Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

BACKGROUND: Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

CONCLUSIONS/SIGNIFICANCE: Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.

FKBPL and Peptide Derivatives: Novel Biological Agents That Inhibit Angiogenesis by a CD44-Dependent Mechanism

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action.

Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status.

Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype.

Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues Conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to (he dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds Conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1.3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo ill a number of human tumor xenograft models.

The Anti-Migratory Effects of FKBPL and Its Peptide Derivative, AD-01: Regulation of CD44 and the Cytoskeletal Pathway

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity and and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cells

Despite recent therapeutic advances, the response rates to chemotherapy for patients with metastatic colon cancer remain at approximately 50% with the fluoropyrimidine, 5-fluorouracil (5-FU), continuing to serve as the foundation chemotherapeutic agent for the treatment of this disease. Previous studies have demonstrated that overexpression of thymidylate synthase (TS) is a key determinant of resistance to 5-FU-based chemotherapy. Therefore, there is a significant need to develop alternative therapeutic strategies to overcome TS-mediated resistance. In this study, we demonstrate that the histone deacetylase inhibitors (HDACi) vorinostat and LBH589 significantly downregulate TS gene expression in a panel of colon cancer cell lines. Downregulation of TS was independent of p53, p21 and HDAC2 expression and was achievable in vivo as demonstrated by mouse xenograft models. We provide evidence that HDACi treatment leads to a potent transcriptional repression of the TS gene. Combination of the fluoropyrimidines 5-FU or FUdR with both vorinostat and LBH589 enhanced cell cycle arrest and growth inhibition. Importantly, the downstream effects of TS inhibition were significantly enhanced by this combination including the inhibition of acute TS induction and the enhanced accumulation of the cytotoxic nucleotide intermediate dUTP. These data demonstrate that HDACi repress TS expression at the level of transcription and provides the first evidence suggesting a direct mechanistic link between TS downregulation and the synergistic interaction observed between HDACi and 5-FU. This study provides rationale for the continued clinical evaluation of HDACi in combination with 5-FU-based therapies as a strategy to overcome TS-mediated resistance.

MErCuRIC1: A Phase I study of MEK1/2 inhibitor PD-0325901 with cMET inhibitor crizotinib in RASMT and RASWT (with aberrant c-MET) metastatic colorectal cancer (mCRC) patients.

Background: RAS is mutated (RASMT) in ~55% of mCRC, and phase III studies have shown that patients harbouring RAS mutations do not benefit from anti-EGFR MoAbs. In addition, ~50% of RAS Wild Type (RASWT) will not benefit from the addition of an EGFR MoAb to standard chemotherapy. Hence, novel treatment strategies are urgently needed for RASMT and > 50% of RASWT mCRC patients. c-MET is overexpressed in ~50-60%, amplified in ~2-3% and mutated in ~3-5% of mCRC. Recent preclinical studies have shown that c-MET is an important mediator of resistance to MEK inhibitors (i) in RASMT mCRC, and that combined MEKi/METi resulted in synergistic reduction in tumour growth in RASMT xenograft models (1). A number of recent studies have highlighted the role of c-MET in mediating primary/secondary resistance to anti-EGFR MoAbs in mCRC, suggesting that patient with RASWT tumours with aberrant c-MET (RASWT/c-MET+) may benefit from anti-c-MET targeted therapies (2). These preclinical data supported the further clinical evaluation of combined MEKi/METi treatment in RASMT and RASWT CRC patients with aberrant c-MET signalling (overexpression, amplification or mutation; RASWT/c-MET+). Methods: MErCuRIC1 is a phase I combination study of METi crizotinib with MEKi PD-0325901. The dose escalation phase, utilizing a rolling six design, recruits 12-24 patients with advanced solid tumours and aims to assess safety/toxicity of combination, recommended phase II (RPII) dose, pharmacokinetics (PK) and pharmacodynamics (PD) (pERK1/2 in PBMC and tumour; soluble c-MET). In the dose expansion phase an additional 30-42 RASMT and RASWT/c-MET mCRC patients with biopsiable disease will be treated at the RPII dose to further evaluate safety, PK, PD and treatment response. In the dose expansion phase additional biopsy and blood samples will be obtained to define mechanisms of response/resistance to crizotinib/PD-0325901 therapy. Enrolment into the dose escalation phase began in December 2014 with cohort 1 still ongoing. EudraCT registry number: 2014-000463-40. (1) Van Schaeybroeck S et al. Cell Reports 2014;7(6):1940-55; (2) Bardelli A et al. Cancer Discov 2013;3(6):658-73. Clinical trial information: 2014-000463-40.

A bioavailable cathepsin S nitrile inhibitor abrogates tumor development

BACKGROUND: Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.

METHODS: Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.

RESULTS: We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.

CONCLUSIONS: In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

Sustained inhibition of deacetylases is required for the antitumor activity of the histone deactylase inhibitors panobinostat and vorinostat in models of colorectal cancer

Despite compelling preclinical data in colorectal cancer (CRC), the efficacy of HDACIs has been disappointing in the clinic. The goal of this study was to evaluate the effectiveness of vorinostat and panobinostat in a dose- and exposure-dependent manner in order to better understand the dynamics of drug action and antitumor efficacy. In a standard 72 h drug exposure MTS assay, notable concentration-dependent antiproliferative effects were observed in the IC50 range of 1.2-2.8 μmol/L for vorinostat and 5.1-17.5 nmol/L for panobinostat. However, shorter clinically relevant exposures of 3 or 6 h failed to elicit any significant growth inhibition and in most cases a >24 h exposure to vorinostat or panobinostat was required to induce a sigmoidal dose-response. Similar results were observed in colony formation assays where ≥ 24 h of exposure was required to effectively reduce colony formation. Induction of acetyl-H3, acetyl-H4 and p21 by vorinostat were transient and rapidly reversed within 12 h of drug removal. In contrast, panobinostat-induced acetyl-H3, acetyl-H4, and p21 persisted for 48 h after an initial 3 h exposure. Treatment of HCT116 xenografts with panobinostat induced significant increases in acetyl-H3 and downregulation of thymidylate synthase after treatment. Although HDACIs exert both potent growth inhibition and cytotoxic effects when CRC cells were exposed to drug for ≥ 24 h, these cells demonstrate an inherent ability to survive HDACI concentrations and exposure times that exceed those clinically achievable. Continued efforts to develop novel HDACIs with improved pharmacokinetics/phamacodynamics, enhanced intratumoral delivery and class/isoform-specificity are needed to improve the therapeutic potential of HDACIs and HDACI-based combination regimens in solid tumors.

The CXCR2 antagonist, SCH-527123, shows antitumor activity and sensitizes cells to oxaliplatin in preclinical colon cancer models

Colorectal cancer is the second most common cause of cancer-related death in the United States. Recent studies showed that interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) are significantly upregulated in both the tumor and its microenvironment, and act as key regulators of proliferation, angiogenesis, and metastasis. Our previous study showed that IL-8 overexpression in colorectal cancer cells triggers the upregulation of the CXCR2-mediated proliferative pathway. The aim of this study was to investigate whether the CXCR2 antagonist, SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin both in vitro and in vivo. SCH-527123 showed concentration-dependent antiproliferative effects in HCT116, Caco2, and their respective IL-8-overexpressing variants colorectal cancer cell lines. Moreover, SCH-527123 was able to suppress CXCR2-mediated signal transduction as shown through decreased phosphorylation of the NF-κB/mitogen-activated protein kinase (MAPK)/AKT pathway. These findings corresponded with decreased cell migration and invasion, while increased apoptosis in colorectal cancer cell lines. In vivo results verified that SCH-527123 treatment decreased tumor growth and microvessel density when compared with vehicle-treated tumors. Importantly, these preclinical studies showed that the combination of SCH-527123 and oxaliplatin resulted in a greater decrease in cell proliferation, tumor growth, apoptosis, and angiogenesis that was superior to single-agent treatment. Taken together, these findings suggest that targeting CXCR2 may block tumor proliferation, migration, invasion, and angiogenesis. In addition, CXCR2 blockade may further sensitize colorectal cancer to oxaliplatin treatment.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.