COL8A1 enhances the invasion/metastasis in MDA-MB-231 cells via the induction of IL1B and MMP1 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis and a lack of specific targets and targeted therapeutics. Previously, we have reported that COL8A1, which is highly expressed in the mesenchymal stem-like (MSL) subtype of TNBC, facilitates TNBC growth via FAK/Src activation. Furthermore, we have found that COL8A1 enhances the invasion and metastasis of MDA-MB-231 cells, classified into MSL. However, the mechanism of invasion and metastasis by COL8A1 remains unclear. Here, we investigated the biological function of COL8A1 on the invasion and metastasis of MDA-MB-231 cells. METHODS: The invasion and metastasis of MDA-MB-231 cells were evaluated using three-dimensional (3D) culture methods and xenograft mouse models. DNA microarray analysis examined the gene expression in COL8A1-overexpressing MDA-MB-231 cells and control cells. Gene expression was verified using RT-qPCR. RESULTS: COL8A1-deficient cells showed little or no metastasis, whereas forced expression of COL8A1 in MDA-MB-231 cells, the MSL subtype of TNBC cell lines, significantly promoted distant metastasis after tumor resection. As with in vivo, 3D invasion assay revealed that COL8A1 increased the invasion capacity of MDA-MB-231 and Hs578T cells, classified into the MSL subtype of TNBC. DNA microarray analysis for COL8A1-overexpressing cells indicated that COL8A1 induces interleukin 1B (IL1B) and matrix metalloproteinase-1 (MMP1) expression, both of which are correlated with COL8A1 expression in the mesenchymal subtypes of TNBC, and the Kaplan-Meier plotter provided evidence that the prognosis in the MSL subtype was strongly associated with both gene expressions and COL8A1 expression. Pharmacological inhibitor treatment showed that COL8A1 regulated IL1B and MMP1 expression through a different pathway. Moreover, the knockdown of each gene expression reduced the invasion capacity of COL8A1-overexpressing MDA-MB-231 and Hs578T cells. CONCLUSION: Our findings indicate that COL8A1-induced IL1B and MMP1 enhanced the invasion and metastasis of the MSL subtype of TNBC. Considering our previous findings that COL8A1 promotes tumor growth, COL8A1 may be a prognostic and practical therapeutic target in TNBC.

COL8A1 facilitates the growth of triple-negative breast cancer via FAK/Src activation.

PURPOSE: Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, and treatment options are limited because of the lack of signature molecules and heterogeneous properties of cancer. COL8A1 expression is higher in breast cancer than in normal tissues and is strongly correlated with worse overall survival in patients with breast cancer. However, the biological function of COL8A1 on cancer progression is not fully understood. In this study, we investigated the biological function of COL8A1 on TNBC progression. METHODS: COL8A1-deficient cells were generated using the CRISPR-Cas9 system. The tumor growth and metastasis of TNBC cells were evaluated using three-dimensional culture (3D) methods and xenograft mouse models. The activation of focal adhesion kinase (FAK)/Src by COL8A1 in TNBC cells was evaluated by immunoblotting. RESULTS: COL8A1 expression was primarily distributed into TNBC cell lines. Further, relapse-free survival in TNBC patients with the MSL subtype was strongly associated with the COL8A1 expression. MDA-MB-231 and Hs578T cells, classified as the MSL subtype, strongly express COL8A1, and COL8A1 protein expression was induced by hypoxia in both cell lines. Loss of COL8A1 expression inhibited spheroid /tumor growth and metastasis in vitro and in vivo. Further, exogenous COL8A1 promoted TNBC growth via the FAK/Src activation. Finally, the spheroid growth of MDA-MB-231 and Hs578T cells was inhibited by defactinib, a FAK inhibitor, without cytotoxicity. CONCLUSION: These results indicate that COL8A1-mediated FAK/Src activation produces a more aggressive phenotype in TNBC, and its target inhibition may be an efficacious treatment for TNBC.

Rapid and efficient synthesis of a novel cholinergic muscarinic M1 receptor positive allosteric modulator using flash chemistry.

Continuous flow-flash synthesis of a 2-bromobenzaldehyde derivative 18 as a key intermediate of a novel cholinergic muscarinic M1 positive allosteric modulator 1 bearing an isoindolin-1-one ring system as a pharmacophore has been achieved using flow microreactors through selective I/Li exchange of 1-bromo-2-iodobenzene derivative 17 with BuLi and subsequent formylation at -40 °C of the highly reactive 2-bromophenyllithium intermediate using DMF, which is difficult to achieve by a conventional batch process due to the conversion of the highly reactive 2-bromophenyllithium intermediate into benzyne even at -78 °C. Late-stage cyclization to give the isoindolin-1-one ring system, through reductive amination of 18 followed by palladium-catalyzed carbonylation with carbon monoxide and intramolecular cyclization, efficiently afforded 1 for its further research and development.

Development of two fluorine-18 labeled PET radioligands targeting PDE10A and in vivo PET evaluation in nonhuman primates.

INTRODUCTION: Phosphodiesterase 10A (PDE10A) is a member of the PDE enzyme family that degrades cyclic adenosine and guanosine monophosphates (cAMP and cGMP). Based on the successful development of [11C]T-773 as PDE10A positron emission tomography (PET) radioligand, in this study our aim was to develop and evaluate fluorine-18 analogs of [11C]T-773. METHODS: [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were synthesized from the same precursor used for 11C-labeling of T-773 in a two-step approach via 18F-fluoromethylation and 18F-fluoroethylation, respectively, using corresponding deuterated synthons. A total of 12 PET measurements were performed in seven non-human primates. First, baseline PET measurements were performed using High Resolution Research Tomograph system with both [18F]FM-T-773-d2 and [18F]FE-T-773-d4; the uptake in whole brain and separate brain regions, as well as the specific binding and tissue ratio between putamen and cerebellum, was examined. Second, baseline and pretreatment PET measurements using MP-10 as the blocker were performed for [18F]FM-T-773-d2 including arterial blood sampling with radiometabolite analysis in four NHPs. RESULTS: Both [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were successfully radiolabeled with an average molar activity of 293 ± 114 GBq/µmol (n=8) for [18F]FM-T-773-d2 and 209 ± 26 GBq/µmol (n=4) for [18F]FE-T-773-d4, and a radiochemical yield of 10% (EOB, n=12, range 3%-16%). Both radioligands displayed high brain uptake (~5.5% of injected radioactivity for [18F]FM-T-773-d2 and ~3.5% for [18F]FE-T-773-d4 at the peak) and a fast washout. Specific binding reached maximum within 30 min for [18F]FM-T-773-d2 and after approximately 45 min for [18F]FE-T-773-d4. [18F]FM-T-773-d2 data fitted well with kinetic compartment models. BPND values obtained indirectly through compartment models were correlated well with those obtained by SRTM. BPND calculated with SRTM was 1.0-1.7 in the putamen. The occupancy with 1.8 mg/kg of MP-10 was approximately 60%. CONCLUSIONS: [18F]FM-T-773-d2 and [18F]FE-T-773-d4 were developed as fluorine-18 PET radioligands for PDE10A, with the [18F]FM-T-773-d2 being the more promising PET radioligand warranting further evaluation.

Development of a series of novel carbon-11 labeled PDE10A inhibitors.

Phosphodiesterase 10A (PDE10A) is a member of the PDE family of enzymes that degrades cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our aim was to label a series of structurally related PDE10A inhibitors with carbon-11 and evaluate them as potential positron emission tomography (PET) radioligands for PDE10A using nonhuman primates. The series consisted of seven compounds based on the 3-(1H-pyrazol-5-yl)pyridazin-4(1H)-one backbone. These compounds were selected from the initial larger library based on a number of parameters such as affinity, selectivity for hPDE10A in in vitro tests, lipophilicity, and on the results of multidrug resistance protein 1 (MDR1)-LLCPK1 and the parallel artificial membrane permeability assays. Seven radioligands (KIT-1, 3, 5, 6, 7, 9, and 12) were radiolabeled with carbon-11 employing O-methylation on the hydroxyl moiety using [(11)C]methyl triflate. In vivo examination of each radioligand was performed using PET in rhesus monkeys; analysis of radiometabolites in plasma also was conducted using HPLC. All seven radioligands were labeled with high (>90%) incorporation of [(11)C]methyl triflate into their appropriate precursors and with high specific radioactivity. Carbon-11 labeled KIT-5 and KIT-6 showed high accumulation in the striatum, consistent with the known anatomical distribution of PDE10A in brain, accompanied by fast washout and high specific binding ratio. In particular [(11)C]KIT-6, named [(11)C]T-773, is a promising PET tool for further examination of PDE10A in human brain.

Evaluation of a novel PDE10A PET radioligand, [(11) C]T-773, in nonhuman primates: brain and whole body PET and brain autoradiography.

Phosphodiesterase 10A (PDE10A) is considered to be a key target for the treatment of several neuropsychiatric diseases. The characteristics of [(11) C]T-773, a novel positron emission tomography (PET) radioligand with high binding affinity and selectivity for PDE10A, were evaluated in autoradiography and in nonhuman primate (NHP) PET. Brain PET measurements were performed under baseline conditions and after administration of a selective PDE10A inhibitor, MP-10. Total distribution volume (VT ) and binding potential (BPND ) were calculated using various kinetic models. Whole body PET measurements were performed to calculate the effective dose of [(11) C]T-773. Autoradiography studies in postmortem human and monkey brain sections showed high accumulation of [(11) C]T-773 in the striatum and substantia nigra which was blocked by MP-10. Brain PET showed high accumulation of [(11) C]T-773 in the striatum, and the data could be fitted using a two tissue compartment model. BPND was approximately 1.8 in the putamen when the cerebellum was used as the reference region. Approximately 70% of PDE10A binding was occupied by 1.8 mg/kg of MP-10. Whole body PET showed high accumulation of [(11) C]T-773 in the liver, kidney, heart, and brain in the initial phase. The radioligand was partly excreted via bile and the gastrointestinal tract, and partly excreted through the urinary tract. The calculated effective dose was 0.007 mSv/MBq. In conclusion, [(11) C]T-773 was demonstrated to be a promising PET radioligand for PDE10A with favorable brain kinetics. Dosimetry results support multiple PET measurements per person in human studies. Further research is required with [(11) C]T-773 in order to test the radioligand's potential clinical applications.

Molecular Imaging of PDE10A Knockout Mice with a Novel PET Radiotracer: [(11)C]T-773.

PURPOSE: [(11)C]T-773 is a new radioligand for positron emission tomography (PET) targeting the phosphodiesterase 10A enzyme (PDE10A). PDE10A is highly expressed in the striatum by medium spiny neurons, and it has been demonstrated to be involved in the regulation of striatal signaling through the reduction of medium spiny neuronal sensitivity towards glutamatergic excitation. PDE10A is associated with Parkinson's disease and different neuropsychiatric disorders such as Huntington's disease, obsessive-compulsive disorders (OCD) and schizophrenia. Studies have indicated that the inhibition of PDE10A may represent a novel therapeutic approach to the treatment of the aforementioned diseases characterized by the reduced activity of medium spiny neurons. An appropriate PET radioligand for PDE10A would help to facilitate drug development and drug evaluation. PROCEDURES: We have evaluated the [(11)C]T-773 ligand in PDE10A knockout mice (heterozygous [HET] and homozygous [HOM]) as well as in normal control animals (WILD) with PET. RESULTS: The regional percent standardized uptake values (%SUV; mean ± SD) in the striatum were 48.2 ± 1.0 (HOM), 63.6 ± 5.3 (HET) and 85.1 ± 6.3 (WILD). Between each animal group the striatal %SUV values were significantly different (p < 0.0001). The striatal BPND values (mean ± SD) were 0.0 ± 0.0 (HOM), 0.14 ± 0.07 (HET) and 0.56 ± 0.15 (WILD). The BPND values were significantly lower in homozygous and heterozygous animals compared to wild type (p < 0.0001). CONCLUSIONS: The novel PDE10A radioligand [(11)C]T-773 shows increased signals with higher levels of PDE10A and acceptable binding in the striatum in control animals compared to knockout mice.

Characterization of the binding properties of T-773 as a PET radioligand for phosphodiesterase 10A.

INTRODUCTION: Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP and is selectively expressed in striatal medium spiny neurons. Recent studies have suggested that PDE10A inhibition is a novel approach for the treatment of disorders such as schizophrenia and Huntington's disease. A positron emission tomography (PET) occupancy study can provide useful information for the development of PDE10A inhibitors. We discovered T-773 as a candidate PET radioligand for PDE10A and investigated its properties by in vitro autoradiography and a PET study in a monkey. METHODS: Profiling of T-773 as a PET radioligand for PDE10A was conducted by in vitro enzyme inhibitory assay, in vitro autoradiography, and PET study in a monkey. RESULTS: T-773 showed a high binding affinity and selectivity for human recombinant PDE10A2 in vitro; the IC50 value in an enzyme inhibitory assay was 0.77nmol/L, and selectivity over other PDEs was more than 2500-fold. In autoradiography studies using mouse, rat, monkey, or human brain sections, radiolabeled T-773 selectively accumulated in the striatum. This selective accumulation was not observed in the brain sections of Pde10a-KO mice. The binding of [(3)H]T-773 to PDE10A in rat brain sections was competitively inhibited by MP-10, a selective PDE10A inhibitor. In rat brain sections, [(3)H]T-773 bound to a single high affinity site of PDE10A with Kd values of 12.2±2.2 and 4.7±1.2nmol/L in the caudate-putamen and nucleus accumbens, respectively. In a monkey PET study, [(11)C]T-773 showed good brain penetration and striatum-selective accumulation. CONCLUSION: These results suggest that [(11)C]T-773 is a potential PET radioligand for PDE10A.

Combinational effects of farnesoid X receptor antagonist and statin on plasma lipid levels and low-density lipoprotein clearance in guinea pigs.

AIMS: We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model. MAIN METHODS: Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment. KEY FINDINGS: Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10mg/kg) or atorvastatin (10mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%. SIGNIFICANCE: Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.

Antidyslipidemic effects of a farnesoid X receptor antagonist in primates.

AIMS: We investigated antidyslipidemic effects of a farnesoid X receptor antagonist compound-T3 in non-human primates as a novel treatment approach for dyslipidemia. MAIN METHODS: Cynomolgus monkeys were fed a high-fat diet over 3 weeks. Drugs were administered to the monkeys for a week, and their plasma and fecal lipid parameters were measured. KEY FINDINGS: Compound-T3 dose-dependently decreased the plasma non-high-density lipoprotein (non-HDL) cholesterol and apolipoprotein B levels in high-fat diet-fed cynomolgus monkeys. The plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, and total fecal bile acid levels increased, suggesting that the hypocholesterolemic effects would be dependent on the activation of cholesterol catabolism in the liver. Compound-T3 significantly increased the plasma levels of HDL cholesterol and apolipoprotein A-I. In this condition, the cholesterol absorption inhibitor ezetimibe significantly decreased the plasma non-HDL cholesterol levels and increased the fecal cholesterol levels without affecting plasma HDL cholesterol and triglyceride levels. Bile acid sequestrant cholestyramine tended to decrease plasma non-HDL cholesterol and increase fecal bile acid levels. The cholesteryl ester transfer protein inhibitor torcetrapib significantly increased plasma HDL cholesterol levels without affecting plasma non-HDL cholesterol and fecal cholesterol levels. SIGNIFICANCE: The results of ezetimibe, cholestyramine, and torcetrapib treatments indicate that our high-fat diet fed monkey model would be a preferred animal model for studying non-statin type antidyslipidemic drugs. Compound-T3 significantly decreased non-HDL cholesterol levels and increased HDL cholesterol levels in the monkey model, suggesting that a farnesoid X receptor antagonist could be a therapeutic option in human dyslipidemia.

Effects of a farnesoid X receptor antagonist on hepatic lipid metabolism in primates.

We aimed to elucidate the mechanism underlying the anti-dyslipidemic effect of compound-T3, a farnesoid X receptor antagonist, by investigating its effects on hepatic lipid metabolism in non-human primates. We administered lipid-lowering drugs for 7 days to cynomolgus monkeys receiving a high-fat diet, and subsequently measured the levels of lipid parameters in plasma, feces, and hepatic tissue fluids. Compound-T3 (0.3 and 3mg/kg p.o.) significantly decreased the plasma levels of non-high-density lipoprotein (non-HDL) cholesterol and apolipoprotein B in a dose-dependent manner. It also decreased the mRNA levels of hepatic small heterodimer partner-1, induced the mRNA expression of hepatic cholesterol 7α-hydroxylase, reduced hepatic cholesterol and triglyceride levels, increased fecal bile acid excretion, and upregulated the expression of hepatic low-density lipoprotein (LDL) receptor. Furthermore, compound-T3 significantly increased plasma HDL cholesterol and apolipoprotein A-I levels. The mRNA expression levels of hepatic apolipoprotein A-I tended to increase after compound-T3 treatment. Compound-T3 also induced accumulation of hepatic bile acids and decreased the mRNA expression levels of the hepatic bile acid export pump. The effects of cholestyramine (300mg/kg p.o.) on the plasma and hepatic lipid parameters were similar to those of compound-T3, and it increased fecal bile acid levels without causing accumulation of hepatic bile acids. These findings suggest that LDL receptor-mediated hepatic LDL incorporation due to cholesterol catabolism catalyzed by cholesterol 7α-hydroxylase decreases plasma non-HDL cholesterol levels. Upregulation of hepatic apolipoprotein A-I mRNA expression may partially contribute to the increase in HDL cholesterol levels mediated by compound-T3.