpirole on ovarian tumor angiogenesis and tumor growth in an in ovo tumor model. We observed that tumor microvessel density decreased at
intermediate doses quinpirole, but that tumor growth and microvessel density were not affected by high doses of quinpirole.
Dose dependency of treatment effect is known for dopamine and used in the clinic: low doses of dopamine at 1-2 µg/kg/minute mainly stimulate DRD1 which results in selective vasodilatation in the kidneys. Higher dopamine doses of 5-10 µg/kg/minute also stimulate β1 adrenergic receptors, and thereby increase cardiac output [8]. The observed difference in preclinical effect between doses quinpirole may
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also be due to the stimulation of various receptors, as DRD2 agonists including quinpirole, bromocriptine and cabergoline not only have affinity for DRD2, but also for other dopamine and serotonin receptors [9]. Activation of some of these receptors is thought to have a pro-angiogenic and pro-tumor effect, contrary to the anti-angiogenic and anti-tumor effect of DRD2 activation [10,11].
Clinical activity of the DRD2 agonist cabergoline was shown in patients with endometriosis. A characteristic of endometriosis is the formation of blood vessels in and around endometriotic lesions [12]. These lesions express DRD2 [13]. First, in an endometriosis mouse model, a decrease in active endometriotic lesions was demonstrated after treatment with cabergoline, which was paralleled by a reduction in newly developed blood vessels and reduced VEGF receptor phosphorylation [14]. Next, in an open-label, randomized trial in 140 endometriosis patients, the effect of 12 weeks cabergoline 0.5 mg orally two times a week on endometrioma size was compared to treatment effect of the luteinizing hormone-releasing hormone agonist triptorelin acetate 3.75 mg subcutaneously once a month. In 64.7% of the patients treated with cabergoline versus 21.7% of the patients treated with triptorelin acetate, endometrioma size was decreased by at least 25%. Side effects were comparable between treatment arms, although a higher number of patients who used cabergoline experienced gastrointestinal complaints such as nausea, vomiting and constipation [15].
Translating this to cancer treatment will be a major challenge. Given the affinity of the aforementioned DRD2 agonists for other dopamine and serotonin receptors, optimal dose finding and development of highly selective DRD2 agonists would be critical. Furthermore, it has become clear over the last decade that inhibition of angiogenesis prolongs progression free survival in several tumor types, but rarely translates into prolongation of overall survival.
Tumor typing
In order to assess in which patients treatment with drugs targeting dopamine or serotonin receptors can be potentially beneficial, we evaluated dopamine and serotonin receptor expression in tumors.
Immunohistochemistry is laborious for high volume screening. Functional genomic mRNA profiling is a method that can be used to screen a large set of tumor samples from publicly available databases for mRNA overexpression of selected genes, compared to healthy tissue [16]. Next to this method, we analyzed dopamine and serotonin receptor presence and location at the protein level with immunohistochemistry in small samples of different tumor types.
In chapter 4, we observed overexpression of 5-HTR1B, 5-HTR2B, DRD1 and DRD2 in rare tumor types such as ependymoma, synovial sarcoma, and astrocytoma
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contamination by careful blood collection, rapid sample handling, and drug plus diet restrictions. Free plasma and platelet serotonin concentrations were analyzed with LC-MS/MS. Compared to matched healthy individuals, the free plasma serotonin concentration was 10-fold lower and platelet serotonin concentration was 14-fold lower in 64 SSRI users. Free plasma and platelet serotonin concentrations were lower if the patient used a higher SSRI dose or a SSRI with a higher binding affinity for the serotonin transporter. Concentrations of serotonin’s metabolite 5-HIAA was only slightly elevated in plasma of SSRI users compared with healthy individuals, suggesting that an increased break-down of serotonin to 5-HIAA did not cause the low serotonin concentrations observed.
Serotonin is not only converted to 5-HIAA, but also to melatonin. Previous studies used ELISA to demonstrate that melatonin is present in platelets. We hypothesized that analysis of melatonin with LC-MS/MS, which is a more refined method, would confirm this interesting observation. Therefore, in chapter 7, we compared melatonin
concentrations in platelets measured with LC-MS/MS and ELISA. In 20 healthy volunteers, blood was drawn at 8:00 AM as melatonin secretion displays a circadian rhythm. Melatonin concentrations were analyzed in platelet rich plasma (PRP) and platelet poor plasma (PPP) as a proxy for melatonin concentrations in platelets, because measurement in isolated platelets is prone to artifacts. Contrary to previous results, we did not detect melatonin in platelets as melatonin concentrations did not differ between PRP and PPP. This observation emphasizes the importance of well validated assays, which in this study changed the view on the presence of melatonin in platelets.
DISCUSSION AND FUTURE PERSPECTIVES
Dopamine agonists as anti-angiogenic agents
Various preclinical studies described an inhibitory effect of DRD2 agonists on tumor angiogenesis and tumor growth. In one of our studies, we evaluated the effect of the DRD2 agonist quinpirole on ovarian tumor angiogenesis and tumor growth in an in ovo tumor model. We observed that tumor microvessel density decreased at
intermediate doses quinpirole, but that tumor growth and microvessel density were not affected by high doses of quinpirole.
Dose dependency of treatment effect is known for dopamine and used in the clinic: low doses of dopamine at 1-2 µg/kg/minute mainly stimulate DRD1 which results in selective vasodilatation in the kidneys. Higher dopamine doses of 5-10 µg/kg/minute also stimulate β1 adrenergic receptors, and thereby increase cardiac output [8]. The observed difference in preclinical effect between doses quinpirole may
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also be due to the stimulation of various receptors, as DRD2 agonists including quinpirole, bromocriptine and cabergoline not only have affinity for DRD2, but also for other dopamine and serotonin receptors [9]. Activation of some of these receptors is thought to have a pro-angiogenic and pro-tumor effect, contrary to the anti-angiogenic and anti-tumor effect of DRD2 activation [10,11].
Clinical activity of the DRD2 agonist cabergoline was shown in patients with endometriosis. A characteristic of endometriosis is the formation of blood vessels in and around endometriotic lesions [12]. These lesions express DRD2 [13]. First, in an endometriosis mouse model, a decrease in active endometriotic lesions was demonstrated after treatment with cabergoline, which was paralleled by a reduction in newly developed blood vessels and reduced VEGF receptor phosphorylation [14]. Next, in an open-label, randomized trial in 140 endometriosis patients, the effect of 12 weeks cabergoline 0.5 mg orally two times a week on endometrioma size was compared to treatment effect of the luteinizing hormone-releasing hormone agonist triptorelin acetate 3.75 mg subcutaneously once a month. In 64.7% of the patients treated with cabergoline versus 21.7% of the patients treated with triptorelin acetate, endometrioma size was decreased by at least 25%. Side effects were comparable between treatment arms, although a higher number of patients who used cabergoline experienced gastrointestinal complaints such as nausea, vomiting and constipation [15].
Translating this to cancer treatment will be a major challenge. Given the affinity of the aforementioned DRD2 agonists for other dopamine and serotonin receptors, optimal dose finding and development of highly selective DRD2 agonists would be critical. Furthermore, it has become clear over the last decade that inhibition of angiogenesis prolongs progression free survival in several tumor types, but rarely translates into prolongation of overall survival.
Tumor typing
In order to assess in which patients treatment with drugs targeting dopamine or serotonin receptors can be potentially beneficial, we evaluated dopamine and serotonin receptor expression in tumors.
Immunohistochemistry is laborious for high volume screening. Functional genomic mRNA profiling is a method that can be used to screen a large set of tumor samples from publicly available databases for mRNA overexpression of selected genes, compared to healthy tissue [16]. Next to this method, we analyzed dopamine and serotonin receptor presence and location at the protein level with immunohistochemistry in small samples of different tumor types.
In chapter 4, we observed overexpression of 5-HTR1B, 5-HTR2B, DRD1 and DRD2 in rare tumor types such as ependymoma, synovial sarcoma, and astrocytoma
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with functional genomic mRNA profiling. Translation of these findings to the clinic, in the hypothetical situation that targeting one of these receptors would have strong antitumor activity, will be a challenge. Performing studies in patients with rare tumors is difficult due to the low number of patients that fit the criteria for inclusion. Basket studies can be a solution to this problem. For such studies, patients are selected based on tumor characteristics rather than on tumor type [17]. A successful example of a basket study is the European organization for Research and Treatment or Cancer (EORTC) 90101 CREATE trial, in which patients with six different tumor types with specific alterations in the anaplastic lymphoma kinase (ALK) and/or MET pathway in tumor cells were treated with crizotinib. Six of 12 ALK-positive patients with advanced myofibroblastic tumors achieved a complete or partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) [18].
Indoleamine 2,3-dioxygenase (IDO)
IDO is an enzyme which is overexpressed in many tumor types. IDO converts tryptophan, the precursor of serotonin, to kynurenine. Consequently, tryptophan reserves become depleted and concentrations of tryptophan’s metabolite kynurenine are elevated [19]. Reduced availability of tryptophan could lead to a decrease in serotonin production. In the study described in chapter 5, we observed increased plasma kynurenine/tryptophan ratios in RCC and pNET patients compared to healthy individuals, which may indicate that IDO activity is enhanced. High IDO activity contributes to an immunosuppressive tumor microenvironment, which is one of the hallmarks of cancer [2].
The inhibitory effect of IDO on the immune system is dual. First, enhanced IDO activity in the tumor causes depletion of tryptophan in the tumor microenvironment and thereby decreases proliferation rates of CD8+ T-cells and natural killer cells. Second, regulatory T-cells are activated by enhanced IDO activity and induce suppression of the immune response [19].
Several IDO inhibitors have been developed and have been tested in preclinical and clinical studies, mostly in combination with immune checkpoint inhibitors. However, after promising phase I and phase II trials in various tumor types [20], a phase III trial with the IDO1 inhibitor epacadostat in combination with the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab failed to show benefit over pembrolizumab alone in metastatic melanoma patients. Therefore, many ongoing studies with IDO1 inhibitors were halted [21]. Currently, other strategies for the use of IDO1 inhibitors and other interventions in the tryptophan-kynurenine pathway are evaluated for treatment of cancer [22].
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11. Borcherding DS, Tong W, Hugo ER, et al. Expression and therapeutic targeting of dopamine receptor-1 (D1R) in breast cancer. Oncogene 2016; 35: 3103-3113.
12. Taylor RN, Yu J, Torres PB, et al. Mechanistic and therapeutic implications of angiogenesis in endometriosis. Reprod Sci 2009; 16: 140-146.
13. Novella-Maestre E, Carda C, Ruiz-Sauri A, et al. Identification and quantification of dopamine receptor 2 in human eutopic and ectopica endometrium: a novel molecular target for endometriosis therapy. Biol Reprod 2010; 83: 866-873.
14. Novella-Maestre E, Carda C, Noquera I, et al. Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis. Hum Reprod 2009; 24: 1025-1035.
15. Hamid AM, Madkour WA, Moawad A, et al. Does cabergoline help in decreasing endometrioma size compared to LHRH agonist? A prospective randomized study.
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with functional genomic mRNA profiling. Translation of these findings to the clinic, in the hypothetical situation that targeting one of these receptors would have strong antitumor activity, will be a challenge. Performing studies in patients with rare tumors is difficult due to the low number of patients that fit the criteria for inclusion. Basket studies can be a solution to this problem. For such studies, patients are selected based on tumor characteristics rather than on tumor type [17]. A successful example of a basket study is the European organization for Research and Treatment or Cancer (EORTC) 90101 CREATE trial, in which patients with six different tumor types with specific alterations in the anaplastic lymphoma kinase (ALK) and/or MET pathway in tumor cells were treated with crizotinib. Six of 12 ALK-positive patients with advanced myofibroblastic tumors achieved a complete or partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) [18].
Indoleamine 2,3-dioxygenase (IDO)
IDO is an enzyme which is overexpressed in many tumor types. IDO converts tryptophan, the precursor of serotonin, to kynurenine. Consequently, tryptophan reserves become depleted and concentrations of tryptophan’s metabolite kynurenine are elevated [19]. Reduced availability of tryptophan could lead to a decrease in serotonin production. In the study described in chapter 5, we observed increased plasma kynurenine/tryptophan ratios in RCC and pNET patients compared to healthy individuals, which may indicate that IDO activity is enhanced. High IDO activity contributes to an immunosuppressive tumor microenvironment, which is one of the hallmarks of cancer [2].
The inhibitory effect of IDO on the immune system is dual. First, enhanced IDO activity in the tumor causes depletion of tryptophan in the tumor microenvironment and thereby decreases proliferation rates of CD8+ T-cells and natural killer cells. Second, regulatory T-cells are activated by enhanced IDO activity and induce suppression of the immune response [19].
Several IDO inhibitors have been developed and have been tested in preclinical and clinical studies, mostly in combination with immune checkpoint inhibitors. However, after promising phase I and phase II trials in various tumor types [20], a phase III trial with the IDO1 inhibitor epacadostat in combination with the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab failed to show benefit over pembrolizumab alone in metastatic melanoma patients. Therefore, many ongoing studies with IDO1 inhibitors were halted [21]. Currently, other strategies for the use of IDO1 inhibitors and other interventions in the tryptophan-kynurenine pathway are evaluated for treatment of cancer [22].
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12. Taylor RN, Yu J, Torres PB, et al. Mechanistic and therapeutic implications of angiogenesis in endometriosis. Reprod Sci 2009; 16: 140-146.
13. Novella-Maestre E, Carda C, Ruiz-Sauri A, et al. Identification and quantification of dopamine receptor 2 in human eutopic and ectopica endometrium: a novel molecular target for endometriosis therapy. Biol Reprod 2010; 83: 866-873.
14. Novella-Maestre E, Carda C, Noquera I, et al. Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis. Hum Reprod 2009; 24: 1025-1035.
15. Hamid AM, Madkour WA, Moawad A, et al. Does cabergoline help in decreasing endometrioma size compared to LHRH agonist? A prospective randomized study.
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16. Fehrmann RS, Karjalainen JM, Krajewska M, et al. Gene expression analysis identifies global gene dosage sensitivity in cancer. Nat Genet 2015; 47: 115-125.
17. Tao JJ, Schram AM, Hyman DM. Basket studies: redefining clinical trials in the era of genome-driven oncology. Annu Rev Med 2018; 69: 319-331.
18. Schöffski P, Sufliarsky J, Gelderblom H, et al. Crizotinib in patients with advanced, inoperable inflammatory myofibroblastic tumours with and without anaplastic lymphoma kinase gene alterations (European Organisation for Research and Treatment of Cancer 90101 CREATE): a multicenter, single-drug, prospective, non-randomised phase 2 trial. Lancet Respir Med 2018; 6: 431-441.
19. Liu M, Wang X, Wang L, et al. Targeting the IDO1 pathway in cancer: from bench to bedside. J Hematol Oncol 2018; 11: 100.
20. Luther C, Swami U, Zhang J, et al. Advanced stage melanoma therapies: detailing the present and exploring the future. Crit Rev Oncol Hematol 2019; 133: 99-111.
21. Komiya T, Huang CH. Updates in the clinical development of epacadostat and other indoleamine 2,3-dioxygenase 1 inhibitors (IDO1) for human cancers. Front Oncol 2018; 8: 423.
22. Labadie BW, Bao R, Luke JJ. Reimagining IDO pathway inhibition in cancer immunotherapy via downstream focus on the tryptophan-kynurenine-aryl hydrocarbon axis. Clin Cancer Res 2019; 25: 1462-1471.
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16. Fehrmann RS, Karjalainen JM, Krajewska M, et al. Gene expression analysis identifies global gene dosage sensitivity in cancer. Nat Genet 2015; 47: 115-125.
17. Tao JJ, Schram AM, Hyman DM. Basket studies: redefining clinical trials in the era of genome-driven oncology. Annu Rev Med 2018; 69: 319-331.
18. Schöffski P, Sufliarsky J, Gelderblom H, et al. Crizotinib in patients with advanced, inoperable inflammatory myofibroblastic tumours with and without anaplastic lymphoma kinase gene alterations (European Organisation for Research and Treatment of Cancer 90101 CREATE): a multicenter, single-drug, prospective, non-randomised phase 2 trial. Lancet Respir Med 2018; 6: 431-441.
19. Liu M, Wang X, Wang L, et al. Targeting the IDO1 pathway in cancer: from bench to bedside. J Hematol Oncol 2018; 11: 100.
20. Luther C, Swami U, Zhang J, et al. Advanced stage melanoma therapies: detailing the present and exploring the future. Crit Rev Oncol Hematol 2019; 133: 99-111.
21. Komiya T, Huang CH. Updates in the clinical development of epacadostat and other indoleamine 2,3-dioxygenase 1 inhibitors (IDO1) for human cancers. Front Oncol 2018; 8: 423.
22. Labadie BW, Bao R, Luke JJ. Reimagining IDO pathway inhibition in cancer immunotherapy via downstream focus on the tryptophan-kynurenine-aryl hydrocarbon axis. Clin Cancer Res 2019; 25: 1462-1471.
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