Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

An amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) is mitogenic for osteoblast-like cells

A peptide mitogen for cultured osteoblast-like cells was purified from serum-free conditioned culture medium of a human prostatic cancer cell line, PC-3. Based on amino acid sequencing and estimated molecular weight, this peptide was identified as an NH2-terminal fragment of urokinase-type plasminogen activator (uPA). Recombinant high molecular weight (HMW) uPA and the NH2-terminal growth factor domain (GFD) of uPA, but not low molecular weight (LMW) uPA (lacking the NH2-terminal region) stimulated [3H] thymidine incorporation and proliferation in osteoblast-like cells, and specific, competitive binding sites for HMW, but not LMW, uPA were demonstrable. These studies demonstrate the production of a mitogenic NH2-terminal fragment of uPA by a human prostatic cancer cell line which may be of importance in the pathogenesis of osteoblastic metastases.     

p75 neurotrophin receptor inhibits invasion and metastasis of gastric cancer

The p75 neurotrophin receptor (p75NTR) is a focus for study at present. However, its function in gastric cancer was not elucidated. Here, we investigated its relation with metastasis of gastric cancer. By immunohistochemistry, we found that the positive rate of p75NTR expression in metastatic gastric cancer was 15.09% (16 of 106), which was lower compared with nonmetastatic gastric cancer (64.15%; 68 of 106). The average staining score in nonmetastatic gastric cancer was significantly higher than in metastatic gastric cancer (1.21 +/- 0.35 versus 0.23 +/- 0.18; P<0.01). p75NTR protein level was also lowly expressed in the highly liver-metastatic gastric cancer cell line XGC9811-L compared with other gastric cancer cell lines by Western blotting. It could also significantly inhibit the in vitro adhesive, invasive, and migratory and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN45 by reducing urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 proteins and by increasing tissue inhibitor of matrix metalloproteinase (TIMP)-1 protein. Further studies showed that p75NTR could suppress the nuclear factor-kappaB (NF-kappaB) signal. SN50, a specific inhibitor of NF-kappaB, which could inhibit in vitro invasive and migratory abilities of gastric cancer cells, reduced expression of uPA and MMP9 proteins and increased expression of TIMP1 protein. Taken together, p75NTR had the function of inhibiting the invasive and metastatic abilities of gastric cancer cells, which was mediated, at least partially, by down-regulation of uPA and MMP9 proteins and up-regulation of TIMP1 protein via the NF-kappaB signal transduction pathway. Our studies suggested that p75NTR may be used as a new potential therapeutic target in metastatic gastric cancer.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Plasmin produces an E-cadherin fragment that stimulates cancer cell invasion

Matrix metalloproteases from the cell surface cleave an 80 kDa E-cadherin fragment (sE-CAD) that induces invasion of cancer cells into collagen type I and inhibits cellular aggregation. Conditioned media from MDCKts.srcCl2 cells at 40 degrees C and 35 degrees C, PCm.src5 and COLO-16 cells at 37 degrees C contained spontaneously released sE-CAD; these 48 h old conditioned media were capable of inhibiting E-cadherin functions in a paracrine way. Here we show direct cleavage of the extracellular domain of E-cadherin by the serine protease plasmin. sE-CAD released by plasmin inhibits E-cadherin functions as evidenced by induction of invasion into collagen type I and inhibition of cellular aggregation. This functional inhibition by sE-CAD was reversed by aprotinin or by immunoadsorption on protein Sepharose 4 fast flow beads with antibodies against the extracellular part of E-cadherin. Our results demonstrate that plasmin produces extracellular E-cadherin fragments which regulate E-cadherin function in cells containing an intact E-cadherin/catenin complex.     

PTEN inhibits the invasion and metastasis of gastric cancer via downregulation of FAK expression

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in inhibiting tumor growth and metastasis. However, the mechanism by which PTEN restricts gastric cancer progression and metastasis remains largely elusive. Here we demonstrated that PTEN overexpression or knockdown in gastric cancer cells led to the downregulation or upregulation of focal adhesion kinase (FAK), and decreased or increased cell invasion, respectively. Moreover, FAK overexpression could rescue the inhibition of cell invasion by PTEN. These results were further confirmed in orthotropic gastric cancer nude mice model. In addition, in human gastric cancer tissues, PTEN protein level was conversely correlated with FAK protein level. Mechanistically, we found that PTEN inhibited PI3K/NF-κB pathway and inhibited the DNA binding of NF-κB on FAK promoter. Taken together, our data reveal a novel mechanism that PTEN inhibits the growth and invasion of gastric cancer via the downregulation of FAK expression and suggest that exploiting PTEN/PI3K/NF-κB/FAK axis is a promising approach to treat gastric cancer metastasis.     

MFAP3L activation promotes colorectal cancer cell invasion and metastasis

An abundance of microfibril-associated glycoprotein 3-like (MFAP3L) significantly correlates with distant metastasis in colorectal cancer (CRC), although the mechanism has yet to be explained. In this study, we observed that MFAP3L knock-down resulted in reduced CRC cell invasion and hepatic metastasis. We evaluated the cellular location and biochemical functions of MFAP3L and found that this protein was primarily localized in the nucleus of CRC cells and acted as a protein kinase. When EGFR translocated into the nucleus upon stimulation with EGF, MFAP3L was phosphorylated at Tyr287 within its SH2 motif, and the activated form of MFAP3L phosphorylated ERK2 at Thr185 and Tyr187. Moreover, the metastatic behavior of CRC cells in vitro and in vivo could be partially explained by activation of the nuclear ERK pathway through MFAP3L phosphorylation. Hence, we experimentally demonstrated for the first time that MFAP3L likely participates in the nuclear signaling of EGFR and ERK2 and acts as a novel nuclear kinase that impacts CRC metastasis.     

Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G₀/G₁ phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.     

MicroRNA-221 promotes colorectal cancer cell invasion and metastasis by targeting RECK

MicroRNAs (miRNAs) have recently emerged as regulators of metastasis. We provide insight into the behavior of miR-221 in colorectal cancer (CRC) metastasis by showing that miR-221 is significantly upregulated in metastatic CRC cell lines and tissues. miR-221 overexpression enhances, whereas miR-221 depletion reduces CRC cell migration and invasion in vitro and metastasis in vivo. We identify RECK as a direct target of miR-221, reveal its expression to be inversely correlated with miR-221 in CRC samples and show that its re-introduction reverses miR-221-induced CRC invasiveness. Collectively, miR-221 is an oncogenic miRNA which may regulate CRC migration and invasion through targeting RECK.     

Directed cell invasion and migration during metastasis

Metastasis requires tumor cell dissemination to different organs from the primary tumor. Dissemination is a complex cell motility phenomenon that requires the molecular coordination of the protrusion, chemotaxis, invasion and contractility activities of tumor cells to achieve directed cell migration. Recent studies of the spatial and temporal activities of the small GTPases have begun to elucidate how this coordination is achieved. The direct visualization of the pathways involved in actin polymerization, invasion and directed migration in dissemination competent tumor cells will help identify the molecular basis of dissemination and allow the design and testing of more specific and selective drugs to block metastasis.     

Selective localization of receptors for urokinase amino-terminal fragment at substratum contact sites of an in vitro-established line of human epidermal cells.

We have shown the presence of surface receptors for the amino-terminal fragment (ATF) of human urokinase-type plasminogen activator (u-PA) on an in vitro-established cell line of human epidermal origin by both radio-binding assays with human 125I-u-PA-ATF and transmission electron microscopy of a gold-u-PA complex. On the basis of cross-linking experiments with 125I-u-PA-ATF and subsequent autoradiography of the gels we have observed that such receptors are not spontaneously released into the culture medium. The treatment with phosphatidylinositol-specific phospholipase C induces the release of the receptor, which behaves as a glycosyl phosphatidyl inositol(GPI)-anchored protein. Phase-partitioning experiments on cell lysates have shown that the receptor partitions into the detergent phase. By detaching cell monolayers with the chelating agent EDTA we have prepared the cell-substratum contact sites of these cells, which represent only the 3.5% of the surface membrane of monolayered cells. Such plasma membrane remnants are highly selected since they contain about 43% of total u-PA-ATF binding sites. Such binding sites show the same biochemical and morphological characteristics of u-PA-ATF receptors observed in the monolayered cells, thus indicating that u-PA is selectively concentrated at the level of cell-substratum contacts. This is likely to enable directional proteolysis for cell migration and invasion.     

Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumor cell metastasis.Key words: in vivo imaging, photobleaching, photoactivation, biomarkersMetastasis is a complex process consisting of interactions between cancer cells and their surrounding extra-cellular matrix and stroma. To give rise to a secondary tumor, a primary tumor cell undergoes alterations to its cell-cell and cell-ECM contacts, allowing it to breach the basement membrane and intravasate into the vasculature or the lymphatic system. A tumor cell must survive in the circulation before extravasating at a secondary site and initiating new tumor growth and the development of its own blood supply. Imaging this process in live animals under native physiological conditions is inherently difficult due to poor sample stability, tissue penetration and autofluorescence of the tissue. However, new advances in fluorescent imaging, including the continued development of green fluorescent protein (GFP) and its variants, have facilitated the observation of this process and shed light on some key mechanisms that determine how and why cells metastasise. The use of fluorescent probes for in vivo imaging can be divided into two types (1) ‘passive’ markers or reporters used for direct visualization and tracking of cell movement in relation to extracellular structures and (2) more complex, ‘active’ reporters or biosensors for monitoring detailed processes such as biochemical activity or protein-protein interactions during metastasis.^1,2 In some cases there can be overlap between both types of imaging which will be addressed here.The majority of early intravital imaging studies focused on the stages of metastasis that occur after dissemination from the primary tumor and predominantly used a ‘passive’ reporter approach to assess tumor cell behavior. Models of circulating tumor cells have allowed for analysis at the single cell level of tumor cell velocity, persistence, shape change and interactions with the ECM and stroma in secondary tissue.^3–5 The use of fluorescently-labelled cells has also revealed some limiting factors that cause the arrest of cancer cells in target tissue such as trapping in small capillary networks due to tumor cell size or adhesion to surrounding vessel walls.^6,7 Furthermore, experimental models of metastasis such as intra-splenic, intra-cardial and tail vein injections in combination with fluorescently-tagged tumor cells has provided information on the colonisation, extravasation and dormancy of tumor cells in secondary sites (Fig. 1 and refs. ^{5, 8} and ⁹). Collectively, along with the rapid increase in tissue specific expression of GFP in mouse cancer models,¹⁰ a wealth of information on different steps of the metastatic process has begun to emerge.Open in a separate windowFigure 1(A) Whole body optical imaging of mCherry-expressing SW 620 colon cancer cell metastases after approximately six weeks post intra-splenic injection. Images were obtained using the Olympus OV100 whole body imaging system with an Olympus MT10, 150 w, Xenon light source, using a low magnification objective (macro lens) with a magnification of 0.14× and numerical aperture of 0.04. (B) mCherry expressing SW 620 colon cancer cells colonizing the liver 30 mins after intra-splenic injection. 1 × 10⁶ cells were injected into the spleen of an anesthetised CD-1 nude mouse and the incision sealed using ‘Clay Adams’ vetinary clips (VetTec). The mouse was placed on a heat pad for 30 mins then sacrificed. An incision was made in the abdomen to expose the liver and images of fluorescent cells within the liver were obtained using a 0.8× (0.22 NA) objective lens with variable zoom on the Olympus OV100.The departure of individual cells away from solid primary tumors into the blood stream has been a more difficult process to study using intravital imaging. It is a rare, sporadic event, requiring long acquisition and the inherent density and complex nature of the tumor tissue poses problems for imaging. Overcoming autofluorescence and light scattering has recently been improved due to advances in fluorophores^1,11 and the combined use of long-term multiphoton microscopy¹² has allowed greater resolution and tissue penetration than before. Multiphoton imaging can also provide additional detail regarding the interaction between cells and the surrounding extracellular environment using second harmonic signal generation (SHG) from collagen, elastin and other matrix proteins found in connective tissue.^13,14 In this regard, imaging the interaction of cancer cells with extracellular matrix has revealed distinct modes of cell locomotion adopted by cancer cells in vivo, such as ameboid or mesenchymal invasion, that depend upon the topography or density of the surrounding matrix.^3,13,15 A greater understanding of the initial cell movement and interaction with the extracellular environment will enhance our ability to pin-point cell-ECM targets that may be of clinical relevance in the future.Concurrent with the use of GFP as a ‘passive’ marker, a number of techniques have been developed that facilitate the visualization and localisation of GFP-tagged fusion proteins to quantify changes in protein expression, mobility and sub-cellular interactions during various processes in vitro. These include photobleaching (PB), photoactivation (PA), fluorescence resonance energy transfer (FRET) and fluorescent life time imaging microscopy (FLIM).^2,16,17 The adaptation of these techniques for in vivo imaging to examine the activity of key molecules will provide new ‘active’ markers or reporters that can be correlated with biological processes important in disease progression such as migration, proliferation and cell death. Other fluorescent probes such as MMPsense or Apotrace that measure ‘active’ processes such as metalloproteinase activity or apoptosis have also recently been used in animals.^18,19 In this way we can get closer to understanding how subcellular components or signal transduction pathways interact in real-time. The improved spatial and temporal detail will facilitate the ‘when and where’ we should target metastatic cancer cells for therapy.¹²In our recent paper we have adapted two techniques, photobleaching and photoactivation, for in vivo imaging and used them to assess the potential of E-cadherin as a molecular biosensor for cell migration in live tumors.²⁰ E-cadherin-based cell-cell contacts are prominent sites of remodelling during early stages of epithelial to mesenchymal transition (EMT). The disruption or deregulation of E-cadherin-based adhesions leads to the collapse of normal epithelial architecture that precedes the initial intravasation of cells from tumors.^21–23 In vitro photobleaching analysis of E-cadherin can be used as an ‘active’ molecular read-out of cell migration, as cells within a stationary colony show significantly reduced E-cadherin mobility compared to collectively migrating cells.²⁰ Moreover, as demonstrated in Figure 2 (reviewed in ref. ²⁰), E-cadherin mobility can also be spatially regulated within a population of tumor cells, as cells at the rear of a wound show impaired E-cadherin mobilisation compared to cells at the leading edge of the wound. This suggests a gradient of E-cadherin mobilisation within the local environment of a tumor may exist and could potentially be used in the future to map areas of weakened cell-cell adhesion from which cells are more likely to migrate. In vivo analysis of E-cadherin dynamics showed that changes in the mobility of E-cadherin can also be used as an ‘active’ marker of cell behavior in live animals, and may be useful in predicting cell mobilisation from primary tumors.²⁰Open in a separate windowFigure 2FRAP of GFP-E-cadherin at the rear or front of a wound heal assay. (A and B) Schematic and representative images of a wound heal assay depicting the area of cells selected for E-cadherin-based cell-cell junction FRAP analysis (red broken line). (C and D) Representative images of FRAP experiments performed at the rear or front of a wound heal assay respectively. White solid arrows represent area of photobleaching at the rear and white broken arrows represent area of photobleaching at the front of the wound. Red arrows indicate dynamics of cells at the front of the wound. Cells were classed to be at the front of the wound within the first three cells from the wound border (reviewed in ref. ²⁰).We also demonstrated the subcellular tracking of plasma membrane dynamics in vivo using the membrane-targeting sequence of H-Ras fused to photoactivatable-GFP.^24,25 Importantly, both the dynamics of cell-cell junctions, as visualised using E-cadherin:GFP, and the dynamics of the plasma membrane, which also plays a fundamental role in cell invasion and metastasis, are significantly different in vivo than in vitro.²⁰ Critically, this raises the possibility that many signalling axes and networks may function differently in vivo and therefore care must be taken when correlating in vitro information to the live setting. Lastly, we demonstrated the benefits of in vivo imaging in the assessment of molecular-based targeted therapeutics by using the Src inhibitor dasatinib, which impaired E-cadherin cell mobility in vivo but not in vitro.^20,26In the context of previous intravital imaging studies, our work suggests that we are at the beginning of a new stage of intravital imaging in which ‘active’ probes can help predict the efficacy of novel therapeutic treatments and also provide a context dependent read-out of oncogene-induced biological behavior in live animals. Importantly, not all molecules are adaptable for this type of in vivo imaging. Careful selection of candidate molecular markers that demonstrate clear changes attributable to a biological function, for example, subcellular relocalization or compartmentalisation, will be ideally suited for this type of intravital examination in the future.Here we have adopted two key fluorescent imaging techniques typically used in vitro and combined them with a fundamental biological question in vivo. The adaptation of other techniques for in vivo imaging such as FRET or FLIM-FRET probes will provide a detailed pixel by pixel map of the activity and behavior of key signalling proteins in live animals.^2,27 The use of these ‘active’ probes in vivo may hold further surprises concerning differences in molecular behavior in live animals compared to the traditional ‘snap-shot’ approach in vitro. Finally, one of the major challenges of in vivo imaging during drug discovery is the need for repeated imaging of the same animal in the presence or absence of drugs. The continued development of optical windows and observation chambers for non-invasive real-time imaging will facilitate this and allow for the assessment of drug response at the single cell level.²⁸ This, when combined with the subcellular optical techniques described here, will prove very useful in the future for in vivo imaging when evaluating the aetiology of the disease or during the drug discovery process.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

MicroRNA let-7f inhibits tumor invasion and metastasis by targeting MYH9 in human gastric cancer

Background

MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.

Methodology/Principal

Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels.

Conclusions/Significance

Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.     

LINC01133 inhibits breast cancer invasion and metastasis by negatively regulating SOX4 expression through EZH2

Mounting evidence highlights long non‐coding RNAs (lncRNAs) as crucial regulators in multiple types of biological processes and contributing to tumourigenesis. LINC01133, located in chromosome 1q23.2, was a recently identified novel lncRNA with a length of 1154nt. It was involved in the development of colorectal cancer and non‐small cell lung cancer. However, its clinical relevance, biological functions and potential molecular mechanism in breast cancer are still unclear. In this study, we found that the LINC01133 expression was significantly down‐regulated in breast cancer samples and was associated with progression and poor prognosis of breast cancer. Further experiments demonstrated that overexpression of LINC01133 inhibited invasion and metastasis in breast cancer both in vitro and in vivo. Mechanistic investigations revealed that LINC01133 repressed SOX4 expression by recruiting EZH2 to SOX4 promoter. Moreover, rescue experiments further confirmed that LINC01133 functional acted as an anti‐oncogene, at least partly, via repressing SOX4 in breast cancer. Taken together, these findings imply that LINC01133 could serve as a novel prognostic biomarker and potential therapeutic target for breast cancer.     

Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis

Cancer-associated fibroblasts (CAFs) play crucial roles in tumor progression, given the dependence of cancer cells on stromal support. Therefore, understanding how CAFs communicate with endometrial cancer cell in tumor environment is important for endometrial cancer therapy. Exosomes, which contain proteins and noncoding RNA, are identified as an important mediator of cell–cell communication. However, the function of exosomes in endometrial cancer metastasis remains poorly understood. In the current study we found that CAF-derived exosomes significantly promoted endometrial cancer cell invasion comparing to those from normal fibroblasts (NFs). We identified a significant decrease of miR-148b in CAFs and CAFs-derived exosomes. By exogenously transfect microRNAs, we demonstrated that miR-148b could be transferred from CAFs to endometrial cancer cell through exosomes. In vitro and in vivo studies further revealed that miR-148b functioned as a tumor suppressor by directly binding to its downstream target gene, DNMT1 to suppress endometrial cancer metastasis. In endometrial cancer DNMT1 presented a potential role in enhancing cancer cell metastasis by inducing epithelial–mesenchymal transition (EMT). Therefore, downregulated miR-148b induced EMT of endometrial cancer cell as a result of relieving the suppression of DNMT1. Taken together, these results suggest that CAFs-mediated endometrial cancer progression is partially related to the loss of miR-148b in the exosomes of CAFs and promoting the transfer of stromal cell-derived miR-148b might be a potential treatment to prevent endometrial cancer progression.