Sortilin/Neurotensin Receptor-3 in Cancer: History
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Jean Mazella

The multifunctional role of sortilin in cancers development The development of cancerous tumors is known to be the consequence of the overexpression of growth factors. Unfortunately, when treated with radiotherapy or chemical therapy, some tumors can metastasize as a result of the weakening of cancer cell–cell interactions in the tumor tissue, leading to the dissemination of cancer cells in the circulation. Both mechanisms of cancer growth and metastasis are regulated by a large panel of circulating activators from several neuropeptides to membrane-bound factors released by matrix metalloprotease (MMP)-dependent shedding, such as Epidermal Growth Factor Receptor (EGFR) ligands. One of the most studied neuropeptides involved in cancer progression is neurotensin (NTS), the three known receptors of which (two G-protein coupled receptors, NTSR1 and NTSR2, and a type I receptor, NTSR3) are expressed in numerous cancers and particularly in digestive cancers. Interestingly, NTSR3, also previously identified as Sortilin, is shed from the plasma membrane, leading to the release of a soluble form of sortilin (sSortilin). However, growing evidence indicates the emerging role of membrane-bound Sortilin/NTSR3 and its soluble counterpart in cancer cell proliferation and dissemination.

  • sortilin
  • neurotensin
  • neurotensin receptor-3
  • soluble sortilin
  • colorectal cancer

1. The Membrane-Bound Sortilin/-3Neurotensin (NTS) Receptor(3NTSR3)

1.1. The Role of Membrane Sortilin/NTSR3 in the Signaling and Trafficking of Neurotrophin Receptors

Neurotrophins (NTs) are growth factors that control a series of functions in the nervous system. The mature forms of Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF), as well as those of NT4/5 and NT3, are involved in Trk-dependent neuronal cell survival, whereas their unmatured forms are responsible for cell death through p75NTR [1]. In fact, all the functions described above for NT receptors necessitate their association with Sortilin/NTSR3, which was well described in a previous review [2]. Focusing on colorectal cancer cells, the interaction of Sortilin/NTSR3 with either TrkB or p75NTR, both expressed in colorectal cancer cells, triggers opposite functions. On the one hand, BDNF, the secretion of which is activated by Sortilin/NTSR3 [3], induces cell proliferation and displays anti-apoptotic effects through TrkB [4]. One the other hand, exogenous pro-BDNF induced colorectal cancer cell apoptosis through Sortilin/NTSR3 as a co-receptor of p75NTR, the high-affinity receptor for pro-neurotrophins, suggesting a mechanism of Sortilin/NTSR3 action that can counterbalance cell survival [4].

1.2. The Role of Membrane Sortilin/NTSR3 in the Signaling and Trafficking of Neurotensin Receptors

NTS and its receptors NTSR1 and Sortilin/NTSR3 are significantly overexpressed in colorectal cancer cells when compared to the surrounding normal epithelium, an observation that can potentially be used as a prognostic biomarker associated with more advanced colorectal cancer and poorer disease-free survival [5].
In the human colonic adenocarcinoma cell line HT29, Sortilin/NTSR3 is co-expressed with the G-protein coupled receptor NTSR1. Immunoprecipitation experiments provided evidence for endogenous complex formation between these two receptors. It has also been demonstrated that the NTSR1–Sortilin/NTSR3 complex is internalized on NTS stimulation [6]. More interestingly, the interaction of Sortilin/NTSR3 with NTSR1 modulates both the NTS-induced phosphorylation of mitogen-activated protein (MAP) kinases and the phosphoinositide (PI) turnover mediated by NTSR1 [7], suggesting that Sortilin/NTSR3 may act as a co-receptor to participate in true NTS signaling. To further examine the functionality of Sortilin/NTSR3 trafficking in HT29 cells, the internalization of the Sortilin/NTSR3–NTS complex was followed from the plasma membrane to the trans-Golgi network (TGN), where NTS was bound to a lower molecular form of the receptor compared to the form found at the cell surface or on early endosomes [7]. This result suggested that the signaling and transportation functions of Sortilin/NTSR3 may be mediated through different molecular forms of the protein, a high-molecular-weight membrane form responsible for NTS endocytosis and a low-molecular-weight intracellular form responsible for the sorting of internalized NTS to the TGN. Once again, the role of Sortilin/NTSR3 in HT29 proliferation appears rather essential in the regulation of the action of NTS to modulate cancer cell proliferation.
In the same colorectal cell line, a study from Navarro et al. demonstrated that NTS-induced proliferation was dependent on the internalization of the Sortilin/NTSR3-NTS complex [8]. Inhibition of the internalization process affected NTS-induced Erk1/2 phosphorylation and cell proliferation, whereas the peptide-induced activation of phospholipase C was unaffected, indicating that the two intracellular pathways activated by NTS in HT29 cells (phospholipase C and MAP kinases) are independent. This can be explained by distinct conformational structures formed by the associated NTSR1 and Sortilin/NTSR3, leading to either G-protein activation or to the process of sequestration.

2. The Soluble Form of Sortilin/NTSR3

2.1. Shedding of the Cell Surface Sortilin/NTSR3

The shedding of Sortilin/NTSR3 was not stimulated by NTS itself, but the amount of shed protein (sSortilin/NTSR3) recovered in the extracellular medium was enhanced when the internalization process was blocked by hyperosmolar sucrose suggesting an accumulation of the protein at the cell surface and also an increase in the amount of shed protein in these conditions. The shedding process of Sortilin/NTSR3 is activated in a concentration- and time-dependent manner by PMA (Phorbol 12-Myristate 13-Acetate), a protein with a molecular weight of 100 kDa, which is slightly lower than that detected in crude homogenates (110 kDa). PMA acts as an activator of MMPs via the PKC pathway in several types of cells such as neurons, microglial cells, and cancer cells [9]. In the same way, other PKC activators such as carbachol or PGE2 [10] increased the shedding of Sortilin/NTSR3 [11]. Note that other members of the Vps10p receptor family, SorLA and SorCS1-3, are also shed [12][13].

2.2. Binding and Internalization Properties of sSortilin/NTSR3

The shed Vps10p proteins could display their own activities as ligands or could serve as transporters/protectors to avoid the proteolytic degradation of their ligands. Binding experiments performed on HT29 cell homogenates using 125I-radiolabeled proteins showed that sSortilin/NTSR3 specifically bound to HT29 membranes with an affinity of 5 nM but not to the other NTS receptors [11]. Although in numerous cancer cell systems, NTS signaling depends on EGFR activation [14][15], this is not the case in HT29 cells, since sSortilin/NTSR3 is unable to compete with EGF on the Epidermal Growth Factor Receptor (EGFR), and reciprocally, EGF is unable to compete with sSortilin/NTSR3 on its binding sites [11]. These results indicate that sSortilin/NTSR3 recognizes a specific receptor in HT29 cells that is neither sortilin nor EGFR.
After binding to a specific receptor, sSortilin/NTSR3 is rapidly and efficiently sequestrated at 37 °C into HT29 cells by a mechanism dependent on hyperosmolar sucrose [11]. Following its internalization, 60–70% of the sequestered protein is recovered into lysosomes and degraded. The remaining non-degraded sSortilin/NTSR3 could be sorted to recycling vesicles or to other cellular compartments to trigger unidentified functions. The intracellular fate of sSortilin/NTSR3 appears to follow the same sorting to lysosomes that the membrane-bound Sortilin/NTSR3 undergoes [16].

2.3. Cell Functions of sSortilin/NTSR3 in HT29 Cells

sSortilin/NTSR3 induces plasma membrane translocation of PKCα and consequently increases the intracellular concentration of calcium at low concentrations (10 nM) [11]. It was shown that the effect of sSortilin/NTSR3 on calcium concentrations can be desensitized, a mechanism frequently observed by the internalization and uncoupling of functional receptors such as G-protein coupled receptors [17] and the low-density lipoprotein lipase receptor family [18].
In HT29 cells, sSortilin/NTSR3 rapidly and transiently activates Akt phosphorylation through the upstream phosphorylation of the complex focal adhesion kinase FAK-Src [11]. The activation of the phosphatidylinositol 3-kinase (PI3 kinase) pathway is an important step to induce calcium release from the intracellular stores (for a review, see [19]), a pathway involved in the development of colorectal cancers [20]. It is important to note that the activation of the FAK pathway is involved in survival mechanisms, and especially in a variety of distinct cancer cell development and metastasis processes [21][22].

2.4. Morphological Changes of HT29 Cells Induced by sSortilin/NTSR3

The activation of the focal adhesion kinase (FAK) pathway is known to be correlated with numerous cellular processes such as cell spreading, adhesion, migration, and survival [23]. In HT29 cells, the shape and the morphology on sSortilin/NTSR3 incubation were investigated to determine the role of the protein in the regulation of cancer cell detachment [24].
The geometric distribution (polygon classes) of cells, assessed using labeling with fluorescent anti-E-cadherin antibodies, illustrates that resting confluent HT29 cells presented a geometric distribution corresponding to 46% hexagons, a distribution in agreement with several other resting cells [25][26]. Interestingly, sSortilin/NTSR3-treated HT29 cells displayed a significant reduction (to 30%) in the proportion of hexagons in favor of pentagons, as well as an increase in the cell surface [24].
The modifications by sSortilin/NTSR3 of the actin cytoskeleton and the cell shape, as well as its ability to activate FAK, were likely linked to the cell–matrix contact weakening, which can lead to cell migration. However, HT29 cells are non-migrating cells [27]; therefore, the role of sSortilin/NTSR3 could correspond to involvement in the first step of a mechanism responsible for cell detachment.
Could the reorganization of the cell shape by sSortilin/NTSR3 be in agreement with the modifications of the architecture of ultra-structural components such as desmosomes and intermediate filaments? [24]. To answer this question, the number and structure of desmosomes have been analyzed. Desmosomes are formed by plaque densities and bundles of intermediate filaments. These structures are involved in cell–cell adhesion by connecting the proteins forming plaque densities to the interfilaments’ cytoskeleton. The desmosomes are important to ensure tissue integrity and to maintain homeostasis [28]. In fact, sSortilin/NTSR3 decreases the average number of desmosomes per cell and modifies the architecture of desmosomes, thus weakening the cell–cell and cell–matrix interactions. The disorganization of desmosomes may contribute to the weakening of the cell barrier, which can allow for the crossing of growth factors leading to tissue dysfunction, particularly in the development or progression of human epithelial cancer cells (for reviews see [29][30]).
The marked changes observed in the sSortilin/NTSR3-treated HT29 cell morphology were correlated with the decreased expression of E-cadherin and a series of integrin family members, proteins implicated in cell–cell junctions or cell adhesion [24][31]. A decrease in or loss of integrin couples has already been described in colonic epithelial cells [32][33] in association with a poor prognosis.
Cell detachment from the plates has previously been observed in resting colonic cancer cells including HCT116, HT29, and SW620 cell lines. The action of sSortilin/NTSR3 in the weakening of cell–cell contact and cell–matrix interactions may be part of a mechanism responsible for the initial steps leading to cancer cell detachment and diffusion from primary tumors to healthy non-tumoral cells, thus facilitating metastasis .

3. Another Crucial Function of Sortilin/NTSR3: Possible Role in the Field of Cancer

Involvement of Sortilin/NTSR3 in the Membrane Expression of TREK-1OK

A previous study demonstrated the interaction between the two proteins sortilin and TREK-1, in which mice with deletions of the sortilin (sort1) or TREK-1 (kcnk2) genes displayed a similar phenotype of resistance to depressive-like behavior during resignation tests such as the forced swimming test (FST) and the tail suspension test (TST) [34]. TREK-1 belongs to the family of two-pore-domain potassium channels, which play important roles in neuroprotectioTablen, pain, analgesia, and depression [35][36][37]. As one of the first functions identified for Sortilin/NTSR3 was to address numerous proteins from the intracellular compartments to the plasma membrane or lysosomes [38][39], it was crucial to determine whether sortilin and TREK-1 were associated, and if they were, how sortilin was involved in the sorting of TREK-1. This hypothesis was firstly confirmed by demonstrating that the TREK-1 channel expression at the plasma membrane of COS-7 cells was strongly enhanced by the co-expression of Sortilin/NTSR3 [34], and secondly, by observing that the brain of sort1−/− mice had an altered TREK-1 function due to a dramatically lower expression of the channel at the plasma membrane of neurons [40]. Therefore, the regulation of the functional expression of TREK-1 could be of importance in a series of human cancers, including prostate [41][42] and endometrial [43] cancers, in which the overexpression of the potassium channel appears to be responsible for tumor development. The ability of spadin, a shorter analog of the pro-peptide (PE) released from the maturation of Sortilin/NTSR3 to block the activity of TREK1, indicates that it could possibly be used as a tool to decrease the proliferation of cancer cells.

This entry is adapted from the peer-reviewed paper 10.3390/ijms231911888

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