Invasive Phenotype Of Tumor Cells Research Articles

Biomimetic “nano-spears” for CAFs-targeting: splintered three “shields” with enhanced cisplatin anti-TNBC efficiency

The treatment dilemma of triple-negative breast cancer (TNBC) revolves around drug resistance and metastasis. Cancer-associated fibroblasts (CAFs) contribute to cisplatin (Cis) resistance and further metastasis in TNBC, making TNBC a difficult-to-treat disease. The dense stromal barrier which restricts drug delivery, invasive phenotype of tumor cells, and immunosuppressive tumor microenvironment (TME) induced by CAFs serve as three “shields” for TNBC against Cis therapy. Here, we designed a silybin-loaded biomimetic nanoparticle coated with anisamide-modified red blood cell membrane (ARm@SNP) as a “nanospear” for CAFs-targeting, which could shatter the “shields” and significantly exhibit inhibitory effect on 4T1 cells in combination with Cis both in vitro and in vivo. The ARm@SNP/Cis elicited 4T1 tumor growth arrest and destroyed three “shields” as follows: disintegrating the stromal barrier by inhibiting blood vessels growth and the expression of fibronectin; decreasing 4T1 cell invasion and metastasis by affecting the TGF-β/Twist/EMT pathway which impeded EMT activation; reversing the immunosuppressive microenvironment by increasing the activity and infiltration of immunocompetent cells. Based on CAFs-targeting, ARm@SNP reversed the resistance of Cis, remodeled the TME and inhibited invasion and metastasis while significantly improving the therapeutic effect of Cis on 4T1 tumor-bearing mice, providing a promising approach for treating intractable TNBC.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

556 Background: Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Methods: Here, we generated genomic, transcriptomic, and T cell repertoire data coupled with digital pathology and digital spatial profiling of 257 primary, 28 relapsed, and 176 metastatic regions from 182 HCC patients. Results: Primary tumors rich in hypoxia signatures tended to give rise to polyclonal dissemination, which was associated with a poor clinical outcome. Genomic divergence between primary and metastatic HCC was high, and early dissemination was prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases was associated with decreased T cell reactivity, which may have resulted from disruptions to neoantigen presentation. We identified macroevolutionary somatic copy number alterations as highly selected events driving metastasis. Furthermore, we found that subclonal Wnt pathway mutations were not selected during spreading, whereas subclones without Wnt mutations showed a strong selective advantage for metastasis and were characterized by a reactive microenvironment rich in activated fibroblasts favoring an invasive tumor cell phenotype. Finally, metastases without Wnt mutations exhibited higher enrichment of immunosuppressive B cells that mediated terminal exhaustion of CD8+ T cells via HLA-E – NKG2A checkpoint axis. Conclusions: Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Identification and Characterization of Tunneling Nanotubes Involved in Human Mast Cell FcεRI-Mediated Apoptosis of Cancer Cells.

Simple SummaryMast cells (MCs) are ubiquitously found in most tissues and in and around tumors. Their role in cancer pathogenesis remains an open area of investigation, and their interactions with tumor cells has not been explored. Here, a novel mechanism of communication between human MCs and tumor cells involving tunneling nanotubes (TnT) and other membrane structures is described. The formation of these communication structures is dependent on MC receptors interacting with tumor antigens through tumor-specific immunoglobulins and results in tumor-killing mediators from MC entering the tumor cells. This mechanism underlying the MC killing of tumor cells has important implications in understanding cancer pathogenesis.Mast cells (MCs) are found in practically all tissues where they participate in innate and adaptive immune responses. They are also found in and around tumors, yet their interactions with cancer cells and the resulting impact on cancer cell growth and metastasis are not well understood. In this study, we examined a novel mechanism of IgE-FcεRI-mediated, intercellular communication between human adipose-derived mast cells (ADMC) and cancer cells. The formation of heterotypic tunneling nanotubes (TnT) and membrane structures between MCs and tumor cells in vitro was examined using microscopy and a diverse array of molecule-specific indicator dyes. We show that several MC-specific structures are dependent on the specific interactions between human tumor IgE-sensitized MCs and antigens on the tumor cell surface. The formation of TnT, membrane blebs and other MC-specific structures paralleled FcεRI-degranulation occurring within 30 min and persisting for up to 24 h. The TnT-specific adhesion of FcεRI-activated MCs to tumor cells was characterized by the transport of the MC granule content into the tumor cells, including tryptase and TNF-α. This interaction led to apoptosis of the tumor cells, which differs from previous studies examining tissue cells within the cancer microenvironment. The formation of heterotypic TnT results in stimulation of an invasive tumor cell phenotype and increased tumor cell invasion and chemoresistance of the cancer cells. These studies describe a heretofore-unrecognized mechanism underlying IgE-mediated interactions and FcεRI-activated MC-mediated killing of tumor cells through the formation of TnT.

Abstract A038: uPA/uPAR/MMP9 signaling hindered by heme oxygenase-1 in prostate cancer: Impact on cell invasiveness

Abstract Cell motility plays a crucial role in cancer metastasis. The actin cytoskeleton behaves as the cellular engine behind this process and is governed by complex signaling transduction cascades. Cancer cells have shown a variety of alterations both in the expression and localization of cytoskeleton-regulating proteins, which increase their capacity to migrate and invade homing organs. Heme oxygenase 1 (HO-1) is a stress response protein and a critical mediator of cellular homeostasis. Although HO-1’s role in cancer has been controversial, we have previously shown that its pharmacologic or genetic upregulation is associated with a less aggressive phenotype in PCa. HO-1 inhibits cell proliferation, migration, and invasion; impairs tumor growth and angiogenesis in vivo; and downregulates the expression of target genes associated with inflammation. We have also demonstrated that HO-1 is implicated in prostate cancer (PCa) cell adhesiveness and zippering (an increment in the number of filopodia between cells as well as a decrease in the levels of MMP9 (RTqPCR, Zimography, and Microarray analysis) favoring a more adhesive phenotype. However, the molecular mechanism behind this modulation was not yet deciphered. In this work we first assessed the presence of MMP9 in filopodia-like protrusions between neighboring cells to rule out that these increased protrusions were invadopodia structures. HO-1 was induced pharmacologically (hemin 80 uM, 24 h), fixed with PFA and stained with anti-HO-1 and anti-MMP9 antibodies. PCa cells were imaged by confocal microscopy. We observed that MMP9 did not colocalize with HO-1 and was absent at the leading edge of PCa cells. It is broadly known that MMP9 and the plasminogen activator urokinase (uPA) are important factors for cancer invasion and metastasis, both of them responsible for degrading the extracellular matrix (ECM). To assess whether the forced expression of HO-1 modulated the MMP9/uPA pathway, we studied the expression of several inhibitors and activators of this cascade through a transcriptomics approach. The RNAseq profiling for both PCa cells overexpressing HO-1 pharmacologically or genetically showed a direct regulation of the critical factors in the uPA/uPAR cascade, such as the downregulation of the axis activators uPA/uPAR (PLAU/PLAUR) and tPA (PLAT), and the upregulation of the axis inhibitors CPB2 (thrombin activator of fibrinolysis inhibitor), SERPINF2 (alpha 2 anti- plasmin), and F12 (factor XIIA). Our findings were further validated by an uPA ELISA revealing that under the same experimental conditions, the levels of this urokinase secreted to the conditioned media were significantly reduced. Altogether, our work delineates a molecular axis by which HO-1 potentially shuts down the acquisition of an invasive tumor cell phenotype, crucial for PCa cancer metastasis. Citation Format: Alejandra V. Paez, Javier H. Cotignola, Elba S. Vazquez, Geraldine Gueron. uPA/uPAR/MMP9 signaling hindered by heme oxygenase-1 in prostate cancer: Impact on cell invasiveness [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A038.

Abstract 4269: Interstitial fluid pressure alters cell motility and collective invasion via EMT marker expression in an engineered model of a human breast tumor

Abstract We developed a three-dimensional (3D) engineered model of a solid human breast tumor to study the effects of interstitial fluid pressure (IFP) on collective invasion and the expression levels of epithelial-mesenchymal transition (EMT) markers. Many solid tumors exhibit elevated IFP; as these tumors grow, intra-tumoral vascular and lymphatic vessels collapse. The non-functioning lymphatic system impairs drainage, and immature hyperpermeable blood vessels cause fluid to accumulate within the interstitial space. As a result, IFP rises steeply beyond the tumor periphery and plateaus at pressures as high as 50 mm Hg above normal at the tumor core. This pressure profile, in turn, leads to outward fluid flow from the core of the tumor. IFP has been shown to affect the migratory behavior of individual cells in 3D cell culture models, though its role in collective cancer invasion remains unknown. Moreover, the underlying molecular mechanisms linking IFP to changes in cell motility remain unclear. We sought to address these questions using our engineered model. Our 3D culture model consists of an aggregate of MDA-MB-231 breast cancer cells (mimicking a solid tumor) embedded within a 3D collagen gel that is flanked by two media reservoirs. The IFP profile experienced by the cancer cells is established by altering the heights of the media reservoirs on either side of the collagen, creating a hydrostatic pressure gradient. Transcript levels of EMT markers in the aggregates subjected to a variety of pressure profiles were determined using quantitative real-time PCR. Expression of these markers was also manipulated ectopically through the creation of stable cell lines. Time-lapse imaging and cell tracking were used to determine the persistence and motility of individual cells within the aggregates. We found that the direction of IFP-induced flow determines the invasive phenotype of tumor cells. Additionally, high expression levels of both mesenchymal (Snail1, vimentin) and epithelial (E-cadherin, keratin-8) markers were characteristic of collectively invading aggregates, suggesting that partial EMT is important for collective invasion. Ectopic expression and knockdown of EMT markers revealed that they are necessary and sufficient for collective invasion in response to IFP. Time-lapse imaging analysis demonstrated that IFP and EMT marker expression also affect the motility and persistence of individual cells within the aggregates, further confirming that IFP is an important regulator of collective invasion. In conclusion, we used a robust culture model of a human breast tumor to gain insight into the mechanisms guiding collective invasion from primary tumors in response to IFP; IFP alters expression levels of EMT markers, thereby regulating collective invasion. Citation Format: Alexandra S. Piotrowski-Daspit, Joe Tien, Celeste M. Nelson. Interstitial fluid pressure alters cell motility and collective invasion via EMT marker expression in an engineered model of a human breast tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4269.

Abstract 5006: A 3-dimensional cell invasion assay compatible with high content screening

Abstract The transition from non-invasive phenotype to invasive phenotype of tumor cells marks the switch from a benign tumor that can be successfully treated via surgery to a more malignant form of cancer. Understanding the mechanisms underlying this hallmark event, which enables tumor cells to invade through extracellular matrix, is critical for discovering pathways and new targets to develop anti-metastatic strategies. Previously, we developed a cell based assay for the study of cell migration in 96-well plates. A self-dissolving biocompatible gel (BCG) is utilized to form uniformly sized, cell-free detection zones in collagen I coated 96-well plates. When cells are seeded into these wells, they pattern in an annular monolayer surrounding the BCG. Once the BCG dissolves, an overlay of collagen I is applied to the assay wells, following which cells can invade in 3-dimensions into the detection zone previously occupied by the BCG. This “Oris™ Pro” cell invasion assay presents a straightforward, accessible and quantifiable cell method to study cell invasion, and can be used for example to evaluate candidate drugs targeting tumor invasion. Microscopic visual assessment of cell invasion in the presence of inhibitors is made by use of cell stains at multiple time-points. In this study we demonstrate the use of this assay platform for robust and reproducible analysis of HT-1080 and MDA- MB-231 cell invasion in 3-D. The presentation will discuss (i) factors that influence cell invasion, including density of collagen overlay and time; (ii) microscopic monitoring of cell invasion in the presence of potential inhibitors throughout the duration of the experiment; and (iii) the qualitative and quantitative readouts that can be obtained using this 3-dimensional cell invasion assay. Citation Format: Jennifer A. Fronczak, Matthew S. Gajeski, Erica L. Beckman, Laura Vollmer, Andreas Vogt, Gopal Krishnan. A 3-dimensional cell invasion assay compatible with high content screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5006. doi:10.1158/1538-7445.AM2014-5006

Potentiation of tumor cell invasion by co-culture with monocytes accompanying enhanced production of matrix metalloproteinase and fibronectin

Macrophages are a major population of immune cells, and those that infiltrate into tumor tissues and affect the malignant behavior of tumor cells are called tumor-associated macrophages (TAMs). We previously reported that human peripheral blood monocytes could be induced in vitro to differentiate into TAM-like cells by co-culture with tumor cells. In the present study, we characterized changes in the invasive phenotype of tumor cells after co-culture with monocytes, and found that MKN1 gastric carcinoma cells acquired higher invasive potential into Matrigel-reconstituted basement membranes, accompanied by enhanced production of matrix metalloproteinase (MMP)-9. The increased invasiveness was inhibited in the presence of an arginyl-glycyl-aspartic acid peptide, suggesting that the process is dependent on the integrin-extracellular matrix interaction. We also found that these cells secreted fibronectin into the culture medium and expressed α5 integrin on their surface at higher levels after the co-culture with monocytes for 5days. The conditioned medium of monocytes also potentiated MKN1 cell invasion; however, the potentiation was lowered by the depletion of tumor necrosis factor (TNF)-α from the conditioned medium with an antibody-protein G-Sepharose conjugate. In addition, the treatment of MKN1 cells with TNF-α promoted invasion of these cells, as well as secretion of MMP-9 and fibronectin. These results suggest that TNF-α secreted from monocytes is, at least in part, involved in the changes in invasive phenotype of tumor cells during co-culture with monocytes.

Mitochondrial redox signaling and cancer invasiveness

The concept that invasive cancer is associated with increased levels of reactive oxygen species (ROS) generated by mitochondria is consistent with an ROS-mediated signaling mechanism. As a tumor grows, it encounters adverse microenvironments, one of which is low oxygen (hypoxia), which selects tumor cells with characteristics of increased invasiveness. Hypoxic environments select for tumor cells with stabilized HIF1 apha, a transcription factor that regulates genes coding for pro-tumor cytokines that signal stromal cells such as macrophages and fibroblasts to support an invasive tumor cell phenotype. HIF1 alpha-mediated switches in the energy production of tumor cells from OXPHOS to glycolysis, as well as age-associated decreases in the metabolic rate of the host, enhance invasive qualities of tumor cells. An increase in environmental oxygen in combination with a mitochondrial targeted catalase mimetic and a metabolism booster may be of interest to investigate as a treatment strategy for invasive cancer.

SPARC functions as an anti-stress factor by inactivating p53 through Akt-mediated MDM2 phosphorylation to promote melanoma cell survival

Aberrant expression of Secreted Protein Acidic and Rich in Cysteine (SPARC)/osteonectin has been associated with an invasive tumor cell phenotype and poor outcome in human melanomas. Although it is known that SPARC controls melanoma tumorigenesis, the precise role of SPARC in melanoma cell survival is still unclear. Here, we show that SPARC has a cell-autonomous survival activity, which requires Akt-dependent regulation of p53. Suppression of SPARC by RNA interference in several human melanoma cells and xenografted A375 tumors triggers apoptotic cell death through the mitochondrial intrinsic pathway and activation of caspase-3. Cell death induced by depletion of SPARC is dependent on p53 and induction of Bax, and results in the generation of ROS. Stabilization of p53 in SPARC-depleted cells is associated with a decrease in Akt-mediated activating phosphorylation of MDM2. Inhibition of Akt signaling pathway is important for the observed changes as overexpression of constitutively active Akt protects cells against apoptosis induced by SPARC depletion. Conversely, increased expression of SPARC stimulates Akt and MDM2 phosphorylation, thus facilitating p53 degradation. Finally, we show that overexpression of SPARC renders cells more resistant to the p53-mediated cytotoxic effects of the DNA-damaging drug actinomycin-D. Our study indicates that SPARC functions through activation of Akt and MDM2 to limit p53 levels and that acquired expression of SPARC during melanoma development would confer survival advantages through suppression of p53-dependent apoptotic pathways.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Lysosomal Acid Hydrolases of the Cathepsin Family Are Novel Targets of INSL3 in Human Thyroid Carcinoma Cells

Insulin-like peptide 3 (INSL3) is present in hyperactive and neoplastic thyrocytes, but the functional role of this relaxin-like peptide hormone during carcinogenesis in the thyroid gland is currently unknown. We generated new cell models of stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 expressing and secreting bioactive human INSL3. These transfectants displayed higher intracellular ATP levels, but INSL3 failed to act as a promoter of growth. The acquisition of an invasive tumor cell phenotype with local tissue invasion represents the beginning of a number of events leading to metastasis, the major cause of fatal outcome in cancer patients. Here we demonstrate a function of INSL3 in elastin degradation, which is considered an early step during basal membrane penetration and tissue invasion by tumor cells. INSL3 markedly increased the production of the lysosomal enzymes cathepsin-L and cathepsin-D. Enhanced secretion of the elastinolytic cathepsin-L was associated with increased elastinolytic activity of FTC-133-INSL3 transfectants. Thus, we provide the first evidence that the INSL3 peptide can promote early tumor cell invasiveness in human thyroid carcinoma cells by enhancing their metabolic activity and elastin-degrading potential.

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44(+) cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positive and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44(+) CSC-like cells invade Matrigel through an EMT, while in contrast, CD44(-) cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44(+)CD24(-) prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Collagen density promotes mammary tumor initiation and progression

BackgroundMammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen. Thus, the influence of the extracellular matrix on breast carcinoma development and the underlying molecular mechanisms are not understood.MethodsTo study the effects of collagen density on mammary tumor formation and progression, we utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue. Imaging of the tumors and tumor-stromal interface in live tumor tissue was performed with multiphoton laser-scanning microscopy to generate multiphoton excitation and spectrally resolved fluorescent lifetimes of endogenous fluorophores. Second harmonic generation was utilized to image stromal collagen.ResultsHerein we demonstrate that increased stromal collagen in mouse mammary tissue significantly increases tumor formation approximately three-fold (p < 0.00001) and results in a significantly more invasive phenotype with approximately three times more lung metastasis (p < 0.05). Furthermore, the increased invasive phenotype of tumor cells that arose within collagen-dense mammary tissues remains after tumor explants are cultured within reconstituted three-dimensional collagen gels. To better understand this behavior we imaged live tumors using nonlinear optical imaging approaches to demonstrate that local invasion is facilitated by stromal collagen re-organization and that this behavior is significantly increased in collagen-dense tissues. In addition, using multiphoton fluorescence and spectral lifetime imaging we identify a metabolic signature for flavin adenine dinucleotide, with increased fluorescent intensity and lifetime, in invading metastatic cells.ConclusionThis study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. Furthermore, the imaging techniques and signature identified in this work may provide useful diagnostic tools to rapidly assess fresh tissue biopsies.

PTEN gene transfer suppresses the invasive potential of human malignant gliomas by regulating cell invasion-related molecules

Loss of function of the tumor suppressor gene PTEN is more frequently encountered in high-grade malignant gliomas than in low-grade gliomas. High-grade gliomas are characterized by their extremely invasive behavior, suggesting that PTEN is one of the important regulators of cell motility and that alterations of its coding gene contribute to a much more invasive tumor cell phenotype. In order to clarify a role of PTEN in glioma invasion, we introduced the wild-type PTEN gene into human malignant glioma cell lines and investigated their motile and invasive activity in a brain slice model that presents circumstances analogous to normal brain conditions in vivo. In addition, we analyzed biochemical and molecular changes resulting from the transfer of PTEN in the glioma cells. Infection of recombinant replication-defective adenovirus vector containing the wild-type PTEN cDNA (Ad5CMV-PTEN) significantly inhibited the cell migration and invasion activities of PTEN-mutated glioma cell lines in in vitro migration and chemoinvasion assays. In an organotypic brain slice model, co-culture of glioma spheroids and rat brain slices demonstrated that Ad5CMV-PTEN transfected cells failed to invade surrounding normal brain tissues. Ad5CMV-PTEN transfer into the glioma cell lines lacking the wild-type gene product decreased the levels of matrix metalloproteinase (MMP)-2 mRNA and inhibited the enzymatic activities of MMP-2 and MMP-9. In contrast, mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-2 was upregulated by the PTEN gene transfer. Introduction of PTEN gene in glioma cell lines markedly reduced the levels of Rac-GTP and Cdc42-GTP, activated forms of these small GTP-binding proteins, and decreased the phosphorylation levels of focal adhesion kinase. These results suggest that PTEN inhibits glioma cell invasion in two ways: suppressing proteolysis of the extracellular matrix by MMPs and modulating the migratory activity of glioma cells to a less motile nature by inactivating two Rho-family GTP-binding proteins, Rac and Cdc42.

Transcriptional expression of the genes implicated in angiogenesis and tumor invasion in cervical carcinomas

Objective. Co-expression patterns of the genes implicated in angiogenesis and tumor invasion in cervical carcinoma cells were investigated together with invasive activity of tumor cells. Transcript levels of those genes were also compared between tumor cells and normal cervical tissues. Methods. Real-time quantitative RT-PCR analysis was conducted on selected 11 genes (total VEGF-A, VEGF 121, VEGF 165, VEGF 189, VEGF-B, C and D, bFGF, dThdPase, MMP-2 and uPA) using 11 cervical carcinoma cell lines and 14 normal cervical tissues. Protein expression of VEGF-C and MMP-2 and invasive activity of tumor cells were evaluated for each cell line by sandwich ELISA and haptoinvasion assay, respectively. Results. Gene co-expression analysis revealed the significant correlation between angiogenic factors and proteinases in malignant but not in normal cervical samples. Gene or protein expression levels of VEGF-C and MMP-2 were well correlated with the number of invaded tumor cells. VEGF-A splicing variants were increased in malignant compared to normal cervical samples but not associated with the invasive activity of the cells. Conclusion. VEGF-C and MMP-2 were closely related to invasive phenotype of tumor cells, whereas VEGF-A isoforms were considered to be involved in cervical carcinogenesis.

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells.

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.

Enhanced tenascin-C expression and matrix deposition during Ras/TGF-beta-induced progression of mammary tumor cells.

Overexpression of tenascin-C (TN-C) in breast carcinomas has been associated with a migratory or even invasive tumor cell phenotype. The mechanisms regulating expression and matrix deposition of TN-C in normal and cancerous breast tissues are, however, little understood. Here, we demonstrate that mouse mammary epithelial cells (EpH4) transformed by oncogenic Ha-Ras (EpRas) overexpress TN-C, which accumulates in the cytoplasm. When EpRas cells undergo epithelial-mesenchymal transition (EMT) in response to TGFbeta1, they secrete TN-C into the culture medium. In EpRas cells undergoing TGFbeta1-induced EMT in three-dimensional (3D)-collagen gel cultures, TN-C was deposited into an extracellular matrix (ECM) already containing fibronectin and perlecan. Under less physiological 2D plastic cultures, EpRas cells undergoing EMT failed to deposit TN-C into an (apparently incomplete) ECM. Ras-downstream signaling was dissected by pharmacological inhibitors and effector-specific Ras mutants (V12S35, V12C40), specifically inhibiting or activating ERK/MAPK or PI3K signaling, respectively. We showed that TN-C overexpression required a hyperactive ERK/MAPK-signaling pathway, while elevated PI3K signaling did not enhance TN-C expression. Similarly, tumors induced by cells exhibiting hyperactive ERK/MAPK signaling showed expression of TN-C in the tumor cells themselves, while only endothelial cells expressed TN-C in tumors caused by the V12C40 mutant (incapable of EMT in vivo). Taken together, our data indicate that hyperactive ERK/MAPK signaling causes enhanced expression of TN-C, while its secretion is induced by TGFbeta1 and both signals cooperate in TN-C matrix deposition. Importantly, both signals also cooperate to induce EMT in vitro and tumor progression/metastasis in vivo.

Role of matrix metalloproteinases 2 and 9 in determination of invasive potential of pancreatic tumors.

Our findings indicate that expression of matrix metalloproteinase-2 is an important prognostic marker of invasive potential of pancreatic tumors. Expression of this marker in neuroendocrine tumors (particularly in gastrinomas) can be used as a differential diagnostic criterion characterizing more malignant phenotype of tumors with the presence of ductal component. On the contrary, weak expression matrix metalloproteinase-9 is characteristic of less invasive phenotype of tumor cells (neuroendocrine and solid pseudopapillary tumors, mucinous cystadenomas). Co-expression of matrix metalloproteinases 2 and 9 is an unfavorable prognostic sign.

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma

Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases--cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human tumors. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor-infiltrated lymph nodes. Cathepsin B activity was determined in lung tumors, lung parenchyma, and tumor cell-infiltrated and noninfiltrated regional lymph nodes of the same patient. The authors investigated 35 cancer patients suffering from nonsmall cell lung carcinoma. Cathepsin B throughout activity was measured by cleavage of the fluorogenic substrate Z-Arg-Arg-AMC at pH 6.0. The median specific cathepsin B activity was highest in tumors, followed by the infiltrated lymph nodes, noninfiltrated lymph nodes, and lung parenchyma. The authors showed a significant 1.8-fold increase in cathepsin B activity in tumor-infiltrated lymph nodes compared with noninfiltrated regional lymph nodes and a 4.5-fold increase in lung tumor tissue compared with lung parenchyma. High cathepsin B activity, both in tumors and tumor cell-infiltrated lymph nodes, indicated poor prognosis for overall survival. Immunohistochemical analysis showed the presence of cathepsin B in histiocytes and tumor cells but not in lymphocytes of lymph node tissue. The authors' findings on higher cathepsin B levels in tumor cell-infiltrated lymph nodes show that increased level of cathepsin B activity is characteristic of the invasive tumor cell phenotype. This corroborates the hypothesis, that tumor cell associated cathepsin B may play a role in lymphogenic metastasis. The authors' results support the use of lymph node associated cathepsin B as a prognostic factor for survival of patients with lung carcinoma.