Malignant progression in cancer requires populations of tumor-initiating cells (TICs

The encapsulation of mRNA in nanosystems as gene vaccines for immunotherapy purposes has experienced an exponential increase in recent years. Despite the many advantages envisaged within these approaches, their application in clinical treatments is still limited due to safety issues. These issues can be attributed, in part, to liver accumulation of most of the designed nanosystems and to the inability to transfect immune cells after an intravenous administration. In this context, this study takes advantage of the known versatile properties of the oligopeptide end-modified poly (β-amino esters) (OM-PBAEs) to complex mRNA and form discrete nanoparticles. Importantly, it is demonstrated that the selection of the appropriate end-oligopeptide modifications enables the specific targeting and major transfection of antigen-presenting cells (APC) in vivo, after intravenous administration, thus enabling their use for immunotherapy strategies. Therefore, with this study, it can be confirmed that OM-PBAE are appropriate systems for the design of mRNA-based immunotherapy approaches aimed to in vivo transfect APCs and trigger immune responses to fight either tumors or infectious diseases.

In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial-mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e-CSCs) from non-CSCs expressing a stable EMT. We have found that the e-CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e-CSCs consistent with the metabolomics and fluxomics analysis that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics and transcriptomics analysis allowed us to identify key metabolic players regulated at the post-transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis.

Supplementary key words ceramide • metastasis • inhibitorsCancer cells develop a lipogenic phenotype that supports the energy and membrane synthesis requirements associated with the enhanced proliferation and survival under stress inherent to malignant progression ( 1, 2 ). The recognition of the importance of this phenotype in cancer has led to the use of enzymes of lipid metabolism as markers to monitor neoplastic progression and response to therapy, as well as to the development of drugs targeted at key lipogenic enzymes, such as fatty acid synthase (FASN) ( 2, 3 ). The excess fatty acid synthesis that results from the coordinated activation of lipogenic enzymes in many types of cancer leads to the accumulation of palmitate, which needs to be further processed by the cells due to the toxic effects of its accumulation. One pathway that Abstract Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confi rm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specifi c AC inhibitors could act by counteracting critical growth properties of SAF2008-00706 and SAF2011-22444 (to G.F.) Press, February 18, 2013 DOI 10.1194 Acid ceramidase as a therapeutic target in metastatic prostate cancer Abbreviations: AC, acid ceramidase; CMH, ceramide monohexoside; MDR1, multidrug resistant protein 1; NC, neutral ceramidase; PC, prostate cancer; PIN, prostate intraepithelial neoplasia; S1P, sphingosine-1-phosphate. This work was supported by Ministry of Science and Innovation Grants ; Agència de Gestió d'Ajuts Universitaris i de Recerca de la Generalitat de Catalunya Grant 2009SGR1072 (to G.F.); Ministry of Science and Innovation Grants SAF2008-04136-C02-01 and SAF2011-24686 (to T.M.T.); Ministry of Economy and Competitivity Grant SAF2012-40017-C02-01 (to T.M.T.); Agència de Gestió d'Ajuts Universitaris i de Recerca de la Generalitat de Catalunya Grant 2009SGR1482 (to T.M.T.); Xarxa de Bancs de Tumours de Catalunya-Pla Director d'Oncologia and Fondo Europeo de Desarrollo...

BackgroundTumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination.MethodsM and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer.ResultsComparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases.ConclusionsThe matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-237) contains supplementary material, which is available to authorized users.

Metabolic reprogramming, a crucial cancer hallmark, shifts metabolic pathways such as glycolysis, tricarboxylic acid cycle or lipogenesis, to enable the growth characteristics of cancer cells. Here, we provide evidence that transketolase-like 1 (TKTL1) orchestrates aerobic glycolysis, fatty acid and nucleic acid synthesis, glutamine metabolism, protection against oxidative stress and cell proliferation. Furthermore, silencing of TKTL1 reduced the levels of sphingolipids such as lactosylceramide (a sphingolipid regulating cell survival, proliferation and angiogenesis) and phosphatidylinositol (which activates PI3K/Akt/mTOR signaling). Thus, in addition to its well-known roles in glucose and amino acid metabolism, TKTL1 also regulates lipid metabolism. In conclusion, our study provides unprecedented evidence that TKTL1 plays central roles in major metabolic processes subject to reprogramming in cancer cells and thus identifies TKTL1 as a promising target for new anti-cancer therapies.

HER3 (ERBB3) is a catalytically inactive pseudokinase of the HER receptor tyrosine kinase family, frequently overexpressed in prostate and other cancers. Aberrant expression and mutations of 2 other members of the family, EGFR and HER2, are key carcinogenic events in several types of tumors, and both are wellvalidated therapeutic targets. In this study, we show that HER3 is required to maintain the motile and invasive phenotypes of prostate (DU-145) and breast (MCF-7) cancer cells in response to the HER3 ligand neuregulin-1 (NRG-1), epidermal growth factor (EGF) and fetal bovine serum. Although MCF-7 breast cancer cells appeared to require HER3 as part of an autocrine response induced by EGF and FBS, the response of DU-145 prostate cancer cells to these stimuli, while requiring HER3, did not appear to involve autocrine stimulation of the receptor. DU-145 cells required the expression of HER3 for efficient clonogenicity in vitro in standard growth medium and for tumorigenicity in immunodeficient mice. These observations suggest that prostate cancer cells derived from tumors that overexpress HER3 are dependent on its expression for the maintenance of major attributes of neoplastic aggressiveness, with or without cognate ligand stimulation. ' UICCKey words: HER3; ERBB3; invasiveness; tumorigenicity; prostate; breast The 3 transmembrane tyrosine kinases and 1 pseudokinase of the HER family, EGFR/ERBB1, HERs2/ERBB2, HER4/ERBB4 and HER3/ERBB3, are key regulators of the growth, survival, differentiation, motility and invasiveness of many cell types.1 Their activities are engaged upon binding of polypeptide ligands, of which more than 13 are known in humans.1 Although all HER ligands are structurally related to EGF, individual ligands preferentially bind to, and activate, specific HERs. Ligand binding induces conformational changes in the receptors, exposing a dimerization surface through which they associate with other HER proteins, forming homodimers or heterodimers.2 These specific associations transduce additional conformational changes to the cytoplasmic domains of the receptors, eventually exposing and activating their tyrosine kinase catalytic domains.2 Of the 4 HER proteins, HER2 lacks the capacity to capture ligands, and is constitutively poised to associate, through its dimerization surface, either with the 2nd HER2 molecule, or with the EGFR or HER3 after they adopt active conformations upon ligand binding.2 The pseudokinase HER3 lacks a tyrosine kinase catalytic center. Binding of NRG-1 by HER3 causes its prompt heterodimerization with HER2 and activation of the kinase activity of the latter, which in turn phosphorylates the HER3 cytoplasmic domain at specific tyrosine residues.3-5 HER3 can also form heterodimeric complexes with ligand-bound EGFR or HER4 1 , resulting in its transactivation. Several of the induced phosphosites on HER3 recruit and activate phosphatidylinositol-3 0 kinase (PI3K), directly coupling this receptor to AKT-dependent pathways that promote cell motility, invasion and survival. 3,4 ...

BackgroundSeveral pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-κB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev.Methodology/Principal FindingsBy applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-κB by TNF-α and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells.Conclusions/SignificanceThis is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.