Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Modulation of Dendritic Cell Function via Nanoparticle-Induced Cytosolic Calcium Changes.

Calcium nanoparticles have been investigated for applications, such as drug and gene delivery. Additionally, Ca2+ serves as a crucial second messenger in the activation of immune cells. However, few studies have systematically studied the effects of calcium nanoparticles on the calcium levels and functions within immune cells. In this study, we explore the potential of calcium nanoparticles as a vehicle to deliver calcium into the cytosol of dendritic cells (DCs) and influence their functions. We synthesized calcium hydroxide nanoparticles, coated them with a layer of silica to prevent rapid degradation, and further conjugated them with anti-CD205 antibodies to achieve targeted delivery to DCs. Our results indicate that these nanoparticles can efficiently enter DCs and release calcium ions in a controlled manner. This elevation in cytosolic calcium activates both the NFAT and NF-κB pathways, in turn promoting the expression of costimulatory molecules, antigen-presenting molecules, and pro-inflammatory cytokines. In mouse tumor models, the calcium nanoparticles enhanced the antitumor immune response and augmented the efficacy of both radiotherapy and chemotherapy without introducing additional toxicity. Our study introduces a safe nanoparticle immunomodulator with potential widespread applications in cancer therapy.

Inhibition of N-myristoyltransferase activity promotes androgen receptor degradation in prostate cancer.

BACKGROUND: Even though prostate cancer (PCa) patients initially respond to androgen deprivation therapy, some will eventually develop castration resistant prostate cancer (CRPC). Androgen receptor (AR) mediated cell signaling is a major driver in the progression of CRPC while only a fraction of PCa becomes AR negative. This study aimed to understand the regulation of AR levels by N-myristoyltransferase in PCa cells. METHODS: Two enantiomers, (1S,2S)- d-NMAPPD and (1R,2R)- d-NMAPPD (LCL4), were characterized by various methods (1 H and 13 C NMR, UHPLC, high-resolution mass spectra, circular dichroism) and evaluated for the ability to bind to N-myristoyltransferase 1 (NMT1) using computational docking analysis. structure-activity relationship analysis of these compounds led to the synthesis of (1R,2R)-LCL204 and evaluation as a potential NMT1 inhibitor utilizing the purified full length NMT1 enzyme. The NMT inhibitory activity wase determined by Click chemistry and immunoblotting. Regulation of NMT1 on tumor growth was evaluated in a xenograft tumor model. RESULTS: (1R,2R)- d-NMAPPD, but not its enantiomer (1S,2S)- d-NMAPPD, inhibited NMT1 activity and reduced AR protein levels. (1R,2R)-LCL204, a derivative of (1R,2R)- d-NMAPPD, inhibited global protein myristoylation. It also suppressed protein levels, nuclear translocation, and transcriptional activity of AR full-length or variants in PCa cells. This was due to enhanced ubiquitin and proteasome-mediated degradation of AR. Knockdown of NMT1 levels inhibited tumor growth and proliferation of cancer cells. CONCLUSION: Inhibitory efficacy on N-myristoyltransferase activity by d-NMAPPD is stereospecific. (1R,2R)-LCL204 reduced global N-myristoylation and androgen receptor protein levels at low micromolar concentrations in prostate cancer cells. pharmacological inhibition of NMT1 enhances ubiquitin-mediated proteasome degradation of AR. This study illustrates a novel function of N-myristoyltransferase and provides a potential strategy for treatment of CRPC.

Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.

Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex-based CRISPR systems have gained momentum due to their reduction of off-target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell-derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs-based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus-like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA-coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single-chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs-based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs-based delivery of CRISPR RNP in gene therapy and disease treatment.

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.

Long-chain fatty acyl-CoA synthetase 1 promotes prostate cancer progression by elevation of lipogenesis and fatty acid beta-oxidation.

Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Long-chain fatty acyl-CoA synthetases (ACSLs), a group of rate-limiting enzymes in fatty acid metabolism, catalyze the bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl-CoAs. In this study, systematical analysis of ACSLs levels and the amount of fatty acyl-CoAs illustrated that ACSL1 were significantly associated with the levels of a broad spectrum of fatty acyl-CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl-CoAs including C16:0-, C18:0-, C18:1-, and C18:2-CoA, triglycerides and lipid accumulation in cancer cells. Mechanistically, ACSL1 modulated mitochondrial respiration, ß-oxidation, and ATP production through regulation of CPT1 activity. Knockdown of ACSL1 inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo. Our study implicates ACSL1 as playing an important role in prostate tumor progression, and provides a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.

Long-Chain Acyl-CoA Synthetase 4-Mediated Fatty Acid Metabolism Sustains Androgen Receptor Pathway-Independent Prostate Cancer.

Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen-AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen-AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen-AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. IMPLICATIONS: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway-independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.

Heparan sulfate inhibits transforming growth factor ß signaling and functions in cis and in trans to regulate prostate stem/progenitor cell activities.

Prostate stem/progenitor cells (PrSCs) are responsible for adult prostate tissue homeostasis and regeneration. However, the related regulatory mechanisms are not completely understood. In this study, we examined the role of heparan sulfate (HS) in PrSC self-renewal and prostate regeneration. Using an in vitro prostate sphere formation assay, we found that deletion of the glycosyltransferase exostosin 1 (Ext1) abolished HS expression in PrSCs and disrupted their ability to self-renew. In associated studies, we observed that HS loss inhibited p63 and CK5 expression, reduced the number of p63+- or CK5+-expressing stem/progenitor cells, elevated CK8+ expression and the number of differentiated CK8+ luminal cells and arrested the spheroid cells in the G1/G0 phase of cell cycle. Mechanistically, HS expressed by PrSCs (in cis) or by neighboring cells (in trans) could maintain sphere formation. Furthermore, HS deficiency upregulated transforming growth factor ß (TGFß) signaling and inhibiting TGFß signaling partially restored the sphere-formation activity of the HS-deficient PrSCs. In an in vivo prostate regeneration assay, simultaneous loss of HS in both epithelial cell and stromal cell compartments attenuated prostate tissue regeneration, whereas the retention of HS expression in either of the two cellular compartments was sufficient to sustain prostate tissue regeneration. We conclude that HS preserves self-renewal of adult PrSCs by inhibiting TGFß signaling and functions both in cis and in trans to maintain prostate homeostasis and to support prostate regeneration.

Dietary palmitate cooperates with Src kinase to promote prostate tumor progression.

Numerous genetic alterations have been identified during prostate cancer progression. The influence of environmental factors, particularly the diet, on the acceleration of tumor progression is largely unknown. Expression levels and/or activity of Src kinase are highly elevated in numerous cancers including advanced stages of prostate cancer. In this study, we demonstrate that high-fat diets (HFDs) promoted pathological transformation mediated by the synergy of Src and androgen receptor in vivo. Additionally, a diet high in saturated fat significantly enhanced proliferation of Src-mediated xenograft tumors in comparison with a diet high in unsaturated fat. The saturated fatty acid palmitate, a major constituent in a HFD, significantly upregulated the biosynthesis of palmitoyl-CoA in cancer cells in vitro and in xenograft tumors in vivo. The exogenous palmitate enhanced Src-dependent mitochondrial ß-oxidation. Additionally, it elevated the amount of C16-ceramide and total saturated ceramides, increased the level of Src kinase localized in the cell membrane, and Src-mediated downstream signaling, such as the activation of mitogen-activated protein kinase and focal adhesion kinase. Our results uncover how the metabolism of dietary palmitate cooperates with elevated Src kinase in the acceleration of prostate tumor progression.

Nanoparticle-Laden Macrophages for Tumor-Tropic Drug Delivery.

Macrophages hold great potential in cancer drug delivery because they can sense chemotactic cues and home to tumors with high efficiency. However, it remains a challenge to load large amounts of therapeutics into macrophages without compromising cell functions. This study reports a silica-based drug nanocapsule approach to solve this issue. The nanocapsule consists of a drug-silica complex filling and a solid silica sheath, and it is designed to minimally release drug molecules in the early hours of cell entry. While taken up by macrophages at high rates, the nanocapsules minimally affect cell migration in the first 6-12 h, buying time for macrophages to home to tumors and release drugs in situ. In particular, it is shown that doxorubicin (Dox) as a representative drug can be loaded into macrophages up to 16.6 pg per cell using this approach. When tested in a U87MG xenograft model, intravenously (i.v.) injected Dox-laden macrophages show comparable tumor accumulation as untreated macrophages. Therapy leads to efficient tumor growth suppression, while causing little systematic toxicity. This study suggests a new cell platform for selective drug delivery, which can be readily extended to the treatment of other types of diseases.

Stromal Gli signaling regulates the activity and differentiation of prostate stem and progenitor cells.

Interactions between cells in the stroma and epithelium facilitate prostate stem cell activity and tissue regeneration capacity. Numerous molecular signal transduction pathways, including the induction of sonic hedgehog (Shh) to activate the Gli transcription factors, are known to mediate the cross-talk of these two cellular compartments. However, the details of how these signaling pathways regulate prostate stem and progenitor cell activity remain elusive. Here we demonstrate that, although cell-autonomous epithelial Shh-Gli signaling is essential to determine the expression levels of basal cell markers and the renewal potential of epithelial stem and progenitor cells, stromal Gli signaling regulates prostate stem and progenitor cell activity by increasing the number and size of prostate spheroids in vitro Blockade of stromal Gli signaling also inhibited prostate tissue regeneration in vivo The inhibition of stromal Gli signaling suppressed the differentiation of basal and progenitor cells to luminal cells and limited prostate tubule secretory capability. Additionally, stromal cells were able to compensate for the deficiency of epithelial Shh signaling in prostate tissue regeneration. Mechanistically, suppression of Gli signaling increased the signaling factor transforming growth factor ß (TGFß) in stromal cells. Elevation of exogenous TGFß1 levels inhibited prostate spheroid formation, suggesting that a stromal Gli-TGFß signaling axis regulates the activity of epithelial progenitor cells. Our study illustrates that Gli signaling regulates epithelial stem cell activity and renewal potential in both epithelial and stromal compartments.

Pharmacologically targeting the myristoylation of the scaffold protein FRS2α inhibits FGF/FGFR-mediated oncogenic signaling and tumor progression.

Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling facilitates tumor initiation and progression. Although currently approved inhibitors of FGFR kinase have shown therapeutic benefit in clinical trials, overexpression or mutations of FGFRs eventually confer drug resistance and thereby abrogate the desired activity of kinase inhibitors in many cancer types. In this study, we report that loss of myristoylation of fibroblast growth factor receptor substrate 2 (FRS2α), a scaffold protein essential for FGFR signaling, inhibits FGF/FGFR-mediated oncogenic signaling and FGF10-induced tumorigenesis. Moreover, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppressed FRS2α myristoylation and decreased the phosphorylation with mild alteration of FRS2α localization at the cell membrane. B13 inhibited oncogenic signaling induced by WT FGFRs or their drug-resistant mutants (FGFRsDRM). B13 alone or in combination with an FGFR inhibitor suppressed FGF-induced WT FGFR- or FGFRDRM-initiated phosphoinositide 3-kinase (PI3K) activity or MAPK signaling, inducing cell cycle arrest and thereby inhibiting cell proliferation and migration in several cancer cell types. Finally, B13 significantly inhibited the growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo In summary, our study suggests a possible therapeutic approach for inhibiting FGF/FGFR-mediated cancer progression and drug-resistant FGF/FGFR mutants.

Paracrine Fibroblast Growth Factor Initiates Oncogenic Synergy with Epithelial FGFR/Src Transformation in Prostate Tumor Progression.

Cross talk of stromal-epithelial cells plays an essential role in both normal development and tumor initiation and progression. Fibroblast growth factor (FGF)-FGF receptor (FGFR)-Src kinase axis is one of the major signal transduction pathways to mediate this cross talk. Numerous genomic studies have demonstrated that expression levels of FGFR/Src are deregulated in a variety of cancers including prostate cancer; however, the role that paracrine FGF (from stromal cells) plays in dysregulated expression of epithelial FGFRs/Src and tumor progression in vivo is not well evaluated. In this study, we demonstrate that ectopic expression of wild-type FGFR1/2 or Src kinase in epithelial cells was not sufficient to initiate prostate tumorigenesis under a normal stromal microenvironment in vivo. However, paracrine FGF10 synergized with ectopic expression of epithelial FGFR1 or FGFR2 to induce epithelial-mesenchymal transition. Additionally, paracrine FGF10 sensitized FGFR2-transformed epithelial cells to initiate prostate tumorigenesis. Next, paracrine FGF10 also synergized with overexpression of epithelial Src kinase to high-grade tumors. But loss of the myristoylation site in Src kinase inhibited paracrine FGF10-induced prostate tumorigenesis. Loss of myristoylation alters Src levels in the cell membrane and inhibited FGF-mediated signaling including inhibition of the phosphotyrosine pattern and FAK phosphorylation. Our study demonstrates the potential tumor progression by simultaneous deregulation of proteins in the FGF/FGFRs/Src signal axis and provides a therapeutic strategy of targeting myristoylation of Src kinase to interfere with the tumorigenic process.

Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first).

Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

In vivo growth and responses to treatment of renal cell carcinoma in different environments.

Renal cell carcinoma is the most common type of kidney cancer in adults and is associated with poor prognosis. The hydrodynamic cell delivery technique was employed in this study to establish tumor growth in mouse lung, liver and kidneys. We demonstrate that RencaLuc cells exhibit different growth rates and responses to the cancer treatment of 5-florouracil and cytokine gene therapy when growing in different organs. The tumor growth rate was faster in the kidneys compared to that in the lung and liver. The liver is the second-best organ in support of tumor growth. Tumors in the liver and lung respond to 5-florouracil treatment but are less responsive in the kidneys. IL-12 gene therapy resulted in whole-body tumor suppression and prolonged animal survival. IFN-ß gene therapy was effective in suppressing tumor growth in the liver but not effective for those in the lung and kidneys. These results suggest that kidney cancer cells, once metastasized in different organs, show different growth patterns and respond differently to treatment. Our data also imply that an animal model with multi-organ tumor growth is critical for development of a new strategy for treatment of tumors when metastasis is suspected. At the same time, the results also provide direct evidence in support of the usefulness of the hydrodynamic tail vein injection as a tool for establishment of tumor growth in the lung, liver and kidneys.