Silvia Peppicelli

Extracellular acidity, a "reappreciated" trait of tumor environment driving malignancy: perspectives in diagnosis and therapy

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.

Acidic pH via NF-κB favours VEGF-C expression in human melanoma cells.

Malignant melanomas are characterized by the ability of early metastatic dissemination to regional lymph nodes and the detection of sentinel lymph node metastases serves as an important prognostic parameter. There is clear evidence that melanoma cells and stromal cells of tumor environment can induce lymphangiogenesis, e.g. growth of lymphatic vessels, and this phenomenon is correlated with lymph node metastases. Vascular endothelial growth factor (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 demonstrate that an acidic extracellular pH promotes the expression of VEGF-C in A375P melanoma cells and in melanoma cells isolated from a human spontaneous metastatic lesion, through the NF-κB transcription factor. We also demonstrate 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-κB activation. Furthermore, we show that esomeprazole abrogates the enhanced VEGF-C expression in tumor cells grown in a acidic medium and stimulated by IL-1β. On the whole, the present study reveals 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.

Inhibition of uPAR-TGFβ crosstalk blocks MSC-dependent EMT in melanoma cells

The capacity of cancer cells to undergo epithelial-to-mesenchymal transition (EMT) is now considered a hallmark of tumor progression, and it is known that interactions between cancer cells and mesenchymal stem cells (MSCs) of tumor microenvironment may promote this program. Herein, we demonstrate that MSC-conditioned medium (MSC-CM) is a potent inducer of EMT in melanoma cells. The EMT profile acquired by MSC-CM-exposed melanoma cells is characterized by an enhanced level of mesenchymal markers, including TGFβ/TGFβ-receptors system upregulation, by increased invasiveness and uPAR expression, and in vivo tumor growth. Silencing TGFβ in MSC is found to abrogate ability of MSC to promote EMT characteristics in melanoma cells, together with uPAR expression, and this finding is strengthened using an antagonist peptide of TGFβRIII, the so-called P17. Finally, we demonstrate that the uPAR antisense oligonucleotide (uPAR aODN) may inhibit EMT of melanoma cells either stimulated by exogenous TGFβ or MSC-CM. Thus, uPAR upregulation in melanoma cells exposed to MSC-medium drives TGFβ-mediated EMT. On the whole, TGFβ/uPAR dangerous liaison in cancer cell/MSC interactions may disclose a new strategy to abrogate melanoma progression.Key messageMesenchymal stem cell (MSC)-conditioned medium induces EMT-like profile in melanoma.MSC-derived TGFβ promotes uPAR and TGFβ/TGFβ-receptor upregulation in melanoma.TGFβ gene silencing in MSCs downregulates uPAR expression and EMT in melanoma.uPAR downregulation prevents MSC-induced EMT-like profile in melanoma cells.Inhibition of the dangerous TGFβ/uPAR relationship might abrogate melanoma progression.

The metabolically-modulated stem cell niche: a dynamic scenario regulating cancer cell phenotype and resistance to therapy.

This Perspective addresses the interactions of cancer stem cells (CSC) with environment which result in the modulation of CSC metabolism, and thereby of CSC phenotype and resistance to therapy. We considered first as a model disease chronic myeloid leukemia (CML), which is triggered by a well-identified oncogenetic protein (BCR/Abl) and brilliantly treated with tyrosine kinase inhibitors (TKi). However, TKi are extremely effective in inducing remission of disease, but unable, in most cases, to prevent relapse. We demonstrated that the interference with cell metabolism (oxygen/glucose shortage) enriches cells exhibiting the leukemia stem cell (LSC) phenotype and, at the same time, suppresses BCR/Abl protein expression. These LSC are therefore refractory to the TKi Imatinib-mesylate, pointing to cell metabolism as an important factor controlling the onset of TKi-resistant minimal residual disease (MRD) of CML and the related relapse. Studies of solid neoplasias brought another player into the control of MRD, low tissue pH, which often parallels cancer growth and progression. Thus, a 3-party scenario emerged for the regulation of CSC/LSC maintenance, MRD induction and disease relapse: the “hypoxic” versus the “ischemic” vs. the “acidic” environment. As these environments are unlikely constrained within rigid borders, we named this model the “metabolically-modulated stem cell niche.”

Inflammatory cytokines induce vascular endothelial growth factor‑C expression in melanoma‑associated macrophages and stimulate melanoma lymph node metastasis

Lymph node colonization by tumor cells is one of the key determinants of melanoma staging and prognosis, and tumor-associated macrophages (TAMs) are the predominant type of inflammatory cell in the tumor environment which secretes vascular endothelial growth factor (VEGF)-C, the most potent lymphangiogenic growth factor. In the present study, to elucidate the mechanism involved in VEGF-C expression in TAMs, murine peritoneal macrophages were co-cultivated with syngeneic B16 melanoma cells to mimic the reciprocal interactions between tumor cells and macrophages found in spontaneous tumors. In the present study, upon contact with tumor cells, macrophages were found to express a higher level of VEGF-C which was associated with an increase in the expression of IL-1β and TNF-α and their receptors. Antibodies against the IL-1β and TNF-α receptors were added to media that had been conditioned by the macrophage-tumor cell co-cultures and inhibition of VEGF-C was observed in macrophages co-cultivated with the tumor cells. Furthermore, when IL-1β and TNF-α were used at a non-toxic level, they enhanced peritoneal lymph node colonization by melanoma cells. Thus, in the present study, macrophagic IL-1β and TNF-α were observed to promote VEGF-C expression in TAMs, as well as melanoma lymph node metastasis, suggesting that inhibiting the signaling between tumor cells and TAMs may be required to inhibit lymphangiogenesis and lymph node metastasis.

Extracellular acidity strengthens mesenchymal stem cells to promote melanoma progression

Mesenchymal stem cells (MSC) participate to tumor stroma development and several evidence suggests that they play a role in facilitating cancer progression. Because melanoma often shows extracellular pH low enough to influence host cell as tumor cell behavior, the aim of this study is to elucidate whether acidity affects cross talk between MSC and melanoma cells to disclose new liaisons promoting melanoma progression, and to offer new therapeutic opportunities. We found that MSC grown in a low pH medium (LpH-MSC) stimulate melanoma xenografts more than MSC grown in a standard pH medium. LpH-MSC express a higher level of TGFβ that is instrumental of epithelial-to-mesenchymal transition (EMT)-like phenotype induction in melanoma cells. LpH-MSC profile also shows a switching to an oxidative phosphorylation metabolism that was accompanied by a forced glycolytic pathway of melanoma cells grown in LpH-MSC-conditioned medium. Metformin, an inhibitor of mitochondrial respiratory chain was able to reconvert oxidative metabolism and abrogate TGFβ expression in LpH-MSC. In addition, esomeprazole, a proton pump inhibitor activated in acidosis, blocked TGFβ expression in LpH-MSC through the downregulation of IkB. Both agents, metformin and esomeprazole, inhibited EMT profile in melanoma cells grown in LpH-MSC medium, and reduced glycolytic markers. Thus, acidosis of tumor microenvironment potentiates the pro-tumoral activity of MSC and orchestrates for a new potential symbiosis, which could be target to limit melanoma progression.

Metformin is also effective on lactic acidosis-exposed melanoma cells switched to oxidative phosphorylation

ABSTRACT Low extracellular pH promotes in melanoma cells a malignant phenotype characterized by an epithelial-to-mesenchymal transition (EMT) program, endowed with mesenchymal markers, high invasiveness and pro-metastatic property. Here, we demonstrate that melanoma cells exposed to an acidic extracellular microenvironment, 6.7±0.1, shift to an oxidative phosphorylation (Oxphos) metabolism. Metformin, a biguanide commonly used for type 2 diabetes, inhibited the most relevant features of acid-induced phenotype, including EMT and Oxphos. When we tested effects of lactic acidosis, to verify whether sodium lactate might have additional effects on acidic melanoma cells, we found that EMT and Oxphos also characterized lactic acid-treated cells. An increased level of motility was the only gained property of lactic acidic-exposed melanoma cells. Metformin treatment inhibited both EMT markers and Oxphos and, when its concentration raised to 10 mM, it induced a striking inhibition of proliferation and colony formation of acidic melanoma cells, both grown in protons enriched medium or lactic acidosis. Thus, our study provides the first evidence that metformin may target either proton or lactic acidosis-exposed melanoma cells inhibiting EMT and Oxphox metabolism. These findings disclose a new potential rationale of metformin addition to advanced melanoma therapy, e.g. targeting acidic cell subpopulation.

CD63 Tetraspanin Is a Negative Driver of Epithelial-to-Mesenchymal Transition in Human Melanoma Cells

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

Context-dependent miR-204 and miR-211 affect the biological properties of amelanotic and melanotic melanoma cells

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 vemurafenibs 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 vemurafenibs 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.

Acidity of Microenvironment as a Further Driver of Tumor Metabolic Reprogramming

In the last decade, experimental research has intensely focused on metabolic reprogramming of tumor cells, which contributes to cancer cell adaptation and survival in different and hostile microenvironments. Metabolic reprogramming consists of the switch of tumor cells from aerobic or anaerobic glycolysis to oxidative phosphorylation. A comprehensive vision of the metabolic scenario involving functionally different tumor cell subpopulations was proposed as a necessary premise to the design of new strategies of diagnosis and therapy. Special focus has been put on the role of acidosis of certain tumor regions, a very important although frequently neglected aspect.Despite the progresses in cancer therapy, the escaping of tumor cancer cells from host defense and relapse of disease still represent main issues in tumor-bearing patients. Indeed, malignant cells are provided with a tremendous plasticity that they exploit to survive, replicate and invade in stressed microenvironments. Such plasticity allows cancer cells to easily modify their properties, including metabolism, switching back and forth from aerobic or anaerobic glycolysis to oxidative phosphorylation (OxPhos). It is well ascertained that a suitable metabolic profile of cancer cells is necessary to sustain tumor growth, local invasion and distant colonization. Thus, cancer metabolism needs to be considered in view of the design of new strategies to control tumor progression.