Lung Metastasis Assay Research Articles

Abstract 119: Overexpression SOCS-1 may inhibit invasion and metastasis of cervical cancer

Abstract Purpose: To investigate whether the overexpression of SOCS-1 affects radiosensitivity, invasion, and metastasis in xenograft nude mouse model. Methods: We established stable SOCS-1-overexpressing ME-180 cell line with pLNCX2-mSOCS1-Myc. Also we established xenograft nude mouse model for tumor growth delay experiment. Immunohistochemistry was employed to elucidate the pattern of expression of VEGF, CD-31, MMP-2, and TUNEL assay for apoptosis in SOCS-1 overexpressing ME-180 cells. Tumor cells were injected to right thigh of mouse to form cancer mass. Lung metastasis assay was performed in day 45 and 60. Metastatic nodules in both lungs were obtained and counted for statistical analysis. Results: Tumor growth delay after 5 Gy radiation was observed in SOCS overexpressing tumor compared to wild type tumor but the difference did not show the statistical significance (p>0.05, Mann-Whitney U test). Immunohistochemistry showed that decreased MMP-2 expression, decreased VEGF expression, decreased CD-31 expression, and significantly elevated index of apoptotic body in SOCS-1 overexpressing tumor. Lung metastasis assay showed statistically significant decrement of metastatic nodule count in SOCS-1 overexpressing tumor (p<0.05, Mann-Whitney U test). Conclusion: Decrement of radiosensitivity was statistically insignificant in SOCS-1 overexpressing tumor while invasion, angiogenesis, and metastasis were inhibited. Also apoptosis was increased in SOCS-1 overexpressing tumor. Citation Format: Moon-Hong Kim, Wonwoo Kim, Miae Kang, Hyesil Seol, Jae-Hoon Jeong. Overexpression SOCS-1 may inhibit invasion and metastasis of cervical cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2014-119

Effect of recombinant adenovirus harboring snake venom cystatin on invasion and metastasis of human hepatocellular carcinoma cells MHCC97H

Objective To construct recombinant adenovirus harboring snake venom cystatin(Ad/sv-cystatin)and to investigate its effect on invasion and metastasis(in vivo and in vitro)of human hepatocellular carcinoma(HCC)MHCC97Hcells.Methods The recombinant Ad/sv-cystatin harboring sv-cystatin was constructed to infect MHCC97Hcells.Then the growth of MHCC97Hcells was assessed by CCK-8.Transwell matrigel assay was used to assess MHCC97Hcell migration and invasiveness in vitro.Spontaneous lung metastasis assays were used to examine the effects of Ad/sv-cystatin on the invasion and metastasis of MHCC97Hcells invivo.Results Recombinant Ad/sv-cystatin harboring sv-cystatingene could infect MHCC97Hcells.Ad/svcystatin significantly inhibited the growth,migration and invasion of MHCC97Hcells in vitro compared with control and Ad/null groups.Intra-tumoral injection of Ad/sv-cystatin significantly inhibited the lung metastasis of MHCC97Hcells in nude mice compared with control and Ad/null-treated mice.Conclusion It is indicated that recombinant Ad/sv-cystatin can suppress MHCC97Hcell growth,invasion and metastasis in vitro and in vivo,showing apotential for gene therapy of HCC.

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 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 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.

MiR-93 enhances angiogenesis and metastasis by targeting LATS2

Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17–92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93’s effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.

MiR-29c suppresses invasion and metastasis by targeting TIAM1 in nasopharyngeal carcinoma

Based on microarray analysis, we previously reported that miR-29c is significantly downregulated in nasopharyngeal carcinoma (NPC). However, little is known about the effect and molecular mechanisms of action of miR-29c deregulation during the development and progression of NPC. Quantitative RT-PCR demonstrated that miR-29c was significantly downregulated in NPC cell lines and clinical specimens. Wound healing, Transwell migration and lung metastasis assays demonstrated that ectopic expression of miR-29c inhibited NPC cell migration and invasion in vitro and suppressed the formation of lung metastases in vivo. T cell lymphoma invasion and metastasis 1 (TIAM1) was confirmed as a miR-29c target gene using luciferase reporter assays, quantitative RT-PCR and Western blotting. Ectopic expression of TIAM1 significantly promoted the migration and invasion of SUNE-1 cell line stably overexpressing miR-29c. The prognostic value of TIAM1 was analyzed in 217 NPC patients using immunohistochemistry. Strikingly, patients with high TIAM1 expression had poorer overall, disease-free and distant metastasis-free survival than patients with low TIAM1 expression. Furthermore, multivariate Cox regression analysis revealed that TIAM1 could serve as an independent prognostic factor in NPC. The newly identified miR-29c/TIAM1 pathway further elucidates the molecular mechanisms regulating invasion and metastasis in NPC, and may provide novel prognostic and treatment strategies for NPC patients.

Knockdown of endothelin A receptor expression inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model

Previous in vitro studies suggest that the endothelin A receptor (ETAR) could be a potential therapeutic target for osteosarcoma (OS) metastasis. In the present study, we assessed for the first time the role of ETAR in OS proliferation and pulmonary metastasis in vivo. MG-63 human OS cells were stably transduced with ETAR shRNA or scramble control shRNA and injected into the tibia of nude mice to generate an orthotopic xenograft OS model. The mice were divided into three groups (n=10 each group): i) the untransduced control group, where the mice were injected with untransduced MG-63 cells; ii) the scramble control group, where the mice were injected with cells stably transduced with the scramble control shRNA; iii) the ETAR-shRNA group, where the mice were injected with cells stably transduced with ETAR shRNA. The ETAR shRNA knocked down more than 75% of endogenous ETAR expression and significantly inhibited invasion, but not proliferation/viability of MG-63 cells in vitro. In the orthotopic xenograft OS mouse model, no significant difference in the tumor volume was observed over time among the untransduced control, the scramble control and the ETAR-shRNA groups. However, a combination of clinical signs, organ examinations and quantitative clonogenic lung metastasis assays showed that lung metastasis in the ETAR-shRNA group was significantly lower than that in the control groups. Immunohistochemical analyses revealed that the matrix metalloproteinase-2 (MMP-2) expression was significantly reduced in the ETAR-shRNA group compared with the control groups. The results were confirmed with western blot analyses using primary tumor tissues or the stably transduced MG-63 cells. In conclusion, we demonstrated in an orthotopic xenograft OS model that ETAR is critical for OS pulmonary metastasis, but not for tumor growth. This study provides the first in vivo evidence suggesting an important role for ETAR in OS pulmonary metastasis.

Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an invitro gelatinous protein (Matrigel) invasion and an invivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Invitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 ± 4.74) than in RH10Gy-SnonR (30.6 ± 3.85) cells, and lowest in McA-RH7777 (11.4 ± 3.56) cells. The same pattern was observed invivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 ± 5.38), RH10Gy-SnonR (22.17 ± 4.26), and McA-RH7777 (8.3 ± 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-α and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of metastasis-related cytokines and factors from NPCs after irradiation may be a possible mechanism for the radiation-induced invasiveness and metastatic potential of HCC.

Anti-angiogenesis effect of essential oil from Curcuma zedoaria in vitro and in vivo

Curcuma zedoaria (Berg.) Rosc., a traditional Chinese herb, was used widely but absolutely prohibited for the pregnant in clinic. Based on that there is abundant angiogenesis in endometrium and placenta during gestation period, we hypothesized that some components from it could inhibit angiogenesis and then damaged the supply of oxygen and nutrition to the embryo, which finally led to gestation failure. This study was set to demonstrate whether essential oil, major components of Curcuma zedoaria had anti-angiogenic effect. Essential oil of Curcuma zedoaria (EO-CZ) was abstracted by steam distillation extraction. Cell proliferation assay and two angiogenic models, rat aortic ring assay and chick embryo chorioallantoic membrane assay were presented. Furthermore, melanoma growth and experimental lung metastasis assay in mice were performed to evaluate its anti-angiogenesis effect in vivo. Immunohistochemical analysis and enzyme-linked immunosorbent assay (ELISA) were used to respectively detect the expression of CD34 and matrix metalloproteinases (MMPs). EO-CZ exhibited anti-proliferative effect on B16BL6 and SMMC-7721 cells, the IC(50), respectively was 41.8 μg/ml and 30.7 μg/ml, and on HUVEC (Human Umbilical Vein Endothelial Cells) cells with IC(50) of far more than 120 μg/ml. Both 20 μg/ml and 40 μg/ml EO-CZ indicated significant suppression on sprouting vessels of aortic ring and formation of microvessels in chick embryo chorioallantoic membrane in vitro. Moreover, solid melanoma grown in left oxter of mice was obviously inhibited after oral intake of 100 and 200 mg/kg of EO-CZ a day for 28 days, and CD34 expression indicating angiogenesis in melanoma reduced significantly compared with control; melanoma metastatic nodules in lung were detected to be inhibited, as well as MMP-2 and MMP-9 expression in serum. Essential oil, a fat-soluble fraction of Curcuma zedoaria, presented anti-angiogenic activity in vitro and in vivo, resulting in suppressing melanoma growth and lung metastasis. And this was associated with down-regulating MMPs.

Polymeric nanospheres modified with YIGSR peptide for tumor targeting

YIGSR peptide anchored pegylated nanospheres (YIGSR-SN) loaded with 5-fluorouracil (5-FU) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells over-expressing laminin receptors on and around tumor tissue and thus for assessing their targetability. Pegylated nanosphere (SN) without peptide conjugate were used for comparison. The average size of all nanosphere preparations prepared was ∼108 nm and maximum drug entrapment was 68.5 ± 5.2%. In vitro endothelial cell binding of nanospheres exhibited 8-fold higher binding of YIGSR-SN to HUVEC in comparison to the SN. Spontaneous lung metastasis and angiogenesis assays show that YIGSR peptide anchored nanospheres are significantly (p ≤ 0.05) effective in the prevention of lung metastasis and angiogenesis compared to free 5-FU and SN. In therapeutic experiments, 5-FU, SN, and YIGSR-SN were administered intravenously on day 4 at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing BALB/c mice resulting in effective regression of tumors in YIGSR-SN compared with free 5-FU and SN. Results indicate that YIGSR peptide anchored pegylated nanospheres bearing 5-FU are significantly (p ≤ 0.05) active against primary tumor and metastasis than the non-targeted pegylated nanospheres and free drug. Thus, YIGSR peptide anchored pegylated nanospheres hold potential of targeted cancer chemotherapeutics.

Liposomes modified with YIGSR peptide for tumor targeting

YIGSR peptide anchored sterically stabilized liposomes (YIGSR-SL) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells overexpressing laminin receptors on and around tumor tissue and thus for assessing their targetabilty. In vitro endothelial cell binding of liposomes exhibited 7-fold higher binding of YIGSR-SL to HUVEC in comparison to the nontargeted sterically stabilized liposomes (SL). Spontaneous lung metastasis and angiogenesis assays show that YIGSR peptide anchored liposomes are significantly (P ≤ 0.01) effective in the prevention of lung metastasis and angiogenesis compared to free 5-fluorouracil (5-FU) and SL. YIGSR-SL was very effective in regression of tumors in BALB/c mice bearing B16F10 melanoma cells. Results indicate that YIGSR peptide anchored sterically stabilized liposomes bearing 5-FU are significantly (P ≤ 0.01) active against primary tumor and metastasis than the SL and free drug. Thus, YIGSR peptide anchored sterically stabilized liposomes hold potential of targeted cancer chemotherapeutics.

Transmembrane Interactions Are Needed for KAI1/CD82-Mediated Suppression of Cancer Invasion and Metastasis

In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and alpha3beta1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis.

A highly invasive human glioblastoma pre-clinical model for testing therapeutics

Animal models greatly facilitate understanding of cancer and importantly, serve pre-clinically for evaluating potential anti-cancer therapies. We developed an invasive orthotopic human glioblastoma multiforme (GBM) mouse model that enables real-time tumor ultrasound imaging and pre-clinical evaluation of anti-neoplastic drugs such as 17-(allylamino)-17-demethoxy geldanamycin (17AAG). Clinically, GBM metastasis rarely happen, but unexpectedly most human GBM tumor cell lines intrinsically possess metastatic potential. We used an experimental lung metastasis assay (ELM) to enrich for metastatic cells and three of four commonly used GBM lines were highly metastatic after repeated ELM selection (M2). These GBM-M2 lines grew more aggressively orthotopically and all showed dramatic multifold increases in IL6, IL8, MCP-1 and GM-CSF expression, cytokines and factors that are associated with GBM and poor prognosis. DBM2 cells, which were derived from the DBTRG-05MG cell line were used to test the efficacy of 17AAG for treatment of intracranial tumors. The DMB2 orthotopic xenografts form highly invasive tumors with areas of central necrosis, vascular hyperplasia and intracranial dissemination. In addition, the orthotopic tumors caused osteolysis and the skull opening correlated to the tumor size, permitting the use of real-time ultrasound imaging to evaluate antitumor drug activity. We show that 17AAG significantly inhibits DBM2 tumor growth with significant drug responses in subcutaneous, lung and orthotopic tumor locations. This model has multiple unique features for investigating the pathobiology of intracranial tumor growth and for monitoring systemic and intracranial responses to antitumor agents.

Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma

Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.

Antimetastatic Effect of an Orally Active Heparin Derivative on Experimentally Induced Metastasis

Orally active anticancer drugs have great advantages for the treatment of cancer. Compelling data suggest that heparin exhibits critical antimetastatic effects via interference with P-selectin-mediated cell-cell binding. However, heparin should be given parenterally because it is not orally absorbed. Here, we evaluated the inhibitory effect of orally absorbable heparin derivative (LHD) on experimentally induced metastasis. We developed LHD, which is a chemical conjugate of low molecular weight heparin and deoxycholic acid, and measured the plasma concentration of LHD after oral administration. To evaluate the antimetastatic effect of LHD, we carried out experimental lung metastasis assays in vivo using murine melanoma or human lung carcinoma cells and interruption assay between murine melanoma cells and activated platelets and human umbilical vascular endothelial cells in vitro. In mice, the plasma concentration was approximately 7 microg/mL at 20 minutes after oral administration of LHD (10 mg/kg), indicating that bleeding was not induced at this dose. Interestingly, we found that LHD dramatically attenuated metastasis experimentally induced by murine melanoma or human lung carcinoma cells and that its antimetastatic activity was attributed to the interruption of the interactions between melanoma cells and activated platelets and between melanoma cells and human umbilical vascular endothelial cells by blocking selectin-mediated interactions. Furthermore, it prevented tumor growth in secondary organs. On the basis of these findings, the present study shows the possibility of LHD as a suitable first-line anticancer drug that can be used for preventing metastasis and recurrence because it has therapeutic potential as an antimetastatic drug, has lower side effects, and can be orally absorbed.

TPM3-ALK expression induces changes in cytoskeleton organisation and confers higher metastatic capacities than other ALK fusion proteins

Translocations of the anaplastic lymphoma kinase (ALK) gene result in the production of a number of oncogenic ALK fusion proteins implicated in tumour development. We have previously shown that X-ALK fusion proteins have differential effects on the proliferation, transformation, and invasion properties of NIH3T3 cells in vitro. In the present study, we have investigated the metastatic potential of various X-ALK expressing cell lines using an experimental lung metastasis assay. We have shown that TPM3-ALK expression bestows higher metastatic capacities than other X-ALK fusion proteins and in addition, that TPM3-ALK fusion protein expression specifically induces changes in cell morphology and cytoskeleton organisation. Co-immunoprecipitation studies demonstrate a specific interaction between TPM3-ALK and endogenous tropomyosin. Together the specific actions of TPM3-ALK on the cytoskeleton organisation offer an interesting hypothesis with respect to the higher cell motility and metastatic potential of this fusion protein.

Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Galphaq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment.

Increased plasma MMP9 in integrin α1‐null mice enhances lung metastasis of colon carcinoma cells

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin alpha1-null mice, a genetic model for increased plasma levels of MMP9 and MMP9-generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202-7). We show that while the number of lung colonies in integrin alpha1-null mice was significantly increased compared to their wild-type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well-vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.

The Low Molecular Weight Cyclin E Isoforms Augment Angiogenesis and Metastasis of Human Melanoma Cells In vivo

Abstract Immunohistochemical analysis has consistently shown that cyclin E is up-regulated in human malignant melanomas in vivo. Here we analyzed such expression in more detail and show that cyclin E is overexpressed and present in low molecular weight (LMW) isoforms in metastatic melanoma and in a subset of primary invasive melanoma tumor tissues, but not in benign nevi. Human metastatic melanoma cell lines, but not normal melanocytes, also expressed the LMW cyclin E forms. The biological significance of these findings was established by showing that overexpression of two LMW cyclin E forms named cyclin E truncated 1 [cyclinE(T1)] and cyclin E truncated 2 [cyclinE(T2)] in a low tumorigenic and non-metastatic primary cutaneous melanoma cell line generated angiogenic tumors with prominent perineural invasion compared with full-length cyclin E. In addition, cyclin E(T1)– and cyclin E(T2)–expressing melanoma cells displayed a dramatic increase in the incidence and number of metastases in an experimental lung metastasis assay. Together, these results indicate that the LMW cyclin E forms are functional and likely act as regulators of human melanoma tumor progression and invasion.