Human Fibrosarcoma HT1080 Cells Research Articles

The Mechanisms of Stimulation of Migration and Invasion of Tumor Cells by Extracellular Heat Shock Protein 90 (eHsp90) in vitro

Extracellular heat shock protein 90 (eHsp90) plays an important role in cell motility, invasion, and metastasis of tumor cells. eHsp90 stimulates migration and invasion of cells via interaction with surface receptors, which is accompanied by the activation of multiple cell motility-related signaling pathways. In addition, еHsp90 promotes cell invasion by the activation of extracellular matrix metalloproteinases. The role of different receptors, intracellular signaling pathways, and matrix metalloproteinases in the еHsp90-dependent migration and invasion of different types of cells has been investigated insufficiently. In this study, we demonstrated that HER2 is involved in the еHsp90-mediated stimulation of migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells in vitro. eHsp90-induced migration and invasion of cells are accompanied by the activation of ERK1/2-, IKK/NF-κB-, FAK-, ROCK1- and Src-mediated signaling pathways and by the limited activation of JNK, while the p38-mediated signaling cascade is not activated. eHsp90 also stimulates PI3K-Akt signaling pathway in А-172 cells, while in НТ1080 cells Akt is activated regardless of PI3K. It has been established that matrix metalloproteinases are involved in the eHsp90-dependent stimulation of invasion of А-172 and НТ1080 cells in vitro.

Preparation and biological evaluation of a novel pH-sensitive poly (β-malic acid) conjugate for antitumor drug delivery.

Poly (β‑malic acid), referred to as PMLA, has been synthesized and introduced as a polymeric drug carrier due to its desirable biological properties. In the present study, a novel pH‑sensitive polymer‑drug conjugate based on PMLA, PMLA‑Hz‑doxorubicin (DOX), was prepared, and another conjugate, PMLA‑ami‑DOX, was synthesized as a comparison. The structures, conjugation efficiency, and drug release properties of the prodrugs were determined. The cytotoxicity and cell uptake were assessed using the HT1080 human fibrosarcoma cell line as an invitro cell model. The release of DOX in the two conjugates were pH‑dependent in PBS buffer at a pH of 5.6, 6.0, 6.8 and 7.4. The quantity of drug released increased with the decrease in pH, and PMLA‑ami‑DOX released twice as much as PMLA‑Hz‑DOX (12h). The cytotoxicity of PMLA‑Hz‑DOX at pH 7.4 was lower than that of free DOX and increased with the decrease in pH, indicating that the cytotoxicity of PMLA‑Hz‑DOX was pH‑sensitive. Flow cytometry and confocal experiments confirmed the efficiency of the PMLA‑Hz‑DOX conjugate. Therefore, bonding DOX to PMLA via an acid‑sensitive hydrazone bond may be used to reduce its toxic side effects on normal tissues while responding to tumor pH and releasing the drug.

Triggered radiosensitizer delivery using thermosensitive liposomes and hyperthermia improves efficacy of radiotherapy: An in vitro proof of concept study

IntroductionTo increase the efficacy of chemoradiation and decrease its toxicity in normal tissue, a new concept is proposed, local radiosensitizer delivery, which combines triggered release of a radiosensitizer from thermosensitive liposomes with local hyperthermia and radiotherapy. Here, key aspects of this concept were investigated in vitro I) the effect of hyperthermia on the enhancement of radiotherapy by ThermoDox (thermosensitive liposome containing doxorubicin), II) the concentration dependence of the radiosensitizing effect of doxorubicin and III) the sequence of doxorubicin, hyperthermia and radiotherapy maximizing the radiosensitizing effect.MethodsSurvival of HT1080 (human fibrosarcoma) cells was measured after exposure to ThermoDox or doxorubicin for 60 minutes, at 37 or 43°C, with or without irradiation. Furthermore, cell survival was measured for cells exposed to different doxorubicin concentrations and radiation doses. Finally, cell survival was measured after applying doxorubicin and/or hyperthermia before or after irradiation. Cell survival was measured by clonogenic assay. In addition, DNA damage was assessed by γH2AX staining.ResultsExposure of cells to doxorubicin at 37°C resulted in cell death, but exposure to ThermoDox at 37°C did not. In contrast, ThermoDox and doxorubicin at 43°C resulted in similar cytotoxicity, and in combination with irradiation caused a similar enhancement of cell kill due to radiation. Doxorubicin enhanced the radiation effect in a small, but significant, concentration-dependent manner. Hyperthermia showed the strongest enhancement of radiation effect when applied after irradiation. In contrast, doxorubicin enhanced radiation effect only when applied before irradiation. Concurrent doxorubicin and hyperthermia immediately before or after irradiation showed equal enhancement of radiation effect.ConclusionIn vitro, ThermoDox resulted in cytotoxicity and enhancement of irradiation effect only in combination with hyperthermia. Therefore hyperthermia-triggered radiosensitizer release from thermosensitive liposomes may ultimately serve to limit toxicities due to the radiosensitizer in unheated normal tissue and result in enhanced efficacy in the heated tumor.

Lysophosphatidic acid receptor-2 (LPA2) and LPA5 regulate cellular functions during tumor progression in fibrosarcoma HT1080 cells

Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) regulate a variety of malignant properties in cancer cells. In the present study, we investigated the roles of LPA receptors in the promotion of cellular functions during tumor progression in fibrosarcoma cells. To obtain long-term anticancer drug treated cells, human fibrosarcoma HT1080 cells were treated with methotrexate (MTX) and cisplatin (CDDP) for 6 months. LPAR2 and LPAR5 expressions were significantly higher in MTX-treated (HT-MTX) cells than in HT1080 cells. The cell motile and invasive activities of HT-MTX cells were significantly elevated compared with HT1080 cells. Although LPAR5 expression was increased in MTX and CDDP treated (HT-M-C) cells, no change of LPAR2 expression was observed. The cell motile and invasive activities of HT-M-C cells were lower than those of HT1080 cells. Moreover, to evaluate whether LPA receptors promote cell invasive activity, highly invasion (HT1080-M6) cells were established from HT1080 cells. The cell invasive activity of HT1080-M6 cells was approximately 4.5 times higher than HT1080 cell invasion. LPAR2 expression was markedly elevated in HT1080-M6 cells compared with HT1080 cells. The high cell invasion activity of HT1080-M6 cells was significantly suppressed by an antagonist of LPA2, H2L5186303. These results suggest that LPA2 acts as a key regulator of malignant properties in HT1080 cells.

Abstract 5163: Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration

Abstract Focal adhesion (FA) turnover has been demonstrated to play an important role in controlling cell migration and cancer metastasis. FA disassembly was shown to be regulated by endocytosis during cell migration. Rabs are a kind of GTPase that participate in various cellular vesicle transports; however, whether the Rabs play a role in mediating vesicle transport for FA formation is not clear. This study focuses on Rab11, which plays a role in endosome recycling, and examines whether Rab11 has regulatory function in FA formation during cell migration. Wild-type and deficient Rab11 were transfected into human HT1080 fibrosarcoma cells, the cell migration ability was determined by trans-well assay, the localization of Rab11 and focal adhesion molecules were monitored by confocal microscopy and the in vivo study was carried out with subcutaneous xenograft mouse model to evaluate the effect of Rab11 on tumor growth. Results showed Rab11 deficiency inhibited sarcoma cell migration; the Rab11 was also found colocalized with recycled β1 integrin and affected focal adhesion formation. The live image results demonstrated the inactive GDP form Rab11 has no effect on random cell migration; however, the Rab11 knockdown inhibited random cell migration. We further used immunofluorescence and immunoprecipitation and identified the physical interaction of Rab11 and FAK. In the in vivo study, wild-type Rab11 transfected cells increased the tumor volume in xenograft mouse, but not in Rab11-deficient cells. Taken together, the results suggested Rab11 affected cell migration by regulating focal adhesion dynamics through integrin recycling and the effect of Rab11 may be through protein-protein interaction other than GTP activation. Citation Format: Ling-Yi Kao, Wan-Chen Wei, Hsiang-Ling Chiu, Yi-Che Wu, Wei-Ting Chao. Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5163.

Abstract 2135: Complex roles of discoidin domain receptors (DDRs) in tumor growth and experimental metastasis: role of collagen I in DDR-mediated tumor growth

Abstract DDR1 and DDR2 constitute a unique set of receptor tyrosine kinases that signal in response to collagen. Evidence indicates that DDRs play key roles in cancer progression. However, a comparison of the effects of DDR1 and DDR2 on tumor growth and metastasis within the same cellular context has not been done. To accomplish this goal, we developed a doxycycline (DOX) regulated system (Tet-Off) to express human DDR1b or DDR2 in the human HT1080 fibrosarcoma cell line. The cells were then examined for their ability to form subcutaneous (s.c.) tumors, or experimental lung metastases after intravenous (i.v.) injection in SCID mice. To induce or repress DDR expression in vivo, mice were fed a regular diet or a diet with DOX, respectively. Because collagen I (COL I) is a ligand for both DDR1 and DDR2, a set of mice were inoculated s.c. with HT1080 cells suspended in COL I with or without DDR induction in –DOX and +DOX mice, respectively. Growth of s.c. tumors was evaluated by measuring tumor volumes and lung metastases were quantitated by Alu qPCR and histological examination. In the absence of COL I, expression of either DDR1b or DDR2 had no impact on s.c. tumor growth rate, when compared to cells without DDR induction (+DOX). However, in the presence of COL I, tumor growth rates were significantly higher only in HT1080 cells with induced expression of DDR1b or DDR2 (p<0.001 for both), when compared to control cells inoculated with COL I into +DOX mice (no DDR induction). This suggests that a pro-oncogenic effect of DDR1b and DDR2 can be readily uncovered only in tumors growing within a COL I-rich environment. Because the Hippo tumor suppressor pathway has been shown to be regulated by extracellular matrix, homogenates of COL I tumors generated by cells with or without DDR induction were examined for levels and activation of Hippo pathway components. These analyses were consistent with Hippo pathway inactivation in tumors generated by DDR1b- and DDR2-expressing cells in a COL I matrix, consistent with their higher rate of tumor growth. Experimental metastasis assays revealed that induction of DDR2 expression had no impact on lung metastatic burden. In contrast, induction of DDR1b expression dramatically suppressed HT1080 lung metastasis formation, compared to control cells (no DDR1b induction). Collectively, these results suggest that: 1. Ectopic expression of DDRs in HT1080 cells accelerates tumor growth only when co-inoculated with COL I, consistent with a pro-tumorigenic effect of activated receptors within a collagen matrix. 2. A DDR/COL I axis supports HT1080 tumor growth, in part, by inactivation of the Hippo tumor suppressor pathway, suggesting that COL I-initiated DDR signaling regulates the Hippo pathway in vivo. 3. DDR1b, but not DDR2, is a potent suppressor of lung metastasis in the HT1080 model. Taken together, these studies provide novel insights into the complex roles of DDRs in cancer. Citation Format: Benjamin D. Wasinski, R. Daniel Bonfil, Anjum Sohail, Seong Ho Kim, Lisa Polin, Allen-Dexter Saliganan, Mohamad Bouhamdan, Sayed Nabi, Hyeong-Reh C. Kim, Marco Prunotto, Rafael A. Fridman. Complex roles of discoidin domain receptors (DDRs) in tumor growth and experimental metastasis: role of collagen I in DDR-mediated tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2135.

A heptameric peptide isolated from the marine microalga Pavlova lutheri suppresses PMA-induced secretion of matrix metalloproteinase-9 through the inactivation of the JNK, p38, and NF-κB pathways in human fibrosarcoma cells

In this study we isolate a heptameric peptide from the marine microalga Pavlova lutheri and examine its inhibitory effect and mechanism of action on the matrix metalloproteinase-9 (MMP-9) protein in human fibrosarcoma HT1080 cells. Through consecutive rounds of purification we isolated a potent MMP-9 inhibitory peptide composed of seven amino acids: Leu-Leu-Ala-Pro-Pro-Glu-Arg (796.4 Da). We show that the mRNA and protein expression levels of MMP-9 are reduced in the presence of this purified peptide. Furthermore, we show that this peptide inhibits PMA-induced translocation of nuclear factor-κB (NF-κB) by suppressing IκB-α phosphorylation and that it suppresses the phosphorylation of the c-Jun N-terminal (JNK) and p38 mitogen-activated protein kinases (MAPKs). Our results indicate that this peptide, purified from P. lutheri, has the potential to be a valuable natural preventive agent against MMP-related diseases.

New Terpenoids from Chamaecyparis formosensis (Cupressaceae) Leaves with Modulatory Activity on Matrix Metalloproteases 2 and 9.

Chamaecyparis formosensis is Taiwan’s most representative tree, and has high economic value. To date, only a few active chemical constituents have been reported for C. formosensis. In this study, 37 secondary metabolites, including three new compounds (1–3), were extracted from the leaves of C. formosensis. The compounds isolated from the ethyl acetate layer were used at different concentrations to treat HT-1080 human fibrosarcoma cells and to evaluate their effects on matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) expression. Based on extensive analysis of data from high-resolution mass spectrometry (HR-MS) as well as nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV) spectroscopy, the new compounds were identified as 11,12-dihydroxyisodaucenoic acid (1), 12-hydroxyisodaucenoic acid (2), and 1-oxo-2α,3β-dihydroxytotarol (3). Known compounds 4–37 were identified by comparing their spectroscopic data with data reported in the literature. Biological activity tests by gelatin zymographic analysis revealed that seven compounds, including new compound 2, have no cytotoxic effect on HT-1080 cells and were found to increase MMP-2 or MMP-9 expression by 1.25- to 1.59-fold at lower concentrations of 10–50 µM. These naturally derived regulatory compounds could potentially serve as a novel pharmaceutical basis for medical purposes.

Platelet-derived growth factor-C functions as a growth factor in mouse embryonic stem cells and human fibrosarcoma cells

BackgroundPlatelet-derived growth factor-C (PDGF-C) has been shown to be involved in several biological processes, such as embryonic development, wound healing and angiogenesis, as well as in diseases including tumor formation and fibrotic diseases. However, its role in fibrosarcoma and embryonic stem (ES) cells has not been elucidated.MethodsThe expression level of PDGF-C was measured using RT-PCR. The activity of PDGF-C was suppressed using RNA interference or a neutralizing antibody and the effect on cell growth was examined using the WST and soft agar assays.ResultsIn the tumor cell lines studied, the highest level of PDGF-C expression was in human HT1080 fibrosarcoma cells. In ES cells, it was highly expressed in the self-renewal state but not in the differentiated state. PDGF-C knockdown suppressed anchorage-dependent and -independent growth of HT1080 and ES cells. In addition, the suppression of PDGF-C activity by a neutralizing antibody retarded ES cell growth.ConclusionOur results suggest that PDGF-C plays an important role in the proliferation of fibrosarcoma and ES cells.

Genetically modified human type II collagen for N- and C-terminal covalent tagging

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

In vitro anticancer effects of a RAGE inhibitor discovered using a structure-based drug design system.

Receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor implicated in the pathogenesis of certain types of cancer. In the present study, papaverine was identified as a RAGE inhibitor using the conversion to small molecules through optimized-peptide strategy drug design system. Papaverine significantly inhibited RAGE-dependent nuclear factor κ-B activation driven by high mobility group box-1, a RAGE ligand. Using RAGE- or dominant-negative RAGE-expressing HT1080 human fibrosarcoma cells, the present study revealed that papaverine suppressed RAGE-dependent cell proliferation and migration dose-dependently. Furthermore, papaverine significantly inhibited cell invasion. The results of the present study suggested that papaverine could inhibit RAGE, and provided novel insights into the field of RAGE biology, particularly anticancer therapies.

Cell invasiveness in sarcomas: a possibly useful clinical correlation

The prognosis of each individual patient affected by sarcoma, including those with low histopathologic grading, cannot be reliably predicted at the time of surgery. We have developed an in vitro cell invasion assay on early primary cell cultures derived from surgically removed sarcomas. Primary cell cultures were subjected to in vitro cell invasion assays by using Boyden chambers, filters coated with matrigel and fetal bovine serum as a source of chemoattractant. For each primary cell culture, the sarcoma cell invasion index was determined in comparison with the percentage of human fibrosarcoma HT1080 cell invasion extent. The cell invasion index of 7 different sarcomas was evaluated in respect to the outcome of the disease, after a follow-up ranging from 14 to 48 months. Data evidenced that a low cell invasion index (39.7% +/- 8.9) was retained by tumor cells derived from patients with no progression of the disease and with a longer interval of disease-free survival (21 +/- 0.8 months). However, an increase in cell invasion index (61% +/- 5) was retained by tumor cells derived from patients with progression of the disease and with a shorter disease-free survival (9 +/- 3 months). Overall, although only 7 cases were analyzed, a statistically significant correlation was found between disease-free survival and cell invasion index (P = 0.003). Our data support the possibility that cell invasion assays performed in vitro on cells derived from human sarcomas may be predictive of a more aggressive form of the disease.

Prodigiosin R2, a new prodigiosin from the roseophilin producer Streptomyces griseoviridis 2464-S5.

Roseophilin (2) is a unique prodigiosin-related compound produced by Streptomyces griseoviridis 2464-S5, and is characterized by a central furan ring and a bicyclic alkyl chain. During a search for biosynthetic intermediates of 2, a new metabolite designated prodigiosin R2 (1) was isolated from the culture of the roseophilin producer. The molecular formula of 1 was established as C27H35N3O by high-resolution FAB-MS. The structure of 1 was determined by NMR spectroscopic analyses as a prodigiosin derivative with the same bicyclic alkyl chain as 2. Prodigiosin R2 (1) showed potent cytotoxicity against HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells with IC50s of 0.41 and 0.82 μM, respectively.

Abstract B204: Integrin β1 plays a crucial role in vasculogenic mimicry formation

Abstract Tumor metastasis is caused by multiple processes, such as epithelial-mesenchymal transition, lymph node metastasis, and angiogenesis. In recent years, vasculogenic mimicry (VM) is focused on the association with tumor malignancy. VM is a unique phenomenon, a blood vessel-like network structure formed only by cancer cells without vascular endothelial cells, and is observed in extracellular matrix (ECM) rich region. Although previous studies suggested that VM plays a crucial role in tumor metastasis, a detailed mechanism of VM formation has not been fully understood. Latest studies indicate that cell surface proteins such as integrins and cadherins contribute to VM formation. In this study, we focused on transmembrane cell surface receptor, integrin β1. Integrin β1 is an important factor for interactions between cell-cell and cell-ECM, and mediates several cell signalings. Therefore, we investigated the importance of integrin β1 in VM formation. We assessed VM formation capability as network structure formed by cancer cells on matrigel. Since previous studies revealed that Ca2+ regulates integrin activities, we treated with EGTA and CaCl2 to assess whether Ca2+ is necessary for VM formation. We performed integrin β1 gene knockout (KO) by using CRISPR/Cas9 system in human fibrosarcoma HT1080 cells, and assessed VM formation capability of the KO cells. In addition, we established integrin β1-rescued HT1080 cells by reintroduction of Cas9-resistant integrin β1-expressing vector to integrin β1-KO HT1080 cells to confirm the importance of integrin β1 in VM formation. Furthermore, we also established integrin β1-KO human breast cancer MDA-MB-231 cells to verify that integrin β1 is necessary for VM formation in other human cancer cell lines. As a result, chelation of Ca2+ by treatment with EGTA inhibited network formation in HT1080 cells, and cotreatment with CaCl2 rescued this effect, suggesting that integrins might be important for VM formation. In integrin β1-KO HT1080 cells, which is confirmed the completely knockout of integrin β1 by Western blot, network formation was abrogated. Additionally, integrin β1-rescued HT1080 cells exhibited network formation as well as parental HT1080 cells. These results suggested that integrin β1 is a critical factor in VM formation. Furthermore, integrin β1-KO MDA-MB-231 cells also did not form network structure, suggesting that integrin β1 plays a crucial role in VM formation in general human cancer cell lines. Taken together, our study provides new knowledge of the association between VM and integrin β1, and inhibition of integrin β1 may be a novel therapeutic approach for metastatic cancer. Citation Format: Ryota Kawahara, Yuki Niwa, Siro Simizu. Integrin β1 plays a crucial role in vasculogenic mimicry formation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B204.

1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] Inhibits MMP-9 Expression by Regulating NF-κB and MAPKs Signaling Pathways in HT1080 Human Fibrosarcoma Cells.

Hippocampus is a traditional medicine in China, which can be used for treating tumors, aging, fatigue, thrombosis, inflammation, hypertension, prostatic hyperplasia, and other diseases. 1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] from seahorse (Hippocampus kuda Bleeler) has been shown to suppress proinflammatory responses. In the present study, SE1 potently inhibited gelatin digestion by MMP-9 induced by phorbol 12-myristate 13-acetate (PMA) and migration of human fibrosarcoma HT1080 cells in dose-dependent manner. Moreover, western blot analysis and immunofluorescence analysis have been studied on MAPKs (ERK1/2, p38 kinase and JNK) and NF-κB (p65 and IκB), which refer to the clear molecular mechanism. The results indicated that SE1 significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPK: p38 kinase and JNK) and NF-κB. Finally, molecular docking result showed SE1 interacts with TYR245 and HIS226 of MMP-9 by hydrogen bond and Pi-Pi bond to suppress MMP-9 activity. This data suggested that the SE1 may possess therapeutic and preventive potential for the treatment of MMP-9 related disorders.

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials.

Effect of Agmatine Sulfate on Modulation of Matrix Metalloproteinases via PI3K/Akt-1 in HT1080 Cells

The purpose of this study was to investigate the mechanism by which agmatine sulfate induces an anti-metastatic effect in human HT1080 fibrosarcoma cells, by affecting matrix metalloproteinases (MMPs). For the experiments, we used a non-toxic concentration of agmatine, below 512 μM, that was determined using an MTT assay. The effect of agmatine sulfate on metastasis was gelatin zymography, western blot, immunofluorescence staining and cell invasion assay. Agmatine sulfate inhibited MMP-2 activity stimulated by phenazine methosulfate (PMS). Furthermore, the expression level of MMP-2 stimulated by PMS, was decreased, but the expression level of TIMP-1 was increased in the presence of agmatine sulfate. Moreover, it was observed that the expression levels of ERK and p38 were increased, but those of PI3K and Akt-1 associated with the modulation of MMP-2 were decreased in this study. Furthermore, agmatine sulfate decreased the invasion level of human fibrosarcoma cells stimulated by VEGF. These results suggest that agmatine sulfate could inhibit metastasis through inhibition of MMP-2 via the PI3K/Akt-1 signaling pathway.

Abstract 3161: TPX-0005, an ALK/ROS1/TRK inhibitor, overcomes multiple resistance mechanisms by targeting SRC/FAK signaling

Abstract ALK, ROS1, and TRK inhibitors have achieved marked efficacy in treating cancer patients expressing abnormal ALK, ROS1 or NTRK genes. However, the initial success of these therapies is rapidly overshadowed with the development of acquired resistance. In addition, approximately 30-40% of ALK + or ROS1+ NSCLC patients fail to respond to initial crizotinib treatment, representing intrinsic resistance. Target gene amplification, acquired resistance mutations, bypass signaling, epithelial-mesenchymal transition (EMT) and metastasis are the common resistance mechanisms. None of the current ALK, ROS1 or TRK inhibitors can overcome bypass or EMT-based resistance when applied as a single agent therapy. TPX-0005 is a potent kinase inhibitor against WT and mutated ALK, ROS1 and TRK family kinases, especially the clinically significant solvent front mutations. At clinically relevant concentrations, TPX-0005 also inhibits JAK2, SRC and FAK that are important targets in modulating multiple resistance mechanisms. H2228 lung cancer cell line endogenously expresses EML4-ALK v3 and was reported to be resistant to ALK inhibitor TAE684. Upregulation of the bypass signaling kinase EGFR, EMT marker vimentin, and cancer stem-like marker CD44 was reported in H2228 cells, likely leading to intrinsic resistance to ALK inhibitors. In the anti-proliferation assay, both crizotinib and ceritinib were resistant in H2228 cells with IC50 values around 1000 nM, however, TPX-0005 was able to overcome the intrinsic resistance with an IC50 of 100 nM. In H2228 cells, TPX-0005 suppressed the phosphorylation of SRC, FAK and paxillin with IC50 values in a range of 80-100 nM, and downregulated the expression of EGFR, CD44, and vimentin with IC50 values around 100 nM. In addition, TPX-0005 inhibited the phosphorylation of the oncogenic transcription/translation factor YB-1 with an IC50 value around 100 nM in H2228 cells. YB-1 is involved in many aspects of gene expression control that lead to tumor cell growth and drug resistance, including modulation of EGFR upregulation, EMT, and cancer stemness. Therefore, it was postulated that inhibition of SRC/FAK by TPX-0005 suppressed the phosphorylation of YB-1, leading to the downregulation of EGFR, CD44 and vimentin, and eventually to anti-proliferation effect on H2228 cells. TPX-0005 demonstrated an in vitro anti-metastatic activity by inhibiting cell migration in both H2228 cells and HT1080 human fibrosarcoma cells. Taken together, the potent kinase inhibitory activities against SRC/FAK signaling provide a unique polypharmacology profile to TPX-0005 for combatting multiple resistance mechanisms simultaneously, including a broad spectrum of acquired mutations, bypass signaling, EMT, cancer stemness, and metastasis. Citation Format: Dayong Zhai, Wei Deng, John Huang, Evan Rogers, J. Jean Cui. TPX-0005, an ALK/ROS1/TRK inhibitor, overcomes multiple resistance mechanisms by targeting SRC/FAK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2017-3161

Abstract 1044: Development of a miRNA-based signature to predict human cancer metastasis

Abstract Metastasis is the primary cause of death for cancer patients, and invasive cancer cell migration is required at multiple steps during metastasis (e.g. intravasation and extravasation). Since microRNAs (miRNAs) have been implicated as key regulators of metastatic spread of cancer cells, we sought to develop a miRNA signature that can be used to predict cancer metastasis. We hypothesized that the miRNAs that are functionally required for invasive cell migration could serve as biomarkers to predict human cancer metastasis. We developed an intravital imaging-based approach combined with NGS to screen for miRNAs that are required for invasive cell migration of human HT1080 fibrosarcoma cells. The screen-identified miRNAs were validated in a panel of in vitro and in vivo assays for invasive migration. Microarray analysis identified genes downregulated in transfected HT1080 cells. Publically available databases were used to correlate the expression of screen-identified miRNAs and the progression of multiple human cancers. We used a qRT-PCR approach combined with machine learning to develop a miRNA signature to predict metastasis in a 66-patient cohort of prostate cancer. We identified over twenty novel miRNAs that regulate directional cancer invasion. Microarray analysis in HT1080 cells revealed that the altered expression of metastasis-regulating miRNAs is associated with 50% reduction in gene expression of migration and adhesion gene network components such as integrin a4, CDC42, and transgelin). We evaluated the potential of screen-identified miRNAs to serve as biomarkers to predict cancer metastasis. Using publically available databases, we found that majority of screen-identified miRNAs are dysregulated in multiple human cancer types (e.g. prostate, breast, ovarian and lung) and the expression of these miRNAs correlate directly with patient disease progression. We analyzed the expression of the top two screen-identified miRNAs in plasma samples from the PCa patient cohort. A signature was generated using a weighted K-nearest neighbor algorithm that provided a ROC area under the curve of 0.79 for predicting metastatic disease. We identified a panel of novel metastasis-regulating miRNAs that is functionally involved in human cancer metastasis. These miRNAs have the potential to serve as both biomarkers to predict metastasis and potentially as therapeutic targets to block metastasis. Citation Format: Lian Willetts, Konstantin Stoletov, Juan Jovel, Emma Woolner, John D. Lewis. Development of a miRNA-based signature to predict human cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1044. doi:10.1158/1538-7445.AM2017-1044

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.