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Blood vessels are the most important way for cancer cells to survive and diffuse in the body, metastasizing distant organs. During the process of tumor expansion, the neoplastic mass progressively induces modifications in the microenvironment due to its uncontrolled growth, generating a hypoxic and low pH milieu with high fluid pressure and low nutrients concentration. In such a particular condition, reactive oxygen species play a fundamental role, enhancing tumor proliferation and migration, inducing a glycolytic phenotype and promoting angiogenesis. Indeed, to reach new sources of oxygen and metabolites, highly aggressive cancer cells might produce a new abnormal network of vessels independently from endothelial cells, a process called vasculogenic mimicry. Even though many molecular markers and mechanisms, especially in gastric cancer, are still unclear, the formation of such intricate, leaky and abnormal vessel networks is closely associated with patients’ poor prognosis, and therefore finding new pharmaceutical solutions to be applied along with canonical chemotherapies in order to control and normalize the formation of such networks is urgent.

Lactic acidosis characterizes the tumor microenvironment (TME) and is involved in the mechanisms leading to cancer progression and dissemination through the reprogramming of tumor and local host cells (e.g., endothelial cells, fibroblasts, and immune cells). Adipose tissue also represents a crucial component of the TME which is receiving increasing attention due to its pro-tumoral activity, however, to date, it is not known whether it could be affected by the acidic TME. Now, emerging evidence from chronic inflammatory and fibrotic diseases underlines that adipocytes may give rise to pathogenic myofibroblast-like cells through the adipocyte-to-myofibroblast transition (AMT). Thus, our study aimed to investigate whether extracellular acidosis could affect the AMT process, sustaining the acquisition by adipocytes of a cancer-associated fibroblast (CAF)-like phenotype with a pro-tumoral activity. To this purpose, human subcutaneous adipose-derived stem cells committed to adipocytes (acADSCs) were cultured under basal (pH 7.4) or lactic acidic (pH 6.7, 10 mM lactate) conditions, and AMT was evaluated with quantitative PCR, immunoblotting, and immunofluorescence analyses. We observed that lactic acidosis significantly impaired the expression of adipocytic markers while inducing myofibroblastic, pro-fibrotic, and pro-inflammatory phenotypes in acADSCs, which are characteristic of AMT reprogramming. Interestingly, the conditioned medium of lactic acidosis-exposed acADSC cultures was able to induce myofibroblastic activation in normal fibroblasts and sustain the proliferation, migration, invasion, and therapy resistance of breast cancer cells in vitro. This study reveals a previously unrecognized relationship between lactic acidosis and the generation of a new CAF-like cell subpopulation from adipocytic precursor cells sustaining tumor malignancy.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERβ, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERβ in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERβ in male cells and EMT was strongly promoted. An inverse relationship between ERβ expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERβ expressing cell subpopulations and ERβ receptor silencing. Finally, we found that ERβ regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERβ regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

Oleocanthal, a minor polar compound in extra-virgin olive (EVO) oil, contains anticancer properties, which should be encouraged in its use in oncology. Gastric Cancer (GC), a very aggressive human cancer, is often diagnosed at advanced stages, when surgery is substituted or supported by chemotherapy (CT). However, CT frequently fails due to the patient’s resistance to the treatment. Thus, the aim of this study is to verify whether an OC-enriched EVO oil extract fraction (OCF) may be useful in order to overcome a resistance to GC. We evaluated the OCF effects on an AGS gastric adenocarcinoma cell line wild type (AGS wt) and on its subpopulations resistant to 5-fluorouracil (5FUr), Paclitaxel (TAXr) or cisplatin (CISr). We found that a 60 µM dose of the OCF acts on the AGS wt, 5FUr and TAXr, leading to the cell cycle inhibition and to a ROS production, but not on CISr cells. Resistance of CISr to the OCF seems to be due to higher levels of antioxidant-enzymes that can counteract the OCF-induced ROS production. Moreover, using the OCF plus 5-fluorouracil, Paclitaxel or cisplatin, we found a potentiating effect compared with a mono-treatment in all resistant GC cells, including CISr. In conclusion, the use of the OCF in the management of GC has shown very interesting advantages, opening-up the possibility to evaluate the efficacy of the OCF in vivo, as a valid adjuvant in the treatment of resistant GC.

Background: BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue. Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells. Methods: We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction. We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis. Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib. Findings: We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation. Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition. Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells. Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients. Interpretation: We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance. Funds: Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze. Keywords: Vemurafenib, Acquired resistance, Melanoma, uPAR, EGFR

Abstract CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is today one of the most reliable method for gene-editing, supporting previous gene therapies technologies such as TALEN, Meganucleases and ZFNs. There is a growing up number of manuscripts reporting several successful gene-edited cancer cell lines, but the real challenge is to translate this technique to the clinical practice. While treatments for diseases based on a single gene mutation is closer, being possible to target and repair the mutant allele in a selective way generating specific guide RNAs (gRNAs), many steps need to be done to apply CRISPR to face cancer. In this review, we want to give a general overview to the recent advancements in the delivery systems of the CRISPR/Cas9 machinery in cancer therapy.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR “rescue” expression cell lines as well as we promoted the expression of only its 3’UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3’UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a “molecular sponge”. In particular miR146a, by binding PLAUR 3’ UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.

Abstract Near infrared (NIR)‐resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP‐loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC‐loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.

Abstract Background Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or “Warburg effect”), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma. Methods To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay. Results In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption. Conclusions SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

Abstract Background Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. Methods A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. Results In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Conclusion Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the “amoeboid angiogenesis”. uPAR is indispensable for ECs and ECFCs to perform an efficient amoeboid angiogenesis. Therefore, uPAR silencing or the block of its integrin-interaction, together with standard treatment against VEGF, could be a possible solution for angiogenesis inhibition.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins (Cas) is an adaptive immune system in archaea and most bacteria. By repurposing these systems for use in eukaryote cells, a substantial revolution has arisen in the genome engineering field. In recent years, CRISPR-Cas technology was rapidly developed and different types of DNA or RNA sequence editors, gene activator or repressor, and epigenome modulators established. The versatility and feasibility of CRISPR-Cas technology has introduced this system as the most suitable tool for discovering and studying the mechanism of specific genes and also for generating appropriate cell and animal models. SOX genes play crucial roles in development processes and stemness. To elucidate the exact roles of SOX factors and their partners in tissue hemostasis and cell regeneration, generating appropriate in vitro and in vivo models is crucial. In line with these premises, CRISPR-Cas technology is a promising tool for studying different family members of SOX transcription factors. In this review, we aim to highlight the importance of CRISPR-Cas and summarize the applications of this novel, promising technology in studying and decoding the function of different members of the SOX gene family.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

To date, 5-Fluorouracil (5FU) is a major component of several chemotherapy regimens, thus its study is of fundamental importance to better understand all the causes that may lead to chemoresistance and treatment failure. Given the evident differences between prognosis in Asian and Caucasian populations, triggered by clear genetic discordances and given the extreme genetic heterogeneity of gastric cancer (GC), the evaluation of the most frequent mutations in every single member of the 5FU conversion and activation pathway might reveal several important results. Here, we exploited the cBioPortal analysis software to query a large databank of clinical and wide-genome studies to evaluate the components of the three major 5FU transformation pathways. We demonstrated that mutations in such ways were associated with a poor prognosis and reduced overall survival, often caused by a deletion in the TYMP gene and amplification in TYMS. The use of prodrugs and dihydropyrimidine dehydrogenase (DPD) inhibitors, which normally catabolizes 5FU into inactive metabolites, improved such chemotherapies, but several steps forward still need to be taken to select better therapies to target the chemoresistant pools of cells with high anaplastic features and genomic instability.

Gastric cancer (GC) is turning out today to be one of the most important welfare issues for both Asian and European countries. Indeed, while the vast majority of the disease burden is located in China and in Pacific and East Asia, GC in European countries still account for about 100,000 deaths per year. With this review article, we aim to focus the attention on one of the most complex cellular pathways involved in GC proliferation, invasion, migration, and metastasis: the MAP kinases. Such large kinases family is to date constantly studied, since their discovery more than 30 years ago, due to the important role that it plays in the regulation of physiological and pathological processes. Interactions with other cellular proteins as well as miRNAs and lncRNAs may modulate their expression influencing the cellular biological features. Here, we summarize the most important and recent studies involving MAPK in GC. At the same time, we need to underly that, differently from cancers arising from other tissues, where MAPK pathways seems to be a gold target for anticancer therapies, GC seems to be unique in any aspect. Our aim is to review the current knowledge in MAPK pathways alterations leading to GC, including H. pylori MAPK-triggering to derail from gastric normal epithelium to GC and to encourage researches involved in MAPK signal transduction, that seems to definitely sustain GC development.

Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB.

In the original version of this article [1], published on 18 December 2018, there was 1 incorrect author name.

The application of a cell-based growth inhibition on a library of skeletally different glycomimetics allowed for the selection of a hexahydro-2H-furo[3,2-b][1,4]oxazine compound as candidate inhibitors of MDA-MB-231 cell growth. Subsequent synthesis of analogue compounds and preliminary biological studies validated the selection of a valuable hit compound with a novel polyhydroxylated structure for the modulation of the breast carcinoma cell cycle mechanism.