Phenotype Of Metastatic Cells Research Articles

Extracellular vesicles from cancer cells are key players of metastatic cell phenotype induction

Extracellular vesicles from cancer cells are key players of metastatic cell phenotype induction

Protein Kinase A (PKA) as a Master Regulator of the Early Metabolic Reprogramming in Bone Metastatic Prostate Cancer Cells

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P < .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

Linc00941 Is a Novel Transforming Growth Factor β Target That Primes Papillary Thyroid Cancer Metastatic Behavior by Regulating the Expression of Cadherin 6.

Background: Papillary thyroid cancers (PTCs) are common, usually indolent malignancies. Still, a small but significant percentage of patients have aggressive tumors and develop distant metastases leading to death. Currently, it is not possible to discriminate aggressive lesions due to lack of prognostic markers. Long noncoding RNAs (lncRNAs), which are selectively expressed in a context-dependent manner, are expected to represent a new landscape to search for molecular discriminants. Transforming growth factor β (TGFβ) is a multifunctional cytokine that fosters epithelial-to-mesenchymal transition and metastatic spreading. In PTCs, it triggers the expression of the metastatic marker Cadherin 6 (CDH6). Here, we investigated the TGFβ-dependent lncRNAs that may cooperate to potentiate PTC aggressiveness. Methods: We used a genome-wide approach to map enhancer (ENH)-associated lncRNAs under TGFβ control. Linc00941 was selected and validated using functional in vitro assays. A combined approach using bioinformatic analyses of the thyroid cancer (THCA)-the cancer genome atlas (TCGA) dataset and RNA-seq analysis was used to identify the processes in which linc00941 was involved in and the genes under its regulation. Correlation with clinical data was performed to evaluate the potential of this lncRNA and its targets as prognostic markers in THCA. Results: Linc00941 was identified as transcribed starting from one of the TGFβ-induced ENHs. Linc00941 expression was significantly higher in aggressive cancer both in the TCGA dataset and in a separate validation cohort from our institution. Loss of function assays for linc00941 showed that it promotes response to stimuli and invasiveness while restraining proliferation in PTC cells, a typical phenotype of metastatic cells. From the integration of TCGA data and linc00941 knockdown RNA-seq profiling, we identified 77 genes under the regulation of this lncRNA. Among these, we found the prometastatic gene CDH6. Linc00941 knockdown partially recapitulates the effects observed upon CDH6 silencing, promoting cell cytoskeleton and membrane adhesions rearrangements and autophagy. The combined expression of CDH6 and linc00941 is a distinctive feature of highly aggressive PTC lesions. Conclusions: Our data provide new insights into the biology driving metastasis in PTCs and highlight how lncRNAs cooperate with coding transcripts to sustain these processes.

Proteomic analysis of Tn-bearing glycoproteins from different stages of melanoma cells reveals new biomarkers

Cutaneous melanoma, the most aggressive form of skin cancer, responds poorly to conventional therapy. The appearance of Tn antigen-modified proteins in cancer is correlated with metastasis and poor prognoses. The Tn determinant has been recognized as a powerful diagnostic and therapeutic target, and as an object for the development of anti–tumor vaccine strategies. This study was designed to identify Tn-carrying proteins and reveal their influence on cutaneous melanoma progression. We used a lectin-based strategy to purify Tn antigen-enriched cellular glycoproteome, the LC-MS/MS method to identify isolated glycoproteins, and the DAVID bioinformatics tool to classify the identified proteins. We identified 146 different Tn-bearing glycoproteins, 88% of which are new. The Tn-glycoproteome was generally enriched in proteins involved in the control of ribosome biogenesis, CDR-mediated mRNA stabilization, cell-cell adhesion and extracellular vesicle formation. The differential expression patterns of Tn-modified proteins for cutaneous primary and metastatic melanoma cells supported nonmetastatic and metastatic cell phenotypes, respectively. To our knowledge, this study is the first large-scale proteomic analysis of Tn-bearing proteins in human melanoma cells. The identified Tn-modified proteins are related to the biological and molecular nature of cutaneous melanoma and may be valuable biomarkers and therapeutic targets.

Proteomic approach to understand metastatic spread.

The majority of tumor-related deaths are due to metastasis. Despite the clinical importance of understanding metastasis, we lack knowledge of the molecular mechanisms underlying tumor cell spreading and cell survival far from the primary tumor. Elucidating the molecular characteristics of highly metastatic carcinoma cells would help identify biomarkers or therapeutic targets relevant to predicting or combatting metastasis, and for this the phenotype of metastatic cells could be much more important than their genotype. Hence, proteomic approaches have wide potential utility. This review discusses possibilities of analyzing metastasis-specific protein patterns in a range of sample types, including in vitro and in vivo cancer models, and tissues and biological fluids from patients. Proteome approaches can identify proteins involved in regulating the metastatic capacities of tumors.

Mo1401 Serum Metabolomics As the Diagnostic Application for Early Diagnosis of Pancreatic Cancer

manipulating the activity of BMP signalling in vitro. Methods: We performed an immunohistochemical analysis of the expression of BMP signalling components (BMPReceptors and SMADs) in a tissue microarray of 41 patients with pancreatic ductal adenocarcinoma (PDAC) and correlated this to patients' survival. We also compared the expression levels of these components in 51 primary pancreatic tumours with 17 metastases. shRNA against SMAD4 and a plasmid encoding SMAD4 was used to investigate the effect of SMAD4 loss in vitro. The BMPReceptor expression was manipulated using siRNA against in a panel of pancreatic cancer cell lines and effects were analyzed using proliferation and invasion assays. Results: Loss of SMAD4 in patients with PDAC is associated with significantly worse survival (p= 0.025). Moreover, SMAD4 loss is more frequent in the metastases samples compared to the primary tumours (65% vs. 47%). Loss of SMAD4 induces transformation of cancer cells into more aggressive mesenchymal cell type with properties of the cancer stem cells, which proliferate slower and are more resistant to chemotherapy. Importantly, in a group of patients with intact positive SMAD4 expression loss of BMPR1a correlates significantly with a poor prognosis (p=0.02). Activation of BMP signalling in SMAD4 positive cancer cells leads to reduced proliferation, migration and invasion. The importance of BMP Receptor loss is confirmed by showing an increase in proliferation and invasion of pancreatic cancer cells after siRNA mediated knockdown of the BMPReceptors or opposite effect after restitution of BMPR. Conclusion: Loss of BMPR1a and SMAD4 results in poor survival in patients with pancreatic cancers. Inactivation of the BMP pathway increases aggressive tumourigenic properties of pancreatic cancer cells, while activation of the BMP pathway in pancreatic cancer reduces invasive metastatic phenotype of cells. Our data suggest that the BMP pathway can be an attractive target for future therapeutic interventions.

Abstract 2704: Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells.

Abstract Introduction. We have previously demonstrated that the expression levels of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness (basal-like, triple-negative and mesenchymal phenotype) of human breast cancer. The present study was performed to establish stably transfected cell lines and study the functional role of CHST11 in human breast cancer cells. Methods. MCF7 cells were transfected with a plasmid vector containing the CHST11 coding sequence. Stable transfectants, MCF-7-CHST11, were established. Flow cytometry and real-time PCR were used for expression analysis. Metastatic phenotype of cells was characterized through conduction of proliferation and migration assays. Morphology and growth pattern of MCF-7-CHST11 cells were monitored and compared to parental MCF-7 cells. Results. We observed that MCF-7-CHST11 reacted strongly with anti-chondroitin sulfate A (CS-A) antibody and recombinant P-selectin. CS-A is the immediate product of CHST11 activity. CHST11 expression significantly affected cell proliferation as MCF-7-CHST11 cells grew much faster. Migration assay using transwell chamber showed high migratory potential for MCF-7-CHST11 cells as compared to the parental MCF-7 cells. The significant migratory advantage was not proliferation dependent. The CHST11expression affected the morphology of cells and cell spreading behavior consistent with more efficient anchorage independent growth. Colony formation in soft agar, Invasion and adhesion assays are being performed at the present time. We are planning more experiments to study mechanisms involved. Conclusions. The data confirm that the CHST11 products on the cell surface can serve as P-selectin ligands. The results indicate that the CHST11 expression can affect metastatic phenotype of cancer cells by promoting cellular proliferation and migratory potential. Citation Format: Alice Behrens, Fariba Jousheghany, Aiwei Yao-Borengasser, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Ectopic expression of CHST11 promotes proliferation and migration of human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2704. doi:10.1158/1538-7445.AM2013-2704

Regulation of the metastatic cell phenotype by sialylated glycans

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

Caveolin-1-Enhanced Motility and Focal Adhesion Turnover Require Tyrosine-14 but Not Accumulation to the Rear in Metastatic Cancer Cells

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.

Calcium in tumour metastasis: new roles for known actors

In most cases, metastasis, not the primary tumour per se, is the main cause of mortality in cancer patients. In order to effectively escape the tumour, enter the circulation and establish secondary growth in distant organs cancer cells must develop an enhanced propensity to migrate. The ubiquitous second messenger Ca²⁺ is a crucial regulator of cell migration. Recently, a number of known molecular players in cellular Ca²⁺ homeostasis, including calcium release-activated calcium channel protein 1 (ORAI1), stromal interaction molecule 1 (STIM1) and transient receptor potential (TRP) channels, have been implicated in tumour cell migration and the metastatic cell phenotype. We discuss how these developments have increased our understanding of the Ca²⁺ dependence of pro-metastatic behaviours.

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

BackgroundPreviously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.MethodsRetroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.ResultsWe have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.ConclusionsPresented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.

Interactions of α2-HS-glycoprotein (fetuin) with MMP-3 and murine squamous cell carcinoma cells

Alpha2-HS glycoprotein (fetuin) is a major plasma glycoprotein predominantly synthesized by the liver. We have previously demonstrated that human fetuin produced by hepatocellular carcinoma cells can activate the matrix metalloproteinase MMP-9 (gelatinase-B). Stromelysin-1 (MMP-3) is over-expressed in murine skin tumors and is associated with a metastatic cell phenotype. We hypothesize that fetuin plays a role in tumor progression of cell types which by themselves do not have the ability to express fetuin. Immunohistochemical staining revealed that fetuin surrounds the tumor cells in murine squamous cell carcinoma tumor specimens, similar to the expression pattern previously seen for MMP-3. The physical association of fetuin and MMP-3 was demonstrated by immunoprecipitation of radiolabeled fetuin by antibodies to MMP-3. Fetuin facilitates the conversion of pro-MMP-3 to its active form, although this effect is indirect. The association of iodinated fetuin to the cell surface of intact cultured cells derived from a murine tumor with squamous (B9) and spindle (A5) morphologies was determined by binding experiments and Scatchard analysis. Fetuin binds with Bmax values in the range of 1.26-2.1 (mean = 1.7) fmol/1 x 10(5) cells for A5 cells, and 1.5-1.7 (mean = 1.6) fmol/1 x 10(5) cells for B9 cells. The mean KD was 0.46+/-0.19 nmol for both A5 and B9 cells. Our data therefore are consistent with the model that fetuin binds to the cell surface of tumor cells and acts to localize and anchor other molecules important during tumor progression to the plasma membrane.

A Potential Role for Interleukin-8 in the Metastatic Phenotype of Breast Carcinoma Cells

This study shows a strong correlation between the metastatic potentials of breast carcinoma cell lines and their ectopic expression of interleukin-8 (IL-8). Correlations exist for both constitutive and induced levels of IL-8 released. A correlation was also observed between cell morphology, metastatic potential, and IL-8 profile. Metastatic lines are fusiform in appearance, whereas, nonmetastatic lines are epithelioid. The metastatic potential of two breast carcinoma lines was examined using an orthotopic model of spontaneous metastasis. Metastatic cells formed rapidly growing, poorly differentiated primary tumors that metastasized. Nonmetastatic cells formed rapidly growing differentiated primary tumors that did not produce detectable metastases. Comparison of IL-8 expression by the parental cells and cell cultures developed from primary and metastatic tumors, demonstrates that IL-8 released by cultured cells from the primary tumor is higher than that of the parental cells, and IL-8 released by cultured cells derived from the metastatic lung tumors is greater than that released by cultured cells derived from the primary tumor. These data demonstrate a strong correlation between the metastatic phenotype of a cell and its IL-8 expression, suggesting a role for IL-8 in promoting the metastatic potential of breast tumor cells.

Microenvironment-induced cancer metastasis

Purpose : To present and evaluate clinical data suggesting that cancer metastasis may be induced by the microenvironment of the primary tumour and to discuss possible mechanisms of microenvironment-induced metastasis, based on a critical review of relevant data from studies of experimental tumours and cells in culture. Conclusions : Low oxygen tension in the primary tumour is associated with metastasis in soft tissue sarcoma, cervix carcinoma and carcinoma of the head and neck. Multiple mechanisms may be involved in hypoxia-induced metastasis. Thus, hypoxia followed by reoxygenation may induce point mutations and DNA strand breakage leading to deletions, amplifications and genomic instability. Hypoxia may also provide a physiological pressure in tumours selecting for metastatic cell phenotypes. Moreover, hypoxia may induce a temporary increase in the expression of gene products involved in the metastatic cascade, either through gene amplifications or through normal physiological processes by activating oxygen sensors, hypoxia signal transduction pathways and DNA transcription factors. Low glucose concentration, high lactate concentration and low extracellular pH may induce metastasis by similar mechanisms as hypoxia. Tumour reoxygenation during radiation therapy may promote microenvironment-induced metastasis by rescuing hypoxic or nutritionally deprived metastatic cells from dying. Ionizing radiation can elicit a stress response in tumour cells similar to that elicited by hypoxia. Radiation therapy may therefore adversely affect the rate of metastasis in patients who do not achieve control of the primary tumour by enhancing the expression of gene products of importance in metastasis.

337 Invasiveness of endothelial cells: Role in tumor associated angiogenesis

Neoangiogenesis, the formation of new capillary vessels, is essential for tumor growth and the formation of metastases. This process requires that the activated endothelial cells invade their own basement membrane. We have used the reconstituted basement membrane Matrigel model (Albini et al., Cancer Res. 47: 3239, 1987) to study endothelial cell (EC) invasion in vitro . Supernatants (CM) of cells from Kaposi's sarcoma (KS), a highly angiogenic neoplasm strongly induce EC invasiveness. The activity of gelatinase-A, an enzyme closely linked with the invasive phenotype of metastatic cells, is enhanced in endothelial cells treated with KS-CM. Exogenous TIMP-2 is capable of suppressing endothelial cell invasion stimulated by KS products. Inhibition is also induced by a synthetic peptide from the propeptide region of gelatinase A, containing a conserved unpaired cysteine residue. bFGF (a product of KS cells) as well as KS-CM are strongly angiogenic in the matrigel sponge model in vivo . TIMP-2 can suppress bFGF induced neovascularization of the sponges and inhibits KS products associated angiogenesis. Histological examination shows that no endothelial cells were present in the TIMP-2 treated bFGF sponges, confirming that endothelial cell invasiveness is necessary for tumor angiogenesis. We are now attempting to use N-acetyl-cysteine, a chemopreventive agent which also interferes with metalloprotease activity, as potential antiangiogenic drug ( AIDS & CNR grants )

Phenotype of metastatic cells as target for anti-metastatic interventions

Phenotype of metastatic cells as target for anti-metastatic interventions