Sigma receptor ligands: novel applications in cancer imaging and treatment

Sigma receptoren (subtypes sigma-1 en sigma-2) zijn unieke bindingsplaatsen die een aanzienlijk hogere expressie vertonen in kanker dan in gezond weefsel. Hoewel de endogene agonisten niet met zekerheid geïdentificeerd werden, zijn vele kunstmatige liganden beschikbaar voor onderzoek van de normale en pathofysiologische functie van sigma receptoren. Van therapeutische doseringen van zulke verbindingen (met name sigma-1 antagonisten en sigma-2 agonisten) is bekend dat zij de proliferatie en overleving van kankercellen doen afnemen maar nauwelijks invloed hebben op normale cellen. Het onderzoek dat in dit proefschrift wordt beschreven was gericht op de verdere ontwikkeling van sigma liganden voor diagnostische beeldvorming en als chemotherapeutica in de oncologie. We presenteren gegevens die erop wijzen dat competitie door steroïdhormonen variabiliteit van de opname van sigma liganden tijdens PET-imaging kan veroorzaken. Met name progesteron competeert met 11C-SA4503 voor binding aan sigma receptoren in kankercellen. We tonen ook aan dat sigma-1 receptoren in spontane hypofysetumoren tot overexpressie komen en dat 11C-SA4503-PET een geschikte methode kan zijn om zulke laesies aan te tonen. We rapporteren dat het anti-kankereffect van sigma liganden wordt bepaald door de sigma-2 receptorbezetting in kankercellen. Cytotoxiciteit gaat gepaard met opvallende, vroege veranderingen van het metabolisme in zulke cellen. Meting van de sigma-2 receptor bezetting in menselijke tumoren d.m.v. PET kan wellicht worden gebruikt om de therapeutische dosis te voorspellen. Een combinatie van sigma liganden en cytokine-gebaseerde therapie werd getest en bleek robuste anti-kanker effecten te veroorzaken in melanoma cellijnen en in primair materiaal van ovariumcarcinoom afkomstig van kankerpatiënten. Sigma receptors (subtypes sigma-1 and sigma-2) are unique binding sites showing a considerably higher expression in cancerous compared to non-cancerous tissues. Although the endogenous agonists have not been identified with certainty, many artificial ligands are available to study the function of sigma receptors in health and disease. Therapeutic doses of such compounds (particularly sigma-1 antagonists and sigma-2 agonists) are known to decrease proliferation and survival of cancer cells, but minimally affect non-cancer cells. Research described in this thesis was aimed at further development of sigma ligands as diagnostic imaging agents and therapeutic drugs in oncology. We present evidence indicating that competition by steroid hormones accounts for variability of sigma ligand uptake during PET imaging. Specifically, progesterone competes with 11C-SA4503 for binding to sigma-1 receptors in cancer cells. We also show that sigma-1 receptors are overexpressed in spontaneous pituitary tumors and that 11C-SA4503-PET may be a suitable method for detection of such lesions. We report that sigma receptor occupancy in cancer cells determines the observed anti-cancer effect of sigma ligands. Cytotoxicity is accompanied by striking, early changes of metabolism in such cells. Assessment of sigma-2 receptor occupancy in human tumors with PET may be employed for prediction of the therapeutic dose. A combination of sigma ligands and cytokine-based therapy was tested and found to display robust anti-cancer effects in melanoma cell lines and in patient-derived primary samples of ovarian carcinoma.

Potential applications for sigma receptor ligands in cancer diagnosis and therapy

AbstractSigma receptors (sigma-1 and sigma-2) represent two independent classes of proteins. Their endogenous ligands may include the hallucinogen N,N-dimethyltryptamine (DMT) and sphingolipid-derived amines which interact with sigma-1 receptors, besides steroid hormones (e.g., progesterone) which bind to both sigma receptor subpopulations. The sigma-1 receptor is a ligand-regulated molecular chaperone with various ion channels and G-protein-coupled membrane receptors as clients. The sigma-2 receptor was identified as the progesterone receptor membrane component 1 (PGRMC1). Although sigma receptors are over-expressed in tumors and up-regulated in rapidly dividing normal tissue, their ligands induce significant cell death only in tumor tissue. Sigma ligands may therefore be used to selectively eradicate tumors. Multiple mechanisms appear to underlie cell killing after administration of sigma ligands, and the signaling pathways are dependent both on the type of ligand and the type of tumor cell. Recent evidence suggests that the sigma-2 receptor is a potential tumor and serum biomarker for human lung cancer and an important target for inhibiting tumor invasion and cancer progression. Current radiochemical efforts are focused on the development of subtype-selective radioligands for positron emission tomography (PET) imaging. Right now, the mostpromising tracers are [18F]fluspidine and [18F]FTC-146 for sigma-1 receptors and [11C]RHM-1 and [18F]ISO-1 for the sigma-2 subtype. Nanoparticles coupled to sigma ligands have shown considerable potential for targeted delivery of antitumor drugs in animal models of cancer, but clinical studies exploring this strategy in cancer patients have not yet been reported. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Advanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to <b>1</b>. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and <b>2</b>. activate apoptotic TRAIL-signaling.</p> <p>Results</p> <p>Treatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.</p> <p>Conclusions</p> <p>Anti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.</p

An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients

Simple Summary Treatment of patients diagnosed with advanced pulmonary adenocarcinoma depends on the presence of genomic aberrations that are targetable for a specific tyrosine kinase inhibitor. Subsequent treatment lines depend on presence of mutations that are associated with emerging resistance. These aberrations include a variety of gene activating mutations, including single nucleotide variants, small insertion-deletions, exon skipping events, and gene fusions. At this moment different assays are used to detect these aberrations in the clinic. In this paper we introduce a novel method that can detect these genomic alterations in a single, RNA-based, assay. The design of the all-in-one assay is flexible allowing addition of new targets in subsequent designs. We show that this all-in-one assay has a high accuracy even on formalin-fixed-paraffin-embedded tissue samples, making it readily applicable in a clinical diagnostic setting. The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with >50 K unique reads and a DV200 >30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive

Steroid hormones affect binding of the sigma ligand C-11-SA4503 in tumour cells and tumour-bearing rats

PURPOSE: Sigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist (11)C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels. METHODS: (11)C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1 h or 5 min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone. RESULTS: Binding of (11)C-SA4503 to C6 cells was increased (~50%) upon removal and decreased (~60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC(50) progesterone 33 nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of (11)C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected. CONCLUSIONS: The binding of (11)C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids