TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling.

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

Abnormal erythroid maturation leads to microcytic anemia in the TSAP6/Steap3 null mouse model.

Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3(-/-) animals. In addition, progression from the proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice.

Lessons from tumor reversion for cancer treatment.

PURPOSE OF REVIEW: Tumor reversion is the biological process by which highly tumorigenic cells lose at great extent or entirely their malignant phenotype. The purpose of our research is to understand the molecular program of tumor reversion and its clinical application. We first established biological models of reversion, which was done by deriving revertant cells from different tumors. Secondly, the molecular program that could override the malignant phenotype was assessed. Differential gene-expression profiling showed that at least 300 genes are implicated in this reversion process such as SIAH-1, PS1, TSAP6, and, most importantly, translationally controlled tumor protein (TPT1/TCTP). Decreasing TPT1/TCTP is key in reprogramming malignant cells, including cancer stem cells. RECENT FINDINGS: Recent findings indicate that TPT1/TCTP regulates the P53-MDM2-Numb axis. Notably, TPT1/TCTP and p53 are implicated in a reciprocal negative-feedback loop. TPT1/TCTP is a highly significant prognostic factor in breast cancer. Sertraline and thioridazine interfere with this repressive feedback by targeting directly TPT1/TCTP and inhibiting its binding to MDM2, restoring wildtype p53 function. Combining sertraline with classical drugs such as Ara-C in acute myeloid leukemia may be also beneficial. SUMMARY: In this review, we discuss some of these reversion pathways and how this approach could open a new route to cancer treatment.

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene.

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the "normal" allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother.

Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila.

Regulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.

Dysregulation of TCTP in Biological Processes and Diseases.

Translationally controlled tumor protein (TCTP), also called histamine releasing factor (HRF) or fortilin, is a multifunctional protein present in almost all eukaryotic organisms. TCTP is involved in a range of basic cell biological processes, such as promotion of growth and development, or cellular defense in response to biological stresses. Cellular TCTP levels are highly regulated in response to a variety of physiological signals, and regulatory mechanism at various levels have been elucidated. Given the importance of TCTP in maintaining cellular homeostasis, it is not surprising that dysregulation of this protein is associated with a range of disease processes. Here, we review recent progress that has been made in the characterisation of the basic biological functions of TCTP, in the description of mechanisms involved in regulating its cellular levels and in the understanding of dysregulation of TCTP, as it occurs in disease processes such as cancer.

Introduction: How We Encountered TCTP and Our Purpose in Studying It.

In this brief introduction, we describe our encounter with TCTP. Back in 2000, we discovered TCTP in two quite different ways: first, we looked at protein partners of TSAP6 and one of them was TCTP. Then, in collaboration with Sidney Brenner, we performed a high-throughput differential screening comparing the parental cancer cells with revertants. The results indicated that TCTP was of the most differentially expressed genes. These two approaches were carried out only months apart. They guided our research and led to the discoveries of drugs that inhibit the function of TCTP. Much of the preclinical data on sertraline as an inhibitor of TCTP in cancer were obtained with Judith Karp at Johns Hopkins. This drug is now given in combination with Ara-C to patients in a phase I clinical trial for Acute Myeloid Leukemia. We will here detail how all this happened in our lab while working around one central project: tumor reversion.

Targeting TCTP with Sertraline and Thioridazine in Cancer Treatment.

We have initially demonstrated in knocking down experiments that decreasing TCTP in cancer cells leads in some tissues to cell death while in others to a complete reorganization of the tumor into architectural structures reminiscent of normal ones. Based on these experiments and a series of other findings confirming the key role of TCTP in cancer, it became important to find pharmacological compounds to inhibit its function, and this became for us a priority. In the present text, we explain in detail the experiments that were performed and the perspectives of sertraline in cancer treatment, as this became today a reality with a clinical study that started in collaboration with Columbia University and Johns Hopkins University.

Acetylation of translationally controlled tumor protein promotes its degradation through chaperone-mediated autophagy.

Translationally controlled tumor protein (Tpt1/TCTP) is a multi-functional cytosolic protein whose cellular levels are finely tuned. TCTP regulates protein behavior by favoring stabilization of protein partners or on the contrary by promoting degradation of others. TCTP has been shown to be transcriptionally and translationally regulated, but much less is known about its degradation process. In this study, we present evidence that chaperone-mediated autophagy (CMA) contributes to TCTP regulation. CMA allows lysosomal degradation of specific cytosolic proteins on a molecule-by-molecule basis. It contributes to cellular homeostasis especially by acting as a quality control for cytosolic proteins in response to stress and as a way of regulating the level of specific proteins. Using a variety of approaches, we show that CMA degradation of TCTP is Hsc70 and LAMP-2A dependent. Our data indicate that (i) TCTP directly interacts with Hsc70; (ii) silencing LAMP-2A in MEFs using siRNA leads to inhibition of TCTP downregulation; (iii) TCTP is relocalized from a diffuse cytosolic pattern to a punctate lysosomal pattern when CMA is upregulated; (iv) TCTP is degraded in vitro by purified lysosomes. Importantly, using lysine-mutated forms of TCTP, we show that acetylation of Lysine 19 generates a KFERQ-like motif and promotes binding to Hsc70, lysosome targeting and TCTP degradation by CMA. Altogether these results indicate that TCTP is degraded by chaperone-mediated autophagy in an acetylation dependent manner.

TCTP contains a BH3-like domain, which instead of inhibiting, activates Bcl-xL.

Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members' structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11-31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP(+/-) mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.

TPT1/ TCTP-regulated pathways in phenotypic reprogramming.

Evolutionary conserved and pleiotropic, the TPT1/TCTP gene (translationally controlled tumor protein, also called HRF, fortilin), encodes a highly structured mRNA shielded by ribonucleoproteins and closely resembling viral particles. This mRNA activates, as do viruses, protein kinase R (PKR). The TPT1/TCTP protein is structurally similar to mRNA-helicases and MSS4. TPT1/TCTP has recently been identified as a prognostic factor in breast cancer and a critical regulator of the tumor suppressor p53 and of the cancer stem cell (SC) compartment. Emerging evidence indicates that TPT1/TCTP is key to phenotypic reprogramming, as shown in the process of tumor reversion and possibly in pluripotency. We provide here an overview of these diverse functions of TPT1/TCTP.

Reciprocal repression between P53 and TCTP.

Screening for genes that reprogram cancer cells for the tumor reversion switch identified TCTP (encoding translationally controlled tumor protein) as a crucial regulator of apoptosis. Here we report a negative feedback loop between P53 and TCTP. TCTP promotes P53 degradation by competing with NUMB for binding to P53-MDM2-containing complexes. TCTP inhibits MDM2 auto-ubiquitination and promotes MDM2-mediated ubiquitination and degradation of P53. Notably, Tctp haploinsufficient mice are sensitized to P53-dependent apoptosis. In addition, P53 directly represses TCTP transcription. In 508 breast cancers, high-TCTP status associates with poorly differentiated, aggressive G3-grade tumors, predicting poor prognosis (P < 0.0005). Tctp knockdown in primary mammary tumor cells from ErbB2 transgenic mice results in increased P53 expression and a decreased number of stem-like cancer cells. The pharmacological compounds sertraline and thioridazine increase the amount of P53 by neutralizing TCTP's action on the MDM2-P53 axis. This study links TCTP and P53 in a previously unidentified regulatory circuitry that may underlie the relevance of TCTP in cancer.

The molecular programme of tumour reversion: the steps beyond malignant transformation.

How cells become malignant has preoccupied scientists for over a century. However, the converse question is also valid: are tumour cells capable of reverting from their malignant state? Askanazy's studies in 1907 indicated that teratoma cells could differentiate into normal somatic tissues and current evidence indicates that some tumour cells have acquired the molecular circuitry that results in the negation of chromosomal instability, translocations, oncogene activation and loss of tumour suppressor genes. Studying these extremely rare events of tumour reversion and deciphering these pathways, which involve SIAH1, presenilin 1, TSAP6 and translationally controlled tumour protein (TCTP), could lead to new avenues in cancer treatment.

Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1.

Tumor reversion is the process by which some cancer cells lose their malignant phenotype. This study was aimed at defining some of the molecular and phenotypic properties of this process. Biological models of tumor reversion were isolated from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent. Differential gene expression analysis was performed between the parental malignant cells and their revertants or alternatively between these parental cells and their SIAH-1 transfectant counterparts. These SIAH-1 transfectants have a suppressed malignant phenotype and were used as a control for a viral-free system. Two hundred sixty-three genes were found to be either activated or inhibited during the reversion process, as confirmed by Northern blot analysis or quantitative PCR. Of these, 32% were differentially expressed in all systems, irrespective of whether parvovirus-selected, SIAH-1 overexpressing, or p53 mutant or wild-type cell lines were used, suggesting the existence of a universal mechanism underlying tumor reversion. Translationally Controlled Tumor Protein (tpt1/TCTP) has the strongest differential expression, down-regulated in the reversion of U937- and SIAH-1-overexpressing cells. Inhibition of TCTP expression by anti-sense cDNA or small interfering RNA molecules results in suppression of the malignant phenotype and in cellular reorganization, similar to the effect of SIAH-1. Hence, tumor reversion can be defined at the molecular level, not just as the reversal of malignant transformation, but as a biological process in its own right involving a cellular reprogramming mechanism, overriding genetic changes in cancer, by triggering an alternative pathway leading to suppression of tumorigenicity.

Genomic organization and expression of mouse Tpt1 gene.

The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny. TCTP is implicated in cell growth, acute allergic response, and apoptosis. In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome. In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes. Only three of the seven genes can produce a protein of the expected molecular weight. We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity. We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.

TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine-releasing factor via a nonclassical pathway.

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.

Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons.

Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-alpha, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-alpha diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms.