A novel view on LMW-PTP involvement in tumorigenesis: from apoptosis resistance to metabolic reprogramming

Protein tyrosine phosphorylation in eukaryotes is a key mechanism for cellular control, since it is involved in several processes, such as cellular metabolism, proliferation, differentiation and oncogenic transformation. A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs) (Alonso et al., 2004). The PTP superfamily comprises almost 70 enzymes that, despite very limited sequence similarity, share a common CX5R active-site motif and an identical catalytic mechanism. LMW-PTPs are a group of cytosolic enzymes of 18 kDa that are widely expressed in different tissues. They are represented by two most abundant isoforms (arised from mutually exclusive alternative splicing), named fast and slow, according to their electrophoretic mobility (Tabernero et al., 2008; Wo et al., 1992; Dissing et al., 1991; Xing et al., 2007). LMW-PTP interacts with several receptor tyrosine kinases and docking proteins that may be involved in cancer progression, but the identification of functionally relevant interactors is not yet conclusive. Several works have shown how LMW-PTP overexpression is associated with human tumorigenesis, especially in breast, colon and kidney. In fact, results obtained with a wide array of human carcinomas indicate a significant increase in the expression of LMW-PTP in tumor tissue and a correlation between higher expression of LMW-PTP on one hand and worse prognosis and reduced survival on the other (Malentacchi et al., 2005). LMW-PTP acts also as a positive regulator of tumor onset and growth in in vivo animal models. (Chiarugi et al., 2004). Moreover there are many findings that LMW-PTP plays a key role in chemoresistance: Ferreira et al., demonstrated how the phosphatase overexpression induce resistance towards vincristine and imatinib, in a leukemia cell lines (Ferreira et al., 2012). Our studies were conducted on a Melanoma cell line, A375, to study in depth the role of LMW-PTP in skin tumor onset, in order to elucidate the molecular mechanism and pathways in which this enzyme is involved and also in the view of identifying new possible therapeutic targets. Using transient silencing technique, we tested the apoptotic response of A375 cells: citofluorimetric analysis showed that upon phosphatase silencing, treatment with 5FU increase cell mortality up to 50%. In agreement with these results, western blot analysis of apoptotic markers (Caspase3, Bcl2 and Bim) confirmed that LMW-PTP silencing sensitize cancer cells towards chemotherapic drugs. Looking for a link between LMW-PTP and apoptosis, we speculate that the phosphatase exerts its action through Cav1-Bcl2 pathways. In fact the phosphorylation on Tyr14 of Cav-1 leads to Bcl2 degradation, increasing apoptosis. Our data demonstrate how LMW-PTP knocking-down lead to Cav-1 Tyr phosphorylation, and Bcl-2 down-regulation. Several studies have demonstrated how tumor cells can develop chemoresistance increasing the activity or the expression level of membrane transporters that actively expel chemotherapy drugs from inside. Our experiments suggest that LMW-PTP may confer resistance against anti-tumoral drugs, increasing the activity of some of these membrane transporters, thus limiting the toxic effect of chemotherapy. One of the most important treatment for Melanoma is Radiotherapy. Using therapeutic doses of radiation (2Gy), we demonstrated that silenced cells were more responsive to this treatment, with respect to control samples, confirming that LMW-PTP plays a key-role not only in chemio- but also in radio-resistance. Furthermore resistant cancer cells, usually, show common phenotypes: one of this features is self-renewal capability. Colony formation assay demonstrates that melanoma cells are able to reform new colonies, even when cells are exposed to a damage, such as 5FU treatment or 2Gy irradiation. When LMW-PTP is knocked-down and cells treated with 5FU or radiated, we observed no colony formation: we can assume that LMW-PTP silencing leads to the loss of some characteristics of self- renewal, fundamental for metastasis. LMW-PTP exerts its action also through some molecules implicated in cell migration and adhesion. Adhesion and Detachment assay showed that LMW-PTP silencing lead cells to be more adherent to a substrate. Accordingly Wound Healing Assay demonstrated that melanoma cells are able to migrate and refill the wound very quickly. We tested also the Invasiveness of A375 cells: silenced cells showed a decreased ability to invade, respect to untreated cells: in fact LMW-PTP down-regulation lead to a MMP-9 reduction. Considering the importance of LMW-PTP in tumor onset we investigate the possibility to inhibit this enzyme as a new therapeutic approach.. Previous work demonstrated that a natural compound, Morin, is an enzymatic inhibitor of LMW-PTP. Morin is not toxic for Melanoma cells, but its combination with 5FU, causes a strong increase of apoptotic cells. Interestingly, this sensitization is not reproducible in non tumoral cell line, such as C2C12 myoblast: this co-treatment could be specific for cancer cells. Furthermore our studies demonstrate that Morin doesn’t act only as an inhibitor of LMW-PTP: western blot analysis showed that the flavonol lead to phosphatase degradation, in a dose and time-dependent manner. This down-regulation may be due to different mechanism, but since this effect start 2h after Morin incubation, we hypothesized that a proteasome activation may be involved. Incubation with Morin together with a proteasome inhibitor (MG132), confirmed our hypothesis. The enhancement of chemotherapic action, obtained with Morin is reproducible even with Radiation therapy. A375 cells pre-treated with Morin and then radiated with 2Gy, decrease dramatically their viability. Moreover irradiation exposition didn’t influence self-renewal capability of Melanoma cells: on the contrary, Morin treatment before irradiation is able to affect the formation of new colonies after the treatment. LMW-PTP is involved in cell-adhesion, migration and invasion: in fact Morin can affect this markers. After Morin treatment Melanoma cells are less able to migrate and invade; furthermore cells increase the number of focal adhesions. To better understand the role of LMW-PTP in tumor onset, we looked for new substrates. To analyze this aspect we studied the phospho-proteomic profile of silenced Melanoma cells. Through these analysis we identified different proteins showing an higher levels of Tyr phosphorylation, upon LMW-PTP silencing. One of the new substrates identified with this experimental approach is Annexin-A1, a protein involved in apoptosis: this finding suggest a further possible link between LMW-PTP and apoptosis resistance, a link that will be further investigated. More interestingly, four of the proteins identified with the proteomic analysisbelong to glycolysis pathway, PKM2, α-Enolase, GAPDH and TIM. To confirm this results we performed immune and co-immunoprecipitations: these analyses confirmed that the mentioned enzymes get in contact with LMW-PTP and, presumably, are direct substrates of the phosphatase. It is well known that the “Warburg effect” causes alteration in cancer cell energetic metabolism, leading cells to consume large quantity of glucose, metabolizing it predominantly through glycolysis, and producing high level of lactate. Considering that four of these substrates are involved in glycolysis pathway, we tested some metabolic parameters. When LMW-PTP is silenced A375 cells consume less O2, and consequently their glucose up-take is higher, producing more lactate respect to controls. Pyruvate kinase controls the final and rate-limiting reaction of glycolysis: PKM2 undergoes conformational conversion between a tetrameric/full active and a dimeric/less active state. The conversion to a less active state, induced by Tyr phosphorylation, confers to PKM2 “non-metabolic” abilities. Indeed, PKM2 translocates into the nucleus and acts as a transcriptional co-activator of β-catenin and hypoxia-inducible factor 1α (HIF-1α), cooperating to control cell proliferation and glucose catabolism, respectively. Western Blot Analysis of the Glucose transporter GLUT-1 and Hesokinase II, confirmed our previous data. When LMW-PTP is down-regulated both proteins had an increased expression level, explaining, at least in part, the glycolytic metabolism showed by silenced cells. Moreover LMW-PTP influences not only the Tyr phosphorylation state of PKM2, but also its expression level: in fact when the phosphatase is down-regulated PKM2 protein level is higher. In conclusion, our results demonstrated that LMW-PTP plays a key role in chemo and radio-resistance acquisition of Melanoma cells: in fact when the phosphatase is knocked-down cells are more responsive to therapy. Considering that gene silencing cannot be used in patients, the discovery that Morin is able to reproduce the same phenotype, open new possibilities for therapies. Moreover LMW-PTP seems to influence metabolism of Melanoma cells, a parameter often deregulated in cancer cells. Further studies will be conducted to better characterize the role of this enzyme, and its role in the regulation of tumor metabolism.