Lactate supports cell-autonomous ECM production to sustain metastatic behavior in prostate cancer.

Extracellular matrix (ECM) is a major component of the tumor environment, promoting the establishment of a pro-invasive behavior. Such environment is supported by both tumor- and stromal-derived metabolites, particularly lactate. In prostate cancer (PCa), cancer-associated fibroblasts (CAFs) are major contributors of secreted lactate, able to impact on metabolic and transcriptional regulation in cancer cells. Here, we describe a mechanism by which CAF-secreted lactate promotes in PCa cells the expression of genes coding for the collagen family. Lactate-exploiting PCa cells rely on increased α-ketoglutarate (α-KG) which activates the α-KG-dependent collagen prolyl-4-hydroxylase (P4HA1) to support collagen hydroxylation. De novo synthetized collagen plays a signaling role by activating discoidin domain receptor 1 (DDR1), supporting stem-like and invasive features of PCa cells. Inhibition of lactate-induced collagen hydroxylation and DDR1 activation reduces the metastatic colonization of PCa cells. Overall, these results provide a new understanding of the link between collagen remodeling/signaling and the nutrient environment exploited by PCa.

Lactate: A Metabolic Driver in the Tumour Landscape.

The presence of lactate in human tumours has been long neglected, confined to the role of a waste product derived from glycolysis and as a biomarker of malignancy. More recently, lactate has been rediscovered as signalling molecule that plays important roles in the regulation of the metabolic pathways, the immune response, and cell-to-cell communication within the tumour microenvironment. This review examines recent discoveries about the functional role of lactate in shaping the behaviour and the phenotype of tumour and tumour-associated cells, and describes potential clinical approaches to target lactate transport and metabolism in tumours.

Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling.

BACKGROUND: In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity. METHODS: We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies. RESULTS: Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF. CONCLUSIONS: We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

Stromal-induced mitochondrial re-education: Impact on epithelial-to-mesenchymal transition and cancer aggressiveness.

Metabolic reprogramming as well as the flexible utilisation of fuel sources by tumour cells has been considered not only intrinsic to malignant cells but also sustained by resident and/or recruited stromal cells. The complexity of tumour-stroma cross-talk is experienced by neoplastic cells through profound changes in the own metabolic machinery. In such context, mitochondria are dynamic organelles that receive, orchestrate and exchange a multiplicity of stromal cues within the tumour cells to finely regulate key metabolic and signalling pathways, allowing malignant cells to adapt and thrive in an ever-changing environment. In this review, we focus on how tumour mitochondria are coached by stromal metabolic supply and how this re-education sustains tumour malignant traits.

Targeted DNA oxidation by LSD1-SMAD2/3 primes TGF-ß1/ EMT genes for activation or repression.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor ß1 (TGF-ß1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-ß1 during EMT initiation and establishment. TGF-ß1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-ß1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-ß1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-ß1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-ß1 and the priming role of DNA oxidation that marks TGF-ß1-induced and -repressed genes involved in the EMT.

DNMT3A epigenetically regulates key microRNAs involved in epithelial-to-mesenchymal transition in prostate cancer.

Epithelial-to-mesenchymal transition (EMT) is involved in prostate cancer (PCa) metastatic progression, and its plasticity suggests epigenetic implications. Deregulation of DNA methyltransferases (DNMTs) and several microRNAs (miRNAs) plays a relevant role in EMT, but their interplay has not been clarified yet. In this study, we provide evidence that DNMT3A interaction with several miRNAs has a central role in an ex vivo EMT PCa model obtained via exposure of PC3 cells to conditioned media from cancer-associated fibroblasts. The analysis of the alterations of the miRNA profile shows that miR-200 family (miR-200a/200b/429, miR-200c/141), miR-205 and miR-203, known to modulate key EMT factors, are down-regulated and hyper-methylated at their promoters. DNMT3A (mainly isoform a) is recruited onto these miRNA promoters, coupled with the increase of H3K27me3/H3K9me3 and/or the decrease of H3K4me3/H3K36me3. Most interestingly, our results reveal the differential expression of two DNMT3A isoforms (a and b) during ex vivo EMT and a regulatory feedback loop between miR-429 and DNMT3A that can promote and sustain the transition towards a more mesenchymal phenotype. We demonstrate the ability of miR-429 to target DNMT3A 3'UTR and modulate the expression of EMT factors, in particular ZEB1. Survey of the PRAD-TCGA dataset shows that patients expressing an EMT-like signature are indeed characterized by down-regulation of the same miRNAs with a diffused hyper-methylation at miR-200c/141 and miR-200a/200b/429 promoters. Finally, we show that miR-1260a also targets DNMT3A, although it does not seem to be involved in EMT in PCa.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.

miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

BACKGROUND: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance. METHODS: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines. RESULTS: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines. CONCLUSIONS: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

Correction: miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Treatment with Cannabinoids as a Promising Approach for Impairing Fibroblast Activation and Prostate Cancer Progression.

Endo-, phyto- and synthetic cannabinoids have been proposed as promising anti-cancer agents able to impair cancer cells' behavior without affecting their non-transformed counterparts. However, cancer outcome depends not only on cancer cells' activity, but also on the stromal cells, which coevolve with cancer cells to sustain tumor progression. Here, we show for the first time that cannabinoid treatment impairs the activation and the reactivity of cancer-associated fibroblasts (CAFs), the most represented stromal component of prostate tumor microenvironment. Using prostate cancer-derived CAFs, we demonstrated that WIN 55-212.2 mesylate, a synthetic full agonist of cannabinoid receptors (CBs) 1 and 2, downregulates α-smooth muscle actin and matrix metalloprotease-2 expression, and it inhibits CAF migration, essential features to ensure the activated and reactive CAF phenotype. Furthermore, by impairing stromal reactivity, WIN 55-212.2 mesylate also negatively affects CAF-mediated cancer cells' invasiveness. Using selective antagonists of CBs, we proved that CAFs response to WIN 55-212.2 mesylate is mainly mediated by CB2. Finally, we suggest that endocannabinoids self-sustain both prostate tumor cells migration and CAFs phenotype by an autocrine loop. Overall, our data strongly support the use of cannabinoids as anti-tumor agents in prostate cancer, since they are able to simultaneously strike both cancer and stromal cells.

ß3-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance.

ß-adrenergic signaling is known to be involved in cancer progression; in particular, beta3-adrenoreceptor (ß3-AR) is associated with different tumor conditions. Currently, there are few data concerning ß3-AR in myeloid malignancies. Here, we evaluated ß3-AR in myeloid leukemia cell lines and the effect of ß3-AR antagonist SR59230A. In addition, we investigated the potential role of ß3-AR blockade in doxorubicin resistance. Using flow cytometry, we assessed cell death in different in vitro myeloid leukemia cell lines (K562, KCL22, HEL, HL60) treated with SR59230A in hypoxia and normoxia; furthermore, we analyzed ß3-AR expression. We used healthy bone marrow cells (BMCs), peripheral blood mononuclear cells (PBMCs) and cord blood as control samples. Finally, we evaluated the effect of SR59230A plus doxorubicin on K562 and K562/DOX cell lines; K562/DOX cells are resistant to doxorubicin and show P-glycoprotein (P-gp) overexpression. We found that SR59230A increased cancer cell lines apoptosis especially in hypoxia, resulting in selective activity for cancer cells; moreover, ß3-AR expression was higher in malignancies, particularly under hypoxic condition. Finally, we observed that SR59230A plus doxorubicin increased doxorubicin resistance reversion mainly in hypoxia, probably acting on P-gp. Together, these data point to ß3-AR as a new target and ß3-AR blockade as a potential approach in myeloid leukemias.

Stromal-induced downregulation of miR-1247 promotes prostate cancer malignancy.

Cancer progression is strictly dependent on the relationship between tumor cells and the surrounding stroma, which supports cancer malignancy promoting several crucial steps of tumor progression, including the execution of the epithelial to mesenchymal transition (EMT) associated with enhancement in cell invasion, resistance to both anoikis and chemotherapeutic treatments. Recently it has been highlighted the central role of microRNAs (miRNAs) as regulators of tumor progression. Notably, in several tumors a strong deregulation of miRNAs is observed, supporting proliferation, invasion, and metabolic reprogramming of tumor cells. Here we demonstrated that cancer-associated fibroblasts induce a downregulation of miR-1247 in prostate cancer (PCa) cells. We proved that miR-1247 repression is functional for the achievement of EMT and increased cell invasion as well as stemness traits. These phenomena contribute to promote the metastatic potential of PCa cells as demonstrated by increased lung colonization in in vivo experiments. Moreover, as a consequence of miR-1247 downregulation, we observed a correlated increased expression level of neuropilin-1, a miR-1247 target involved as a coreceptor in the epidermal growth factor receptor signaling. Taken together, our data highlight miR-1247 as a potential target for molecular therapies aimed to block the progression and diffusion of PCa.

Conjugation of a GM3 lactone mimetic on carbon nanotubes enhances the related inhibition of melanoma-associated metastatic events.

GM3-ganglioside is known to be involved in melanoma proliferation. In order to modulate metastatic-related events, we have functionalized multi-walled carbon nanotubes (MWCNTs) with multiple copies of a GM3-lactone mimetic. The MWCNTs proved to guarantee the appropriate spatial arrangement of the mimetic allowing a stronger inhibition of migration and invasiveness of human melanoma (A375) cells compared to other multivalent constructs reported before. In addition, the effect of the multivalent tubular conjugate on the inhibition of specific tyrosine kinases, which are associated with the ganglioside complexes within the membrane domains, was demonstrated. Finally, the short-term fate of the conjugate was assessed, for the first time, by means of the 1H NMR relaxometry technique by exploiting the signal arising from the CNTs.

Mesenchymal Stem Cells are Recruited and Activated into Carcinoma-Associated Fibroblasts by Prostate Cancer Microenvironment-Derived TGF-ß1.

Tumor stromal cells can supply appropriate signals that may develop aggressive phenotypes of carcinoma cells and establish a complex scenario which culminates in metastasis. Recent works proposed that bone marrow-derived mesenchymal stem cells (MSC) are recruited to primary tumors. However, the exact functions of these cells in the tumor microenvironment are not well characterized, as it is reported that MSC can either promote or inhibit tumor progression. In the present study, we aim at investigating the signaling molecules which regulate the interplay between MSC, prostate carcinoma (PCa) cells and two important cellular types constituting the tumor-associated stroma, macrophages and fibroblasts, during their progression toward malignancy. We identified TGF-ß1 as a crucial molecule able to attract MSC recruitment both to PCa cells as well as to tumor stroma components. Moreover, PCa- and tumor stroma-secreted TGF-ß1 is important to induce MSC transdifferentiation into carcinoma-associated fibroblast (CAF)-like cells. Consequently, the CAF-like phenotype acquired by MSC is central to promote tumor progression related effects. Thus, tumor-educated MSC enhance PCa invasiveness compared to nonactivated MSC. Additionally, differing from normal MSC, CAF-like MSC perform vascular mimicry and recruit monocytes, which can be further polarized to M2 macrophages within the PCa environment. Our findings indicate a prominent role for TGF-ß1 in MSC mobilization and activation strengthened by the fact that the blockade of TGF-ß1 signaling impairs MSC promotion of PCa progression. Stem Cells 2016;34:2536-2547.

The metabolic gene HAO2 is downregulated in hepatocellular carcinoma and predicts metastasis and poor survival.

BACKGROUND & AIMS: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. METHODS: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. RESULTS: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. CONCLUSION: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC.

Anoikis molecular pathways and its role in cancer progression.

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonizing of distant organs. As anchorage-independent growth and epithelial-mesenchymal transition, two features associated with anoikis resistance, are vital steps during cancer progression and metastatic colonization, the ability of cancer cells to resist anoikis has now attracted main attention from the scientific community. Cancer cells develop anoikis resistance due to several mechanisms, including change in integrins' repertoire allowing them to grow in different niches, activation of a plethora of inside-out pro-survival signals as over-activation of receptors due to sustained autocrine loops, oncogene activation, growth factor receptor overexpression, or mutation/upregulation of key enzymes involved in integrin or growth factor receptor signaling. In addition, tumor microenvironment has also been acknowledged to contribute to anoikis resistance of bystander cancer cells, by modulating matrix stiffness, enhancing oxidative stress, producing pro-survival soluble factors, triggering epithelial-mesenchymal transition and self-renewal ability, as well as leading to metabolic deregulations of cancer cells. All these events help cancer cells to inhibit the apoptosis machinery and sustain pro-survival signals after detachment, counteracting anoikis and constituting promising targets for anti-metastatic pharmacological therapy. This article is part of a Special Section entitled: Cell Death Pathways.

Tumor microenvironment: bone marrow-mesenchymal stem cells as key players.

Tumor progression is a multistep phenomenon in which tumor-associated stromal cells perform an intricate cross-talk with tumor cells, supplying appropriate signals that may promote tumor aggressiveness. Among several cell types that constitute the tumor stroma, the discovery that bone marrow-derived mesenchymal stem cells (BM-MSC) have a strong tropism for tumors has achieved notoriety in recent years. Not only are the BM-MSC recruited, but they can also engraft at tumor sites and transdifferentiate into cells such as activated fibroblasts, perivascular cells and macrophages, which will perform a key role in tumor progression. Whether the BM-MSC and their derived cells promote or suppress the tumor progression is a controversial issue. Recently, it has been proposed that proinflammatory stimuli can be decisive in driving BM-MSC polarization into cells with either tumor-supportive or tumor-repressive phenotypes (MSC1/MSC2). These considerations are extremely important both to an understanding of tumor biology and to the putative use of BM-MSC as "magic bullets" against tumors. In this review, we discuss the role of BM-MSC in many steps in tumor progression, focusing on the factors that attract BM-MSC to tumors, BM-MSC differentiation ability, the role of BM-MSC in tumor support or inhibition, the immunomodulation promoted by BM-MSC and metastatic niche formation by these cells.

Angiopoietin-like 7, a novel pro-angiogenetic factor over-expressed in cancer.

Angiopoietin-like (ANGPTL) proteins are secreted proteins showing structural similarity to members of the angiopoietin family. Some ANGPTL proteins possess pleiotropic activities, being involved in cancer lipid, glucose energy metabolisms, and angiogenesis. ANGPTL7 is the less characterized member of the family whose functional role is only marginally known. In this study, we provide experimental evidences that ANGPTL7 is over-expressed in different human cancers. To understand the role played by ANGPTL7 in tumor biology, we asked whether ANGPTL7 is endogenously expressed by malignant cells or in response to environmental stimuli. We found that ANGPTL7 is marginally expressed under standard growth condition while it is specifically up-regulated by hypoxia. Interestingly, the protein is secreted and partially associated with the exosomal fraction, suggesting that it could be found in the systemic circulation of oncologic patients and act in an endocrine way. Moreover, we found that ANGPTL7 exerts a pro-angiogenetic effect on human differentiated endothelial cells by stimulating their proliferation, motility, invasiveness, and capability to form capillary-like networks while it does not stimulate progenitor endothelial cells. Finally, we showed that ANGPTL7 promotes vascularization in vivo in the mouse Matrigel sponge assay, thereby accrediting this molecule as a pro-angiogenic factor.

Tumor-stroma metabolic relationship based on lactate shuttle can sustain prostate cancer progression.

BACKGROUND: Cancer cell adopts peculiar metabolic strategies aimed to sustain the continuous proliferation in an environment characterized by relevant fluctuations in oxygen and nutrient levels. Monocarboxylate transporters MCT1 and MCT4 can drive such adaptation permitting the transport across plasma membrane of different monocarboxylic acids involved in energy metabolism. METHODS: Role of MCTs in tumor-stroma metabolic relationship was investigated in vitro and in vivo using transformed prostate epithelial cells, carcinoma cell lines and normal fibroblasts. Moreover prostate tissues from carcinoma and benign hypertrophy cases were analyzed for individuating clinical-pathological implications of MCT1 and MCT4 expression. RESULTS: Transformed prostate epithelial (TPE) and prostate cancer (PCa) cells express both MCT1 and MCT4 and demonstrated variable dependence on aerobic glycolysis for maintaining their proliferative rate. In glucose-restriction the presence of L-lactate determined, after 24 h of treatment, in PCa cells the up-regulation of MCT1 and of cytochrome c oxidase subunit I (COX1), and reduced the activation of AMP-activated protein kinase respect to untreated cells. The blockade of MCT1 function, performed by si RNA silencing, determined an appreciable antiproliferative effect when L-lactate was utilized as energetic fuel. Accordingly L-lactate released by high glycolytic human diploid fibroblasts WI-38 sustained survival and growth of TPE and PCa cells in low glucose culture medium. In parallel, the treatment with conditioned medium from PCa cells was sufficient to induce glycolytic metabolism in WI-38 cells, with upregulation of HIF-1a and MCT4. Co-injection of PCa cells with high glycolytic WI-38 fibroblasts determined an impressive increase in tumor growth rate in a xenograft model that was abrogated by MCT1 silencing in PCa cells. The possible interplay based on L-lactate shuttle between tumor and stroma was confirmed also in human PCa tissue where we observed a positive correlation between stromal MCT4 and tumor MCT1 expression. CONCLUSIONS: Our data demonstrated that PCa progression may benefit of MCT1 expression in tumor cells and of MCT4 in tumor-associated stromal cells. Therefore, MCTs may result promising therapeutic targets in different phases of neoplastic transformation according to a strategy aimed to contrast the energy metabolic adaptation of PCa cells to stressful environments.

Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells.

BACKGROUND: Cellular plasticity confers cancer cells the ability to adapt to microenvironmental changes, a fundamental requirement for tumour progression and metastasis. The epithelial to mesenchymal transition (EMT) is a transcriptional programme associated with increased cell motility and stemness. Besides EMT, the mesenchymal to amoeboid transition (MAT) has been described during tumour progression but to date, little is known about its transcriptional control and involvement in stemness. The aim of this manuscript is to investigate (i) the transcriptional profile associated with the MAT programme and (ii) to study whether MAT acquisition in melanoma cancer cells correlates with clonogenic potential to promote tumour growth. RESULTS: By using a multidisciplinary approach, we identified four different treatments able to induce MAT in melanoma cells: EphA2 overexpression, Rac1 functional inhibition using its RacN17 dominant negative mutant, stimulation with Ilomastat or treatment with the RhoA activator Calpeptin. First, gene expression profiling identified the transcriptional pathways associated with MAT, independently of the stimulus that induces the MAT programme. Notably, gene sets associated with the repression of mesenchymal traits, decrease in the secretion of extracellular matrix components as well as increase of cellular stemness positively correlate with MAT. Second, the link between MAT and stemness has been investigated in vitro by analysing stemness markers and clonogenic potential of melanoma cells undergoing MAT. Finally, the link between MAT inducing treatments and tumour initiating capability has been validated in vivo. CONCLUSION: Taken together, our results demonstrate that MAT programme in melanoma is characterised by increased stemness and clonogenic features of cancer cells, thus sustaining tumour progression. Furthermore, these data suggest that stemness is not an exclusive feature of cells undergoing EMT, but more generally is associated with an increase in cellular plasticity of cancer cells.