Fibrosarcoma Line Research Articles

Binding Mechanisms and Therapeutic Activity of Heterocyclic Substituted Arylazothioformamide Ligands and Their Cu(I) Coordination Complexes.

Finding new sources of biologically active compounds for anticancer or antimicrobial therapies remains an active area of research. Azothioformamides (ATFs) with a 1,3 N=N-C=S heterodiene backbone are a new class of biologically active compounds that chelate metals (e.g., Cu) forming stable ATF metal coordination complexes. In this study, ATF ligands were prepared with pyrrolidine, piperidine, N-methylpiperazine, and morpholine substituents on the formamide as to add more heterocyclic drug-like character for biological studies. Formamide derivatives were then complexed with various Cu(I) salts to form coordination complexes. Cu(I) salts were selected as to create potential bioactive compounds with less toxicity. Binding association constants of each Cu(I) salt to ATF ligands were extrapolated from UV-vis titration studies and were corroborated with DFT calculations using a hybrid functional B3LYP method. It was observed that the smaller pyrrolidine functionalized ATFs bound to the Cu(I) salts had stronger binding than any of the larger six-membered-ring heterocycles with association values in the 104 - 105 M-1 range. The ATF-Cu(I) salt coordination complexes were then evaluated for antimicrobial activity against two bacteria (Staphylococcus aureus, Escherichia coli), one yeast (Candida albicans), four human cancer lines (A-549, K-562, HT-1080, MDA-MB-231), and two normal human lines (MRC-5, HFF). The ATF ligands themselves were inactive against all microbes and most human lines except K-562 cells, which were sensitive to three of the four ligands (IC50's = 7.0-25.5 μM). Most ATF-Cu(I) complexes showed low to medium micromolar activity against Candida albicans (IC50's 2.6-24.8 μM) and Staphylococcus aureus (IC50's = 3.4-37.7 μM), with increasing activity corresponding to complexes with higher binding association constants. The antiproliferative properties of ATF-Cu(I) metal salt complexes against mammalian cells were mixed, with low to medium micromolar activity across all cell lines. Notably, several ATF-Cu(I) salt coordination complexes showed submicromolar activity against the HT-1080 fibrosarcoma line (0.52-0.69 μM). The results demonstrate promising activity of ATF-Cu(I) complexes, particularly with pyrrolidine as the formamide component. These studies suggest that the stronger binding association values correlate to higher levels of biological activity.

Abstract 3974: Investigating doxorubicin resistance in fibrosarcoma

Abstract Cytotoxic chemotherapy has been the backbone of medical oncology for nearly a century for multiple cancer types. However, when treating advanced or metastatic disease toxicity, side effects, and acquired resistance often limit the duration of therapy. While randomized control trials have traditionally served as the proving ground for first and subsequent line therapies, rare tumor subtypes including soft tumor sarcoma (STS) provide unique challenges for developing robust treatment data. By developing human based in vitro models, we can investigate potential therapeutic mechanisms for STS. Fibrosarcoma is one distinct histologic STS subtype that relies heavily on anthracycline treatment in the first line setting, however, there are limited options once resistance develops. Anthracyclines, including doxorubicin, exert their effect through a variety of mechanisms including reactive oxygen species, DNA-adduct formation, and inhibiting topoisomerase II, histone eviction, calcium and iron homeostasis, and ceramide overproduction. However, there have also been multiple mechanisms of doxorubicin resistance including disruption of cell membrane, alteration of cell surface molecules, inactivation of drugs, gene amplification, activating alternative signaling pathways, and affecting cellular repair mechanisms. In order to investigate the role of anthracycline resistance in fibrosarcoma lines we acquired two human derived fibrosarcoma lines SW684 and HT-1080 from American Type Culture Collection (ATCC). Characterization of HT-1080 using publicly available datasets demonstrated that TP53, CDK2NA and NF1 serve as major driver mutations while SW684 relies on NRAS and IDH1 mutations. We further characterized these cell lines in vitro and noted that HT-1080 has a quick doubling time of 16 hours while SW684 has a doubling time of 91 hours. Additionally, after doxorubicin treatment, we found that HT-1080 is sensitive with an IC50 of 5.594 nanomolar while SW684 is relatively doxorubicin resistant with an IC50 of 28.915 micromolar. While proliferative rate may play a role in the sensitivities of these cell lines further characterization of the mechanism leading to doxorubicin resistance is needed. To that end, we are using a CRISPR-Cas9 approach for further genome wide screening of these fibrosarcoma lines in the presence of doxorubicin. Exploring the genetic mechanisms that result in development of doxorubicin resistance may reveal novel, targetable mechanisms expanding treatment durability in this rare STS. Citation Format: Rodney Dixon Dorand, Donna Dang, Elizabeth J. Davis, Ben Ho Park. Investigating doxorubicin resistance in fibrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3974.

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

DRES-05. PREDICTORS OF SENSITIVITY TO COMBINED TEMOZOLOMIDE AND PARP INHIBITOR IN GLIOMA

Abstract Agents targeting the PARP enzyme family are under active development for the treatment of gliomas. PARP inhibitors (PARPi) can enhance the effect of temozolomide (TMZ) in IDH wild-type glioblastomas, and in addition recent studies have shown that PARP inhibitors can be selectively lethal in IDH1 mutant cancers. Here, we sought to identify predictors of sensitivity and resistance to PARP inhibition in gliomas. We treated 4 IDH1 mutant and 8 IDH wild-type glioma lines, as well as IDH1 mutant fibrosarcoma line HT1080, with TMZ, the PARPi olaparib, or the combination, and assessed cellular growth and survival. In dose response assessments, 7 out of 13 lines were sensitive to olaparib monotherapy (3 IDH1 mutant and 4 IDH wild-type). Combination with TMZ resulted in 6 of 13 lines responding to dual therapy, with an additive effect seen in 5 PARPi monotherapy sensitive gliomas. Notably, 5 of the 6 lines responsive to the combination harbored CDKN2A deletion, compared to none of the non-responsive lines (p=.0021). Treatment with CDK inhibitor palbociclib partially reversed sensitivity to TMZ+PARPi in HT1080, supporting a mechanistic basis for this association. PARPi sensitivity in glioma lines can be augmented by the addition of TMZ. CDKN2A deletion may serve as a potential biomarker identifying tumors sensitive to this combination therapy.

Human mesenchymal stromal cells do not promote recurrence of soft tissue sarcomas in mouse xenografts after radiation and surgery

Background. Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. Methods. Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow–derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γcnull mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. Results. MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. Discussion. These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients.

Abstract 5854: BT5528, a Bicycle Toxin Conjugate targeting EphA2 has potent anti-tumor activity without bleeding or coagulation abnormalities in preclinical models

Abstract Ephrin receptor A2 (EphA2) is a member of the Ephrin receptor family of cell-cell junction proteins and is both highly overexpressed in several solid tumors and associated with poor prognosis in patients. EphA2 is considered a high-value target for cancer therapeutics, but development of an antibody drug conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events, including bleeding toxicity were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumor penetration. BT5528 was selected from a panel of >75 Bicycle Toxin Conjugates (BTCs) with variations in Bicycle binder, molecular spacer, cleavable linker and toxin payload, based on in vivo efficacy and tolerability. BT5528 is effective in EphA2-expressing xenograft models, with efficacy seen from 1mg/kg qw and complete tumor regression at 3mg/kg qw. Rapid, complete regression of tumors is seen across a range of EphA2-expressing cell lines, including HT1080 fibrosarcoma line, MDA-MB-231 triple negative breast cancer line and NCI-H1975 lung cancer line and NSCLC PDX models. In order to test the benefit of the enhanced tumor penetration of BTCs, NSCLC PDX tumors were grown to >1000mm3 before commencing treatment. Complete regression of tumor was seen following 3 weeks of dosing. The bleeding events reported in early clinical trials of MEDI-547 (EphA2 ADC) were consistent with effects on the coagulation system in preclinical models. Dose-limiting toxicology in non-human primate (NHP) was reportedly Disseminated Intravascular Coagulation (DIC) and changes were seen in APTT and D-Dimer, with associated elevations in liver enzymes. Toxicology studies of BT5528 in rat and NHP showed no laboratory or clinical evidence of DIC, with normal levels of platelets, D-Dimer, APTT, and other coagulation-related parameters. Bicycle Toxin Conjugates (BTC) targeting EphA2 show potent antitumor activity in a range of solid tumor xenograft models. The BTCs show profound efficacy, without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway. Citation Format: Gavin Bennett, Philip Huxley, Amy Brown, Gemma Mudd, Peter U. Park, Nicholas Keen. BT5528, a Bicycle Toxin Conjugate targeting EphA2 has potent anti-tumor activity without bleeding or coagulation abnormalities in preclinical models [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 5854.

Ultrasound-stimulated microbubble enhanced low-dose radiation response

We have recently demonstrated that mechanical perturbation of endothelial cells from ultrasound-stimulated microbubbles (USMB) results in enhanced tumor radiosensitivity at low 2 Gy doses of radiation. Our hypothesis is that USMB-based endothelial membrane perturbations produce ceramide via a sphingomyelinase (ASMase) pathway, and act synergistically with radiation to enhance overall tumor response. Here, we investigate the role of the SMase-ceramide pathway on USMB-based endothelial radiosensitization. Experiments were carried out in wild type (C57BL/6) and ASMase knockout mice, implanted with a fibrosarcoma line (MCA-129). Animals were treated with radiation doses varying from 0 to 8 Gy alone, or in combination with ultrasound-stimulated microbubbles. Treatment response was assessed with Doppler ultrasound vascularity index acquired at 3, 24, and 72 hrs using a VEVO770 preclinical ultrasound system. Staining using ISEL, ceramide, and CD31 immunohistochemistry of tumor sections was used to complement res...

ATPS-852-HYDROXYGLUTARATE DEPLETION IS NOT SUFFICIENT TO INHIBIT GROWTH OF SEVERAL PROGRESSIVE IDH1 MUTANT SOLID CANCER TYPES

Targeted agents that inhibit 2-hydroxyglutarate (2-HG) production by mutant IDH1 have shown considerable promise in acute leukemia. To examine the potential effects of 2-HG reduction in IDH1 mutant solid cancers, we treated endogenous IDH1 mutant tumor cells from several tissue types with a well-characterized inhibitor of IDH1 R132H and R132C (IDH1i) in vitro and in vivo. We confirmed potent inhibition of 2-HG formation in vitro in IDH1R132H glioma tumorsphere lines and a fibrosarcoma line, HT1080 (IDH1R132C). Then, we broadly tested 8 endogenous IDH1 mutant cell lines, including 6 IDH1R132H gliomas, HT1080 and a melanoma line E232-2010 (IDH1R132C). IDH1i markedly reduced 2-HG, however did not inhibit in vitro growth of any cell line. We then assessed the effect of IDH1i treatment on mice bearing patient-derived, endogenous IDH1 mutant glioma orthotopic xenografts. Continuous daily oral administration of IDH1i (200 mg BID) achieved marked 2-HG reduction within the tumor. However, we found no significant difference in tumor size or survival of IDH1i-treated versus vehicle-treated mice. Furthermore, neither short-term nor prolonged (>6mo) in vitro IDH1i exposure significantly altered the global trimethylation or dimethylation of histone 3 lysine 9 (H3K9), global trimethylation of H3K27, genomewide distribution of DNA methylation or methylation of the O-6-methylguanine-DNA methyltransferase gene promoter. Paradoxically, long-term in vitro IDH1i incubation resulted in acceleration of cell proliferation and significantly shorter animal survival upon subsequent intracerebral implantation, compared to passage-matched IDH1 mutant glioma cells grown in the absence of IDH1i. Thus, 2-HG levels can be uncoupled from cell proliferation in several IDH1 mutant solid cancer types, and the epigenetic alterations induced by mutant IDH1 are not reversed solely by depletion of 2-HG, even after many cell divisions. Alternative combination therapeutic strategies may be needed for successful growth inhibition of progressive mutant IDH1 solid cancers.

Abstract 1812: Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide

Abstract We have recently demonstrated that mechanical perturbation of endothelial cells from ultrasound-stimulated microbubbles (USMB) results in enhanced tumour radiosensitivity. When exposed to high-power ultrasound, microbubbles release energy in the form of local mechanical forces on the membranes of surrounding endothelial cells. Upon irradiation, rapid vascular shutdown occurs followed by extensive secondary tumour cell death. Our hypothesis is that USMB-based endothelial membrane perturbations produce ceramide via a sphingomyelinase (ASMase) pathway, and act synergistically with radiation to enhance overall tumour response. Here we investigate the role of the SMase-ceramide pathway on USMB-based endothelial radiosensitization. Experiments were carried out in wild type (C57BL/6) and ASMase knockout mice, implanted with a fibrosarcoma line (MCA-129). Animals were treated with radiation doses varying from 0-8 Gy alone, or in combination with USMB. In addition, we pre-treated a subset of animals with sphingosine-1-phosphate (S1P). Treatments with USMB consisted of a 16-cycle tone burst at a 500 kHz center frequency and 570 kPa using a 2.86 cm element diameter ultrasound transducer. The total insonification time was 750 ms over 5 minutes. Microbubbles (Lantheus Medical) were injected through the tail-vein resulting in a blood volume concentration of 1% or 3% (v/v). Treatment response was assessed with Doppler ultrasound acquired at 3, 24 and 72 hrs using a VEVO770 preclinical ultrasound system. The vascularity index (VI) was used to quantify power Doppler data. Staining using ISEL, ceramide and CD31 immunohistochemistry of tumour sections was used to complement ultrasound images, and to confirm results. Results suggest a link between USMB-based radiosensitization and ASMase. We observed an average decrease of 30% in the VI by 3 hrs in wild type tumours receiving 8 Gy radiation alone. In contrast, those receiving 8 Gy and USMB resulted in a VI decrease of up to 50%. Similarly, while 2 Gy alone induced minimal effects on the tumour vasculature, combining it with USMB resulted in up to 40% decrease in the VI by 24 hrs. Vascular effects were significant (p < 0.05) and sustained for up to 72 hrs after combined treatments. These resulted in significant cell death. In contrast to wild type animals, ASMase knockout mice, or wild-type mice receiving S1P, were found to be generally resistant to the anti-vascular effects of radiation and USMB. We noted minimal cell death and no vascular shutdown following any of the treatments in those experimental groups. Overall conclusions drawn from this work point to a mechanotransduction-like effect that results in endothelial radiosensitization. Citation Format: Gregory Jan Czarnota, Ahmed El Kaffas, Anoja Giles, Azza Al Mahrouki, Amr Hashim. Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2015-1812

Fibroblast activation protein increases metastatic potential of fibrosarcoma line HT1080 through upregulation of integrin-mediated signaling pathways.

The serine protease fibroblast activation protein (FAP) is selectively expressed on tumour-associated fibroblasts in most human epithelial tumours, as well as on some mesenchymal tumours such as sarcoma. High FAP expression is most often associated with poor outcome and increased metastasis. Here, we compare the in vitro metastatic potential of HT1080 fibrosarcoma cells with and without FAP expression in order to elucidate the mechanism by which FAP may influence metastasis. In the presence of FAP, cells were more adhesive to extracellular matrix proteins and migrated and invaded through Matrigel to a greater degree. The anti-FAP antibody ESC11, which caused internalization of FAP, decreased adhesion and migration, but only when cells expressed FAP. It was also found that blocking activity of integrins β1 and αvβ3 reduced both cell adhesion and migration and this effect was much more marked in FAP-expressing HT1080 cells than mock-transfected HT1080 cells. The expression or activation of intracellular proteins that form part of the downstream signaling of integrins, including integrin-linked kinase, Rac1 and focal adhesion kinase, was also upregulated when FAP was expressed, suggesting that FAP not only upregulates metastatic-like cell behaviours through interaction with integrins, but also influences the intracellular signaling of integrins. This was confirmed using both PI3 kinase and Src kinase inhibitors, which decreased adhesion and migration in FAP-expressing cells, but did not affect mock-transfected HT1080 cells. FAP is therefore a useful target for anti-cancer therapy, as not only is its expression tumour-selective, but its downregulation has the potential to reduce incidence of metastasis.

Competing for the treasure in exceptions

Competing for the treasure in exceptions

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues.

Abstract 5427: Combination of aflibercept (VEGF trap) and docetaxel produces increased antitumor effects associated with enhanced changes to tumor vasculature

Abstract VEGF blockade in combination with chemotherapy has shown increased anti-tumor efficacy in preclinical models and clinical studies. However, the mechanism by which combination treatments result in enhanced effects is not well understood. In this study, we sought to characterize the mechanism for improved effects of aflibercept plus docetaxel on HT1080, a human fibrosarcoma line that is resistant to anti-VEGF treatment. Immunodeficient SCID mice bearing subcutaneous HT1080 tumors (size ∼100 mm3) were treated with either control protein (hFc) plus docetaxel vehicle, aflibercept (25 mg/kg s.c.), docetaxel (4.5 mg/kg i.v.), or the combination. Following three treatments administered every 3-4 days, tumors were left to grow and harvested when the average size of the treatment group reached ∼600mm3. We found that combination treatment with aflibercept plus docetaxel was significantly more efficacious than either single agent. Although treatments with docetaxel alone and in combination with aflibercept both initially regressed tumor size, the combination was significantly more effective at delaying long-term tumor regrowth. In tumors harvested 24 hrs after the last treatment, combination treatment produced a dramatic decrease in the number of proliferating cells and a concomitant increase in pyknotic cells. Aflibercept or docetaxel alone had only modest effects on the vessels in this tumor, whereas the combination treatment resulted in pruned vessels with less branching, some vessels with punctate endothelial cell staining, and an increase in TUNEL-positive endothelial cells. These results suggest that combination of aflibercept plus taxols (docetaxel) causes enhanced effects on the tumor vessels, thus improving efficacy in tumors with vasculature that exhibit resistance to VEGF blockade. Further understanding the mechanistic basis underlying these combination treatments may provide insight into improving current strategies to manage tumors resistant to antiangiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5427.

Tumorigenicity of IL-1α– and IL-1β–Deficient Fibrosarcoma Cells

Analyzing the growth of fibrosarcoma lines derived from IL-1α–, IL-1β–, or IL-1αβ–knockout (−/−) mice in the immunocompetent host revealed that tumor-derived IL-1α and IL-1β exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1–deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1–deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1α−/−β–competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1βcomp lines, IL-1α strengthens anchorage-independent growth, but both IL-1α and IL-1β support drug resistance. Corresponding to the aggressive growth, IL-1βcomp cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated αv/β3 and matrix metalloproteinase expression. Recruitment of myeloid cells requires IL-1β but is regulated by IL-1α, because inflammatory chemokine and cytokine expression is stronger in IL-1α−/−βcomp than in IL-1wt lines. This regulatory effect of tumorderived IL-1α is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1β. Both sarcoma cell–derived IL-1α and IL-1β promote tumor growth. However, IL-1α exerts regulatory activity on the tumor cell–matrix cross-talk, and only IL-1β initiates systemic inflammation.

Opposing effects of fibrosarcoma cell‐derived IL‐1α and IL‐1β on immune response induction

There is evidence that cell-associated IL-1 alpha supports immune response induction. Here we explored the impact of malignant cell-derived IL-1 on immunogenicity, immune response induction and tumor-induced immunosuppression using 3-methylcholanthrene-induced fibrosarcoma lines derived from wild-type (wt), IL-1 alpha-, IL-1 beta- or IL-1a beta-knockout (IL-1 alpha(-/-), IL-1 beta(-/-), IL-1 alphabeta(-/-)) C57BL6 mice. The wt, IL-1 alpha(-/-), IL-1 beta(-/-) and IL-1 alphabeta(-/-) fibrosarcoma lines express MHC class I molecules at a high level. The lines do not differ in their susceptibility toward NK cells, macrophages, and allogeneic CTL, or in their capacity as stimulators of an allogeneic response. However, IL-1 beta(-/-) tumors rarely grow in the syngeneic host, which is the consequence of a strong T helper and CTL response induction by IL-1 alpha-competent, IL-1 beta(-/-) tumors. On the other hand, IL-1 beta-competent, IL-1 alpha(-/-) tumors strongly assist CD11b(+)Gr-1(+) myeloid-derived suppressor cell and regulatory T cell expansion, which both suppress with high efficacy activated T helper cell proliferation and CTL lysis. In IL-1 alphabeta(-/-) tumors, the absence of IL-1 alpha becomes decisive, i.e. despite reduced suppressor cell recruitment, tumor growth was unimpaired due to inefficient immune response induction. Thus, sarcoma cell-derived IL-1 alpha and IL-1 beta do not act in concert. Induction of a strong immune response by IL-1 alpha demands therapeutic exploitation, which may become more efficient if systemic induction of immunosuppression by IL-1 beta can also be circumvented.

Membrane Type 1 Matrix Metalloproteinase (MT1-MMP/MMP-14) Cleaves and Releases a 22-kDa Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Fragment from Tumor Cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.

Tumor-induced mechanical hyperalgesia involves CGRP receptors and altered innervation and vascularization of DsRed2 fluorescent hindpaw tumors

Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer-associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral-derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor–nerve–vessel associations in context with behavioral assessment of tumor-associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers and less densely vascularized than tumors from non-hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post-implantation, the density of protein gene product 9.5 (PGP9.5)-immunoreactive nerves and CD31-immunoreactive blood vessels in tumors decreased, whereas CGRP-immunoreactive nerve density remained unchanged. Importantly, intra-tumor injection of a CGRP1 receptor antagonist (CGRP 8–37) partially blocked the tumor-associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor-induced nociception through a receptor-mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell-axon communication may underlie, at least in part, the occurrence of cancer pain.

Sp1 regulates expression of MET, and ribozyme-induced down-regulation of MET in fibrosarcoma-derived human cells reduces or eliminates their tumorigenicity

Changes in expression of hepatocyte growth factor (HGF) and its receptor, MET, are associated with formation and malignant progression of human tumors. In the present study, 10 of 11 human fibrosarcoma cell lines tested expressed significantly higher levels of MET than were found in a series of normal human fibroblast lines. Still more significant, MET was constitutively phosphorylated in all 11 fibrosarcoma lines, whereas the normal fibroblasts exhibited very low levels of the phosphorylated form. All the cell lines expressed HGF mRNA. To determine the role of MET and/or HGF in tumorigenesis, a fibrosarcoma line expressing high levels of MET protein and low levels of HGF/NK2 mRNA was stably transfected with a hammerhead ribozyme targeting MET. In addition, a fibrosarcoma line expressing high levels of both MET protein and HGF/NK2 mRNA was transfected with a ribozyme targeting MET, or with a ribozyme targeting MET and another targeting HGF. The transfectant cell lines no longer formed tumors, or did so at a greatly reduced frequency and/or longer latency. Because Sp1 is a transcription factor for MET, we assayed the cell lines for their level of Sp1 protein. Sp1 was markedly overexpressed in 7 of the 11 fibrosarcoma lines compared to normal fibroblast lines. Deletion analysis and site-directed mutagenesis of the MET promoter revealed that tandem Sp1 sites in the proximal promoter are critical for transcription of MET. Increased expression of Sp1 in a normal human fibroblast line containing a MET promoter-luciferase construct resulted in a dose-dependent increase in luciferase. Conversely, inhibition of Sp1 binding to DNA in a fibrosarcoma cell line, using an Sp1 decoy, dramatically reduced MET expression. Taken together, these results indicate that in human fibrosarcoma cells, high levels of the phosphorylated form of MET are required for tumor formation and that Sp1 can function to control the level of MET.

Replication of enhancer-deficient amphotropic murine leukemia virus in human fibrosarcoma but not in primary human fibroblasts.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species including humans and is a potential contaminant in MLV vector preparations for human gene transfer studies. In general, MLV replication depends on the expression of viral genes under the control of 75 bp enhancer elements in the long terminal repeat. However, in specific human fibrosarcoma and lymphoma lines replication of amphotropic MLV is possible without these enhancers. Fibrosarcomas are malignant tumors of fibroblast origin. To test the replication potential of intact and enhancerless amphotropic MLV in untransformed cells, infection studies with these viruses were carried out in three types of primary human fibroblasts. Replication of amphotropic MLV is observed in two of three tested fibroblast strains. None of these primary human fibroblasts is permissive for enhancer-deficient MLV, suggesting that replication of this virus may be limited to transformed cells.