Heparanase: A Potential Therapeutic Target in Sarcomas.

Sarcomas comprise a heterogeneous group of rare malignancies of mesenchymal origin including more than 70 subtypes. They may arise in muscle, bone, cartilage and other connective tissues. Their high histological and genetic heterogeneity makes diagnosis and treatment very challenging. Deregulation of heparanase has been found in several sarcoma subtypes and high expression levels have been correlated with poor prognosis in Ewing's sarcoma and osteosarcoma. Altered expression of specific heparan sulfate proteoglycans and heparan sulfate biosynthetic enzymes has also been observed. Advances in molecular pathogenesis of sarcomas have evidenced the critical role of several heparan sulfate binding growth factors and receptor tyrosine kinases, highly interconnected with the microenvironment, in sustaining tumor growth and progression. Interference with heparanase/heparan sulfate functions represents a potential therapeutic approach in sarcoma. In this chapter, we summarize the current knowledge about the biological significance of heparanase expression and its potential as a therapeutic target in subtypes of both soft tissue and bone sarcomas. Particular emphasis is given to the involvement of heparan sulfate proteoglycans and their synthesizing and modifying enzymes in bone physiology and disorders leading up to the pathobiology of bone sarcomas. The chapter also describes the cooperation between exostin loss-of-function and heparanase upregulation in hereditary Multiple Osteochondroma syndrome as a paradigmatic example of constitutive alteration of the heparanase/heparan sulfate proteoglycan system which may contribute to progression to malignant secondary chondrosarcoma. Preclinical evidence of the role of heparanase as a promising therapeutic target in various sarcoma subtypes is finally resumed.

Heparan Sulfate Mimetics in Cancer Therapy: The Challenge to Define Structural Determinants and the Relevance of Targets for Optimal Activity.

Beyond anticoagulation, the therapeutic potential of heparin derivatives and heparan sulfate (HS) mimetics (functionally defined HS mimetics) in oncology is related to their ability to bind and modulate the function of a vast array of HS-binding proteins with pivotal roles in cancer growth and progression. The definition of structural/functional determinants and the introduction of chemical modifications enabled heparin derivatives to be identified with greatly reduced or absent anticoagulant activity, but conserved/enhanced anticancer activity. These studies paved the way for the disclosure of structural requirements for the inhibitory effects of HS mimetics on heparanase, selectins, and growth factor receptor signaling, as well as for the limitation of side effects. Actually, HS mimetics affect the tumor biological behavior via a multi-target mechanism of action based on their effects on tumor cells and various components of the tumor microenvironment. Emerging evidence indicates that immunomodulation can participate in the antitumor activity of these agents. Significant ability to enhance the antitumor effects of combination treatments with standard therapies was shown in several tumor models. While the first HS mimetics are undergoing early clinical evaluation, an improved understanding of the molecular contexts favoring the antitumor action in certain malignancies or subgroups is needed to fully exploit their potential.

Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a ß-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-ß-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

New mechanisms for old drugs: Insights into DNA-unrelated effects of platinum compounds and drug resistance determinants.

Platinum drugs have been widely used for the treatment of several solid tumors. Although DNA has been recognized as the primary cellular target for these agents, there are unresolved issues concerning their effects and the molecular mechanisms underlying the antitumor efficacy. These cytotoxic agents interact with sub-cellular compartments other than the nucleus. Here, we review how such emerging phenomena contribute to the pharmacologic activity as well as to drug resistance phenotypes. DNA-unrelated effects of platinum drugs involve alterations at the plasma membrane and in endo-lysosomal compartments. A direct interaction with the mitochondria also appears to be implicated in drug-induced cell death. Moreover, the pioneering work of a few groups has shown that platinum drugs can act on the tumor microenvironment as well, and potentiate antitumor activity of the immune system. These poorly understood aspects of platinum drug activity sites may be harnessed to enhance their antitumor efficacy. A complete understanding of DNA-unrelated effects of platinum compounds might reveal new aspects of drug resistance allowing the implementation of the antitumor therapeutic efficacy of platinum compound-based regimens and minimization of their toxic side effects.

Non-oncogene dependencies: Novel opportunities for cancer therapy.

Targeting oncogene addictions have changed the history of subsets of malignancies and continues to represent an excellent therapeutic opportunity. Nonetheless, alternative strategies are required to treat malignancies driven by undruggable oncogenes or loss of tumor suppressor genes and to overcome drug resistance also occurring in cancers addicted to actionable drivers. The discovery of non-oncogene addiction (NOA) uncovered novel therapeutically exploitable "Achilles' heels". NOA refers to genes/pathways not oncogenic per sé but essential for the tumor cell growth/survival while dispensable for normal cells. The clinical success of several classes of conventional and molecular targeted agents can be ascribed to their impact on both tumor cell-associated intrinsic as well as microenvironment-related extrinsic NOA. The integration of genetic, computational and pharmacological high-throughput approaches led to the identification of an expanded repertoire of synthetic lethality interactions implicating NOA targets. Only a few of them have been translated into the clinics as most NOA vulnerabilities are not easily druggable or appealing targets. Nonetheless, their identification has provided in-depth knowledge of tumor pathobiology and suggested novel therapeutic opportunities. Here, we summarize conceptual framework of intrinsic and extrinsic NOA providing exploitable vulnerabilities. Conventional and emerging methodological approaches used to disclose NOA dependencies are reported together with their limits. We illustrate NOA paradigmatic and peculiar examples and outline the functional/mechanistic aspects, potential druggability and translational interest. Finally, we comment on difficulties in exploiting the NOA-generated knowledge to develop novel therapeutic approaches to be translated into the clinics and to fully harness the potential of clinically available drugs.

Targeting EZH2 in SMARCB1-deficient sarcomas: Advances and opportunities to potentiate the efficacy of EZH2 inhibitors.

Soft tissue sarcomas (STSs) are rare mesechymal malignancies characterized by distintive molecular, histological and clinical features. Many STSs are considered as predominatly epigenetic diseases due to underlying chromatin deregulation. Discovery of deregulated functional antagonism between the chromatin remodeling BRG1/BRM-associated (BAFs) and the histone modifying Polycomb repressor complexes (PRCs) has provided novel actionable targets. In epithelioid sarcoma (ES), extracranial, extrarenal malignant rhabdoid tumors (eMRTs) and synovial sarcoma (SS), the total or partial loss of the BAF core subunit SMARCB1, driven by different alterations, is associated with PRC2 deregulation and dependency on its enzymatic subunit, EZH2. In these SMARCB1-deficient STSs, aberrant EZH2 expression and/or activity emerged as a druggable vulnerability. Although preclinical investigation supported EZH2 targeting as a promising therapeutic option, clinical studies demonstrated a variable response to EZH2 inhibitors. Actually, whereas the clinical benefit recorded in ES patients prompted the FDA approval of the EZH2 inhibitor tazemetostat, the modest and sporadic responses observed in eMRT and SS patients highlighted the need to deepen mechanistic as well as pharmacological investigations to improve drug effectiveness. We summarize the current knowledge of different mechanisms driving SMARCB1 deficiency and EZH2 deregulation in ES, eMRT and SS along with preclinical and clinical studies of EZH2-targeting agents. Possible implication of the PRC2- and enzymatic-independent functions of EZH2 and of its homolog, EZH1, in the response to anti-EZH2 agents will be discussed together with combinatorial strategies under investigation to improve the efficacy of EZH2 targeting in these tumors.

Combinatorial strategies to potentiate the efficacy of HDAC inhibitors in fusion-positive sarcomas.

Fusion positive (FP) sarcomas are characterized by chromosomal rearrangements generating pathognomonic fusion transcripts and oncoproteins. In Ewing's sarcoma family of tumors (ESFTs), FP-rhabdomyosarcomas (FP-RMS) and synovial sarcomas (SS), the most common and aggressive forms of sarcomas in childhood and adolescence, the oncogenic rearrangements involve transcription cofactors causing widespread epigenetic rewiring and aberrant gene expression. Through the cooperation with histone deacetylases (HDACs) in transcription regulatory complexes, the fusion oncoproteins affect histone acetylation and chromatin remodeling. The participation of HDACs in core mechanisms of sarcoma cell transformation has paved the way to the investigation of HDAC inhibitors (HDACis) for therapeutic intervention. Preclinical studies have provided convincing evidence that HDAC activity abrogation can revert malignant cell features driven by FET-ETS, PAX3/7-FOXO1 or SS18-SSX fusion oncogenes in ESFTs, FP-RMS, or SS models, respectively, resulting in in vitro and in vivo growth inhibition. While clinical trials of HDACi monotherapies led to drug approval in some hematologic malignancies, no significant therapeutic benefit has been reported in solid tumors, including sarcomas. HDACi-based combination therapies with targeted or conventional anticancer agents have shown limited efficacy in early studies recruiting sarcoma patients, although partial responses and disease stabilization have been reported. In these trials, sarcomas were represented, however, as unclassified group in most cases. We summarize, here, studies addressing the role of HDACs in FP-sarcoma pathobiology and HDACi-based rational drug combinations. Finally, we discuss the opportunity of exploiting drug inhibitory profile and expression/function of specific HDAC isoenzymes to harness the full therapeutic potential of HDACis in these sarcoma histotypes.

Beyond targeting amplified MDM2 and CDK4 in well differentiated and dedifferentiated liposarcomas: From promise and clinical applications towards identification of progression drivers.

Well differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS) are tumors of the adipose tissue poorly responsive to conventional cytotoxic chemotherapy which currently remains the standard-of-care. The dismal prognosis of the DDLPS subtype indicates an urgent need to identify new therapeutic targets to improve the patient outcome. The amplification of the two driver genes MDM2 and CDK4, shared by WDLPD and DDLPS, has provided the rationale to explore targeting the encoded ubiquitin-protein ligase and cell cycle regulating kinase as a therapeutic approach. Investigation of the genomic landscape of WD/DDLPS and preclinical studies have revealed additional potential targets such as receptor tyrosine kinases, the cell cycle kinase Aurora A, and the nuclear exporter XPO1. While the therapeutic significance of these targets is being investigated in clinical trials, insights into the molecular characteristics associated with dedifferentiation and progression from WDLPS to DDLPS highlighted additional genetic alterations including fusion transcripts generated by chromosomal rearrangements potentially providing new druggable targets (e.g. NTRK, MAP2K6). Recent years have witnessed the increasing use of patient-derived cell and tumor xenograft models which offer valuable tools to accelerate drug repurposing and combination studies. Implementation of integrated "multi-omics" investigations applied to models recapitulating WD/DDLPS genetics, histologic differentiation and biology, will hopefully lead to a better understanding of molecular alterations driving liposarcomagenesis and DDLPS progression, as well as to the identification of new therapies tailored on tumor histology and molecular profile.

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma.

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.

Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma.

BACKGROUND: Synovial sarcoma (SS) is an aggressive soft tissue tumor with limited therapeutic options in advanced stage. SS18-SSX fusion oncogenes, which are the hallmarks of SS, cause epigenetic rewiring involving histone deacetylases (HDACs). Promising preclinical studies supporting HDAC targeting for SS treatment were not reflected in clinical trials with HDAC inhibitor (HDACi) monotherapies. We investigated pathways implicated in SS cell response to HDACi to identify vulnerabilities exploitable in combination treatments and improve the therapeutic efficacy of HDACi-based regimens. METHODS: Antiproliferative and proapoptotic effects of the HDACi SAHA and FK228 were examined in SS cell lines in parallel with biochemical and molecular analyses to bring out cytoprotective pathways. Treatments combining HDACi with drugs targeting HDACi-activated prosurvival pathways were tested in functional assays in vitro and in a SS orthotopic xenograft model. Molecular mechanisms underlying synergisms were investigated in SS cells through pharmacological and gene silencing approaches and validated by qRT-PCR and Western blotting. RESULTS: SS cell response to HDACi was consistently characterized by activation of a cytoprotective and auto-sustaining axis involving ERKs, EGR1, and the ß-endoglycosidase heparanase, a well recognized pleiotropic player in tumorigenesis and disease progression. HDAC inhibition was shown to upregulate heparanase by inducing expression of the positive regulator EGR1 and by hampering negative regulation by p53 through its acetylation. Interception of HDACi-induced ERK-EGR1-heparanase pathway by cell co-treatment with a MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/roneparstat) enhanced antiproliferative and pro-apoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and nuclear heparanase levels. The combination of SAHA with SST0001 prevented the upregulation of ERK-EGR1-heparanase induced by the HDACi and promoted caspase-dependent cell death. In vivo, the combined treatment with SAHA and SST0001 potentiated the antitumor efficacy against the CME-1 orthotopic SS model as compared to single agent administration. CONCLUSIONS: The present study provides preclinical rationale and mechanistic insights into drug combinatory strategies based on the use of ERK pathway and heparanase inhibitors to improve the efficacy of HDACi-based antitumor therapies in SS. The involvement of classes of agents already clinically available, or under clinical evaluation, indicates the transferability potential of the proposed approaches.

Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line.

BACKGROUND: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1. RESULTS: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin ß1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed. CONCLUSIONS: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

Receptor tyrosine kinases and heparan sulfate proteoglycans: Interplay providing anticancer targeting strategies and new therapeutic opportunities.

The development of pharmacological and biological inhibitors of receptor tyrosine kinases (RTKs) has changed the treatment paradigm of several neoplastic diseases. However, the occurrence of intrinsic and acquired resistance represents a limit to the efficacy of these drugs even in RTK-addicted cancers. The identification of innovative therapeutic approaches and rationale-based drug combinations remains a primary need to improve patients' outcome. Heparan sulfate proteoglycans (HSPGs) at the cell surface and in the extracellular matrix bind to and modulate the biological activity of a great number of heparan sulfate (HS) binding proteins. The participation of HSPGs as accessory molecules in the growth factor-receptor interactions and mechanism of activation of several RTKs provides the basis for developing alternative therapeutic strategies based on targeting HSPGs by antibodies or HS mimetics to interfere with the aberrant oncogenic signaling implicated in the pathobiology of several tumors. Here, we focus on the FGF-FGFR-HSPG and HGF-Met-HSPG axes as paradigmatic examples of the multiple-level interconnections between RTKs and HSPGs influencing cell signaling, gene expression, drug sensitivity, and promoting a permissive microenvironment for tumor growth and progression. In these reciprocal regulations, the HS degrading enzymes heparanase and endosulfatases play key roles contributing to the high structural complexity and heterogeneity of HS chains as well as to the specificity of their interaction with proteins. Actually, heparanase and endosulfatases represent, in turn, promising therapeutic targets. We also report some studies describing the effects of FGFR and Met inhibitors on the expression of genes encoding HSPGs and related enzymes, and discuss about the potential impact of these effects on drug response. Finally, we argue about the need of in-depth investigation of the role of HSPGs and their modifying enzymes in specific tumor pathologies to exploit the opportunity of combination treatments including HS mimetics or HSPG directed antibodies to improve efficacy of RTK inhibitors and overcome drug resistance.

Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma.

Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.

Supersulfated low-molecular weight heparin synergizes with IGF1R/IR inhibitor to suppress synovial sarcoma growth and metastases.

Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.

Novel Benzazole Derivatives Endowed with Potent Antiheparanase Activity.

Heparanase is the sole mammalian enzyme capable of cleaving glycosaminoglycan heparan sulfate side chains of heparan sulfate proteoglycans. Its altered activity is intimately associated with tumor growth, angiogenesis, and metastasis. Thus, its implication in cancer progression makes it an attractive target in anticancer therapy. Herein, we describe the design, synthesis, and biological evaluation of new benzazoles as heparanase inhibitors. Most of the designed derivatives were active at micromolar or submicromolar concentration, and the most promising compounds are fluorinated and/or amino acids derivatives 13a, 14d, and 15 that showed IC50 0.16-0.82 µM. Molecular docking studies were performed to rationalize their interaction with the enzyme catalytic site. Importantly, invasion assay confirmed the antimetastatic potential of compounds 14d and 15. Consistently with its ability to inhibit heparanase, compound 15 proved to decrease expression of genes encoding for proangiogenic factors such as MMP-9, VEGF, and FGFs in tumor cells.

Novel Symmetrical Benzazolyl Derivatives Endowed with Potent Anti-Heparanase Activity.

Heparanase is the only mammalian endo-ß-d-glucuronidase involved in a variety of major diseases. The up-regulation of heparanase expression increases tumor size, angiogenesis, and metastasis, representing a validated target in the anti-cancer field. To date, only a few small-molecule inhibitors have been described, but none have gotten through pre-clinical development. Previously, we explored 2-(4-(4-(bromo-methoxybenzamido)benzylamino)phenyl) benzazole derivatives as anti-heparanase agents, proposing this scaffold for development of broadly effective heparanase inhibitors. Herein, we report an extended investigation of new symmetrical 2-aminophenyl-benzazolyl-5-acetate derivatives, proving that symmetrical compounds are more effective than asymmetrical analogues, with the most-potent compound, 7g, being active at nanomolar concentration against heparanase. Molecular docking studies were performed on the best-acting compounds 5c and 7g to rationalize their interaction with the enzyme. Moreover, invasion assay confirmed the anti-metastatic potential of compounds 5c, 7a, and 7g, proving the inhibition of the expression of proangiogenic factors in tumor cells.

Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1.

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.

Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance.

Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens.

Overcoming ABC transporter-mediated multidrug resistance: The dual role of tyrosine kinase inhibitors as multitargeting agents.

Resistance to conventional and target specific antitumor drugs still remains one of the major cause of treatment failure and patience death. This condition often involves ATP-binding cassette (ABC) transporters that, by pumping the drugs outside from cancer cells, attenuate the potency of chemotherapeutics and negatively impact on the fate of anticancer therapy. In recent years, several tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib, dasatinib, ponatinib, gefitinib, erlotinib, lapatinib, vandetanib, sunitinib, sorafenib) have been reported to interact with ABC transporters (e.g., ABCB1, ABCC1, ABCG2, ABCC10). This finding disclosed a very complex scenario in which TKIs may behave as substrates or inhibitors depending on the expression of specific pumps, drug concentration, affinity for transporters and types of co-administered agents. In this context, in-depth investigation on TKI chemosensitizing functions might provide a strong rationale for combining TKIs and conventional therapeutics in specific malignancies. The reposition of TKIs as antagonists of ABC transporters opens a new way towards anticancer therapy and clinical strategies aimed at counteracting drug resistance. This review will focus on some paradigmatic examples of the complex and not yet fully elucidated interaction between clinical available TKIs (e.g. BCR-ABL, EGFR, VEGFR inhibitors) with the main ABC transporters implicated in multidrug resistance.