Tumors are ecosystems which develop from stem cells endowed with unlimited self-renewal capability and genetic instability, under the effects of mutagenesis and natural selection imposed by environmental changes. Abnormal vascularization, 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 are all factors contributing to reduce pH in the extracellular space of tumors. Evidence is accumulating that acidity is associated with a poor prognosis and participates actively to tumor progression. This review addresses some of the most experimental evidences providing that acidity of tumor environment facilitates local invasiveness and metastatic dissemination, independently from hypoxia, with which acidity is often but not always associated. Clinical investigations have also shown that tumors with acidic environment are associated with resistance to chemotherapy and radiation-induced apoptosis, suppression of cytotoxic lymphocytes, and natural killer cells tumoricidal activity. Therefore, new technologies for functional and molecular imaging as well as strategies directed to target low extracellular pH and low pH-adapted tumor cells might represent important issues in oncology.

Tumor cell plasticity largely depends on epithelial-to-mesenchymal transition (EMT) and its reversion. It was ascertained that EMT characterizes disease progression, including melanoma malignancy. As most solid tumors, melanoma shows extracellular acidosis, we analyse the impact of acidic environment on the EMT development in human melanoma cells. Melanoma cells were exposed to an acidic extracellular environment (pH 6.7) and tested for EMT markers. We found that acidic cells express a significant up-regulation of mesenchymal markers (N-cadherin, Vimentin), transcription factors (Twist, NF-κB) and a significant, although modest, reduction of E-cadherin expression. Acidic cell also express an increased invasiveness through Matrigel associated with an up-regulation of MMP-9 activity. When we injected acidic cells intravenously into immunodeficient animals, we found a number of lung micrometastases not different from non-acidic cells. Indeed, they show a partial G1 cell cycle arrest, which might interfere with the growth of lung colonies. When we investigated the in vitro invasiveness and lung colonization of a mixed population of acidic and non acidic melanoma cells, we found that acidic cells promote in vitro invasiveness of non-acidic cells and this cooperation leads to an higher migration rate than 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. We found evidence that acidity triggers in melanoma cells an EMT program, which although "incomplete", potentiates migration rate and development of lung colonies into immunodeficient host of cells grown in standard pH.

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted “same miRNA family = same function” rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

Oleuropein (Ole), a secoiridoid glucoside present in Olea europaea leaves, gained scientific interest thanks to its several biological properties, including the anticancer one. We verified whether Ole might potentiate the cytotoxicity of conventional drugs used to treat melanoma, disclosing a potentially new therapeutic strategy. We tested the cytotoxic action of Ole alone or in combination with chemotherapeutics on A375 human melanoma cells. We found that Ole was able, at a dose of 500 µM, to stimulate apoptosis, while at a non-toxic dose of 250 µM, it affected cell proliferation and induced the downregulation of the pAKT/pS6 pathway. A dose of 250 µM Ole did not potentiate the effect of Vemurafenib (PLX4032), but it succeeded in increasing the cytotoxic effect of Dacarbazine (DTIC). The major effect was found in the association between Ole and Everolimus (RAD001), also on PLX4032-resistant BRAF melanoma cells, which possibly cooperate in the inhibition of the pAKT/pS6 pathway. Of interest, an olive leaf extract enriched in equimolar Ole was more effective and able to further improve DTIC and RAD001 efficacy on BRAF melanoma cells with respect to Ole alone. Therefore, Ole represents a natural product able to potentiate a wide array of chemotherapeutics against BRAF melanoma cells affecting the pAKT/pS6 pathway.

Cancer cells overexpress CAIX under transient and chronic extracellular acidosis. Acidosis-induced CAIX overexpression is NF-κB mediated and HIF-1α independent. FC16-670A prevents CAIX overexpression and induces acidified cancer cell death.

Drug combination represents one of the most accredited strategies of cancer therapy able to improve drug efficacy and possibly overcome drug resistance. Among the agents used to complement conventional chemotherapy, carbonic anhydrase IX (CAIX) inhibitors appear as one of the most suitable, as markers of hypoxic and acidic cancer cells which do not respond to chemo- and radiotherapy. We performed preclinical in vitro assays to evaluate whether the SLC-0111 CAIX inhibitor co-operates and potentiates the cytotoxic effects of conventional chemotherapeutic drugs in A375-M6 melanoma cells, MCF7 breast cancer cells, and HCT116 colorectal cancer cells. Here, we demonstrate that the SLC-0111 CAIX inhibitor potentiates cytotoxicity of Dacarbazine and Temozolomide currently used for advanced melanoma treatment. SLC-0111 also increases breast cancer cell response to Doxorubicin and enhances 5-Fluorouracil cytostatic activity on colon cancer cells. These findings disclose the possibility to extend the use of CAIX inhibitors in the combination therapy of various cancer histotypes.

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