Modulate Cancer Cell Invasion Research Articles

Obstructive sleep apnea promotes the progression of lung cancer by modulating cancer cell invasion and cancer-associated fibroblast activation via TGFβ signaling

ABSTRACT Objective: Obstructive sleep apnea (OSA) is associated with severity of pneumonia; however, the mechanism by which OSA promotes lung cancer progression is unclear. Methods: Twenty-five lung cancer patients were recruited to investigate the relationship between OSA and cancer-associated fibroblast (CAFs) activation. Lung cancer cells (A549) and WI38 fibroblast cells were used to explore the hypoxia-induced TGFβ expression using qPCR, Western blot, and ELISA. Wound healing and transwell assays were performed to evaluate cancer cell migration and invasion. A549 or A549-Luc + WI38 xenograft mouse models were established to detect the intermittent hypoxia (IH) associated with lung tumor growth and epithelial–mesenchymal transition (EMT) in vivo. Results: OSA promotes CAF activation and enrichment in lung cancer patients. Hypoxia (OSA-like treatment) activated TGFβ signaling in both lung cancer cells and fibroblasts, which promoted cancer cell migration and invasion, and enriched CAFs. IH promoted the progression and EMT process of lung cancer xenograft tumor. Co-inoculation of lung cancer cells and fibroblast cells could further promote lung cancer progression. Conclusions: IH promotes lung cancer progression by upregulating TGFβ signaling, promoting lung cancer cell migration, and increasing the CAF activation and proportion of lung tumors.

Notch/CXCR4 Partnership in Acute Lymphoblastic Leukemia Progression.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Recent advances in chemotherapy have made ALL a curable hematological malignancy. In children, there is 25% chance of disease relapse, typically in the central nervous system. While in adults, there is a higher chance of relapse. ALL may affect B-cell or T-cell lineages. Different genetic alterations characterize the two ALL forms. Deregulated Notch, either Notch1 or Notch3, and CXCR4 receptor signaling are involved in ALL disease development and progression. By analyzing their relevant roles in the pathogenesis of the two ALL forms, new molecular mechanisms able to modulate cancer cell invasion may be visualized. Notably, the partnership between Notch and CXCR4 may have considerable implications in understanding the complexity of T- and B-ALL. These two receptor pathways intersect other critical signals in the proliferative, differentiation, and metabolic programs of lymphocyte transformation. Also, the identification of the crosstalks in leukemia-stroma interaction within the tumor microenvironment may unveil new targetable mechanisms in disease relapse. Further studies are required to identify new challenges and opportunities to develop more selective and safer therapeutic strategies in ALL progression, possibly contributing to improve conventional hematological cancer therapy.

FAK family kinases: The Yin and Yang of cancer cell invasiveness

ABSTRACTProline-rich tyrosine kinase 2 (PYK2, also known as Pyk2) and its closely related focal adhesion kinase (FAK) modulate cancer cell invasion by coordinating the balance between focal adhesion-mediated migration and invadopodia-dependent extracellular matrix invasion. Our recent findings present Pyk2 and FAK as novel mediators of breast cancer invasiveness and as potential targets for blocking breast cancer metastasis.

MicroRNA-34a: A Versatile Regulator of Myriads of Targets in Different Cancers

MicroRNA-34a (miR-34a) is a tumor suppressor that has attracted considerable attention in recent years. It modulates cancer cell invasion, metastasis, and drug resistance, and has also been evaluated as a diagnostic and/or prognostic biomarker. A number of targets of miR-34a have been identified, including some other non-coding RNAs, and it is believed that the modulation of these myriads of targets underlines the versatile role of miR-34a in cancer progression and pathogenesis. Seemingly appealing results from preclinical studies have advocated the testing of miR-34a in clinical trials. However, the results obtained are not very encouraging and there is a need to re-interpret how miR-34a behaves in a context dependent manner in different cancers. In this review, we have attempted to summarize the most recent evidence related to the regulation of different genes and non-coding RNAs by miR-34a and the advances in the field of nanotechnology for the targeted delivery of miR-34a-based therapeutics and mimics. With the emergence of data that contradicts miR-34a’s tumor suppressive function, it is important to understand miR-34a’s precise functioning, with the aim to establish its role in personalized medicine and to apply this knowledge for the identification of individual patients that are likely to benefit from miR-34a-based therapy.

Possibilities of Radiosensitization and Reduced Invasion After Targeting the SCF/c-Kit Pathway in Human Cancer Cells

The SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced proliferation and invasion. Resulting therapy resistance and metastasis often are limiting factors of a successfull radiotherapy. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a reduced cancer cell invasion and whether a significant increased radiosensitivity could be achieved. Experiments were performed using different NSCLC (H23, H226, H460, H520, H1299, H1975, A549) and prostate cancer cell lines (DU 145, PC3, LnCAP) growing either under 2D or 3D conditions. SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cells were irradiated with 2 Gy or 6 Gy Photons. Cell survival was determined by colony formation assay. Invasion was measured using BioCoat™ Matrigel™ invasion chambers. When grown in 3D a 2-3-fold increase in invasion capability was examined in all NSCLC and prostate cancer cell lines except H520. This could be explained by EMT-like changes (enhanced expression of vimentin and N-cadherin and reduction of E-cadherin) when cells were grown in 3D. Just the opposite trends were found for H520 cells. Knock down of SCF was generally found to result in significantly reduced invasion and in only a minor increase of cellular radiosensitivity (2D and 3D). Only in cells characterized by a massive overexpression of SCF (H520) a significant increase of cellular radiosensitivity could be obtained after SCF knock down. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. Photon irradiation did not modulate cancer cell invasion (2D and 3D). For the regulation of tumor cell motility and invasion the SCF/c-Kit pathway appears to take a key position. Inhibition of this pathway significantly reduces the cell invasion of human NSCLC and prostate cancer cells under 2D and 3D conditions. In addition our data indicate that generally cell survival and radioresponse are not strongly determined by the SCF/c-Kit pathway. As a consequence targeting SCF or c-Kit only results in increased radiosensitivity when SCF is strongly over expressed as found for H520 cells.

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments

Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100–200μm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. Statement of SignificanceThreedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression.

FNC, a Novel Nucleoside Analogue, Blocks Invasion of Aggressive Non-Hodgkin Lymphoma Cell Lines Via Inhibition of the Wnt/β-Catenin Signaling Pathway

Chemotherapy is the primary therapy for malignant lymphoma (ML). However, the clinical outcome is still far from satisfactory. Consequently, an understanding of the mechanism of modulating cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. FNC, 2'- deoxy- 2' -β- fluoro -4'- azidocytidine, a novel cytidine analogue, has demonstrated significantly inhibitory effects on proliferation of several non-Hodgkin lymphoma (NHL) cell lines. A previous study indicated that FNC effectively inhibited the growth of Raji and JeKo-1 cells in dose-time dependent effects with IC50 values of 0.2μM and 0.097μM, respectively. This study was focused on investigating the anti-invasive properties of FNC on NHL cells and its potential mechanisms of action. Cell adhesion and transwell chamber assays were utilized to investigate the anti-invasive effects of FNC on Raji and JeKo-1 cells. Real-time PCR and Western blotting were employed to qualify the expression of β-catenin, the glycogen synthase kinase-3 beta (GSK-3β), E-cadherin vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The results revealed that FNC remarkably inhibited the adhesion, migration and invasion of two human aggressive non-Hodgkin lymphoma cell lines in a dose dependent manner. Furthermore, β-catenin, MMP-2, MMP-9, VEGF mRNA and protein levels were decreased after FNC treatment, while GSK-3β and E-cadherin increased. Our studies thus provide evidence and a rationale that FNC may offer an effective chemotherapeutic agent by regulating the invasion and metastasis of aggressive non-Hodgkin lymphoma via inhibition of the Wnt/β-catenin signaling pathway.

β-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α

Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While β-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of β-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of β-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with β-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following β-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered β-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following β-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following β-carotene treatment. In conclusion, the present study provides the first evidence that β-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.

Abstract B165: The antitumor efficacy of eribulin is mediated via suppression of invasion and profound induction of cell death in triple negative breast cancer.

Abstract Eribulin, a microtubule-targeting antitumor drug, is FDA-approved for the treatment of certain patients with advanced or metastatic breast cancer. Some patients treated with this drug have evidence of reduced metastatic dissemination. Triple-negative breast cancer (TNBC), which accounts for approximately 15%-20% of all breast cancers, does not respond to hormone therapies so patients are dependent on chemotherapy. A subset of these patients are intrinsically resistant to chemotherapy, or subsequently relapse and develop metastatic disease, eventually leading to mortality. We explored the mechanistic basis for the antitumor efficacy of eribulin in a panel of triple negative breast cancer cell lines that histologically are classified as basal-like or claudin-low. Specifically, we determined the ability of eribulin to induce cell death versus senescence, and secondly to modulate cancer cell invasion and epithelial to mesenchymal transition (EMT) gene expression. Senescence is a clinically relevant cell fate following chemotherapy that can propel tumor progression and EMT. Eribulin was efficacious at inducing cell death in both subtypes of TNBC, and had a remarkably low propensity to induce senescence in TNBC compared to non-small cell lung cancer and colon cancer cells. A 24-hour treatment with eribulin partially prevented tumor cell invasion in the claudin-low subtype, as assayed by Boyden chamber analysis. Drug-tolerant claudin-low cells that survived high-dose eribulin treatment were also found to have a reduction in EMT markers. In summary, in vitro analysis of cell fate and metastasis indicate that eribulin has an ability to modulate invasiveness and EMT characteristics in preclinical models of TNBC. This research was funded by Eisai Inc. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B165. Citation Format: Jing Hu, Susan Band Horwitz, Hayley M. McDaid. The antitumor efficacy of eribulin is mediated via suppression of invasion and profound induction of cell death in triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B165.

Mechanical regulation of cancer cell apoptosis and autophagy: Roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK

Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm2 induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm2, oscillatory shear stress (OSS) at 0.5±4dyn/cm2 cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V–FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.

Mulberry Leaf Extract Inhibits Invasive Potential and Downregulates Hypoxia-Inducible Factor-1α (HIF-1α) in SK-N-BE(2)C Neuroblastoma Cells

A neuroblastoma is an extracranial solid tumor diagnosed in childhood. Since tumor metastasis is the main cause of death for most neuroblastoma patients, an understanding of the mechanisms that modulate cancer cell invasion is a key to developing more effective chemotherapeutic agents. In the current study, we examined to determine whether mulberry leaf (ML) extract effectively inhibits the invasion potential of neuroblastoma cells in vitro. ML extract was found to suppress cell invasiveness as well as the activity and expression of matrix metalloproteinase-2 (MMP-2) under both normoxia and hypoxia in neuroblastoma. ML extract downregulated the expression of hypoxia-inducible factor-1α (HIF-1α), a well-known regulator of tumor metastasis, and its downstream targets, vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT-1). Taken together, these results suggest that ML extract has chemotherapeutic effects on neuroblastoma cells by regulating invasion potential, thereby controlling the metastasis of neuroblastomas.

Cell surface heat shock protein 90 and tumor metastasis

Heat shock protein 90(Hsp90)is one of the most abundant molecular chaperones whose association is essential to maintain the stability and function of uumerous client proteins,and is the unique target protein for cancer therapy.Over the past years,most attention has been given to the study of the intracelhilar Hsp90.However,a pool of Hsp90 has been described to be present on the cell surface and even to be secreted extracellularly,which plays an important role in modulating cancer cell invasion in vitro and metastasis in vivo. Key words: HSP90 heat-shock proteins; Neoplasm metastasis

INVOLVEMENT OF S100A6 (CALCYCLIN) IN PANCREATIC CANCER PROGRESSION

Introduction: The molecular profiling of pancreatic cancer revealed an over-expression of a member of the S100 family of calcium binding proteins, S100A6. A recent study from our laboratory reported an association between elevated levels of S100A6 in pancreatic ductal adenocarcinoma cells and poor survival in pancreatic cancer patients. In the present study we investigated the effects of siRNA mediated depletion of S100A6 on pancreatic cancer motility and invasion. Furthermore, we determined potential binding partners of S100A6 in pancreatic cancer cells and the mechanism by which S100A6 might modulate cancer cell invasion. Methods: RNA interference was used to reduce S100A6 expression in three pancreatic cancer cell lines. Cell motility and invasion were assessed using Boyden and Matrigel chambers respectively. We also used Proteins co-immunoprecipitated with anti S100A6 followed by two-dimensional gel electrophoresis and MALDI-MS to find out the S100A6 binding proteins. The levels of released MMPs in cell culture supernatant were confirmed by gelatine zymography. Results: We observed that siRNA-mediated suppression of S100A6 protein significantly reduced pancreatic cancer cell motility and invasion. A number of potential S100A6 binding proteins were identified including annexin A2, a mediator of the plasmin/plasminogen activator system, and tropomyosin beta, an actin-binding protein involved in stabilization of actin filaments and control of cell motility. The siRNA for S100A6 reduced the MMP-2 activity as shown by gelatine zymography. Conclusion: These results suggest that S100A6 expression is correlated with the motility and invasion of pancreatic cancer cells. S100A6 might contribute to disease progression by influencing the function of MMP-2.

Fibronectin induces colon cancer MMP-2 activation through a Src- and NfκB-dependent mechanism

Background. MMP-2 is a matrix metalloproteinase implicated in colon cancer invasion. MMP-2 is secreted in latent form (72 kDa) then cleaved into the active form (69 kDa) by membrane-bound type MMP (MT1-MMP). The signals regulating MMP-2 activation are poorly understood. Fibronectin is a major extracellular matrix component of cancer-associated stroma. Because integrin-mediated interactions with fibronectin are implicated to modulate cancer cell invasion, here we tested the hypothesis that fibronectin regulates MMP-2 activation through a Src- and NFκB-dependent signal. Methods. We tested three human colon cancer cell lines, SW480, HCT116, and HT29. Cells were seeded onto noncoated and fibronectin- or laminin-coated plates. MMP-2 activation was assessed by zymography and Western blotting. MT1-MMP expression, Src activation, and NFκB activation were assayed using Western blotting, immunoprecipitation, and EMSA, respectively. We treated cells with anti-integrin blocking antibody, PP2, or PDTC to inhibit cell attachment to fibronectin, Src, or NFκB, respectively. Results. Minimal NFκB activation was detected under baseline conditions in each of the three cell lines. Fibronectin, but not laminin, induced MT1-MMP, Src phosphorylation, and NFκB activation at 6 h in all three cell lines, with concomitant induction of active form of MMP-2. Anti-α4β1, but not α5β1 integrin blocking antibody (2.5 μg/ml) suppressed MT-MMP1 expression (by 84, 80, and 82% for SW480, HCT116, and HT29 respectively, P < 0.05). PP2 (20 μM), and PDTC (100 μM) suppressed Src activation (by 93, 91, and 88% for each cell line, P < 0.05) and NFκB activation (by 95, 92, and 88% for each cell line, P < 0.05), respectively, and they each abolished fibronectin-induced MT1-MMP expression as well as MMP-2 activation. Conclusion. These findings suggested that fibronectin induces MMP-2 activation by MT1-MMP through a α4β1 integrin, -Src-, and NFκB-dependent signal in colon cancer cells. This signaling pathway may represent a therapeutic target for strategies designed to inhibit colon cancer progression.