Role of Chemokine Receptors in Thyroid Cancer and Immunotherapy

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Endocrine-Related F Coperchini et al. Chemokine receptors in 26:8 R465–R478 Cancer thyroid cancer REVIEW Role of chemokine receptors in thyroid cancer and immunotherapy

Francesca Coperchini1, Laura Croce1,2, Michele Marinò3, Luca Chiovato1,4 and Mario Rotondi1,4

1Istituti Clinici Scientifici Maugeri IRCCS, Unit of Internal Medicine and Endocrinology, Laboratory for Endocrine Disruptors, Pavia, Italy 2PHD Course in Experimental Medicine, University of Pavia, Pavia, Italy 3Endocrinology Unit I, Department of Clinical and Experimental Medicine, University Hospital of Pisa, University of Pisa, Pisa, Italy 4Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy

Correspondence should be addressed to L Chiovato: [email protected]

Abstract

Inflammation is currently regarded as an essential component of malignancies. It is Key Words now known that the tumor microenvironment may profoundly influence the biological ff chemokine receptors behavior of cancer cells and ultimately the patient’s outcome. Chemokine and their ff thyroid cancer receptor play a major role in determining the immune phenotype of the cells infiltrating ff immunotherapy the thyroid tumor microenvironment. Experimental evidence shows that both normal ff tumor microenvironment and cancer thyroid cells express specific chemokine receptors. The expression of at least ff CXCL8 some of these receptors exerts several biological effects, which influence the course of the disease. The present review article will take into account the role of the most studied chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, DARC, CCR3, CCR6 and CCR7) in the context of thyroid cancer. This review will focus on current knowledge provided by in vitro and in vivo studies specifically performed on thyroid cancer including (i) expression of chemokine receptors in normal and cancer thyroid cells; (ii) role of chemokine receptors in affecting the biological behavior of thyroid tumors including the metastatic process; (iii) current knowledge about immunotherapies through targeting of chemokine receptors in thyroid cancer. Endocrine-Related Cancer (2019) 26, R465–R478

Introduction

The endocrine and the immune systems are characterized of this immune cell infiltrate determines the balance by a cross-talk aimed at maintaining homeostasis (Kelley between anti-tumor and pro-tumor responses (cell 1988). A share of common ligands (hormones and growth, angiogenesis and metastasis) (Balkwill 2003, cytokines) and their specific receptors is responsible 2012). Differentiated thyroid cancers are the most for this interaction, which occurs, not only in the prevalent endocrine malignancies being in most cases context of endocrine autoimmunity, but also during slow growing, clinically indolent lesions with an overall the development of cancer (Kelley 1988). Inflammatory good prognosis and a low-mortality rate (Haugen et al. molecules and their receptors are able to influence 2017). The association between chronic inflammation cancer survival and progression (Balkwill 2003, Balkwill and thyroid cancer has long been recognized (Mantovani 2012). In particular, the interaction of chemokines and et al. 2010, Allavena et al. 2011). Briefly, a mixture of their receptors mediates immune cell trafficking into the immune cells and soluble mediators, interacting together tumor microenvironment resulting in the recruitment through binding receptors, is present within or nearby the and activation of different cell types. The characteristics tumor, and it is considered to play a major role in tumor

-19-0163 Endocrine-Related F Coperchini et al. Chemokine receptors in 26:8 R466 Cancer thyroid cancer progression and clinical outcome (Cunha et al. 2014). on the concept that different organs produce chemokines, Chemokines are soluble mediators, some of which are which can attract the correspondent chemokine receptor- produced not only by immune cells, but also by normal bearing cancers cells (Fig. 1) (Homey et al. 2002, Zlotnik thyroid cells and by cancer cells within the thyroid tumor et al. 2011). microenvironment (Rotondi et al. 2018). Chemokines regulate the traffic and the behavior of cells by binding seven transmembrane spanning G protein–coupled Thyroid cancer and chemokine receptors receptors (Chow & Luster 2014). The present review deals with the role of chemokine receptors in thyroid cancer, Literature data show that several chemokine receptors play their contribution in the metastatic processes and recent a role in thyroid cancer; these include CXCR1, CXCR2, findings on their role for immunotherapy. CXCR3, CXCR4, CXCR7, DARC, CCR3, CCR6 and CCR7 (Fig. 2). Table 1 summarizes our current knowledge on the role of these receptors in thyroid cancer. Chemokine receptors and cancer

The effects of chemokines on target cells are mediated CXCR1 and CXCR2 by specific receptors (Rossi & Zlotnik 2000, Chow & Luster 2014), which are responsible for the activation of The CXC chemokine receptors 1 and 2 are generally a cascade of cellular responses after binding with their expressed simultaneously. CXCR1 is the receptor for respective ligand (Rossi & Zlotnik 2000). Chemokine CXCL1, CXCL6, CXCL7 and CXCL8 chemokines receptors are composed of seven transmembrane (7TM) (Rotondi et al. 2007, Waugh & Wilson 2008). CXCR1 domains and are further classified as ‘typical’ and is mainly expressed in neutrophils and was originally ‘atypical chemokine receptors’. Nineteen of 23 known characterized by its ability to induce the chemotaxis of chemokine receptors are classified as ‘typical chemokine leukocytes. CXCR1, originally shown to act on multiple receptors’, because they are G protein–coupled receptors. cell types, is involved in invasion and migration of (Legler & Thelen 2018). The remaining four receptors estrogen receptor (ER)-negative breast cancer cells are classified as ‘atypical chemokine receptors’, because (Holmes et al. 1991, 2009, Jiang et al. 2013). In several they share the seven transmembrane domain structure solid tumors, the expression of CXCR1 is positively of conventional chemokine receptors, but do not couple correlated with drug resistance, cell invasion and to G proteins. Consequently, they fail to induce the metastasis (Jiang et al. 2013). CXCR2 binds the CXCL1, classical signaling and downstream cellular responses, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8 acting as scavengers targeting chemokines for lysosomal chemokines. Among them, CXCL8 plays a major role in degradation (Legler & Thelen 2018). Because the thyroid cancer microenvironment (Rotondi et al. 2007, chemokine/chemokine receptors system is typically 2018). CXCR2 was described originally in leukocytes redundant, most chemokine receptors bind two or more and subsequently in non-hematopoietic cells, such as chemokines and some chemokines bind both typical and keratinocytes, neurons and epidermal cells, in regions atypical receptors (Zlotnik et al. 2006, Ulvmar et al. 2011). corresponding to intermediate levels of differentiation Metastatic spread is the primary cause of morbidity (Baggiolini et al. 1989, Tecimer et al. 2000, Waugh & and mortality in cancer patients. Several lines of evidence Wilson 2008). Immunohistochemical analysis of CXCR2 indicate that the metastatic infiltration of distant organs expression revealed that it is universally present in cells is mediated by chemokines and by their cognate receptors of the dispersed neuroendocrine system (cells in the (Balkwill 2012, Caronni et al. 2016). Strong support central and peripheral nervous systems) and in squamous to this notion derives from in vitro and in vivo (animal epithelial cells (Tecimer et al. 2000, Waugh & Wilson models) studies, which demonstrate that blocking specific 2008). The first study investigating these receptors in chemokine/chemokine receptor axes does reduce the thyroid cells demonstrated their expression in monocytes, development of metastasis in several cancers. These granulocytes and infiltrating cells of thyroid cancer and include breast (Williams et al. 2010), prostate (Darash- in para-follicular C cells (Aust et al. 2001). In 2015, Zhao Yahana et al. 2004), lung (Su et al. 2005, Speetjens et al. et al., demonstrated, by immunohistochemistry, the 2009), colorectal (Jung et al. 2017) and gastric (Zhao et al. expression of CXCR1 protein in 70% of 106 follicular 2011) cancer, as well as glioblastoma (Terasaki et al. 2011). thyroid cancers cells (FTCs) and in 27.3% of 128 follicular These data support the so-called ‘homing theory’ based thyroid adenomas (FTAs) (Zhao et al. 2015).

The role of CXCR1 and CXCR2 in promoting in normal and neoplastic thyroid cells, the latter both in metastatic thyroid cancer is still debated. Some but not surgical specimens of PTC and in PTC cell lines (Fang all studies found a cancer-promoting effect. In 2014, et al. 2014). Immunostaining for CXCR1 and CXCR2 Tang et al. analyzed by immunohistochemistry the was performed in 26 PTCs and in 6 normal thyroid expression of CXCR1 in 129 papillary thyroid cancers tissue specimens. Eighty-three percent of PTC specimens (PTCs), 61 hyperplastic thyroid nodules and 118 normal showed a strong staining for CXCR1; the corresponding thyroid tissue specimens (Tang et al. 2014). They found figure for CXCR2 was 71.5%. Virtually no staining the CXCR1 molecule to be highly expressed in PTCs and occurred in normal thyroid tissue specimens. A positive significantly correlated with lymph node metastases Tang( cell membrane staining for both CXCR1 and CXCR2 et al. 2014). Real-time RT-PCR showed an upregulation of was also detected in three PTC cell lines: K1, TPC-1 and the CXCR1 mRNA in PTCs which significantly correlated BCPAP (Fang et al. 2014). At variance with the previous with lymph node metastases (Tang et al. 2014). These study by Tang et al., no association was found between the results prompted subsequent studies aimed at evaluating expression of CXCR1 and CXCR2 and the occurrence of the expression of CXCR1 and CXCR2 in thyroid cancer lymph node metastasis and/or of extra-thyroid extension cells and their involvement in the metastatic process. (Fang et al. 2014). In a further study, Visciano et al. Fang et al. evaluated the expression of CXCR1 and CXCR2 (2015) confirmed the observations byTang et al. (2014),

Figure 1 Representation of the homing theory: cancer cells expressing a given chemokine receptor metastatize from the cancerous mass to distinct organs being attracted by the respective binding chemokine gradient. A full colour version of this figure is available athttps://doi.org/10.1530/ERC-19-0163 .

Figure 2 Representative picture of the thyroid tumor microenvironment showing chemokine receptors expressed by normal and cancerous thyroid cells. The respective binding chemokines are also shown. A full colour version of this figure is available athttps://doi.org/10.1530/ERC-19-0163 . reporting that CXCR1 and CXCR2 were expressed in both (CXCL9, CXCL10 and CXCL11), which are strongly normal and thyroid cancer cells. The negative finding upregulated by IFN-γ stimulation (Loetscher of Fang in normal thyroid cells was probably due to the et al. 1996, Cole et al. 1998, Rotondi et al. rather low number of normal thyroid tissue specimens (6). 2007) and for CXCL4. After IFN-γ stimulation, In a recent study, Cui et al. investigated the involvement thyrocytes secrete CXCR3-binding chemokines, of the CXCL5–CXCR2 axis in epithelial-to-mesenchymal which in turn recruit Th1 lymphocytes-expressing CXCR3 transition (EMT) of PTC (Cui et al. 2018). The activation and secreting IFN-γ. This sequence of events strongly of this chemokine receptor axis promoted the migration supports the concept that IFN-γ inducible chemokines and invasiveness of PTC cells in vitro (Cui et al. 2018). and their receptor CXCR3 play an important role in the The expression of EMT-associated molecules E-cadherin, initiation of autoimmune thyroiditis (AITD) (Rotondi N-cadherin and vimentin was upregulated in response to et al. 2003, 2007). Subsequently, it was demonstrated recombinant human-(rh)-CXCL5 stimulation. As assessed that the binding of CXCR3 by its chemokine ligands by Western blot analysis, treatment with rh-CXCL5 drives the traffic of Th1 cells, CD8+ T cells and NK upregulated the markers of EMT, while, in knocked cells into the tumor microenvironment (Nagarsheth down cells for CXCR2, a downregulation occurred (Cui et al. 2017). CXCR3 expression is upregulated in many et al. 2018). These findings suggest that the interaction tumors, suggesting its pro-tumorigenic role in the of CXCL5 with CXCR2 has a role in the EMT process of inflammatory microenvironment Abron( et al. 2018, thyroid cancer. Susek et al. 2018). In this scenario, we know that different CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) can trigger different and sometimes opposite CXCR3 responses. This is, at least in part, due to the presence of The CXC chemokine receptor 3 is commonly associated two splicing variants of the receptor: CXCR3A involved with Th1 immune responses and Th1-associated in cell proliferation and CXCR3B involved in cell diseases. CXCR3 is the receptor for Th1-type chemokines apoptosis (Urra et al. 2018).

Table 1 Chemokine receptors expression and role in thyroid cancer.

Chemokine Infiltrating immune receptor Binding chemokine/s Thyroid cells expression cells expression Relevance for thyroid cancer CXCR1 CXCL1, CXCL6, CXCL7 Normal human thyroid cells (Tang et al. TAM (Fang et al. Association with human lymph and CXCL8 2014, Visciano et al. 2015) 2014) node metastasis (Tang et al. Rat PTC (Fang et al. 2014) 2014) Human FTC, FTA (Tang et al. 2014, Zhao No association with lymph node et al. 2015) metastasis or extra-thyroid extension in rats (Fang et al. 2014) CXCR2 CXCL1, CXCL2, CXCL3, Rat PTC (Fang et al. 2014) TAM (Fang et al. Promotion of EMT in vitro (Cui CXCL5, CXCL6, 2014) et al. 2018) CXCL7, CXCL8 No association with lymph node metastasis or extra-thyroid extension in rats (Fang et al. 2014) CXCR3 CXCL4, CXCL9-10-11 Normal human thyroid cells, human No data available No data available TPC-1 cell line, non-metastatic human PTC (Urra et al. 2018) CXCR4 CXCL12 Normal human thyroid cells (González TAM (Kim et al. Promotion of rat thyroid cancer et al. 2009), rat RET/PTC thyroid cancer 2016) cell migration (Castellone et al. cell line (Castellone et al. 2004), human 2004) PTC, human ATC, human MTC, human Promotion of in vitro human FTC, human ARO cells (He et al. 2010, anaplastic thyroid cell Torregrossa et al. 2010, 2012, Shin migration, Association with et al. 2013) human metastasis in vivo (Torregrossa et al. 2010, 2012) Promotion of EMT (Hwang et al. 2003) CXCR7 CXCL12 and I-TAC Human PTC (Liu et al. 2014) No data available Association with lymph node metastasis (Liu et al. 2014) DARC CXCL8, CXCL5, CXCL6, Normal human thyroid cells, human No data available No data available CXCL11, CCL2, CCL5, FTA, human FTC, human CVPTC, CCL7, CCL11, CCL14, human FVPTC (Latini et al. 2013) CCL17 CCR3 CCL4, CCL5, CCL7, Normal human thyroid tissue, human No data available No data available CCL11, CCL13, thyroid cancer tissue (González et al. CCL15, CCL24, 2009, Tang et al. 2018) CCL26, CCL28 CCR6 CCL20 Normal human thyroid cell line and Dendritic cell Promotion of invasiveness of primary cultures (Zeng et al. 2014, (Tsuge et al. human thyroid cancer cells in Coperchini et al. 2016), human TPC-1, 2005) vitro (Zeng et al. 2014, BCPAP, FTC-133, and SW1736cells Coperchini et al. 2016) (Zeng et al. 2014, Coperchini et al. Association with metastasis of 2016) thyroid cancer in liver mouse model (Dellacasagrande et al. 2003) CCR7 CCL19 and CCL21 Human PTC, MTC (García-López et al. Lymphocyte and Association with human lymph 2001), primary cultures of papillary dendritic cell node metastases (Zhang et al. human thyroid cancer cells with RET/ (Sancho et al. 2016) PTC rearrangement expression (Zhang 2006) et al. 2016)

ATC, anaplastic thyroid cancer; CVPTC, classical variant of papillary thyroid cancer; EMT, epithelial-to-mesenchymal transition; FTA, follicular thyroid adenoma; FTC, follicular thyroid cancer; FVPTC, follicular variant of papillary thyroid cancer; MTC, medullary thyroid cancer; PTC, papillary thyroid cancer; TAM, tumor-associated macrophages.

The first demonstration that CXCR3 is also involved associated with a better prognosis as assessed by a in cancer was provided by Tsuchiya et al. in 2004. longer survival (Tsuchiya et al. 2004). Similar findings These authors investigated the expression of CXCR3, were obtained in two rare cases of thyroid PTCL, in different subtypes of peripheral-T-cell-lymphoma complicating chronic autoimmune thyroiditis (Koida (PTCL), reporting that the expression of CXCR3 was et al. 2007).

The common belief that the major role of the CXCL12 (Castellone et al. 2004). CXCR4 was also shown CXCR3 chemokine receptor lies in thyroid autoimmune to be expressed in human neoplastic cell lines derived disease rather than in thyroid cancer development from PTC and in anaplastic thyroid cancer (ATC) (Hwang is probably the reason for the lack of systematic data et al. 2003, De Falco et al. 2007). Also in these cell lines, on this issue. The topic was addressed again in 2016, treatment with CXCL12 induced cell proliferation and when Jiskra et al. investigated the expression of some motility which were reverted by the CXCR4 antagonist chemokine receptors in lymphocytes from benign and AMD3100 or by interfering with the CXCR4 mRNA malignant thyroid nodules derived from patients with synthesis (De Falco et al. 2007). Immunohistochemical and without Hashimoto’s thyroiditis (Jiskra et al. 2016). analysis aimed at detecting CXCR4 in thyroid lesions Flow cytometry analysis showed that lymphocytes from showed that this receptor is mainly distributed in the neoplastic nodules had a higher expression of CXCR3 nucleus of thyroid cancer cells, with weak staining in the compared with those from the benign ones (Jiskra cytoplasm. No expression was detected in the adjacent et al. 2016). More recently, Urra et al. characterized the benign tissue. The CXCR4 staining pattern was stronger expression pattern of the CXCR3 variants (CXCR3A in more aggressive thyroid carcinomas, such as poorly and CXCR3B) in benign tumors and PTC (Urra et al. differentiated thyroid carcinoma (100%) and medullary 2018). Interestingly, these two CXCR3 splicing variants thyroid carcinoma (85.7%). A lower expression of CXCR4 have profoundly different function, because CXCR3A was found in the less aggressive variants, such as classic promotes cell viability, proliferation and angiogenesis, PTC (68.8%) and follicular thyroid carcinoma (66.7%). while CXCR3B displays angio-static and apoptotic effects Shin et al. reported a different immunohistochemical (Lasagni et al. 2003). pattern of CXCR4 according to the histologic subtype Immunohistochemistry analysis revealed that total of PTC. Indeed, CXCR4 expression was greater in poorly CXCR3 levels were increased in PTC compared with normal differentiated (81.0%) than in classic PTC (50.0%); low contralateral tissue. CXCR3A and CXCR3B levels were expression levels were also found in follicular (33.9%) also evaluated in non-metastatic PTC and metastatic PTC. and in the diffuse sclerosing variants (14.3%) of PTC The results showed that upregulation of CXCR3A and (Shin et al. 2013). Although at strikingly lower levels, CXCR3B was associated with absence of lymph node CXCR4 is also expressed in benign conditions, including metastasis in PTC, but CXCR3B protein was higher in Hashimoto thyroiditis (15.8%), follicular adenoma (8.0%) metastatic PTC. (Urra et al. 2018). and nodular goiter (6.7%) (He et al. 2010). It seems worth highlighting that the expression of CXCR4 also influences cell traffic in thyroid cancer microenvironment. Indeed, CXCR4 in anaplastic thyroid cancer (ATC) a significant correlation The CXC chemokine receptor 4 binds CXCL12. The role between CXCR4 expression and numbers of tumor- of the CXCL12/CXCR4 signal was originally investigated associated macrophages (TAMs) was reported (Kim et al. in the fields of immunology, of hematopoiesis, of 2016). CXCL12 induced migration in human thyroid lymphocyte homing and of HIV infection (Brelot et al. anaplastic carcinoma cell lines (ARO), which was inhibited 1997, Zou et al. 1998, Franitza et al. 2002). CXCR4 is by an anti-CXCR4 antibody (Hwang et al. 2003). A further by no doubts the most studied chemokine receptor confirmation of the role of CXCR4 in thyroid cancer in thyroid cancer. CXCR4 is not expressed by normal aggressiveness and metastatic behavior stems from the thyroid cells (Castellone et al. 2004). CXCR4 was first study of Gonzalez et al. By using immunohistochemistry detected in a rat thyroid cell line characterized by the and flow cytometry analysis, these authors demonstrated presence of a RET/PTC rearrangement being the level of that CXCR4 is found in nearly 90% of PTC cells, being expression associated with the cell ability to invade and its expression correlated with node metastases. The migrate. In addition, CXCR4 also displayed a relevant expression of CXCR4 was also found to be positively anti-apoptotic effect on cancer cells (Castellone et al. correlated with tumor size in PTC (Werner et al. 2017). 2004). Further confirmation of the pro-tumorigenic Taken together, these findings support the concept that effect of CXCR4 derived from experiments showing that the expression of CXCR4 is positively related to more treatment with a CXCR4-blocking antibody abolished aggressive tumors and/or to those with a poorer prognosis thyroid cancer cell migration induced by treatment with (González et al. 2009).

Based on the above described preclinical data, the downregulation of cell differentiation, by downregulating expression of CXCR4 was searched for in thyroid cancer Notch signaling (Zhang et al. 2015). Furthermore, Zhang cells within the preoperative diagnostic work-up of et al. (2016) recently reported that a high expression of thyroid lesions. In a retrospective study, Torregrossa et al. CXCR7 increases both CXCL8 and VEGF levels, two main evaluated the expression of several markers including molecules with proven pro-tumorigenic effect (Rotondi CXCR4 in thyroid fine-needle aspiration cytologic (FNAC) et al. 2018). specimens obtained from 100 patients who had undergone thyroidectomy for a solitary nodule (Torregrossa et al. 2010). Results of FNAC and immunohistochemistry The CXCR7/CXCR4/CXCL12 axis analysis were related to the final histological diagnosis. They found that CXCR4 was overexpressed in the vast In the previous paragraphs, we focused on the expression of majority of PTCs (92%). On the other hand, taking into a single chemokine receptor in thyroid cancer cells and its account all the lesions classified as benign (by cytology relation with cancer progression and development. However, and post-surgery histology), 96% were negative for CXCR4 some studies focused on the role of a peculiar chemokine receptors combination (CXCL12/CXCR4/CXCR7) (Torregrossa et al. 2010). In addition, the overexpression of CXCR4 was linked to a more aggressive thyroid cancer in driving cancer progression and metastasis in human cancer (Sun 2010). Both and studies phenotype (Torregrossa et al. 2012). CXCR4 is also et al. in vitro in vivo involved in the metastatic process of thyroid cancer. By demonstrated that the role of the CXCL12/CXCR4 axis in cancer progression is mainly to facilitate metastasis using RT-PCR, Wang et al. demonstrated that the levels of TWIST (a marker of EMT) and of the mRNA for CXCR4 and mobilization of cancer cells (Sun et al. 2010). Zhou were significantly greater in PTCs than in normal thyroid et al. found that the expression of CXCR4, CXCL12 tissues and that the levels of both the protein and the and CXCR7 varied in malignant and benign thyroid mRNA for CXCR4 were correlated with the presence of tissue specimens and that the overexpression of CXCR4 and CXCR7 enhanced thyroid cancer cell invasiveness, metastasis (Wang et al. 2013). but not their proliferation (Zhu et al. 2016). The role of CXCL12/CXCR4/CXCR7 was also investigated in CXCR7 medullary thyroid cancer (MTC) cells where it was found to The CXC chemokine receptor 7 mediates cellular influence the biological behavior of neoplastic cells, likely adhesion, migration, proliferation and survival by binding through a regulation of the EMT process (Werner et al. 2017). The CXCR4/CXCR7/CXCL12 axis has a role also in CXCL12 and CXCL11 (Burns et al. 2006, Boldajipour et al. 2008). CXCR7 was found to be expressed by 65.8% of PTC FTC. Werner et al. examined CXCR4/CXCR7 expression in being its level of expression correlated with lymph node 44 FTCs and in the corresponding non-neoplastic thyroid tissue, in 10 distant metastases of FTC and in 18 follicular metastasis (Liu et al. 2012). Subsequently, the same group demonstrated that CXCR7 is markedly expressed in PTC, adenomas. They observed that expression of CXCR4/7 both at the mRNA and the protein level, this expression was associated with larger tumor size, advanced stage at diagnosis and shorter overall and recurrence-free survival. being positively related with tumor progression (Liu et al. 2014). It was also shown that knockdown of CXCR7 in Interestingly, CXCR4 expression was significantly greater PTC cells suppressed their proliferation and invasiveness, in distant metastases as compared with the primary tumor. decreased the expression of some promoting factor for Again, CXCL12 induced cell growth, cell-cycle activation cell growth (cyclin A, CDK2 and PCNA) and increased and EMT, while CXCR4 antagonists significantly reduced the expression of two cell-cycle inhibitors (p21 and p57). FTC invasiveness in vitro (Werner et al. 2018). These changes resulted in S phase arrest and promoted apoptosis. Taken together, these findings pointed toward CCR3 an important role of this receptor in thyroid cancer metastasis (Liu et al. 2014). CC-chemokine receptor 3, a member of the CC receptor The mechanisms involved in the effect of CXCR7 family, binds several chemokines: CCL4, CCL5, CCL7, on tumor progression were studied by Zhang et al. who CCL11, CCL13, CCL15, CCL24, CCL26, CCL28 (Vela et al. showed that CXCR7 regulates growth and metastasis of 2015). CCR3 enhances cellular proliferation, invasion PTC cells via the activation of the PI3K/AKT pathway and migration, mainly through the regulation of ERK and and its downstream NF-κB signaling, as well as via the JNK signaling pathways, in different cancer cell types.

CCR3 was first identified in renal cell carcinomaJöhrer ( virus and in T cells infected with herpes virus (Campbell et al. 2005). Only two studies evaluated its expression et al. 1998, Yanagihara et al. 1998). CCR7 is the chemokine in thyroid cancer (González et al. 2009). Gonzales et al. receptor for CCL19 and CCL21 (Nagarsheth et al. 2017). showed that the expression of CCR3 was higher in PTC CCR7 plays a critical role in lymphocyte and dendritic by immunohistochemistry (2.5-fold versus normal cell trafficking into and within lymph nodes (i.e. the tissue) and by flow cytometry (3.5-fold versus normal preferential metastatic site for PTC and MTC) (Sancho thyroid cells). However, CCR3 was similarly expressed in et al. 2006). Sancho et al., reported that, as evaluated by tumor cells from patients with or without lymph node RT-PCR, the expression of CCR7 was higher in PTC and metastasis. Similar results were recently reported by Tang MTCs than in follicular and poorly differentiated thyroid et al. using a bioinformatic analysis method (Tang et al. carcinomas, being positively correlated with lymph node 2018). These findings do not support a prognostic role of metastases (Sancho et al. 2006). Incubation with CCL21 CCR3 expression in thyroid cancer (González et al. 2009). also promoted proliferation and migration of the CCR7- expressing thyroid tumor cell line TPC-1 (Sancho et al. 2006). Although from a quantitative point of view, CCR7 CCR6 is scarcely detected in PTC cells (5–10%) being absent in CC-chemokine receptor 6 is the sole receptor for the normal thyroid cells (González et al. 2009), its expression, chemokine CCL20. The expression of CCL20 and CCR6 both at the mRNA and at the protein level, was found is increased in many cancers such as colorectal and to be associated with larger tumor size and increased pancreatic cancers being associated with a poor prognosis markers of tumor aggressiveness in PTC (Wagner et al. (Rubie et al. 2010, Frick et al. 2013). In thyroid cancer, 2008). Proliferation assays in primary cultures of PTC cells a role of CCR6 was suggested by the observation that indicated that CCR7 activation by CCL21 is associated dendritic cells expressing CCR6 are found to be densely with a significant increase of cell proliferation (Zhang accumulated in PTC (Tsuge et al. 2005). In a mouse model et al. 2016). Flow cytometry analysis indicated that of liver metastasis, Dellacasagrande et al. demonstrated CCL21/CCR7 interaction significantly increased that CCR6 is overexpressed in metastatic cells the fraction of cells in the G2/M phase of the cell originating from colorectal, thyroid and ovarian cancers. cycle. Western blotting experiments confirmed that This finding would fit with the notion that human liver CCL21/CCR7 interaction significantly upregulated cyclin cells constitutively secrete CCL20, which in turn recruits A, cyclin B1 and cyclin-dependent kinase 1 (CDK1) those cancer cells expressing CCR6 (Dellacasagrande et al. expression (Zhang et al. 2016). 2003). More recently, Zeng et al. showed that thyroid cancer cell lines (TPC-1, BCPAP, FTC-133 and SW1736 cells) and normal thyrocytes (HTori-3) express the CCR6 mRNA and produce the relative protein (Zeng et al. 2014). The atypical receptor DARC Further studies demonstrated that incubation with CCL20 Duffy, also named DARC (Duffy antigen receptor for induces invasiveness and migration of these thyroid cancer chemokines), is a glycosylated membrane protein that cell lines, a phenomenon promptly reverted by knocking belongs to the subfamily of silent chemokine receptors, down CCR6. These experimental results support the which selectively binds angiogenic chemokines, such as concept that the CCL20–CCR6 axis mediates invasiveness CXCL8 (but also CXCL5, CXCL6, CXCL11, CCL2, CCL5, and metastatization of thyroid cancer cells. Moving from CCL7, CCL11, CCL14, CCL17) (Latini et al. 2013). The these data, our group showed by immunocytochemistry expression of DARC was found to be elevated in patients and flow cytometry that the basal expression of CCR6 is with breast cancer. The relationship between DARC and significantly upregulated by TNF- in two thyroid cancer α thyroid cancer was evaluated in a single study by Latini cell lines: TPC-1 and BCPAP (Coperchini et al. 2016). et al. (2013). By using quantitative PCR, DARC expression was searched for in 18 normal thyroid tissues, in 15 FTA and in 139 malignant thyroid tumors. DARC expression CCR7 was higher in benign lesions compared with the malignant The CC-chemokine receptor 7 is expressed on activated ones. Moreover, DARC expression was significantly lower T and B lymphocytes as well as on dendritic cells, being in those tumors with a greater than 10 mm diameter strongly upregulated in B cells infected with Epstein–Barr (Latini et al. 2013). When different subtypes of thyroid

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0163 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 07:19:05AM via free access Endocrine-Related F Coperchini et al. Chemokine receptors in 26:8 R473 Cancer thyroid cancer carcinoma were compared, similar expression levels of tumor angiogenesis, invasiveness and tumor-induced DARC were observed (Latini et al. 2013). DARC expression immunosuppression. Indeed, anti-CXCR4 and anti- was also significantly reduced in PTCs associated with CXCL12 antibodies prevented metastasis, reduced tumor lymphocyte infiltration when compared with normal weight and prevented tumor extravasation in preclinical thyroid tissue. (Latini et al. 2013), suggesting an inverse models (Bertolini et al. 2002, Lapteva et al. 2005, Zlotnik relationship between DARC expression and thyroid 2006). Another good example of immunotherapy through cancer aggressiveness. chemokine receptor blockade is provided by the targeting of the CXCL8-CXCR1/2 axis, which plays an important role in tumor progression and metastasis by regulating cancer stem cell proliferation and self-renewal (Ha et al. Chemokine receptors and immunotherapy 2017). During the past two decades, several CXCR1/2 Effective strategies for immunotherapy are based on the small-molecule inhibitors, CXCL8 release inhibitors and generation of immunity against tumor-specific antigenic neutralizing antibodies against CXCL8 and CXCR1/2 peptides. Given that chemokines and their receptors have have been developed. Several preclinical studies suggest crucial roles in inflammatory human diseases, efforts that combination of CXCR1/2 inhibitors along with other have been made to target chemokine networks in patients targeted therapies, chemotherapies and immunotherapy with autoimmune diseases and chronic inflammation may be effective in treating specific types of cancer (Ha (Nagarsheth et al. 2017). As crucial mediators of cell et al. 2017). migration, chemokines are also an interesting target for Two chemokine receptor-targeted agents, which were therapies aimed at attenuating inflammation without previously used for stem cells mobilization in stem cells inducing generalized immunosuppression in several auto-transplant, are currently studied in humans for their clinical settings, such as cancer, autoimmune disease anti-cancer properties. Reparixin, an allosteric inhibitor and allograph transplant (Rot & von Andrian 2004, of CXCR1 and CXCR2, was demonstrated to be safe and Rotondi et al. 2007, Rotondi & Chiovato 2011). The tolerable in a Phase Ib pilot study in breast cancer patients most successful example of immunotherapy directed in association with paclitaxel (Schott et al. 2017). The to a chemokine receptor is the development of drugs promising oncological response rate lead to the initiation targeting the CXC chemokine receptor 4 (CXCR4) for of a Phase II clinical trial on metastatic triple-negative hematopoietic stem cell mobilization in patients who breast cancer NCT02370238. Plerixafor, an anti-CXCR4 require stem cells auto-transplant. Another example is agent currently approved for hematopoietic stem cells targeting the CC-chemokine receptor 5 (CCR5) in order mobilization in combination with granulocyte colony- to inhibit the entrance of HIV in T cells through CCR5 stimulating factor, is object of a still ongoing Phase I study binding (Johnson et al. 2005, Gulick et al. 2008, Rotondi in advanced ovarian, pancreatic and colorectal cancer & Chiovato 2011). (EUDRACT 2014-000117-31). Targeting chemokine receptors turned out to be Therapies aimed at targeting chemokine receptors a good approach also in several models of cancer can affect the trafficking of different immune cell (Johnson et al. 2005). Interfering with the chemokine subsets and can modify the biological activity of non- system involves several strategies including antibodies, immune cells in tumor microenvironment (Nagarsheth small molecules, modified chemokines, neutralizing et al. 2017). Both direct and indirect manipulations of monoclonal antibodies, binding proteins or chemokine–chemokine receptor signaling pathways pharmacological modulation of chemokine/chemokine could provide antitumoral effects by reshaping receptor interactions (Proudfoot et al. 2003, Johnson its immune and biological phenotypes. Although et al. 2005). These approaches were recently tested in promising results are present, a possible pitfall of different types of cancer with success at least in some immunotherapy directed to chemokine receptors could cases. Targeting of the CCL2, CCL3 or CCL5 signaling be due to the redundancy of the chemokine–chemokine inhibits metastasis and angiogenesis in mouse models of receptor system. On the other hand, it could as well be breast, lung and ovarian cancer (Qian et al. 2011, Long possible that combination therapy with other immune- et al. 2012, Bonapace et al. 2014, Kitamura et al. 2015). modulating drugs could lead to better results in The blockade of CXCR4–CXCL12 signaling reduced cancer therapy.

Table 2 Chemokine receptors targeting for immunotherapy of thyroid cancer.

Compound Pharmacological effect In vivo and in vitro effects in thyroid cancer Reference REPARIXIN Noncompetitive allosteric • Dose-dependent inhibition of CAL62 and 8505C cell Liotti et al. (2017a,b) DF1681Y inhibitor of CXCR1 and CXCR2 growth and induction of apoptosis, with no effect on non-cancerous cell lines • Inhibition of cell-cycle progression with accumulation in G1 phases of 8505C and SW1736 cells • Inhibition of DNA synthesis in cancerous cell lines • Reduction of levels of mRNAs encoding for EMT transcrip- tion factors (SNAIL1/SNAIL, SNAIL2/SLUG, TWIST1, ZEB1) • Reduction of migration of cancerous cell lines • Reduction of stemness markers expression • Reduction of sphere formation • Reduction of xenografted thyroid tumor in nude mice PLERIXAFOR Antagonist of CXCR4 • Inhibition of migration and invasion of papillary thyroid Jung et al. (2016), AMD3100 cancer cell line (BHP10-3) at low doses De Falco et al. (2007) • Inhibition of CXCL12-induced cell proliferation and motility in PTC and ATC cell • Inhibition of BHP10-3M cell proliferation at high doses • Inhibition of formation of xenograft tumors in BALB/c mice

Chemokine receptors and immunotherapy in an inhibition of stemness in thyroid cancer cells (Liotti thyroid cancer et al. 2017b). Interesting results were found not only by targeting these two receptors, but also by modulating the Although most patients respond well to current secretion of one of their ligands, CXCL8, a chemokine therapy, 10–30% of thyroid cancer patients eventually which binds both CXCR1 and CXCR2. By using different develop recurrent disease and tumor metastasis (Ward compounds, it was demonstrated that the inhibition 2014). Immunotherapy aimed at targeting chemokine of CXCL8 secretion in thyroid cancer cells reduces cell receptors was recently considered a possible strategy migration in in vitro experiments (Rotondi et al. 2013, to reduce thyroid cancer progression in these patients. 2015, 2016, Awwad et al. 2018, Coperchini et al. 2019). Currently available data regarding chemokine receptor- Accordingly, thyroid cancer metastasis is reduced and related immunotherapy for thyroid cancer involve survival is prolonged in animal models (Fang et al. three receptors: CXCR1, CXCR2 and CXCR4 (Table 2). 2014). CXCR4 targeting was also evaluated in thyroid Reparixin, an allosteric, noncompetitive, low-molecular- cancer. Jung et al. reported that the antagonist of CXCR4 weight CXCR1–CXCR2 dual inhibitor, was evaluated (AMD3100) had an anti-tumor effect in the PTC cell line for a possible inhibition of thyroid cancer viability and (BHP10-3) (Jung et al. 2016). In particular, AMD3100 stemness. To this purpose, experiments were conducted displayed a dual effect producing an inhibition of thyroid in the 8505C thyroid cancer cell line, which expresses tumor cells migration at low dose and an inhibition of CXCR1 and CXCR2, and in 8505c cells not expressing proliferation at high dose (Jung et al. 2016). CXCR1 and CXCR2 after silencing with small-interfering Although these results should be regarded as RNAs (shCXCR1 and shCXCR2) (Liotti et al. 2017b). The preliminary, it is reasonable to hypothesize that percentage of 8505c apoptotic cells was increased by immunotherapy strategies aimed at targeting chemokine treatment with Reparixin, and completely abolished in receptors in refractory thyroid cancer will be developed in cells silenced for CXCR1 or CXCR2. Moreover, treatment the near future. with Reparixin significantly reduced stemness and EMT marker expression in 8505C thyroid cancer cells, but not in the same cell line silenced for CXCR1 and CXCR2. It is Conclusion interesting to note that the ability of 8505c cells to form spheroids was significantly reduced in shCXCR1 and to The chemokine–chemokine/receptor system is crucially a lesser extent also in shCXCR2 (Liotti et al. 2017a,b). involved in the recruitment and maintenance of immune Taken together, these findings demonstrated the ability of cells within the tumor microenvironment. More than 50 Reparixin to inhibit both CXCR1 and CXCR2 expression chemokine receptors have been identified so far. For nine which, in turn, produces a reduction of viability and of these receptors, there is evidence for their expression

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Received in final form 22 May 2019 Accepted 30 May 2019 Accepted Preprint published online 30 May 2019