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1.
Around 7–17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.  相似文献   

2.
BackgroundOsteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages.ConclusionsConsiderable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.  相似文献   

3.
The SARI (suppressor of AP-1, regulated by IFN) gene, which is also called BATF2, is associated with the risk of several kinds of cancer, and loss of SARI expression is frequently detected in aggressive and metastatic cancer. However, the functional role of SARI in lung adenocarcinoma remains unknown. We have shown that loss of SARI expression initiates epithelial-mesenchymal transition (EMT), which is visualized by repression of E-cadherin and up-regulation of vimentin in lung adenocarcinoma cell lines and in clinical lung adenocarcinoma specimens. Using a human lung xenograft-mouse model, we observed that knocking down endogenous SARI in human carcinoma cells leads to the development of multiple lymph node metastases. Moreover, we showed that SARI functions as a critical protein in regulating EMT by modulating the (GSK)-3β-β-catenin signaling pathway. These results demonstrate the mechanism of SARI function in EMT and suggest that assessment of SARI may serve as a prognostic biomarker and potential therapeutic target for lung adenocarcinoma metastasis.  相似文献   

4.
Side Population (SP) cells, a subset of Hoechst-low cells, are enriched with stem cells. Originally, SP cells were isolated from bone marrow but recently have been found in various solid tumors and cancer cell lines that are clonogenic in vitro and tumorigenic in vivo. In this study, SP cells from lymph node metastatic head and neck squamous cell carcinoma (HNSCC) cell lines were examined using flow cytometry and Hoechst 3342 efflux assay. We found that highly metastatic HNSCC cell lines M3a2 and M4e contained more SP cells compared to the low metastatic parental HNSCC cell line 686LN. SP cells in HNSCC were highly invasive in vitro and tumorigenic in vivo compared to non-SP cells. Furthermore, SP cells highly expressed ABCG2 and were chemoresistant to Bortezomib and etoposide. Importantly, we found that SP cells in HNSCC had abnormal activation of Wnt/β-catenin signaling as compared to non-SP cells. Together, these findings indicate that SP cells might be a major driving force of head and neck tumor formation and metastasis. The Wnt/β-catenin signaling pathway may be an important target for eliminating cancer stem cells in HNSCC.  相似文献   

5.

Background

Radiofrequency ablation (RFA) is one of the curative therapies for hepatocellular carcinoma (HCC), however, accelerated progression of residual HCC after incomplete RFA has been reported more frequently. The underlying molecular mechanism of this phenomenon remains to be elucidated. In this study, we used an incomplete RFA orthotopic HCC nude mouse model to study the invasive and metastatic potential of residual cancer as well as the correlated mechanism.

Methods

The incomplete RFA orthotopic nude mouse models were established using high metastatic potential HCC cell line HCCLM3 and low metastatic potential HCC cell line HepG2, respectively. The changes in cellular morphology, motility, metastasis and epithelial–mesenchymal transition (EMT), and HCC cell molecular markers after in vitro and in vivo incomplete RFA intervention were observed.

Results

Pulmonary and intraperitoneal metastasis were observed in an in vivo study. The underlying pro-invasive mechanism of incomplete RFA appeared to be associated with promoting EMT, including down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. These results were in accordance with the in vitro response of HCC cells to heat intervention. Further studies demonstrated that β-catenin was a pivotal factor during this course and blocking β-catenin reduced metastasis and EMT phenotype changes in heat-treated HCCLM3 cells in vitro.

Conclusion

Incomplete RFA enhanced the invasive and metastatic potential of residual cancer, accompanying with EMT-like phenotype changes by activating β-catenin signaling in HCCLM3 cells.  相似文献   

6.
Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, β1,4N-acetylgalactosaminyltransferese T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewisx and sialyl Lewisa). This modification was associated with elevated expression of the Sda carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo.  相似文献   

7.
8.
Androgen deprivation constitutes the principal therapy for advanced and metastatic prostate cancers. However, this therapeutic intervention usually results in the transition to a more aggressive androgen-independent prostate cancer. The elucidation of molecular alterations during the progression to androgen independence is an integral step toward discovering more effective targeted therapies. With respect to identifying crucial mediators of this transition, we compared the proteomes of androgen-independent (PC3, DU145, PPC1, LNCaP-SF, and 22Rv1) and androgen-dependent (LNCaP and VCaP) and/or normal prostate epithelial (RWPE) cell lines using mass spectrometry. We identified more than 100 proteins that were differentially secreted in the androgen-independent cell lines. Of these, Protein S (PROS1) was elevated in the secretomes of all of the androgen-independent prostate cancer cell lines, with no detectable secretion in normal and androgen-dependent cell lines. Using quantitative PCR, we observed significantly higher (p < 0.05) tissue expression levels of PROS1 in prostate cancer samples, further indicating its importance in prostate cancer progression. Similarly, immunohistochemistry analysis revealed elevation of PROS1 in high grade prostate cancer (Gleason grade ≥8), and further elevation in castration-resistant metastatic prostate cancer lesions. We also observed its significant (p < 0.05) elevation in high grade prostate cancer seminal plasma samples. Taken together, these results show that PROS1 is elevated in high grade and castration-resistant prostate cancer and could serve as a potential biomarker of aggressive disease.  相似文献   

9.
Prostate cancer aggressiveness and metastatic potential are influenced by gene expression and genomic aberrations, features that can be influenced by the 3D structure of chromosomes inside the nucleus. Using chromosome conformation capture (Hi-C), we conducted a systematic genome architecture comparison on a cohort of cell lines that model prostate cancer progression, from normal epithelium to bone metastasis. We describe spatial compartment identity (A-open versus B-closed) changes with progression in these cell lines and their relation to gene expression changes in both cell lines and patient samples. In particular, 48 gene clusters switch from the B to the A compartment, including androgen receptor, WNT5A, and CDK14. These switches are accompanied by changes in the structure, size, and boundaries of topologically associating domains (TADs). Further, compartment changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation. These results suggest that discrete 3D genome structure changes play a deleterious role in prostate cancer progression.   相似文献   

10.
Using cDNA microarrays, we have conducted a systematic characterization of global gene expression in v-raf or v-raf/v-myc transformed rat liver epithelial (RLE) cell lines exhibiting both non-metastatic and metastatic phenotypes. Seven transformed cell lines were compared with the non-transformed parental RLE cell. The hierarchical clustering analysis of gene expression profiles revealed two groups reflecting the in vivo metastatic potential of the cells. Surprisingly, one non-metastatic cell line T1 was co-clustered with metastatic cell lines, suggesting that T1 underwent significant genetic changes. The T1 cell line was further compared against all the metastatic cell lines in order to reveal the critical genes required for metastatic conversion but not expressed in the T1. These data demonstrated that expression of genes involved in apoptosis and immune cell homing were altered in all metastatic cell lines. Survival of both intravasated cells in circulation systems and extravasated cells in a new tissue environment might be critical for the final step in the metastatic process. Our study provides gene expression signatures consistent with two critical events in the metastatic process, namely, the acquisition of early homing capacity and increased survival potential of the tumor cells.  相似文献   

11.
Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Young's modulus of cells. In all cases the Young's moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40–80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.  相似文献   

12.
The non-receptor tyrosine kinase ABL drives myeloid progenitor expansion in human chronic myeloid leukemia. ABL inhibition by the tyrosine kinase inhibitor nilotinib is a first-line treatment for this disease. Recently, ABL has also been implicated in the transforming properties of solid tumors, including triple negative (TN) breast cancer. TN breast cancers are highly metastatic and several cell lines derived from these tumors display high invasive activity in vitro. This feature is associated with the activation of actin-rich membrane structures called invadopodia that promote extracellular matrix degradation. Here, we investigated nilotinib effect on the invasive and migratory properties of different TN breast cancer cell lines. Nilotinib decreased both matrix degradation and invasion in the TN breast cancer cell lines MDA-MB 231 and MDA-MB 468. However, and unexpectedly, nilotinib increased by two-fold the invasive properties of the TN breast cancer cell line BT-549 and of Src-transformed fibroblasts. Both display much higher levels of ABL kinase activity compared to MDA-MB 231. Similar effects were obtained by siRNA-mediated down-regulation of ABL expression, confirming ABL central role in this process. ABL anti-tumor effect in BT-549 cells and Src-transformed fibroblasts was not dependent on EGF secretion, as recently reported in neck and squamous carcinoma cells. Rather, we identified the TRIO-RAC1 axis as an important downstream element of ABL activity in these cancer cells. In conclusion, the observation that TN breast cancer cell lines respond differently to ABL inhibitors could have implications for future therapies.  相似文献   

13.
14.
Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.Approximately half of the patients with pancreatic cancer are initially diagnosed with metastases to distal sites, with the commonest sites being the liver, lung, and peritoneum (1). Therapeutic strategies against metastases could help reduce the high mortality rates associated with this cancer (2). Understanding the nature of metastatic pancreatic cancer at a systems level can enable the discovery of potential targets for the development of targeted therapies.Pancreatic cancer has been shown to be a genetically evolving and heterogeneous disease (35). Clonal diversity and evolution of cancer genomes have also been demonstrated based on the isolation of distinct clonal populations purified directly from patient biopsies by means of flow cytometry followed by genomic characterization (6). A number of reports have documented the adoption of a proteomic approach for the discovery of potential biomarkers in pancreatic cancer (7, 8). However, these studies generally assume pancreatic cancers to be homogeneous, and the emphasis is placed on identifying molecules that are common across a broad array of tumors. There is a lack of studies systematically examining the proteomic changes or signaling pathways across pancreatic cancers to dissect the nature of the heterogeneity of each clone. An excellent setting in which the heterogeneity of tumors can be studied systematically is in a patient harboring metastases to several distant sites. To this end, we chose cells isolated from three metastatic pancreatic lesions of a single patient. The exomes of each tumor site were previously sequenced to study the progression of pancreatic cancer, and the results showed that all cell lines were identical for the genetic status of driver mutations (e.g. KRAS, TP53, and SMAD4) (9). Our hypothesis was that a better understanding of the proteomic consequences of the heterogeneity derived from genetic changes, and possibly other types of alterations, might provide additional opportunities to identify therapeutic targets.In order to precisely quantify differences across the proteomes of multiple metastatic pancreatic cancer lesions, we employed a SILAC-based1 quantitative proteomics strategy combined with high-resolution mass spectrometry (10, 11). Based on changes observed at the whole-proteome level, we found that a class of cell surface receptors showed significant enrichment with the highest alteration of their expression among the three metastatic pancreatic cancer cell lines examined (i.e. peritoneum, lung, and liver). Because the total protein levels provide information about the static levels of proteins and not their activity per se, we decided to examine the activation of phosphorylation-driven pathways, many of which are activated by cell surface receptors. To globally examine tyrosine phosphorylation-based signaling pathways, we carried out mass spectrometric analysis of purified tyrosine phosphorylated peptides enriched using anti-phosphotyrosine antibodies. As a result, we observed differential activation of tyrosine kinases in the three different sites of metastases. For example, Axl receptor tyrosine kinase was found to be hyperphosphorylated in lung and liver metastases relative to peritoneal metastasis. Expression of Axl receptor tyrosine kinase in primary and matched pancreatic cancers on tissue microarrays was validated by immunohistochemistry. Given such unique patterns of activation of pathways, it was possible that tumors derived from different sites could show differences in their sensitivity to pathway inhibitors. To test this, we performed experiments in which we screened cell lines derived from each metastatic site against a panel of small molecule inhibitors. We observed that the three metastatic pancreatic cancers had differential sensitivities to different inhibitors. For example, cells derived from the peritoneal metastasis were highly sensitive to lapatinib, whereas greater sensitivity to the Axl inhibitor R428 was observed in the lung metastasis cell line. Finally, we showed that treatment of mice bearing xenografts from these different pancreatic cancer cell lines with R428, an inhibitor of Axl receptor tyrosine kinase, led to reduction of tumors with evidence of activation of Axl.  相似文献   

15.
Hepatocellular carcinoma (HCC) is a prevalent disease worldwide, and the majority of HCC-related deaths occur due to local invasion and distant metastasis. Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have been hypothesized to be responsible for metastatic disease. Recently, we and others have identified a CSC population from human HCC cell lines and xenograft tumors characterized by their expression of CD133. However, the precise molecular mechanisms by which CD133+ cancer stem-like cells mediate HCC metastasis have not been sufficiently analyzed. Here, we have sorted HCC cells using CD133 as a cancer stem cell (CSC) marker by magnetic-activated cell sorting (MACS) and demonstrated that the CD133+ HCC cells not only possess greater migratory and invasive capacity in vitro but are also endowed with enhanced metastatic capacity in vivo and in human HCC specimens when compared to CD133 HCC cells. Gene expression analysis of the CD133+ and CD133 cells of the HCC line SMMC-7721 revealed that G protein-coupled receptor 87 (GPR87) is highly expressed in CD133+ HCC cells. In this study, we explored the role of GPR87 in the regulation of CD133 expression. We demonstrated that the overexpression of GPR87 up-regulated CD133 expression, promoted CSC-associated migratory and invasive properties in vitro, and increased tumor initiation in vivo. Conversely, silencing of GPR87 expression reduced the levels of CD133 expression. Conclusion: GPR87 promotes the growth and metastasis of CD133+ cancer stem-like cells, and our findings may reveal new targets for HCC prevention or therapy.  相似文献   

16.
Activating mutations in Gαq/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gαq/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gαq-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation GαqQ209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gαq. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gαq, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with GαqQ209L as a partial effector antagonist.  相似文献   

17.
18.
19.

Background

In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2.

Principal Findings

Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.

Conclusions

Cancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression.  相似文献   

20.
In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.  相似文献   

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