Colorectal cancer (CRC) is the second most common cause of cancer-related death in western countries. Approximately one-quarter of newly diagnosed patients for CRC have metastases, and a further 40%-50% experience disease recurrence or develop metastases after all standard therapies. Therefore, understanding the molecular mechanisms involved in the progression of CRC and subsequently developing novel therapeutic targets is crucial to improve management of CRC and patients’ long-term survival. Several tyrosine kinase receptors have been implicated in CRC development, progression and metastasis, including epidermal growth factor receptor (EGFR) and vascular EGFR. Recently, tropomyosin-related kinase B (TrkB), a tyrosine kinase receptor, has been reported in CRC and found to clearly exert several biological and clinical features, such as tumor cell growth and survival in vitro and in vivo, metastasis formation and poor prognosis. Here we review the significance of TrkB and its ligand brain derived-neurotrophic factor in CRC. We focus on their expression in CRC tumor samples, and their functional roles in CRC cell lines and in in vivo models. Finally we discuss therapeutic approaches that can lead to the development of novel therapeutic agents for treating TrkB-expressing CRC tumors.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the western world and the third most common malignancy diagnosed worldwide[1]. With present-day therapies, curative (R0) surgical resection is used in approximately 70%-80% of newly diagnosed patients with localized CRC[2]. However, the overall prognosis remains poor for CRC patients, with a median survival time of approximately 20-24 mo[3-5]. In fact, the remaining 20%-30% of newly diagnosed patients present advanced unresectable metastatic disease, and an additional 40%-50% of patients with initially localized CRC experience disease recurrence despite surgical resection and adjuvant therapy, or develop hepatic and/or lung metastases[5,6].

In recent decades, the development of early detection methods for CRC, together with the evolution of systemic chemotherapy for patients with CRC, has led to a decrease in CRC incidence and mortality worldwide[1]. 5-Fluorouracil (5-FU), discovered in 1957 by Heidelberger et al[7], was for many years, in combination with leucovorin the only treatment for patients with metastatic CRC (mCRC)[8-11]. In the early 2000s, the use of the topoisomerase I inhibitor irinotecan, and subsequently the platinum-containing agent oxaliplatin, were key developments in CRC treatment[5]. These agents were found to significantly prolong the median overall survival (OS) of patients with mCRC from 12.6 mo (5-FU/leucovorin alone as first-line therapy) to 14.8 mo (bolus 5-FU/leucovorin with irinotecan)[12], then from 15 mo (5-FU/leucovorin with irinotecan) to 19.5 mo (FOLFOX; 5-FU/leucovorin with oxaliplatin)[13]. In 2004, Hurwitz et al[14] reported that treatment of CRC patients with bevacizumab (humanized monoclonal antibody against vascular endothelial growth factor) led to a significant increase in OS. Next, in several key trials, monoclonal antibodies to the anti-epidermal growth factor receptor (EGFR), namely cetuximab and panitumumab, were proposed as a novel agents for mCRC treatment. These anti-tyrosine kinase receptor antibodies were the first therapeutic targeted agents in patients with the KRAS wild-type metastatic CRC (mCRC)[5,15,16]. Recently, Regorafenib, the first small-molecule tyrosine kinase inhibitor, was approved as a potential new line of therapy for patients who have progressed after all standard therapies[17].

Expression of the tyrosine kinase receptor TrkB is increasingly reported in CRC, and there is a growing body of evidence that the TrkB/BDNF pathway is an important player in CRC tumor growth and progression. Although mainly recognized for its role in the nervous system, the TrkB/BDNF pathway has been implicated in the pathogenesis and prognosis of several tumors outside of the brain, such as pancreatic[18-20], prostatic[21,22], pulmonary[23,24], ovarian[25], bladder[26] and head and neck cancers[27,28], in addition to choriocarcinoma[29], myeloma[30] and B-lymphocytic leukemia[31,32]. This review will focus on the role of TrkB and its ligand BDNF in promoting CRC progression and survival. In addition, we outline potential therapeutic approaches targeting the TrkB/BDNF pathway.

BDNF is a member of the neurotrophin (NT) family, which is a group of closely related growth factors originally known for their involvement in the differentiation, proliferation, and survival of neural cells in both the central and peripheral nervous systems[33].

The NT family includes NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin 3) and NT-4/5 (neurotrophin 4/5). In 1982, Barde et al[34] first isolated BDNF from pig brain as a survival factor for cultured embryonic chick sensory neurons. NTs are synthesized via the same process of maturation. Indeed, NTs are generated from single protein precursors of approximately 27 kDa called pre-pro-NTs. The latter is composed of a pre-pro-domain, a pro-domain and a mature domain that are sequentially cleaved to obtain mature NTs of approximately 13 kDa. Immature NTs (pre-pro-NTs) are cleaved in the endoplasmic reticulum to generate pro-NTs[35,36]. Pro-NTs are then proteolytically cleaved to mature NTs. Cleavage can occur either intracellularly, by the action of furin or proconvertase, or extracellularly, by the action of plasmin and matrix metalloproteinases (MMPs) such as MMP-3, MMP-7 or MMP-9[36,37]. Accordingly, pro-NTs can be released as soluble forms or processed as mature NTs, each with specific receptor binding properties and physiological consequences. Generally, immature NTs or pro-NTs bind to p75^NTR and sortilin (a member of the VPS10p-domain receptor family) to induce apoptosis via the JNK kinase pathway[38]. In their mature forms, NTs mediate their effects through binding to two structurally unrelated cell membrane receptors, the Trk tyrosine kinase receptors and the common NT receptor p75^NTR[39,40]. BDNF effectively signals through the low affinity receptor (p75^NTR) that belongs to the tumor necrosis factor (TNF) receptor superfamily and through the high-affinity TrkB tyrosine kinase receptor.

TrkB belongs to the Trk family of tyrosine kinase receptors, which consists of an extracellular ligand-binding domain, a transmembrane domain and a cytoplasmic tyrosine kinase domain[41,42]. The extracellular domain of these receptors consists of a leucine-rich domain surrounded by two cysteine rich regions that are thought to be essential for NTs high affinity binding. Near those regions and close to the cell membrane there are two immunoglobulin-like (IG-like) domains that are followed by the transmembrane domain. The IG-like domain directs ligand-binding specificity. The cytoplasmic domain consists of a Shc binding site, a tyrosine kinase domain and a tail region that includes a phospholipase C-gamma binding site (PLC-γ)[43]. Trk was first described in 1986 by Martin-Zanca et al[44] as an oncogene in colonic carcinoma. The Trk family includes three members, TrkA, TrkB and TrkC, each of which is activated by one or more of the four NTs. NGF preferentially binds to TrkA and BDNF and NT-4/5 preferentially bind to TrkB. NT-3 preferentially binds to TrkC but also to TrkA and TrkB to a lesser extent and only in certain cellular contexts[45-48]. The TrkB gene (NTRK2 about 590 kb) is located on chromosome 9q22 and contains 24 exons[43,49]. The TrkB gene can create more than 100 RNA transcripts (due to alternative splicing) and at least 36 of these transcripts are translated, among which there are three major protein isoforms[43,50]. In addition to the full-length TrkB (TrkB-FL, 145 kDa), there are two alternatively spliced TrkB isoforms (95 kDa) that lack the tyrosine kinase domain, TrkB-T1 and TrkB-Shc, which are slightly longer and include the Shc binding site[50]. BDNF binding to the TrkB-FL (full-length) receptor results in its dimerization and subsequent autophosphorylation of tyrosine (Y) Y484 and Y785. Generally, TrkB-FL induces survival and differentiation through the three principle growth factor signaling pathways: Ras/MAPK, PI3Kinase (PI3K)/Akt and PLC-γ/PKC pathways[42,50] (Figure 1). For an expanded review on Trk signaling, see[45,46].

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Figure 1 TrkB-mediated signaling pathways and possible therapeutic targets in cancer. Schematic representation of TrkB receptor, its main downstream signaling pathways, and possible therapeutic targets. Shc: Src homology 2 domain containing transforming protein; Grb2: Growth factor receptor-bound protein 2; Gab1: Grb2 associated binding protein 1; PI3K: Phosphatidylinositol 3-kinase; PDK1: Phosphoinositide-dependent protein kinase 1; mTOR: Mammalian target of rapamycin; mTORC1: mTOR complex 1; SOS: Son of sevenless; MEK: Mitogen-activated protein kinase (MAPK)/ERK kinase; ERK: Extracellular signal-regulated kinase; PLC: Phospholipase C; PKC: Protein kinase C.

TrkB-T1 is abundantly expressed in adult neural cells but also in non-neural cells, yet its biological role in BDNF-mediated signaling remains poorly understood[51]. For many years, both truncated TrkB receptor isoforms were though to act as dominant-negative inhibitors of TrkB-FL and did not signal directly[52-54]. For example, TrkB-T1 or TrkB-Shc can bind to and internalize BDNF or dimerize with TrkB-FL to prevent its autophosphorylation and subsequently inhibit intracellular signaling pathways[50,55]. However, recent studies have demonstrated that TrkB-T1 sequesters and translocate BDNF[56], induces filopodia and neurite outgrowth[57-59], regulates Rho GTPase signaling[60] and intracellular signaling cascades[30,61,62], and mediates cell proliferation[63,64]. Rose et al[65] have demonstrated that astrocytes predominantly express TrkB-T1 and that BDNF-mediated activation of the truncated TrkB-T1 controls calcium release from intracellular stores through an inositol-1,45-phosphate (IP3)-dependent pathway. In addition, Li et al[63] have shown that TrkB-T1 enhanced cell proliferation, promoted formation of colonies in soft agar, stimulated tumor cell invasion and induced liver metastasis in an orthotopic xenograft mouse model of pancreatic cancer. For a recent and extensive review on truncated TrkB, refer to Fenner[55].

Overexpression of the TrkB receptor was first described in human neuroblastoma, which is a childhood malignant tumor derived from primitive cells of the developing sympathetic nervous system[66]. TrkB and BDNF are highly expressed in biologically unfavorable, MYCN-amplified, aggressive neuroblastomas[67]. Indeed, patients with neuroblastoma, whose tumors express elevated levels of TrkB and BDNF, have a poor prognosis[67,68]. It has also been shown that in neuroblastoma tumor cells the TrkB/BDNF pathway is involved in epithelial-mesenchymal-transition-like transformation (EMT), metastasis[69,70], anoikis[71], chemotherapeutic resistance[72,73], and hypoxia via activation of angiogenesis[74]. Thus, BDNF/TrkB signaling in neuroblastoma contributes to tumor growth and an unfavorable outcome in an autocrine/paracrine manner[75].

Activity of the BDNF/TrkB pathway is mainly recognized in the field of neurobiology. However there is now increasing recognition that the TrkB/BDNF pathway regulates important processes and may contribute to the pathogenesis of numerous non-neural carcinomas whose cells express the TrkB receptor, including pancreas[18,19], prostate[21,22], ovarian[76,77], Wilms’ tumors, head and neck[27,28], lung[23,78], breast[79,80], gastric[81,82], and hepatocellular carcinomas[83,84], as well as myelomas[30,85,86] and lymphoid cancers[31,32,87]. Indeed, overexpression of TrkB was found in the highly metastatic pancreatic cancer cells. Moreover, TrkB expression was correlated with perineural invasion, positive retroperitoneal margin, and earlier liver metastasis development in patient samples[19]. In prostate cancer, TrkB and BDNF may be considered as possible diagnostic markers[21]. Furthermore, TrkB therapeutic effects were shown in an in vivo xenograft model using Trk-specific inhibitors, such as CEP-751 or CEP-701. In these studies, results have shown that targeting of the TrkB receptor induces cell death in a cell cycle-independent manner and inhibits tumor growth and metastasis development in prostate cancer[88,89]. In Wilms’ tumors, TrkB overexpression correlated with a higher risk of mortality[90]. The role of the TrkB/BDNF pathway in head and neck squamous cell carcinoma (HNSCC) was also investigated by Kupferman et al[27] who demonstrated that these proteins are expressed in greater than 50% of human HNSCC tumors, but not in the normal upper aerodigestive tract. In vitro and in vivo studies have revealed that TrkB is directly implicated in EMT, tumor growth and invasive behavior of HNSCC. Furthermore, BDNF stimulation of HNSCC cells enhances cell migration and invasion, drug-resistance and anti-apoptotic protein expression[27,28]. BDNF was also described as an autocrine factor in human multiple myeloma cells contributing in tumor progression and survival by activating signaling in the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase-Akt (PI3K/Akt) pathway. In addition, BDNF was able to protect human multiple myeloma (MM) cell lines from apoptosis induced by dexamethasone or bortezomib[30]. Finally, TrkB and BDNF were also reported to be associated with a significant increase in the survival and proliferation of non-Hodgkin lymphoma cells[91] and malignant B cell lines[31,32]. For example, Fauchais et al[31] have shown that endogenous BDNF released under stress culture conditions exerts antiapoptotic effects in human B cell lines. Such autocrine production was linked to the presence of sortilin, an endogenous protein that is able to transport and release BDNF in culture media. In diffuse large B cell lymphoma, in vitro studies have shown that the Trk-inhibitor K252a enhances apoptosis and exhibits additive apoptotic effects with rituximab, a chimeric anti-CD20 monoclonal antibody[32].

Currently, among conventional therapies, advanced CRC remains a major therapeutic challenge with poor overall survival. Therefore, identifying novel molecular mechanisms involved in driving CRC development and progression is crucial for improving clinical management of CRC and patient outcome. TrkB has been shown to associate with CRC local progression, clinical stage, nodal metastasis, distant metastasis, and poor prognosis. Additionally, emerging data suggest that the TrkB/BDNF pathway enhances several biological processes in CRC, including proliferation, invasion, migration, EMT, apoptosis resistance and anoikis resistance. Hence, inhibition of the TrkB/BDNF pathway alone or in combination with other conventional and targeted agents in CRC may hold promise as an important approach to improve patients’ long-term survival.