Hallmarks of tumor environment: role of acidity in melanoma aggressiveness and metastatic dissemination

Tumor microenvironment is a complex system consisting of several important components in constant, reciprocal interactions. Aspects of tumorigenicity such as tumor growth, migration, invasion and metastasis formation, as well as the secretion of soluble factors and the abnormal structure and function of tumor blood vessels depend on these interactions. In this way microenvironment acts as a coconspirator during carcinogenesis and neoplastic progression. Tumor microenvironment is often characterized by an acidic pH due to abnormal vascularisation, reduced lymphatic network, uncontrolled cell growth frequently associated with hypoxia, and extracellular accumulation of glucose metabolites even in the presence of an adequate oxygen level (“the Warburg effect”). Evidence is accumulating that acidity participates to tumor progression and is associated with poor prognosis. Since melanoma, as most solid tumors, often shows extracellular acidosis, in this study we investigate whether acidity is able to induce in melanoma cells different aspects of malignancy such as the epithelial-to-mesenchymal transition (EMT) or vascular endothelial growth factor (VEGF) C expression and whether this is associated with a change in the metabolic profile of acidic cells. In parallel, the effects of acidity on melanoma cell/host cell interactions have been investigated. It is known that during melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an EMT process associated with the acquisition of an invasive and pro-metastatic phenotype. A375M6 melanoma cells were exposed to an acidic extracellular environment (pH 6.6-6.8) and tested for markers and biological properties of EMT. We found that acidic cells express a significant up-regulation of mesenchymal markers (N-cadherin, Vimentin), typical transcription factors (Twist, NF-kB) and a reduction in E-cadherin expression. Acidic cells, also, express an increased MMP-9 activity and invasiveness through Matrigel filters, inhibited by Ilomastat, a MMP inhibitor. Invasiveness and N-cadherin up-regulation in acidic cells were NF-B dependent, suggesting a pivotal role of NF-B transcription factor in acidity-promoted EMT in melanoma cells. We also demonstrated that all these changes are transient and after seven days in standard medium, cells recover their original phenotype. When we injected acidic EMT cells into blood stream of immunodeficient animals, they gave a number of lung micrometastases similar or even lower than non-acidic cells. Acidic cells were found more resistant to an apoptotic stimulus, such as H2O2, whereas they showed a reduction in cell proliferation rate and in cloning efficiency, changes that might interfere with the organ colonization ability. To understand the effective role exerted by acidic cells during melanoma dissemination, we investigated in vitro invasiveness and lung colonization of a mixed population of acidic and non-acidic tumor cells. We found that acidic cells promote in vitro invasiveness of non-acidic cells and this cooperation leads to a migration rate higher than that of acidic cells. Moreover, acidic cells cooperate for a better lung colonization of non-acidic cells that represent the greater part of cells participating to lung micrometastases. A critical early step in the metastatic pathway is the invasion of cancer cells not only into surrounding stroma but also into peritumoral lymphatics. There is clear evidence that melanoma cells can induce lymphangiogenesis and this phenomenon is correlated with lymph node metastases. VEGF-C represents the most potent and well-recognized lymphangiogenic growth factor secreted in tumor milieu by melanoma cells and tumor-associated macrophages, however the mechanism underlying VEGF-C secretion is not completely understood. We demonstrated that an acidic extracellular pH promotes the expression of VEGF-C both in A375P melanoma cells and in melanoma cells isolated from a human spontaneous metastatic lesion, through the NF-kB transcription factor. We also demonstrated that esomeprazole, a proton pump inhibitor which requires acidosis to be activated, is able to prevent VEGF-C expression in acidic melanoma cells by interfering with NF-kB activation. Furthermore, we showed that esomeprazole abrogates the enhanced VEGF-C expression in tumor cells grown in acidic medium and stimulated by IL-1. On the whole, our results reveal that acidity may be considered a strong promoter of VEGF-C expression in melanoma cells and provides a new pharmacological target to limit the development of tumor lymphangiogenesis. We also investigated if acidity could modify the metabolic program of melanoma cells. It is known that acidity in tumor microenvironment is a direct consequence of the metabolism of cancer cells, which use glycolysis even in the areas of ample oxygen supply, but, on the contrary, the effect of the acidity on cell metabolism is less known. Our experiments, conducted using A375M6 melanoma cells, indicate that acidity reduces the glycolytic metabolism, as evidenced by the decreased expression of glucose transporters, reduced lactate efflux and increased lactate uptake, all inverse features compared to those detected in A375M6 grown under hypoxic condition. We also found that the pyruvate kinase M2 (PKM2), a key player in the Warburg effect on cancer cells, is reduced in acidic cells. In order to inhibit the oxidative phosphorylation of acidic melanoma cells we used metformin, the most widely prescribed oral hypoglycemic agent which acts through inhibition of mitochondrial respiration and which has recently received increased attention for its potential antitumorigenic effects. The enhanced invasiveness acquired by melanoma cells exposed to an acidic culture medium was reduced by metformin treatment. Recently, lactate, the end-product of glycolysis, was shown to be associated with metastasis, tumor recurrence, and poor survival, and identified as the keystone of an exquisite symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We hypothesized that tumor microenvironment might facilitate a symbiosis between non-acidic/glycolytic cells that use glucose and produce lactate, and acidic cells that capture the lactate produced by non-acidic cells and oxidize it to produce energy to increase their aggressiveness. We shown that lactate, used by acidic cells for respiration, does not induce an increase of cell proliferation, but stimulates motility of acidic cells, maintaining their high invasiveness and the EMT profile. Lactate also acts on non-acidic cells increasing their motility, invasiveness, and inducing the EMT. We manipulated lactate uptake using the α-cyano-4-hydroxycinnamate (CHC), a drug known to reversibly inhibit MCT1. We found that 10 mM CHC kills acidic cells,0 while has only an antiproliferative effect on non-acid cells, suggesting that glycolytic cells can better tolerate the effects of MCT1 inhibition. The use of lower doses of CHC shows that MCT1 inhibition represses not only the lactate-dependent stimulation of motility and invasiveness, but also the invasiveness of untreated acidic cells. Medium conditioned by control (non-acidic) cells, probably containing lactate, is able to stimulate the motility of acidic cells and CHC blocks this effect, confirming the hypothesis that the lactate produced by non-acidic cells may stimulate acidic cells and that this cross-talk could be inhibited by MCT1 inhibitor. In addition to observing the direct effect of the acidic environment on cancer cells, we also studied how acidity influences the interactions between mesenchymal stem cells (MSC) and tumor cells. It is well known that tumors are composed of malignant tumor cells and non-malignant benign cells, including endothelial cells, myofibroblasts, macrophages, lymphocytes, dendritic cells, and MSC. Large number of MSC are recruited into the stroma of developing tumors and recent evidence suggests that these cells play a role in facilitating cancer progression influencing the behavior and aggressiveness of tumor cells. Our experiments, conducted using A375M6 human melanoma cells and human MSC derived from umbilical cord, demonstrated that condition medium collected from acidic MSC promotes the migratory and invasive abilities of melanoma cells as well as the expression of EMT related genes in a way much more evident compared to the effect of the media conditioned by non-acidic MSC. Whereas the medium conditioned by non-acidic MSC elicits a marked increase in tumor cell proliferation, the medium conditioned by acidic MSC has no significant effect on tumor cell growth; both types of conditioned media (from acidic or non-acidic MSC) induce a mesenchymal morphology of melanoma cells, but neither of them is able to induce resistance to apoptosis. A375M6 cells grown in medium conditioned by acidic MSC show also stimulated expression of TGF-b and TGF-b receptors. We evaluated the expression of several cytokines in MSC grown in acidic medium and we found that the only factor with enhanced expression in acidic MSC was the TGF-bthat we assumed to be the soluble factor secreted by acidic MSC and involved in the stimulated aggressive phenotype of melanoma cells. In effect, we demonstrated that TGF-bis able to increase the invasiveness of melanoma cells and to induce the expression of TGF-b and of EMT markers. In order to inhibit the effect of acidity on MSC and consequently on tumor cells grown in conditioned media, we used esomeprazole. Our experiments indicate that esomeprazole prevents the increase of TGF-b expression and of MMP2 activity in acidic MSC. Moreover, A375M6 cells grown in media conditioned by acidic MSC treated by esomeprazole shown invasiveness and motility comparable with that of cells grown in media conditioned by non-acidic MSC, much lower that invasiveness and motility induced by media conditioned by untreated acidic MSC. Esomeprazole was also able to block the stimulation of EMT in cancer cells grown in conditioned media. Probably, acidic microenvironment activates MSC that, through the TGF-b secretion, stimulates aggressiveness of the neighboring tumor cells. Esomeprazole, counteracting the effect of acidity on MSC, prevents the release of TGF-b and the consequent activation of tumor cells. This study also provides evidence that acidity-induced TGF-b secreted by MSC is able to promote tumor growth in vivo. Subcutaneous co-injection of melanoma cells and acidic/non-acidic MSC in immunodeficient mice resulted in more rapid tumor growth compared with injection with tumor cells alone, but acidic MSC maximally promoted growth of melanoma tumors. When we treated mice with esomeprazole, tumor growth was attenuated, especially in mice injected with acidic MSC. Our findings are in agreement with a large number of results indicating that MSC promote tumor growth in vivo; furthermore, our study explores the relationship between MSC and tumor cells in an acidic microenvironment, proposing esomeprazole, which make acidic MSC incapable of communicating with tumor cells as therapeutic strategy against cancer. In conclusion, acidosis represents a key factor contributing to a more aggressive phenotype of melanoma cells and strategies directed to target acidic environment and/or the intrinsic cell metabolism of low pH-adapted cells are a new and prominent issue in tumor therapy.