N-myristoyltransferase proteins in breast cancer: prognostic relevance and validation as a new drug target.

PURPOSE: N-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood. METHODS: We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706). RESULTS: NMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo. CONCLUSIONS: These results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients.

Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with Ddx1 knockout.

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2-4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2-4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.

Radiosynthesis and Biological Evaluation of [18F]Triacoxib: A New Radiotracer for PET Imaging of COX-2.

Inducible isozyme cyclooxygenase-2 (COX-2) is upregulated under acute and chronic inflammatory conditions, including cancer, wherein it promotes angiogenesis, tissue invasion, and resistance to apoptosis. Due to its high expression in various cancers, COX-2 has become an important biomarker for molecular imaging and therapy of cancer. Recently, our group applied in situ click chemistry for the identification of the highly potent and selective COX-2 inhibitor triacoxib. In this study, we present the radiosynthesis in vitro and in vivo radiopharmacological validation of [18F]triacoxib, a novel radiotracer for PET imaging of COX-2. Radiosynthesis of [18F]triacoxib was accomplished using copper-mediated late-stage radiofluorination chemistry. The radiosynthesis, including radio-HPLC purification, of [18F]triacoxib was accomplished within 90 min in decay-corrected radiochemical yields of 72% (n = 7) at molar activities exceeding 90 GBq/µmol. Cellular uptake and inhibition studies with [18F]triacoxib were carried out in COX-2 expressing HCA-7 cells. Cellular uptake of [18F]triacoxib in HCA-7 cells reached 25% radioactivity/mg protein after 60 min. Cellular uptake was reduced by 63% upon pretreatment with 0.1 mM celecoxib, and 90% of the radiotracer remained intact in vivo after 60 min p.i. in mice. [18F]Triacoxib was further evaluated in HCA-7 tumor-bearing mice using dynamic PET imaging, radiometabolite analysis, autoradiography, and immunohistochemistry. PET imaging revealed a favorable baseline radiotracer uptake in HCA-7 tumors (SUV60min = 0.76 ± 0.02 (n = 4)), which could be blocked by 20% through i.p. pretreatment with 2 mg of celecoxib. Autoradiography and immunohistochemistry experiments further the confirmed blocking of COX-2 in vivo. [18F]Triacoxib, whose nonradioactive analogue was identified through in situ click chemistry, is a novel radiotracer for PET imaging of COX-2 in cancer. Despite a substantial amount of nonspecific uptake in vivo, [18F]triacoxib displayed specific binding to COX-2 in vivo and reinforced the feasibility of optimal structure selection by in situ click chemistry. It remains to be elucidated how this novel radiotracer would perform in first-in-human studies to detect COX-2 with PET.

Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

Elevated growth in breast cancer (BC) activates hypoxia-inducible factor (HIF1α) and downstream, facilitative glucose transporter 1 (GLUT1), which can be visualized with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). GLUT5 (fructose) and GLUT2 (glucose/fructose) might provide alternative targets for BC imaging as to why effects of hypoxia on GLUT1/2/5 levels and function were examined in human BC models. GLUT1/2/5 and HIF1α mRNA was analyzed in BC patient biopsies. In MCF10A, MCF7, and MDA-MB231 cells, [18F]FDG, 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF) and [18F]-fluoroazomycin arabinoside were used in radiotracer experiments, whereas GLUT1/2/5 mRNA was analyzed with real-time PCR and protein levels determined via Western blot/immunohistochemistry. Positron emission tomography imaging was performed in MCF7 and MDA-MB231 tumor-bearing mice. Glucose/fructose/cytochalasin B reduced cellular 6-[18F]FDF uptake by 50%, indicating functional involvement of GLUT2. With GLUT5 staining lower than GLUT1, 6-[18F]FDF revealed lower uptake than [18F]FDG [standardized uptake value (SUV)6-[18F]FDF, 120 min 0.77 ± 0.06 vs. SUV[18F]FDG, 120 min 1.08 ± 0.07] in MDA-MB231 tumors and was blocked by 20% with cytochalasin B after 10 min. Whereas correspondence between 6-[18F]FDF uptake and GLUT5 protein was low, high GLUT2 levels were detected in all cell lines and tumor models. Besides GLUT1, GLUT5 seems to be regulated under hypoxia on the molecular and functional level. Additionally, results strongly support a functional involvement of GLUT2 in fructose metabolism, possibly by compensating for the weaker expression and function of GLUT5 in BC.-Hamann, I., Krys, D., Glubrecht, D., Bouvet, V., Marshall, A., Vos, L., Mackey, J. R., Wuest, M., Wuest, F. Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

Molecular Imaging of GLUT1 and GLUT5 in Breast Cancer: A Multitracer Positron Emission Tomography Imaging Study in Mice.

Use of [18F]FDG-positron emission tomography (PET) in clinical breast cancer (BC) imaging is limited mainly by insufficient expression levels of facilitative glucose transporter (GLUT)1 in up to 50% of all patients. Fructose-specific facilitative hexose transporter GLUT5 represents an alternative biomarker for PET imaging of hexose metabolism in BC. The goal of the present study was to compare the uptake characteristics of selected hexose-based PET radiotracers in murine BC model EMT6. Uptake of 1-deoxy-1-[18F]fluoro-d-fructose (1-[18F]FDF), 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF), 1-deoxy-1-[18F]fluoro-2,5-anhydro-mannitol (1-[18F]FDAM), 2-deoxy-2-[18F]fluoro-d-glucose (2-[18F]FDG), and 6-deoxy-6-[18F]fluoro-d-glucose (6-[18F]FDG) was studied in EMT6 cells, tumors, and muscle and correlated to GLUT1 and GLUT5 expression levels. Fructose-derivative 6-[18F]FDF revealed greater tumor uptake than did structural analog 1-[18F]FDF, whereas 1-[18F]FDAM with locked anomeric configuration showed similar low tumor uptake to that of 1-[18F]FDF. Glucose-derivative 6-[18F]FDG reached maximum tumor uptake at 20 minutes, with no further accumulation over time. Uptake of 2-[18F]FDG was greatest and continuously increasing owing to metabolic trapping through phosphorylation by hexokinase II. In EMT6 tumors, GLUT5 mRNA expression was 20,000-fold lower compared with GLUT1. Whereas the latter was much greater in tumor than in muscle tissue (GLUT1 50:1), the opposite was found for GLUT5 mRNA expression (GLUT5 1:6). GLUT5 protein levels were higher in tumor versus muscle tissue as determined by Western blot and immunohistochemistry. Our data suggest that tumor uptake of fructose metabolism-targeting radiotracers 1-[18F]FDF, 6-[18F]FDF, and 1-[18F]FDAM does not correlate with GLUT5 mRNA levels but is linked to GLUT5 protein levels. In conclusion, our results highlight the importance of detailed biochemical studies on GLUT protein expression levels in combination with PET imaging studies for functional characterization of GLUTs in BC.

Association between cytoplasmic CRABP2, altered retinoic acid signaling, and poor prognosis in glioblastoma.

Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid-binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose-dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA-induced RAR activation. Levels of CRYAB, a small heat shock protein with anti-apoptotic activity, and GFAP, an astrocyte-specific intermediate filament protein, are greatly reduced in CRABP2-depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti-apoptotic pathway, thereby enhancing proliferation and preventing RA-mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients.

CRABP1 is associated with a poor prognosis in breast cancer: adding to the complexity of breast cancer cell response to retinoic acid.

BACKGROUND: Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer. METHODS: CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis. RESULTS: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism. CONCLUSIONS: CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

Calcineurin regulates nuclear factor I dephosphorylation and activity in malignant glioma cell lines.

Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 years. Nuclear factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP; FABP7) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcriptional activity, and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is up-regulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties.

A fatty acid-binding protein 7/RXRß pathway enhances survival and proliferation in triple-negative breast cancer.

FABP7 has been implicated in tumour cell proliferation, cell migration, and poor prognosis in patients with high-grade astrocytoma and melanoma. In this study, we examine FABP7 expression in a cohort of 176 primary breast cancers by gene profiling and tissue microarray immunostaining. We show that FABP7 is significantly up-regulated in triple-negative breast cancer. Elevated FABP7 levels are associated with poor prognosis, absence of oestrogen and progesterone hormone receptors (ER, PR) and HER2, increased cell proliferation, and high tumour grade. Depletion of FABP7 in the ER/PR-negative cell line, MDA-MB-435S, significantly reduced cell growth rate and sensitized the cells to growth inhibition by omega-3 docosahexaenoic acid (DHA). A target of DHA-bound FABP7 in the nucleus is RXRß, a retinoid-activated nuclear receptor that functions as a transcription factor by either homodimerizing or heterodimerizing with other nuclear receptors such as PPARs. Based on our microarray data, RXRß, like FABP7, is an adverse prognostic factor for breast cancer. We propose that the DHA-FABP7-RXRß pathway promotes cell survival/proliferation in triple-negative breast cancer. Targeting this pathway may thus provide an alternate route for the treatment of triple-negative breast cancer.

Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Association of FABP5 expression with poor survival in triple-negative breast cancer: implication for retinoic acid therapy.

Recent studies using animal models suggest that expression of FABP5 drives the stimulation of cell growth observed in estrogen receptor (ER)-negative breast cancer cells on exposure to retinoic acid (RA). The purpose of this study was to investigate the clinicopathological significance of FABP5 in breast cancer and to evaluate FABP5 as a prognostic marker and a possible novel therapeutic target in breast cancer. Gene expression microarray analysis revealed a significant correlation between elevated FABP5 RNA levels and ER/progesterone receptor (PR)-negative status, high tumor grade, and poor prognosis. Tissue microarray analysis demonstrated similar correlations with cytoplasmic FABP5 protein. Based on multivariate proportional regression analysis, cytoplasmic FABP5 is a significant and independent prognostic marker of overall survival and recurrence-free survival in breast cancer. The effects of FABP5 on tumor growth appear to be mediated primarily through cytoplasmic FABP, because no correlation was found between nuclear FABP5 and ER/PR-negative status, recurrence, and survival. FABP5 knockdown in breast cancer cell lines demonstrates a correlation between FABP5 levels and growth response to RA. We propose a model whereby growth-promoting FABP5 competes with growth-inhibiting CRABP2 for RA, with retention of RA in the cytoplasm by FABP5 preventing the inhibition of tumor growth.

Toward in vivo proof of binding of 18F-labeled inhibitor [18F]TRACK to peripheral tropomyosin receptor kinases.

BACKGROUND: Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation 11C and 18F-labeled Trk inhibitors, e.g., [18F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [18F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe. METHODS: In vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [18F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2tm1Bbd/J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples. RESULTS: Uptake of [18F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [18F]TRACK uptake in vitro (IC50 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [18F]TRACK showed uptake into KM12 tumors (SUVmean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [18F]TRACK uptake reached SUVmean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUVmean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p < 0.05) and of 21% in BAT (SUVmean,60 min from 1.32 ± 0.08; n = 5 to 1.59 ± 0.07; n = 6; p < 0.05) respectively. Immunohistochemistry showed TrkB > TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue. CONCLUSION: The present data show that radiotracer [18F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.

DEAD box 1: a novel and independent prognostic marker for early recurrence in breast cancer.

Breast cancer is a heterogeneous disease characterized by diverse molecular signatures and a variable response to therapy. Clinical management of breast cancer is guided by the expression of estrogen and progesterone receptors and HER2 amplification. New prognostic and predictive markers, as well as additional targets for therapy, are needed for more effective management of this disease. Gene expression microarrays were probed with RNAs from 176 primary breast cancer samples and tissue microarrays immunostained with anti-DDX1 antibody, an antibody to DEAD box protein DDX1, a putative RNA-RNA and RNA-DNA unwinding protein normally found in the nucleus. Half of the patient cohort had experienced early relapse despite standard adjuvant therapy, but were otherwise matched for estrogen receptor and HER2 status, stage and duration of follow-up. Here, we identify DDX1 RNA overexpression as an independent prognostic marker for early recurrence in primary breast cancer, with a hazard ratio of 4.31 based on logrank analysis of Kaplan-Meier curves. Elevated levels of DDX1 protein in the cytoplasm also independently correlate with early recurrence with a hazard ratio of 1.90. In conclusion, our data indicate a strong and independent association between poor prognosis and deregulation of the DEAD box protein DDX1. We propose that elevated levels of DDX1 RNA or the presence of DDX1 in the cytoplasm could serve as an effective prognostic biomarker for early recurrence in primary breast cancer.

AP2 transcription factor induces apoptosis in retinoblastoma cells.

The underlying cause of human retinoblastoma is complete inactivation of both copies of the RB1 gene. Other chromosome abnormalities, with the most common being extra copies of chromosome arm 6p, are also observed in retinoblastoma. The RB protein has previously been shown to interact with TFAP2 transcription factors. Here, we show that TFAP2A and TFAP2B, which map to chromosome arm 6p, are expressed in the amacrine and horizontal cells of human retina. TFAP2A RNA can readily be detected in retinoblastoma cell lines and tumors; however, the great majority of retinoblastoma cell lines and tumors are completely devoid of TFAP2A protein and TFAP2B RNA/protein. Transfection of TFAP2A and TFAP2B expression constructs into retinoblastoma cells induces apoptosis and inhibits proliferation. Our results suggest that a consequence of loss of RB1 gene function in retinoblastoma cells is inactivation of TFAP2A and TFAP2B function. We propose that inability to differentiate along the amacrine/horizontal cell lineages may underlie retinoblastoma tumor formation.

PET Imaging of L-Type Amino Acid Transporter (LAT1) and Cystine-Glutamate Antiporter (xc-) with [18F]FDOPA and [18F]FSPG in Breast Cancer Models.

PURPOSE: The present study describes the analysis of amino acid transporters ASCT1, ASCT2, LAT1, and xc- in breast cancer under normoxic and hypoxic conditions. [18F]FDOPA-PET and [18F]FSPG-PET were used as imaging biomarkers to probe L-type amino acid transporter (LAT1) and cystine-glutamate antiporter (xc-) in breast cancer models. PROCEDURES: LAT1 and xc- transporters were studied under normoxic and hypoxic conditions with radiotracers [18F]FDOPA and [18F]FSPG in estrogen receptor-positive (ER+) MCF7 and triple-negative MDA-MB231 cells and in human mammary epithelial MCF10A control cells. Protein expression was analyzed using Western blot and immunohistochemistry. RESULTS: ASCT1 protein expression levels were comparable in all three cell lines, while noticeable ASCT2 expression levels were only found in MCF10A control cells. Higher LAT1 protein expression was detected in ER+ MCF7 cells. High xc- protein expression levels were detected in MDA-MB231 cells. Uptake of [18F]FDOPA through LAT1 was significantly higher in MCF7 versus MDA-MB231 cells, while the uptake of [18F]FSPG through xc- resulted in the opposite confirming expression and functional differences for both amino acid transporters in different breast cancer models. Hypoxia significantly increased [18F]FDOPA uptake in MCF7 cells and [18F]FSPG uptake in MDA-MB231 cells. In vivo PET imaging revealed substantially higher tumor uptake of [18F]FDOPA in MCF7 tumors as well as [18F]FSPG uptake in MDA-MB231 tumors confirming differences detected in vitro. CONCLUSIONS: ER+ breast cancer cells express higher levels of amino acid transporter LAT1, whereas triple-negative breast cancer cells express more xc-. Cellular uptake and PET imaging experiments with [18F]FDOPA and [18F]FSPG confirmed functional LAT1 and xc- expression profiles. There was initial evidence that hypoxia regulates the function of both amino acid transporters in breast cancer. The results further indicated that [18F]FDOPA and [18F]FSPG are suitable radiotracer to distinguish between ER+ and triple-negative breast cancer types.

Tyrosine kinase inhibitor therapy and metabolic remodelling in papillary thyroid cancer.

Targeted therapy is increasingly used to manage metastatic papillary thyroid cancer. The focus of the present study was to examine glucose metabolism and tumor responses for thyroid cancer xenografts expressing the glycolytic pathway modulators platelet-derived growth factor receptor (PDGFR) and BRAFV600E. Radiolabelled glucose derivative [18F]FDG was used to analyze the effects of PDGFR blockade with imatinib, BRAF blockade with vemurafenib, as well as combined PDGFR and BRAF blockade in vitro and in vivo with PET. Dynamic PET data was correlated with immunohistochemistry staining and kinetic analysis for facilitative glucose transporter 1 (GLUT1) and hexokinase-II (HK2). Vemurafenib decreased [18F]FDG uptake in BCPAP cells in vitro; however, it was increased by ~70% with imatinib application to BCPAP cells. This metabolic response to tyrosine kinase inhibition required BRAFV600E as it was not seen in cell lines lacking mutated BRAF (TPC1). In xenografts, imatinib therapy in BCPAP thyroid tumour-bearing mice significantly increased [18F]FDG uptake and retention (>30%) in BCPAP tumours with PDGFRß or both (α+ß) isoforms. Kinetic analysis revealed that the increased glucose uptake is a consequence of increased phosphorylation and intracellular trapping of [18F]FDG confirmed by an increase in HK2 protein expression and activity, but not GLUT1 activity. BRAF inhibition alone, or combined PDGFR and BRAF inhibition, reduced (~60%) [18F]FDG uptake in both types of BCPAP (ß or α+ß) tumours. In terms of tumour growth, combination therapy with imatinib and vemurafenib led to a near abolition of the tumors (~90% reduction), but single therapy for BCPAP with PDGFRα expression was much less effective. In summary, imatinib led to a paradoxical increase of [18F]FDG uptake in xenografts that was reversed through BRAFV600E inhibition. The present data show that metabolic reprogramming in thyroid cancer occurs as a consequence of BRAF-mediated upregulation of HK2 expression that may permit tumour growth with isolated blockade of upstream tyrosine kinase receptors.

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial-to-mesenchymal transition and lipid-derived energy production in prostate cancer cells.

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid-binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial-to-mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid ß-oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12's role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP-PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Differential CRX and OTX2 expression in human retina and retinoblastoma.

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.