Experimental Metastasis Assay Research Articles

Arsenic trioxide inhibits CXCR4-mediated metastasis by interfering miR-520h/PP2A/NF-κB signaling in cervical cancer.

Arsenic apparently affects numerous intracellular signal transduction pathways and causes many alterations leading to apoptosis and differentiation in malignant cells. We and others have demonstrated that arsenic inhibits the metastatic capacity of cancer cells. Here we present additional mechanistic studies to elucidate the potential of arsenic as a promising therapeutic inhibitor of metastasis. The effects of arsenic trioxide (ATO) on human cervical cancer cell lines migration and invasion were observed by transwell assays. In experimental metastasis assays, cancer cells were injected into tail veins of severe combined immunodeficient mice for modeling metastasis. The mechanisms involved in ATO regulation of CXCR4 were analyzed by immunoblot, real-time polymerase chain reaction, and luciferase reporter assays. Immunohistochemistry was utilized to identify PP2A/C and CXCR4 protein expressions in human cervical cancer tissues. ATO inhibited CXCR4-mediated cervical cancer cell invasion in vitro and distant metastasis in vivo. We determined that ATO modulates the pivotal nuclear factor-kappa B (NF-κB)/CXCR4 signaling pathway that contributes to cancer metastasis. Substantiating our findings, we demonstrated that ATO activates PP2A/C activity by downregulating miR-520h, which results in IKK inactivation, IκB-dephosphorylation, NF-κB inactivation, and, subsequently, a reduction in CXCR4 expression. Furthermore, PP2A/C was reduced during cervical carcinogenesis, and the loss of PP2A/C expression was closely associated with the nodal status of cervical cancer patients. Our results indicate a functional link between ATO-mediated PP2A/C regulation, CXCR4 expression, and tumor-suppressing ability. This information will be critical in realizing the potential for synergy between ATO and other anti-cancer agents, thus providing enhanced benefit in cancer therapy.

Suppression of the epithelial-mesenchymal transition by SHARP1 is linked to the NOTCH1 signaling pathway in metastasis of endometrial cancer.

BackgroundMechanisms governing the metastasis of endometrial cancer (EC) are poorly defined. Recent data support a role for Enhancer-of-split and hairy-related protein 1 (SHARP1), a basic helix-loop-helix transcription repressor, in regulating invasiveness and angiogenesis of several human cancers. However, the role of SHARP1 in metastasis of EC remains unclear.MethodsHuman EC cell lines (Ishikawa and HEC-1B) were used. SHARP1 was upregulated by lentivirus transduction, while intracellular domain of NOTCH1 (ICN) were upregulated by transient transfection with plasmids. Effects of SHARP1 on cell migration and invasion were evaluated by wound healing assay and transwell invasion assay. Experimental metastasis assay were performed in nude mice. Effects of SHAPR1 on protein levels of target genes were detected by western blotting. Furthermore, the association between SHARP1 and the NOTCH1/EMT pathway was further verified in EC tissue specimens by immunohistochemical analysis.ResultsOverexpression of SHARP1 in EC cells inhibited cell migration, invasion, and metastasis. Exogenous SHARP1 overexpression affected the proteins levels of genes involved in EMT process and NOTCH1 signaling pathway. Upregulation of ICN in SHARP1-overexpressing Ishikawa cells induced cell migration and an EMT phenotype. Additionally, immunohistochemical analysis demonstrated that SHARP1 protein levels were lower in metastatic EC than in primary tumors, and statistical analysis revealed correlations between levels of SHARP1 and markers of EMT and NOTCH1 signaling pathway in human EC tissue specimen.ConclusionsThis work supports a role for SHARP1 in suppressing EMT and metastasis in EC by attenuating NOTCH1 signaling. Therefore, SHARP1 may be a novel marker for lymphatic metastasis in EC patients.

Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway

Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

The Twist Box Domain Is Required for Twist1-induced Prostate Cancer Metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial-mesenchymal transition (EMT) that promotes cancer metastasis. Structure-function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential.

Abstract PR13: Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors

Abstract Interleukin-4 (IL-4), a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by mediating increased proliferation and survival. The IL-4 Receptor (IL-4R) is overexpressed in human and murine epithelial mammary cancers, and may be a promising target for anti-tumor therapy. Here, we show that loss of IL-4Rα, a component of the IL-4 receptor, in two murine mammary tumor cell lines (R221A and 4T1), reduces tumor cell survival and outgrowth ability, and results in attenuated growth at primary and metastatic sites. RNA interference was used to knock down the expression of IL-4Rα in both cell lines. In support of our hypothesis, reduced IL-4Rα expression in R221A cells nearly abolished tumor take after orthotopic mammary injection in vivo, decreased the median latency of 4T1 primary tumors, and resulted in a significant reduction in tumor size at endpoint. In addition, reduced IL-4Rα expression in both cell lines resulted in a decrease [>2.4 fold] in lung and [>2 fold] liver metastatic tumor burden following experimental metastasis assays in vivo. The decrease in metastatic ability of IL-4Rα knockdown cells was attributed to 1) reduced tumor cell survival as determined by seeding ability (number of tumors) and an increase in apoptosis by caspase-3 positivity, and 2) a reduction in proliferation as determined by outgrowth ability (tumor size) and a decrease in Ki67 positivity. In addition, an overall increase in immune infiltrates (macrophages, neutrophils, and T lymphocytes) within IL-4Rα knockdown tumors was observed, indicating that enhanced clearance of knock down tumor cells could also contribute to the reduction in knock down tumor size visualized at endpoint. Finally, it was determined by western blot analysis that IL-4 ligand stimulates the phosphorylation of AKT (s373) and ERK in R221a and 4T1 cells in vitro. Immunohistochemical analysis of R221a and 4T1 lung metastases showed significantly reduced levels of pAKT (s472) and pERK in IL-4Rα knock down tumors compared to control tumors, implicating a possible role for AKT and ERK in mediating pro-tumor effects IL-4Rα signaling. Collectively, our results demonstrate that the IL-4 receptor promotes the growth of mammary cancer cells at primary and metastatic sites, possibly through activated AKT and ERK signaling, and suggests that available drugs targeting IL4-Rα signaling may have potential for treating primary breast cancers and limiting metastatic disease. This abstract is also presented as poster C51. Citation Format: Katherine T. Venmar, Daniel Hwang, Ashley Dozier, Kathy Carter, Sareena Gillani, Barbara Fingleton. Interleukin-4 receptor α chain regulates the growth of primary and metastatic mammary tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR13.

A Synthetic Podophyllotoxin Derivative Exerts Anti-Cancer Effects by Inducing Mitotic Arrest and Pro-Apoptotic ER Stress in Lung Cancer Preclinical Models

Some potent chemotherapy drugs including tubulin-binding agents had been developed from nature plants, such as podophyllotoxin and paclitaxel. However, poor cytotoxic selectivity, serious side-effects, and limited effectiveness are still the major concerns in their therapeutic application. We developed a fully synthetic podophyllotoxin derivative named Ching001 and investigated its anti-tumor growth effects and mechanisms in lung cancer preclinical models. Ching001 showed a selective cytotoxicity to different lung cancer cell lines but not to normal lung cells. Ching001 inhibited the polymerization of microtubule resulting in mitotic arrest as evident by the accumulation of mitosis-related proteins, survivin and aurora B, thereby leading to DNA damage and apoptosis. Ching001 also activated pro-apoptotic ER stress signaling pathway. Intraperitoneal injection of 2 mg/kg Ching001 significantly inhibited the tumor growth of A549 xenograft, while injection of 0.2 mg/kg Ching001 decreased the lung colonization ability of A549 cells in experimental metastasis assay. These anti-tumor growth and lung colonization inhibition effects were stronger than those of paclitaxel treatment at the same dosage. The xenograft tumor tissue stains further confirmed that Ching001 induced mitosis arrest and tumor apoptosis. In addition, the hematology and biochemistry tests of blood samples as well as tissue examinations indicated that Ching001 treatment did not show apparent organ toxicities in tested animals. We provided preclinical evidence that novel synthetic microtubule inhibitor Ching001, which can trigger DNA damage and apoptosis by inducing mitotic arrest and ER stress, is a potential anti-cancer compound for further drug development.

Abstract 5244: Store-operated Ca2+ entry (SOCE) regulates melanoma progression.

Abstract Background: Melanoma is the worst poor-prognosis skin cancer in the U.S. Although Ca2+ is well recognized as a major second messenger, the role of Ca2+ remains poorly understood in the cancer research field including melanoma. Store-operated calcium entry (SOCE) is the mechanism of Ca2+ entry from the extracellular space in response to Ca2+ depletion in the endoplasmic reticulum (ER). SOCE is regulated by interaction between STIM1 (stromal interaction molecule 1) in the ER and Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane. Our previous studies have demonstrated the presence of SOCE is observed in melanoma cells, and potentially regulates cell migration and cell cycle. In the present study, we extensively examined the effects of SOCE on migration and proliferation in multiple melanoma and melanocyte cell lines as well as on metastasis in mice. Method: Changes in intracellular Ca2+ level were measured by Fluo4-AM, a Ca2+-sensing fluorescent dye. In order to ablate STIM-1 or Orai-1, shRNA for each protein was induced with lentiviral infection in SK-Mel-2, SK-Mel-24 and C8161 cells. Proliferation was measured by the MTT assay. Migration was examined with the Boyden chambers and the scratch assay. Immunocytochemistry was performed to count the number of lamellipodia with F-actin staining. The experimental metastasis assay was performed as follows. Three weeks after the intravenous injection of SK-Mel-2 cells with knockdown of Orai1 or STIM1 in Balb/c nu/nu mice, the lungs were removed and fixed with picric acid. The number of metastatic colonies in the lung surface was counted. Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in 5 primary and 3 metastatic human melanoma cell lines, a mouse melanoma cell line, human and mouse melanocyte cell lines. In addition, metastatic, but not primary, melanoma cell lines showed significantly greater SOCE compared to melanocytes, suggesting that SOCE may positively correlate with malignancy of melanoma. Knockdown of STIM1 inhibited proliferation in metastatic melanoma cells (SK-Mel-2, SK-Mel-24 and C8161) (p<0.01), suggesting that SOCE activates proliferation of melanoma. Inhibition of SOCE with knockdown of Orai1 or STIM1 suppressed migration in cell lines tested (SK-Mel-2 and SK-Mel-24) (p<0.01) and the SOCE inhibitors suppressed it in cell lines (SK-Mel-2 and C8161) (p<0.01 for both cell lines), indicating that SOCE regulates melanoma migration. Indeed, the number of lamellipodia, which reflects the activity of migration, was decreased by deletion of STIM1 or Orai1 (p<0.01). The experimental metastasis assay demonstrated that the number of lung colonies was reduced by knockdown of STIM1 or Orai1 (p<0.01). Conclusion: Our results demonstrated that inhibition of SOCE suppressed proliferation, migration, and metastasis in melanoma, thus SOCE could be a new target for melanoma therapy. Citation Format: Masanari Umemura, Erdene Balijinnyam, Stefan Feske, Mariana S. De Lorenzo, Lai-Hua Xie, Yoshihiro Ishikawa, Kousaku Iwatsubo. Store-operated Ca2+ entry (SOCE) regulates melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2013-5244

Abstract 950: Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models.

Abstract Background. The dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib is approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, several patients do not respond to treatment due to constitutive resistance, and even in responders the occurrence of disease progression is often observed. Recently, the activation of Src TK has been associated with resistance to anti-HER2 drugs, both trastuzumab and lapatinib, in HER2 overexpressing breast cancer cells. However, the molecular mechanisms by which Src activation may affect response to HER2 antagonists are still partially unclear. Methods. We used a panel of HER2 expressing human breast cancer cell lines with different degree of response to lapatinib: SKBR3, BT474 and MDA-MB-361 (high sensitivity), KPL-4 (moderate sensitivity), JIMT-1 (low sensitivity). Moreover, we generated a cell line with acquired resistance to lapatinib, MDA-MB-361-LR (Lapatinib Resistant). On these models, we investigated the effect of Src silencing via small interference RNA (siRNA) or pharmacological inhibition by saracatinib on lapatinib response, both in vitro and in vivo. Results. The combination of Src inhibition with lapatinib synergistically inhibited survival of human breast cancer cells with both intrinsic or acquired resistance to lapatinib, by interfering with signal transducers involved in cell proliferation and survival. Moreover, both saracatinib and Src specific siRNAs were able to reduce migration and invasion capabilities of breast cancer cells, by affecting Focal Adhesion Kinase (FAK) and paxillin activation. In nude mice xenografted with JIMT-1 cells, the combined treatment with saracatinib and lapatinib produced a cooperative antitumor effect with inhibition of tumor growth, interference with signal transduction and prolongation of mice survival. The combination strongly reduced the incidence of lung metastasis, as revealed through experimental metastasis assays. Interestingly, a significant reduction of EGFR/HER2 co-immunoprecipitation was found in resistant cells, thus suggesting that growth signals could be transduced by other type of TKRs dimers in these cells. Moreover, in breast cancer cell lines with both intrinsic or acquired resistance to lapatinib, Src interacted with EGFR rather than with HER2, in opposition to what was observed in sensitive cells. Consistently, EGFR inhibition through cetuximab or EGFR specific siRNAs was able to partially restore lapatinib sensitivity in resistant cells, by interfering with the reciprocal activation of EGFR and Src. Conclusions. Src plays an important role in the onset of resistance to lapatinib in breast cancer cell lines, and its interaction with EGFR may be involved in this event. Therefore, the combined inhibition of Src and EGFR/HER2 axis may represent a potentially effective therapeutic strategy in order to prevent or overcome resistance to lapatinib. Citation Format: Roberta Rosa, Lucia Nappi, Luigi Formisano, Claudia D'Amato, Valentina D'Amato, Vincenzo Damiano, Roberta Marciano, Ana Paula De Maio, Lucia Raimondo, Umberto Malapelle, Giancarlo Troncone, Sabino De Placido, Roberto Bianco. Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2013-950 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5060: Discrepancy between MMP-9 and its fibronectin-like domain in tumor growth and invasion.

Abstract Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as “cancer-associated fibroblasts” (CAFs), primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stromal remodeling, matrix metalloproteinases (MMPs), and MMP-9 in particular, play a prominent role. However, the mechanisms that regulate MMP-9 activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-9 is an important event in its activation, and more generally in tumor growth and invasion. Based on these observations, we addressed the potential association of MMP-9 activity with cell-surface recruitment on human fibroblasts. We show for the first time that recruitment of MMP-9 to the MRC5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, found only in MMP-9 and MMP-2. Functional assays suggest that the active form of MMP-9 triggers α-smooth muscle actin (αSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-β activation. Xenograft experiments using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-9 in NOD/SCID mice revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increases their metastatic proclivity whereas expression of wt MMP-9 either promotes no change or actually reduces the number of metastases. Curiously, xenografts of SW480 colorectal adenocarcinoma cells stably expressing the FN domain of MMP-9 display reduced growth at both the primary (subcutaneous) injection site and the lungs, in experimental metastasis assays, of NOD/SCID mice. This discrepancy is believed to be due to the FN domain that may modulate MMP-9 activity and TGF-β activation in different ways, depending on the tumor cell type. These observations suggest a dual role of MMP-9 and its FN domain in primary tumor growth and metastasis, underscoring the notion that the effect of MMP-9 on tumor cells may depend on their response to TGF-β activation and highlighting possible protective effects of MMPs in tumor progression and invasion. Citation Format: Cynthia Dayer. Discrepancy between MMP-9 and its fibronectin-like domain in tumor growth and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2013-5060

Abstract 3871: Analysis of gene expression patterns downstream of multiple metastatic suppressor genes.

Abstract In recent decades, a distinction has been made between the tumorigenesis and metastasis processes. Metastasis Suppressor Genes (MSGs) are down-regulated in the metastasis compared with the primary tumor. When re-expressed in experimental models, MSGs are able to block spread to distant sites without effecting primary tumor formation. After the discovery of the first MSG, NME1, many others have been identified. To date more than 20 genes have been described to have MSG functions. They include factors involved in cellular proliferation, motility, adhesion, invasion, resistance to apoptosis, and angiogenesis. We hypothesized that common signaling pathways mediate the effects of many MSGs. Using microarray databases of breast cancer cell lines, and siRNA down regulation of 18 MSGs in human MCF-7 breast carcinoma cells (NME1, BRMS1, CD82, CDH1, CDH2, CASP8, MKK4, MKK6, MKK7, MAPK14, GSN, ARHGDIB, AKAP12, DRG1, CD44, PEPB1, RRM1, KISS), we conducted a bioinformatic search for common pathways. Several candidate genes were identified whose expression was inversely related to at least five MSGs. These candidates include UGT1A, PDE5A, SRMS, IL11Ralpha, DNM3, OAS1, BCAR4, and CA9, many of which are previously unknown in the metastasis literature. After stable down-regulation of each candidate gene in the highly aggressive breast cancer cell line MDA-MB-231, in vitro experiments evaluating cell proliferation and motility were performed to validate their link to the metastatic property of tumor cells. Promising data have been obtained with PDE5A. PDE5 (phosphodiesterase type 5A) is a key enzyme involved in the regulation of cGMP-specific signaling pathways in processes such as smooth muscle contraction and relaxation. PDE5A mRNA expression was upregulated by siRNA to 13 MSGs: ARHGDIB, CASP8, CD44, CDH1, CDH2, CDH11, GSN, MKK4, MKK6, MKK7, NME1, PEBP1, RRM1. Stable clones down-regulating PDE5A (MDA-MB-231 shPDE5A) were tested as described above and no difference in cell proliferation was observed, while a 58% reduction in cell motility compared to the empty vector-expressing cells was measured (p=0.0001). An experimental metastasis assay was conducted to determine if altered PDE5A expression functionally contributed to breast cancer metastasis. Tail vein injection of MDA-MB-231 shPDE5A in athymic/nude mice resulted in a 62% reduction in lung metastasis formation compared to the injection of empty-vector expressing cells (p= 0.0004). This result suggests that PDE5A is a new potential therapeutic target in breast cancer metastasis. Citation Format: Natascia Marino, Joshua W. Collins, Changyu Shen, George W. Sledge, Patricia S. Steeg. Analysis of gene expression patterns downstream of multiple metastatic suppressor genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3871. doi:10.1158/1538-7445.AM2013-3871

Abstract 1493: The Twist box is required for Twist1-induced prostate cancer metastasis.

Abstract The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is best known for its roles in cancer by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions, but the importance of these different activities for Twist1 cancer phenotypes are unknown. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. We disrupted the putative transactivation domain in the Twist box of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed biophysical cell traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 expressing Myc-CaP cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Our results show that F191G mutation behaves as loss of function and is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra P. Gajula, Sivarajan T. Chettiar, Russell D. Williams, Saravanan Thiyagarajan, Yoshinori Kato, Khaled Aziz, Ruoqi Wang, Nishant Gandhi, Aaron T. Wild, Farhad Vesuna, Jinfang Ma, Tarek Salih, Jessica Cades, Elana Fertig, Shyam Biswal, Timothy F. Burns, Christine Chung, Charles M. Rudin, Venu Raman, Joseph M. Herman, Russell K. Hales, Steven An, Phuoc T. Tran. The Twist box is required for Twist1-induced prostate cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1493. doi:10.1158/1538-7445.AM2013-1493

Abstract C60: FRIZZLED7 and ERBB3 regulate distinct metastatic properties in melanoma

Abstract Metastasis accounts for a majority of deaths from cancer. Metastatic melanoma is a type of highly aggressive cancer and accounts for ~75% of deaths in patients with skin cancer. The low 5-year survival rate of late-stage melanoma patients (~15%) treated with current methods calls for novel interventions. A constitutively active form of BRAF ( BRAFCA) is present in 60% to 70% of melanoma cases; the remaining 30% express wild type BRAF. Recently, a BRAFCA inhibitor, vemurafenib, has provided some initial benefits in melanoma patients, but resistance quickly developed in these patients. Also, the drug was ineffective in melanomas expressing wild type BRAF, begging for alternative therapeutic strategies. Metastasis is a multistep process, which involves detachment of cancer cells from primary tumor, intravasation into circulation, survival and extravasation into tissue parenchyma, and metastatic growth initiation and expansion. The last two steps are considered rate-limiting steps during metastasis and were examined using the experimental metastasis assay. Our prior studies showed that highly metastatic melanoma cells differentially express a set of 150 genes compared to low metastatic melanoma cells. Among the 150 genes, 44 were positively correlated with poor patient survival and were particularly enriched for genes encoding secreted proteins. We analyzed two of them, FZD7 and ERBB3, in the context of metastatic growth. FRIZZLED7 is a WNT receptor and ERBB3 is a receptor tyrosine kinase. Both were found to be highly expressed in melanomas from patients with poor survival. We showed that shRNA mediated knockdown of FZD7 or ERBB3 reduced the lung colonization ability of melanoma cell lines expressing the constitutively active mutant of BRAF (BRAFCA) in the experimental metastasis assay. In addition, FZD7 or ERBB3 knockdown reduced the metastatic potential of melanoma cells that have developed resistance to BRAF inhibitor. These results provided preclinical evidence for using FZD7 and ERBB3 as intervention entry points to treat both vemurafenib-sensitive and resistant melanomas. We are currently testing whether perturbing these two genes also abolishes metastasis from melanoma cells that carry wild type BRAF in the SKMEL2 cell line, which expresses a wild type BRAF. We are also examining at which step FZD7 and ERBB3 affect metastasis. Our data indicated that, though either FZD7 or ERBB3 knockdown can reduce the number of lung metastases in experimental metastasis assay, only FZD7 knockdown affected the tumor initiating cell frequency and subcutaneous tumor growth. This suggested that ERBB3 and FZD7 may regulate distinct aspects of metastasis. Taken together, we investigated the roles of two cell surface receptors, FZD7 and ERBB3, on melanoma metastasis. Our results revealed signaling pathways that potentially influence distinct steps of metastasis process. Combinatorial therapies that perturb these pathways thus may prove more effective in treating metastatic melanoma than single regimens. Citation Format: Shweta Tiwary, Morgan Preziosi, Sonali Mohanti, Brad Martin, Nathalie Zeitouni, Lei Xu. FRIZZLED7 and ERBB3 regulate distinct metastatic properties in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C60.

Abstract B65: Milky spots are required for ovarian cancer metastatic colonization of peritoneal adipose depots

Abstract Introduction: In ovarian cancer, attachment of cells to the omentum represents a rate-limiting step in metastasis formation. Thus, there is significant interest in identifying the tissue microenvironments involved in cancer cell colonization of this preferred metastatic site. The omentum is composed of translucent membranes and adipose, which contains immune structures known as milky spots. Two general models of omental colonization can be discerned from the published literature. In the “milky spot-driven” model, cancer cell lodging is due to factors produced by milky spots which serve as a colonization niche1. In contrast, the “adipose-driven” model proposes that adipocyte-derived factors are solely responsible for cancer cell lodging2. By taking advantage of peritoneal adipose deposits which either contain or lack milky spots, we set forth to test the hypothesis that these unusual immune structures are required for cancer cell colonization of adipose-rich tissues such as the omentum. Methods: Ovarian cancer cell migration, lodging and growth was assessed in a panel of peritoneal fat depots which either contain milky spots (omentum, splenoportal fat) or lack them (uterine fat, mesentery, and gonadal fat). The presence or absence of milky spots was confirmed by histology and immunohistochemistry (IHC) for CD45. Ovarian cancer cell lodging was evaluated via experimental metastasis assays using SKOV3ip.1 (human) or ID8 (mouse) ovarian cancer cells injected into Athymic Nude or C57BL/6 mice, respectively. At 7 days post injection (dpi) peritoneal fat depots were harvested and evaluated for the presence of cancer cells using histology and pan-cytokeratin IHC. The relative chemoattractant ability of tissue-conditioned media was evaluated by transwell migration assays using SKOV3ip.1 and ID8 cells. Results: The presence of milky spots in omental and splenoportal fat and their absence in uterine, gonadal, and mesenteric fat was confirmed by histology and IHC. In metastasis assays, cancer foci were observed in omental and splenoportal milky spots at 7 dpi. In contrast, cancer cells were never observed in adipose that lacked milky spots; even at 63 dpi. Consistent with this, conditioned media from milky-spot containing tissues had a significantly increased ability to promote ovarian cancer cells migration . Conclusions: In sum, our findings show that milky spots are an absolute requirement for cancer cell lodging on peritoneal adipose. Consistent with this, migration assays show that the presence of milky spots causes a significant enhancement in the ability of peritoneal adipose to promote directed migration. Taken together, these finding suggest that while adipose may secrete a general chemoattrative signal, additional contributions from the milky spots are required for localization and invasion. Current experiments include investigating the chemoattractant(s) used by the milky spots to attract cancer cells and the cell type(s) responsible for their secretion. Competing interests: The authors declare no competing interests.

Abstract B33: Omental milky spots serve as a niche for ovarian cancer metastatic colonization

Abstract Introduction: Metastatic ovarian cancer remains an urgent clinical problem. The homing and invasion of cancer cells into the omentum, the preferred site of ovarian cancer metastasis, is a rate-limiting step in disease progression. We have recently shown that immune cell-containing structures known as milky spots are required for colonization of the omental adipose. Experiments were designed using well-established ovarian cancer models and experimental metastasis assays to answer the following questions: Does cancer cell localization depend upon the immune composition of the milky spots? Do cancer cells utilize the native milky spot microenvironment, or does it undergo remodeling during colonization? Answers to these questions will be the foundation for mechanism-based studies aimed at identifying ovarian cancer-omental interactions that can be targeted therapeutically. Methods: Mouse strains with varying immune-competency were used to: 1) test the effect of immune composition on volume of milky spots within the omentum; 2) determine the requirement of specific immune cell types on ovarian cancer cell lodging and invasion into milky spots; and assess the effect of ovarian cancer cell lodging on milky spot composition. Strains included: C57BL/6 (immunocompetent), Athymic Nude (T cell deficient), Beige Nude (NK and T cell deficient), Rag1-/- (no mature T or B cells, no CD3+ or T cell receptor a-b positive cells), and Igh6-/- (lack mature B cells). Experimental metastasis assays used intraperitoneal injection of 1 x 106 ID8 (mouse) or SKOV3ip.1 (human) ovarian cancer cells into C57BL/6 mice, or immunodeficient mice, respectively. Milky spot volume in naïve omenta was calculated from digital whole-mounts constructed from Giemsa-stained tissue sections. Milky spot composition was evaluated using immunohistochemistry (IHC) for lymphocytes (CD45), T cells (CD3), B cells (B220), macrophages (F4/80), and endothelial cells (CD31). Results: ID8 and SKOV3ip.1 ovarian cancer cells localized to and proliferated in the milky spots. Studies using a panel of immune-deficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Analysis of digital whole mounts found no difference in the volume of milky spots in omenta from C57BL/6, Athymic Nude, Beige Nude, Rag1-/-, Igh6-/- mice. IHC of naïve omenta show the presence of B cells, T cells, and macrophages consistent with the genotype of the mouse. Abundant endothelial cells were present in milky spots corresponding to the dense vasculature that is a conserved feature of milky spots. Finally, evaluation of milky spot composition during metastatic colonization showed an influx of macrophages 24 hours and 3 days post injection (dpi). Staining for activated macrophages in metastases increased from week 1 to week 5; staining persisted until the experimental endpoint. Conclusions: Targeting the host tissue microenviroment is crucial to contain or reduce cancer spread, prolonging patient life. Use of genetic models ruled out a requirement of B, T, and NK cells in ovarian cancer cell homing to milky spots suggesting a role for macrophages in this process. A preliminary study indicates a temporal increase in activated macrophages upon cancer colonization. We speculate that cancer cells co-opt the normal developmental role of macrophages to promote their own growth. Work was supported by grants from the DOD (W81XWH-09-1-0127), NCI/NIH (2RO1CA089569), and Elsa U. Pardee Foundation; A Rivkin Center for Ovarian Cancer Research Pilot Study Award, and funds from the Section of Urology (The University of Chicago) Citation Format: Venkatesh Krishnan, Robert Clark, Michael Schoof, Irving Rodriguez, Marina Chekmareva, Carrie Rinker-Schaeffer. Omental milky spots serve as a niche for ovarian cancer metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B33.

Abstract C53: Tumor progression locus 2 (Tpl2) protein kinase contributes tumor growth and metastasis of renal cell carcinoma

Abstract At least one-third of renal cell carcinoma (RCC) patients are diagnosed with metastases and an additional 20 to 40% of patients develop metastases after radical nephrectomy with curative intent. Although the treatment of advanced RCC has recently evolved to the use of VEGF-targeted antiangiogenic therapies, there have been no significant improvements in the mortality rate of RCC, most likely because currently available therapies are relatively ineffective. A full understanding of the molecular pathogenesis involved in the progression of RCC is important for the development of innovative treatment options. Tumor Progression Locus 2 (Tpl2/MAP3K8), is a serine-threonine kinase with an important physiological role in tumor necrosis factor, interleukin-1, CD40, Toll-like receptor and G protein-coupled receptor-mediated extracellular signal-regulated kinases (ERK1/ERK2), c-Jun NH2-terminal kinases (JNK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38MAPK pathways. Previously identified as an oncogene, its overexpression is reported in many types of cancer and associated with metastatic potential. Inspection of transcriptome profiles registered in the oncomine database indicates that Tpl2 expression is differentially expressed in RCC compared to normal tissue. However, the relevance of Tpl2 in tumor growth and metastasis of RCC and the underlying mechanisms remain to be explored. To investigate a functional role of Tpl2 in the progression of RCC, the molecular effects of Tpl2 inhibition were examined in human RCC preclinical model using Caki-1 cells. In this study, we report a functional role for Tpl2 in RCC tumorigenesis and metastasis. Gene silencing with Tpl2 short hairpin RNA and treatment with a Tpl2 kinase inhibitor remarkably suppressed proliferation, focus formation potential, anoikis-resistance and cell-adhesion as well as in vitro cell migration/invasion capabilities in Caki-1 cells. Cell cycle assay showed that the anti-proliferation effect of Tpl2 shRNA was mediated by arresting cells in the G0/G1 phase. We found that interleukin 1β-induced phosphorylation of ERK or JNK was markedly suppressed by Tpl2 shRNA or Tpl2 kinase inhibitor. Furthermore, the orthotopically xenografted tumor growth of Caki-1 cells was significantly suppressed by Tpl2 knockdown with Tpl2 shRNA. An experimental metastasis assay also shows that Tpl2 inhibition suppressed the ability of Caki-1 cells to form lung metastases in a murine tail vein injection model. In clinical specimens of RCC, immunohistochemistry showed that Tpl2 expression levels in RCC specimens were significantly higher than those in normal renal tissues. These data suggest that Tpl2 may function as an oncogene linked to RCC and potential target for novel therapeutic modalities for advanced RCC. Citation Format: Hye Won Lee, Se Jeong Lee, Mi Young Song, Kyeung Min Joo, Byong Chang Jeong, Do-Hyun Nam. Tumor progression locus 2 (Tpl2) protein kinase contributes tumor growth and metastasis of renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C53.

Abstract B45: The Twist box domain is required for Twist1-induced metastasis of prostate cancer cells

Abstract Purpose: The Twist1 gene has diverse roles during development and pathologic states such as cancer. Twist1 is a bHLH transcription factor that has both repressor and transactivation functions. Twist1 is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through transcriptional repression. The Twist1 transactivation domain has been reported but the functional significance of this domain is still unclear. In cancer, Twist1 is best known for its roles in facilitating tumor progression by inducing an epithelial-mesenchymal transition (EMT) transcriptional program implicated in facilitating tumorigenesis, tumor progression and treatment resistance. We hypothesized Twist1 may mediate these various functions using distinct structural domains and/or motifs. Here we have investigated the role of the Twist box domain of Twist1 in prostate cancer. Methods: We disrupted the putative transactivation domain (TD) of Twist1 by mutating a critical phenylalanine residue (F191) to glycine. We then created stable isogenic prostate cancer cell lines overexpressing wildtype and F191G versions of Twist1. We assessed the role of the Twist box using in vitro and in vivo assays, which mimic the various stages of cancer progression to metastasis. These include loss of homotypic cell-cell contacts, cell migration and invasion, anoikis resistance and soft agar colony formation. We also observed single-cell biophysical traction forces on a fabricated substratum and finally performed experimental lung metastasis assays. Results: The overexpression of Twist1 in prostate cancer cells lead to an EMT biomarker phenotype and the F191G mutant lacked expression of some of these markers. The F191G mutant was deficient for transcriptional activity using promoter reporter based assays. Using single cell measurements we found that Twist1 overexpressing prostate cancer cells exert more force on the substratum than vector control cells. Additional in vitro assays suggest Twist1 can confer cellular properties associated with increased tumor cell aggressiveness including increased migration/invasion, cell death/anoikis resistance and in vitro tumorigenic potential by soft agar colony formation. The Twist box mutant, F191G, displayed compromised activity compared to wildtype Twist1 in many of the in vitro assays described above revealing that the Twist box is necessary for many of the pro-metastatic functions of Twist1. We compared the gene expression profile of Twist1 and F191G overexpressing prostate cancer cells by microarray and observed that the F191G mutant had an expression profile that was similar to wildtype Twist1 but attenuated in key gene sets. Lastly, Twist1 overexpression compared to vector control prostate cancer cells showed an increased frequency of metastatic lung tumors using the experimental lung metastasis assay. Interestingly, Twist1 overexpression also resulted in the appearance of extra-thoracic metastases. The F191G mutant was less able to confer prostate cancer cells the ability to colonize metastatic lesions in the lung and resulted in no extra-thoracic metastases. Conclusions: Our results show that the Twist1-F191G mutant behaves as a loss of function and consequently that the Twist1 box is necessary for Twist1-induced metastasis of prostate cancer cells. Citation Format: Rajendra Gajula, Sivarajan Chettiar, Russell Williams, Saravanan Thiyagarajan, Khaled Aziz, Nishant Gandhi, Aaron Wild, Tarek Salih, Yoshinori Kato, Jessica Cades, Elana Fertig, Christine Chung, Joseph Herman, Russell Hales, Charles Rudin, Steven An, Phuoc T. Tran. The Twist box domain is required for Twist1-induced metastasis of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B45.

Loss of Receptor Protein Tyrosine Phosphatase β/ζ (RPTPβ/ζ) Promotes Prostate Cancer Metastasis

The role of pleiotrophin and its receptors RPTPβ/ζ and Syndecan-3 during tumor metastasis remains unknown. RPTPβ/ζ knockdown initiates EMT, promotes pleiotrophin-mediated migration and attachment through Syndecan-3 and induces in vivo metastasis. RPTPβ/ζ plays a suppressor-like role in prostate cancer metastasis. Boosting RPTPβ/ζ or attenuating Syndecan-3 signaling pathways may lead to more effective therapeutic strategies in treating prostate cancer metastasis. Pleiotrophin is a growth factor that induces carcinogenesis. Despite the fact that many published reports focused on the role of pleiotrophin and its receptors, receptor protein tyrosine phosphatase (RPTPβ/ζ), and syndecan-3 during tumor development, no information is available regarding their function in tumor metastasis. To investigate the mechanism through which pleiotrophin regulates tumor metastasis, we used two different prostate carcinoma cell lines, DU145 and PC3, in which the expression of RPTPβ/ζ or syndecan-3 was down-regulated by the RNAi technology. The loss of RPTPβ/ζ expression initiated epithelial-to-mesenchymal transition (EMT) and increased the ability of the cells to migrate and invade. Importantly, the loss of RPTPβ/ζ expression increased metastasis in nude mice in an experimental metastasis assay. We also demonstrate that RPTPβ/ζ counterbalanced the pleiotrophin-mediated syndecan-3 pathway. While the inhibition of syndecan-3 expression inhibited the pleiotrophin-mediated cell migration and attachment through the Src and Fak pathway, the inhibition of RPTPβ/ζ expression increased pleiotrophin-mediated migration and attachment through an interaction with Src and the subsequent activation of a signal transduction pathway involving Fak, Pten, and Erk1/2. Taken together, these results suggest that the loss of RPTPβ/ζ may contribute to the metastasis of prostate cancer cells by inducing EMT and promoting pleiotrophin activity through the syndecan-3 pathway.

Cullin-3 protein expression levels correlate with breast cancer progression

Cullin-3 is a component of the Cullin-Ring ubiquitin ligase (CRL) family that plays an important role in mediating protein degradation. Deregulation of Cullin-3 expression has been observed in human cancers; however, a role for Cullin-3 in tumor progression has not been previously recognized. Using the MCF10DCIS.com human breast cancer xenograft model, we show that Cullin-3 is increasingly expressed during progression from comedo ductal carcinoma in situ (DCIS) to invasive carcinomas. Cullin-3 protein is not detected in early lesions but is noticeably increased in DCIS tumors and significantly overexpressed in invasive cancers. In experimental metastasis assays, high expression of Cullin-3 was observed in the lung site. Importantly, Cullin-3 staining is detected in human breast cancer tissues, not in normal breast tissues and its expression level positively correlates with tumor stage. These data suggest that Cullin-3 may play an important role in tumor progression from DCIS to invasive cancer and may serve as a biomarker for the diagnosis of aggressive breast cancer.

Dipyridamole prevents triple-negative breast-cancer progression

Dipyridamole is a widely prescribed drug in ischemic disorders, and it is here investigated for potential clinical use as a new treatment for breast cancer. Xenograft mice bearing triple-negative breast cancer 4T1-Luc or MDA-MB-231T cells were generated. In these in vivo models, dipyridamole effects were investigated for primary tumor growth, metastasis formation, cell cycle, apoptosis, signaling pathways, immune cell infiltration, and serum inflammatory cytokines levels. Dipyridamole significantly reduced primary tumor growth and metastasis formation by intraperitoneal administration. Treatment with 15 mg/kg/day dipyridamole reduced mean primary tumor size by 67.5 % (p = 0.0433), while treatment with 30 mg/kg/day dipyridamole resulted in an almost a total reduction in primary tumors (p = 0.0182). Experimental metastasis assays show dipyridamole reduces metastasis formation by 47.5 % in the MDA-MB-231T xenograft model (p = 0.0122), and by 50.26 % in the 4T1-Luc xenograft model (p = 0.0292). In vivo dipyridamole decreased activated β-catenin by 38.64 % (p < 0.0001), phospho-ERK1/2 by 25.05 % (p = 0.0129), phospho-p65 by 67.82 % (p < 0.0001) and doubled the expression of IkBα (p = 0.0019), thus revealing significant effects on Wnt, ERK1/2-MAPK and NF-kB pathways in both animal models. Moreover dipyridamole significantly decreased the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in primary tumors (p < 0.005), and the inflammatory cytokines levels in the sera of the treated mice. We suggest that when used at appropriate doses and with the correct mode of administration, dipyridamole is a promising agent for breast-cancer treatment, thus also implying its potential use in other cancers that show those highly activated pathways.

The molecular mechanism of microRNA-145 to suppress invasion-metastasis cascade in gastric cancer.

Invasion and metastasis are the major features of malignant tumors that are responsible for 90% of cancer-related deaths. Recently, microRNAs have been discovered to have a role in suppressing tumor metastasis. This study's aim was to clarify the roles of miR-145 in gastric carcinomas and its underlying molecular mechanism in regulating tumor metastasis. Here, we demonstrate a stepwise downregulation of miR-145 level in nontumorous gastric mucosa, primary gastric cancers and their secondary metastases. In vitro analysis of miR-145's ectopic expression and loss-of-function suggests that it suppresses gastric cancer cell migration and invasion. In vivo spontaneous metastasis and experimental metastasis assay further confirm its function in suppressing the invasion-metastasis cascade, including impairing local invasion and inhibiting hematogenous metastasis in gastric cancers. Furthermore, we identified a novel mechanism of miR-145 to suppress metastasis. N-cadherin (CDH2) was proved to be a direct target of miR-145, using luciferase assay and western blot. Re-expressing N-cadherin in miR-145-transfected cells reverses their migration and invasion defects. Although not a direct target of miR-145, matrix metallopeptidase 9 (MMP9), but not MMP2, was also significantly decreased in miR-145-expressing cells. We suggest that miR-145 suppresses tumor metastasis by inhibiting N-cadherin protein translation, and then indirectly downregulates its downstream effector MMP9.