Ruolo del microambiente nelle metastasi osteolitiche

Bone is one of the most common sites of distant metastases from breast carcinoma. In lytic bone metastasis, tumor associated-osteoclasts are the main actors of bone resorption. Under physiological conditions, osteoclast formation is activated by cytokines and hormones produced by bone-resident cells, including osteoblasts and mesenchymal stem cells. However, when tumor cells spread to the bone, they disrupt the delicate balance between cells orchestrating osteoclast formation, thereby promoting bone resorption. Current treatments of metastatic bone disease are largely ineffective to improve patient survival, possibly due to their inability to interfere with the metabolic pathways that regulate the behavior and the cross-talk between cancer cells and osteoclasts. To add news insights into the complex pathogenesis of osteolytic metastases, we focused our investigations on the role of lactate in the metabolic symbiosis of bone metastasis. We found that human osteoclasts are characterized by an increased mitochondrial metabolism, that probably supports the biosynthetic needs for osteoclast formation and activation, and that a glycolytic switch support the resorption activity of osteoclasts. Moreover, osteoclasts are able to uptake lactate from the extracellular space, an energy-rich metabolite produced by the glycolytic metabolism of breast carcinoma cells. In osteoclasts, lactate fuels oxidative metabolism, thereby increasing tumor aggressiveness. However, the bone metastatic microenvironment is composed of several types of cells, including tumor-associated stromal cells, bone-resident cells, tumor cells, and cancer-initiating cells. Each cell type takes part to the complex process of metastatic lesions formation. In particular, cancer stem cells play a primary role in tumorigenesis. To gain a more in-depth knowledge on the interplay between cells of tumor bulk, we validated the most stable housekeeping genes for gene expression analysis on cancer stem cells. These findings lay the groundwork for future investigations on the cross-talk between stroma and tumor cells of the metastatic microenvironment.L'osso è uno dei tessuti più comunemente affetti da metastasi da carcinoma mammario, dove tipicamente formano di lesioni osteolitiche caratterizzate da un aumentato riassorbimento osseo da parte degli osteoclasti. In condizioni fisiologiche, l’osteoclastogenesi è regolata da fattori proteici secreti da cellule ossee. Quando le cellule tumorali metastatizzano alle ossa, esse distruggono questo delicato processo, promuovendo l’osteoclastogenesi e il riassorbimento osseo. Le attuali terapie impiegate per curare le metastasi osteolitiche, oltre a presentare una serie di limitazioni, non aumentano la sopravvivenza dei pazienti, probabilmente a causa della loro inadeguatezza nell’interferire con i processi metabolici che regolano il dialogo tra cellule tumorali e osteoclasti. Al fine di far luce sui meccanismi di patogenesi delle metastasi osteolitiche, abbiamo studiato il ruolo del lattato nella simbiosi metabolica del microambiente metastatico, dimostrando innanzitutto che gli osteoclasti umani sono caratterizzati da un aumentato metabolismo mitocondriale rispetto ai loro precursori, che l’attivazione del riassorbimento osseo è regolata da uno switch glicolitico, ma soprattutto che gli osteoclasti utilizzano il lattato, un metabolita ad elevata energia prodotto da metabolismo glicolitico tumorale, per alimentare ulteriormente il metabolismo mitocondriale, determinando di conseguenza un aumento dell’aggressività tumorale. Tuttavia, il microambiente metastatico è composto di diversi tipi di cellule, come le cellule stromali associate al tumore, cellule ossee, cellule tumorali e cellule staminali tumorali. Ciascuno dei suddetti tipi cellulari prende attivamente parte alla formazione di lesioni metastatiche. In particolar modo, le cellule staminali tumorali giocano un ruolo centrale nel processo di carcinogenesi. Al fine di acquisire una conoscenza completa riguardo alle interazioni tra i diversi tipi cellulari, abbiamo vagliato i geni housekeeping comunemente utilizzati in letteratura con lo scopo di identificare i più stabili per le analisi di espressione genica sulle cellule staminali tumorali. Questi risultati pongono le basi per future indagini sul cross-talk tra cellule tumorali e stroma del microambiente metastatico

Prominent role of RAB39A-RXRB axis in cancer development and stemness

In this study, we found that RAB39A, a member of the RAS oncogene family, was selectively expressed in cancer cells of different histotypes, by analyzing gene expression in human osteosarcoma cells and the cancer stem cells (CSCs) and by comparing them with normal cells through global transcriptomics and principal component analyses. We further validated RAB39A as a therapeutic target, by silencing its expression. The silencing impaired cancer stemness and spherogenic ability in vitro, as well as tumorigenesis in vivo. RNA-seq analyses in the silenced spheres suggested that RAB39A is associated downstream with RXRB and KLF4. Notably, RXRB expression was inhibited in RAB39A-silenced CSCs. Induced overexpression of RXRB in RAB39A-silenced cells restored spherogenic ability and tumorigenesis, confirming RXRB as a major effector of RAB39A. Quantitative RT-PCR analysis of 3c400 human cancer tissues showed that RAB39A was highly expressed in sarcomas and in malignancies of lymphoid, adrenal and testicular tissues. Our data provide the rationale for targeting of the RAB39A-RXRB axis as a therapy for aggressive cancers

Continuous enzyme-coupled assay of phosphate- or pyrophosphate-releasing enzymes

A coupled enzyme assay able to monitor the kinetics of reactions catalyzed by phosphate- or pyrophosphate-releasing enzymes is presented here. The assay is based on the concerted action of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase), and xanthine oxidase (XOD). In the presence of phosphate, PNPase catalyzes the phosphorolysis of inosine, generating hypoxanthine, which is oxidized to uric acid by XOD. The uric acid accordingly formed can be spectrophotometrically monitored at 293 nm, taking advantage of a molar extinction coefficient which is independent of pH between 6 and 9. The coupled assay was tested using DNA polymerases as a model system. The activity of Klenow enzyme was quantitatively determined, and it was found in agreement with the corresponding activity determined by traditional methods. Moreover, the continuous coupled assay was used to determine K-m and V-max of Klenow enzyme, yielding values in good agreement with previous observations. Finally, the coupled assay was also used to determine the activity of partially purified DNA polymerases, revealing its potential use to monitor purification of phosphate- or pyrophosphate-releasing enzymes

Respiratory Complex I dysfunction in cancer: from a maze of cellular adaptive responses to potential therapeutic strategies

Mitochondria act as key organelles in cellular bioenergetics and biosynthetic processes producing signals that regulate different molecular networks for proliferation and cell death. This ability is also preserved in pathologic contexts such as tumorigenesis, during which bioenergetic changes and metabolic reprogramming confer flexibility favoring cancer cells survival in a hostile microenvironment. Although different studies epitomize mitochondrial dysfunction as a pro-tumorigenic hit, genetic ablation or pharmacological inhibition of respiratory Complex I causing a severe impairment are associated with a low proliferative phenotype. In this scenario, it must be considered that despite the initial delay in growth, cancer cells may become able to resume proliferation exploiting molecular mechanisms to overcome growth arrest. Here we highlight the current knowledge on molecular responses activated by Complex I-defective cancer cells to bypass physiological control systems and to re-adapt their fitness during microenvironment changes. Such adaptive mechanisms could reveal possible novel molecular players in synthetic lethality with Complex I impairment, thus providing new synergistic strategies for mitochondria-based anti-cancer therapy

Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, shortterm acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance

Pre-clinical Models for Studying the Interaction Between Mesenchymal Stromal Cells and Cancer Cells and the Induction of Stemness

Mesenchymal stromal cells (MSC) have essential functions in building and supporting the tumour microenvironment, providing metastatic niches, and maintaining cancer hallmarks, and it is increasingly evident that the study of the role of MSC in cancer is crucial for paving the way to clinical opportunities for novel anti-cancer therapies. To date, the vast majority of preclinical models that have been used for studying the effect of reactive MSC on cancer growth, metastasis, and response to therapy has been mainly based on in vitro flat biology, including the co-culturing with cell compartmentalization or with cell-to-cell contact, and on in vivo cancer models with different routes of MSC inoculation. More complex in vitro 3D models based on spheroid structures that are formed by intermingled MSC and tumour cells are also capturing the interest in cancer research. These are innovative culture systems tailored on the specific tumour type and that can be combined with a synthetic extracellular matrix, or included in in silico technologies, to more properly mimic the in vivo biological, spatial, biochemical, and biophysical features of tumour tissues. In this review, we summarized the most popular and currently available preclinical models for evaluating the role of MSC in cancer and their specific suitability, for example, in assaying the MSC-driven induction of epithelial-to-mesenchymal transition or of stem-like traits in cancer cells. Finally, we enlightened the need to carefully consider those parameters that might unintentionally strongly affect the secretome in MSC-cancer interplay and introduce confounding variables for the interpretation of results

3D imaging lipidometry in single cell by in-flow holographic tomography

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characteriza- tion in terms of number, size, spatial positioning and relative distribution in the cell volume can shed light on the roles played by LDs. Until now, fluorescence microscopy and transmission electron microscopy are assessed as the gold standard methods for identifying LDs due to their high sensitivity and specificity. However, such methods generally only provide 2D assays and partial measurements. Furthermore, both can be destructive and with low productivity, thus limit- ing analysis of large cell numbers in a sample. Here we demonstrate for the first time the capability of 3D visualization and the full LD characterization in high-throughput with a tomographic phase-contrast flow-cytometer, by using ovarian cancer cells and monocyte cell lines as models. A strategy for retrieving significant parameters on spatial correlations and LD 3D positioning inside each cell volume is reported. The information gathered by this new method could allow more in depth understanding and lead to new discoveries on how LDs are correlated to cellular functions

Prominent role of RAB39A-RXRB axis in cancer development and stemness

In this study, we found that RAB39A, a member of the RAS oncogene family, was selectively expressed in cancer cells of different histotypes, by analyzing gene expression in human osteosarcoma cells and the cancer stem cells (CSCs) and by comparing them with normal cells through global transcriptomics and principal component analyses. Wefurther validated RAB39A as a therapeutic target, by silencing its expression. The silencing impaired cancer stemness and spherogenic ability in vitro, as well as tumorigenesis in vivo. RNA-seq analyses in the silenced spheres suggested that RAB39Ais associated downstream with RXRB and KLF4. Notably, RXRB expression was inhibitedin RAB39A-silenced CSCs. Induced overexpression of RXRB in RAB39A-silenced cells restored spherogenic ability and tumorigenesis, confirming RXRB as a major effector of RAB39A. Quantitative RT-PCR analysis of ~400 human cancer tissues showed that RAB39A was highly expressed in sarcomas and in malignancies of lymphoid, adrenal andtesticular tissues. Our data provide the rationale for targeting of the RAB39A-RXRB axis as a therapy for aggressive cancers

Sarcoma treatment in the era of molecular medicine

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.Peer reviewe