*3.3. Regulation of Protein Degradation*

The mechanisms of regulated degradation of the TCTP protein were reviewed in detail in [34] and were also covered in a review article in this Special Issue in *Cells* [51]. Here, we will just briefly summarise the few cases known today (Table 2). Earlier papers reported the stabilisation of the TCTP (fortilin) protein by the anti-apoptotic protein Mcl-1 [71] and by the small heat shock protein Hsp27 [72], thereby preventing TCTP degradation in specific conditions, such as prostate cancer [73]. It was also

shown that the antimalarial drug dihydroartemisinin (DHA), which is also used as an anti-cancer agent, binds TCTP and targets it for the ubiquitin-proteasome degradation pathway [74]. Another paper reported at the same time that, during mitosis and during meiotic exit, TCTP is partially degraded [75]. Our recent study characterised the pathway for specific TCTP degradation as acetylation-dependent chaperone-mediated autophagy (CMA), which eventually leads to lysosomal degradation of the protein and involves the proteins Hsc70 and LAMP-2A [76].

### **4. Disease Processes Involving Dysregulation of TCTP**

### *4.1. Mechanisms of Cancer Promotion by TCTP*

When we initially discovered that TCTP was overexpressed in a majority of tumors [77], it became clear that its inhibition could potentially result in decreased tumorigenicity. This reprogramming/reversion of cancer cells was found in breast tumor cells, lung cancer, colon carcinoma, and melanoma [77–79]. Typically, decreasing TCTP levels led to a restructuring of the tumor architecture, where breast cancer cells formed again ductal-like structures, reminiscent of normal tissues [77]. This suggested that TCTP regulates a series of oncogenic and tumor suppressor pathways and that its silencing suppresses malignant growth [79]. These aspects of TCTP have been extensively reviewed previously [78]. Since then, it has been confirmed that TCTP is overexpressed in most tumors including clinical samples [5]. The mechanistic way, through which TCTP exerts its action, is most probably by interacting with hundreds of proteins, influencing their function, in different ways [78,80].

As TCTP is a highly conserved protein expressed in all eukaryotic organisms, some of the crucial knowledge has been generated through work in *Drosophila* by K.W. Choi and colleagues. They found that TCTP regulates the TOR pathway through interaction with the dRheb GTPase [81]. Reduction of TCTP levels led to a reduced cell number, along with a smaller organ size. Given the importance of the mTOR pathway in cancer, these results provided one of the links between TCTP and tumorigenicity. These findings in *Drosophila* were further extended by showing the implication of 14-3-3 proteins; their interaction with TCTP and Rheb is necessary for the regulation of TOR [82]. The same group demonstrated that TCTP also regulates genome stability through modulation of dATM, one of the molecular complexes implicated in the DNA damage response [83]. In addition, they showed that TCTP binds to Brahma and negatively regulates its activity [84]. Brahma is the catalytic subunit of the SW1/SNF complex, which modulates chromatin and DNA repair, and which is mutated in more than 20% of human cancers [85]. In an elegant study from Azzam's group [86], it was shown that TCTP forms complexes with ATM and γ-H2AX, suggesting a role in DNA damage and repair following exposure of human cells to low-dose γ-rays. Altogether, these data provide genetic evidence in support of the interaction of TCTP with the TOR-dependent oncogenic pathway, and of its role in maintaining genome stability.

Several studies link TCTP to epithelial to mesenchymal transition (EMT). This biological process is fundamental in early stages of embryonic development, such as the formation of the body plan during gastrulation [87]. Significant knowledge has been generated on the regulation of EMT in different developmental contexts, and evidence for its implication in cancer and metastasis is rapidly progressing [87], but still awaiting confirmation by clinical data. There has also been substantial progress in deciphering the molecular pathways involved in EMT and cancer [87]. The plasticity of cancer cells is key in EMT, especially in reprogramming somatic cells to 'stemness'. Since TCTP plays an important role in tumor reprogramming, it was speculated that it might be part of the EMT induction process. In an interesting study [88], LLC-PK1 kidney epithelial cells were used to test in vitro the potential role of TCTP for the regulation of EMT. The overexpression of TCTP enhanced migration with a reduced expression of E-cadherin and increased expression of transcription factors repressing E-cadherin, such as ZEB1, slug, or twist. Depletion of TCTP reversed the EMT phenotype and suppressed migration. Results suggested that TCTP acts through metalloproteinases, specifically MMP-9, to facilitate cell invasion [88]. In addition, the study provided data indicating

that TCTP regulates pulmonary metastasis of melanoma. Another study [89], this time using A549 lung adenocarcinoma cells, suggested that TCTP was a target of TGF-β1 and necessary for EMT and cytoskeleton reorganisation. Using the same cell line, it was further confirmed that TCTP promotes EMT and tumorigenicity [90,91] by influencing the expression levels of key transcription factors (ZEB1 and alpha SMA), and of miR-200a, miR-141, and miR-424 [90]. A very recent study also showed that TCTP is a key mediator in the induction of EMT by cigarette smoke carcinogens in lung epithelial and non-small-cell lung cancer cells [70]. Overall, these recent reports on the essential role of TCTP in EMT generated substantial support for our original observation [77] that decreased cellular levels of TCTP in cancer cells inhibit tumorigenicity by interfering with migration and invasion.

The capacity of TCTP to regulate diverse pathways, from mTOR and genome integrity to cell migration, has one common feature: cell survival. This is probably best reflected in tumor biology by reshaping tumor organisation and stemness [78], the latter being a conserved TCTP function from developmental biology [92] and tissue maintenance [15] to cancer stem cells [40]. A key aspect of TCTP function as a survival factor is its interaction with both, the anti-apoptotic and pro-apoptotic machinery. It was recently discovered that TCTP contains a BH3-domain, which is a common feature in the Bcl2 family regulators of apoptosis [93]. The crystal structure of a complex of Bcl-xL with a TCTP11–31 deletion variant revealed that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL. Most importantly, TCTP potentiates the anti-apoptotic function of Bcl-xL, which is a unique feature [93]. On the other hand, TCTP has been shown to interact with the p53 tumor suppressor [40,78]. TCTP and p53 are involved in a negative reciprocal feedback loop, in which p53 represses the transcription of TCTP and the latter promotes the degradation of p53 [40]. This negative feedback loop is important for cell fate.

A study in hepatocellular cancer revealed that TCTP overexpression in this cancer induces mitotic defects [41], which is in line with other data showing that TCTP is involved in stabilising the mitotic spindle and is important for orderly mitotic/meiotic progression [94]. Our data also indicate that normal breast stem cells and cancer stem cells have an increased expression of TCTP [40]. As in developmental biology, stemness has to be protected from cell death. Importantly, we observed that breast cancer patients expressing high levels of TCTP in their tumors have a high grade, aggressive malignancy with a poor prognosis [40]. Similar observations have since been published for hepatocellular cancer (HCC) [41], colorectal cancer (CRC) [95], as well as for lung [90], breast [96], and gallbladder [97] cancer. Such elevated levels of TCTP in aggressive malignant disease may contribute to ye<sup>t</sup> another problem, i.e., the enhanced resistance to various treatment modalities. For breast [98], lung [99], and colorectal [100] cancer cells, it has been shown that increased TCTP levels contribute to an increase in radio- and/or chemo-resistance.

Since the beginning of our research, it was obvious that TCTP is a potentially important target for cancer treatment. Sertraline and thioridazine are able to neutralise TCTP and hence, decrease its expression, which leads to apoptosis of cancer cells [79]. Sertraline is now being tested in phase I/II clinical studies [101]. Recently, it was demonstrated that TCTP is a promising target in melanoma, also using sertraline as a drug [102]. In our initial experiments targeting TCTP, we employed anti-histaminic drugs [79]. It has since been shown that anti-histaminic drugs interact with TCTP, and they were suggested as an approach to differentiation therapy [103]. The finding that the anti-malarial drug dihydroartemisinin (DHA), which also has anti-cancer activity [104], binds to and promotes the degradation of human TCTP (fortilin) [74], prompted initial studies to test the use of artemisinin derivatives against TCTP, either alone in gallbladder cancer [97] or in combination therapy against breast cancer [96,105]. Yet another approach, i.e., TCTP-antisense oligonucleotides, is being explored as a strategy against prostate cancer, and this seems to show some promise [106].

### *4.2. TCTP in Cardiovascular and Metabolic Diseases*

Apart from cancer, TCTP dysregulation is also involved in a range of other disease processes (reviewed in [5,107]; for a compilation see Table 3 below). In a recent study, Cai and colleagues reported that TCTP plays a pivotal role in cardiomyocyte survival [46]. Downregulation of TCTP in cardiomyocytes induced cell death with apoptotic and autophagic features. Conversely, cardiomyocyte-specific overexpression of TCTP in mice resulted in decreased susceptibility to doxorubicin-induced cardiac dysfunction. In this case, TCTP acted as a disease-preventing factor. However, in the case of atherosclerosis, two earlier publications demonstrated that TCTP promotes the disease, albeit through two quite di fferent mechanisms. Pinkaew et al. studied the e ffect of heterozygous deficiency of TCTP (fortilin) in mice, in a background of hypercholesteraemia [108]. They arrived at the conclusion that TCTP prevents apoptosis and therefore the reduction of macrophages, which are main contributors to the development of atherosclerosis. Kyunglim Lee's group had previously shown that TCTP overexpression resulted in the inhibition of the Na,K-ATPase, and in the development of systemic hypertension in mice as early as six weeks after birth [109]. In a later paper, they demonstrated that, in ApoE-knockout mice, TCTP overexpression and consequently hypertension accelerated the development of atherosclerotic lesions caused by high-fat and high-cholesterol diet [110].

In a recent review article, K. Lee's group summarised the consequences of the inhibition of the Na,K-ATPase by TCTP. Apart from the development of systemic hypertension, there are additional clinically relevant consequences, i.e., an increased tendency to form lens cataracts in mice and the activation of tumorigenic signalling pathways [111]. Another cardiovascular disease, in which a direct involvement of TCTP has been documented, is pulmonary arterial hypertension (PAH), a lethal disease caused by excessive proliferation of pulmonary endothelial cells (ECs). Using a proteomic approach, Lavoie et al. identified TCTP as one of 22 proteins that are significantly altered in the blood outgrowth ECs (BOECs) of patients with hereditary PAH [112]. Immunostaining revealed a marked increase in TCTP levels particularly in complex lesions of lungs from PAH patients, as well as in a rat model of severe and irreversible PAH. TCTP-knockdown led to an increase in apoptosis and to a reduction of the hyperproliferative phenotype in BOECs from PAH patients. In a recent follow-up study, the group also observed that silencing of TCTP in such BOECs resulted in significant alterations of the morphology and the migration behaviour of these cells [113]. They also demonstrated that TCTP can be transferred from ECs to pulmonary artery smooth muscle cells via exosomes, and in this way, the protein transfers the proliferative phenotype and apoptosis resistance onto neighbouring cell layers, thus playing a core role in the pathobiology of the disease.

Since TCTP is a cytoprotective protein involved in maintaining the cellular homeostasis of specialised cell types, its dysregulation may also play a role in metabolic disease states, such as diabetes. We found that in pancreatic β-cells, TCTP levels are regulated by glucose and that TCTP participates in protecting these cells against apoptosis induced by fatty acids [114]. In a more detailed study, Tsai et al. investigated the adaptation of β-cell mass in mice, both during early development and in insulin-resistant states, and found that TCTP expression correlated with phases of β-cell proliferation and mass expansion [115]. Specific knockout of TCTP in β-cells resulted in decreased growth signalling, β-cell proliferation and mass development, eventually leading to reduced insulin production and hyperglycemia. These observations received additional support by the recent finding that in mouse pancreatic islets, TCTP expression is regulated by the insulin-response element binding protein-1 (IRE-BP1) [31].

One of the pathologies associated with diabetes is nephrotic podocyte hypertrophy, which leads to an increase of glomeruli and to proteinuria. Kim et al. used a mouse model to study the involvement of TCTP in the development of this condition [116]. They found that TCTP knockdown reduced the activation of mTORC1 downstream e ffectors, the overproduction of cyclin-dependent kinases, as well as the size of podocytes and the glomeruli. In *db*/*db* mice, knockdown of TCTP prevented the development of diabetic nephropathy. Another type of hypertrophy, not related to diabetes, where TCTP overexpression was shown to be involved, is skeletal muscle hypertrophy. An in-depth study by Goodman and colleagues elucidated several aspects of TCTP's role and regulation in skeletal muscle, using a range of mouse models [61]. They showed that TCTP is translationally up-regulated via the mTORC1 signalling pathway in skeletal muscle, under both hypertrophic and atrophic conditions. TCTP was su fficient to induce muscle fiber hypertrophy, and the protein may also be involved in inhibiting protein degradation.

Taken together, the various examples of diseases involving TCTP dysregulation (Table 3 below) show that, depending on the specific setting or cell type, the role of TCTP can be either in preventing or in promoting disease processes. TCTP as a cytoprotective protein may be involved in preventing the development of disease, as we see in model studies for cardiomyocytes [46] or pancreatic β-cells [115]. The properties of TCTP as a growth promoting and anti-apoptotic protein can also exacerbate disease processes, e.g., by driving cells into a hyperproliferative state, as in cancer (Section 4.1), PAH [112,113], diabetic nephropathy [116], or muscle hypertrophy [61], by preventing apoptosis, as shown for atherosclerosis [108] and PAH [112,113] or by inhibiting Na,K-ATPase, which leads to hypertension [111].

### *4.3. Allergic and Immune Disorders—TCTP as Histamine Releasing Factor*

The discovery of the activity of TCTP as a histamine-releasing factor (HRF) has spurred a considerable research e ffort aimed at delineating its specific role in triggering cellular responses associated with allergic and other immune disorders. Various aspects of this work were reviewed on earlier occasions [5,57,117,118]; however, since then considerable progress has been made in understanding certain details of the role of TCTP/HRF in the development of various allergic disorders. These recent developments are summarised in a review by T. Kawakami and colleagues in this Special Issue in *Cells* [56], and for further details, the reader is referred to this article. Here, we will just mention a few core points: 1. After some initial controversy about the IgE-binding activity of HRF, it has been clarified that HRF binds to a subset of IgE molecules, in this way triggering histamine release [117]. 2. Several lines of evidence show that it is the dimer of TCTP/HRF, which is the active form for its extracellular activity [118]. The structure of the TCTP dimer has been solved and a model for dimerisation and the IgE binding site was derived from this [119]. A more recent paper also implied the flexible loop of the TCTP/HRF dimer in the activity of the molecule in triggering cytokine release from BEAS2B cells [120]. 3. The role and involvement of HRF in the following allergic disease states has been elucidated, at least in part, either in mouse models or in limited investigations in patients (Table 3): Asthma (reviewed in [56,117,118]), atopic dermatitis [121], food allergy [122,123], and chronic urticaria [124,125]. 4. A number of peptide and other inhibitors of TCTP/HRF showed some promise in alleviating symptoms elicited by this molecule in the context of allergic diseases [56].


**Table 3.** Dysregulation of TCTP in disease processes.

Another paper published in this Special Issue of *Cells* reported quite a di fferent aspect of TCTP's involvement in inflammatory responses. The group of A. Sen ff-Ribeiro in Brazil had previously found that TCTP is part of the venom from the Brown Spider *Loxosceles intermedia*. They have now shown that TCTP is a synergistic factor contributing to the exacerbated inflammatory response elicited by the main toxin of the venom [126].
