Pan-cancer Genomic Analysis of AXL Mutations Reveals a Novel, Recurrent, Functionally Activating AXL W451C Alteration Specific to Myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a common soft tissue sarcoma of the elderly that typically shows low tumor mutational burden, with mutations in TP53 and in genes associated with cell cycle checkpoints ( RB1 , CDKN2A ). Unfortunately, no alterations or markers specific to MFS have been identified and, as a consequence, there are no effective targeted therapies. The receptor tyrosine kinase AXL, which drives cellular proliferation, is targetable by new antibody-based therapeutics. Expression of AXL messenger RNA is elevated in a variety of sarcoma types, with the highest levels reported in MFS, but the pathogenic significance of this finding remains unknown. To assess a role for AXL abnormalities in MFS, we undertook a search for AXL genomic alterations in a comprehensive genomic profiling database of 463,546 unique tumors (including 19,879 sarcomas, of which 315 were MFS) interrogated by targeted next-generation DNA and/or RNA sequencing. Notably, the only genomic alterations recurrent in a specific sarcoma subtype were AXL W451C (n = 8) and AXL W450C (n = 2) mutations. The tumors involved predominantly older adults (age: 44 to 81 [median: 72] y) and histologically showed epithelioid and spindle-shaped cells in a variably myxoid stroma, with 6 cases diagnosed as MFS, 3 as undifferentiated pleomorphic sarcoma (UPS), and 1 as low-grade sarcoma. The AXL W451C mutation was not identified in any non-sarcoma malignancy. A review of publicly available data sets revealed a single AXL W451C-mutant case of UPS that clustered with MFS/UPS by methylation profiling. Functional studies revealed a novel activation mechanism: the W451C mutation causes abnormal unregulated dimerization of the AXL receptor tyrosine kinase through disulfide bond formation between pairs of mutant proteins expressing ectopic cysteine residues. This dimerization triggers AXL autophosphorylation and activation of downstream ERK signaling. We further report sarcomas of diverse histologic subtypes with AXL gene amplifications, with the highest frequency of amplification identified in MFS cases without the W451C mutation. In summary, the activating AXL W451C mutation appears highly specific to MFS, with a novel mechanism to drive unregulated signaling. Moreover, AXL gene amplifications and messenger RNA overexpression are far more frequent in MFS than in other sarcoma subtypes. We conclude that these aberrations in AXL are distinct features of MFS and may aid diagnosis, as well as the selection of available targeted therapies.

Kumquat essential oil decreases proliferation and activates JNK signaling and apoptosis in HT-1080 fibrosarcoma cells.

Kumquats are small citrus fruits produced by the Fortunella japonica tree. In addition to its aroma, kumquat essential oil may have antiproliferative effects; however, research on the effects of kumquat essential oil on human cell lines is limited. This study investigated the effects of kumquat essential oil on the proliferation of three human cell lines (HT-1080 fibrosarcoma cells, HeLa cervical adenocarcinoma cells, and CUA-4 normal human fibroblasts). As the concentration of kumquat essential oil increased, cell proliferation and viability, as measured by MTT activity assays, decreased in all three cell lines. Compared to untreated cells, HT-1080 fibrosarcoma cells exposed to kumquat essential oil exhibited an increased presence of phosphorylated JNK. Apoptosis was also stimulated, as PARP cleavage of treated HT-1080 fibrosarcoma cells was detected. Use of a JNK inhibitor resulted in decreased PARP cleavage in HT-1080 cells following treatment with kumquat EO, suggesting that activity of JNK is implicated in the stress response. The kumquat essential oil constituents limonene and myrcene both independently led to decreased proliferation and apoptosis.

Nuclear factor-κB plays an important role in Tamarixetin-mediated inhibition of matrix metalloproteinase-9 expression.

Flavonoids possess a broad spectrum of pharmacological properties, including anti-cancer, anti-oxidant and immunomodulatory activities. The current study explored the potential of some less-studied flavonoids in inhibiting Matrix Metalloproteinase-9 (MMP-9), a prominent biomarker, upregulated in a variety of cancers and known to promote migration and invasion of cancer cells. Amongst these, Tamarixetin, a naturally occurring flavonoid derivative of Quercetin, demonstrated significant dose-dependent inhibition of MMP-9 expression. Furthermore, a substantial inhibition of migration, invasion and clonogenic potential of HT1080 cells was also observed in the presence of Tamarixetin, which further suggests its role as a potential anti-cancer agent. It is noteworthy that Tamarixetin inhibits nuclear translocation as well the activity of nuclear factor kappa B (NFκB), both of which are functions essential for the activation of MMP-9 in promoting tumorigenesis. Additionally, the endogenous regulators of MMP-9 that tightly control its activity were also modulated by Tamarixetin, as evident from the 1.9 fold increase in the expression of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), with a concomitant 2.2 fold decrease in Matrix Metalloproteinase-14 (MMP-14) expression. The results obtained were further corroborated in three dimensional (3D) tumor models, which showed significant inhibition of MMP-9 activity as well as reduced invasive potential in the presence of Tamarixetin. Taken together, our observations demonstrate for the first time, the anti-invasive potential of Tamarixetin in cancer cells, indicating its possible use as a template for novel therapeutic applications.

Cardiovascular safety of rapidly accelerated fibrosarcoma B-type and/or mitogen-activated extracellular signal-regulated kinase inhibitors: A mixed approach combining a meta-analysis and a pharmacovigilance disproportionality analysis.

BACKGROUND: The risk of cardiovascular adverse events from rapidly accelerated fibrosarcoma B-type (BRAF) and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors is not fully characterized. AIM: To evaluate the cardiovascular adverse events risks related to BRAF and/or MEK inhibitors in randomized placebo-controlled clinical trials and in the real-life setting. METHODS: We used two approaches. First, we conducted a systematic review and meta-analysis of randomized placebo-controlled clinical trials reporting the incidence of cardiovascular adverse events for BRAF and/or MEK inhibitors in cancer patients. Second, we performed a disproportionality analysis, using age- and sex-adjusted reporting odds ratios (arORs) and their 95% confidence intervals (CIs) from the World Health Organization's pharmacovigilance database (VigiBase®) of anticancer drug-associated reports, to investigate real-life data. RESULTS: MEK inhibitors increased the risk of ejection fraction decrease (odds ratio [OR] 3.35, 95% CI 1.58-7.07), peripheral oedema (OR 2.87 95% CI 1.93-4.27) and syncope (OR 6.71, 95% CI 3.00-14.99) compared with placebo in randomized placebo-controlled clinical trials. BRAF and MEK inhibitor combination therapy further increased the risk of ejection fraction decrease. In the disproportionality analysis, we found over-reporting of ejection fraction decrease (arOR 8.42, 95% CI 7.03-10.09), peripheral oedema (arOR 1.39, 95% CI 1.17-1.66), syncope (arOR 1.56, 95% CI 1.22-1.99), torsade de pointes/QT prolongation (arOR 6.13, 95% CI 5.04-7.47) and supraventricular arrhythmias (arOR 1.50, 95% CI 1.21-1.85) for BRAF and MEK inhibitors. BRAF and MEK inhibitors were not associated with hypertension in either approach. CONCLUSIONS: In conclusion, MEK inhibitors increase the risk of ejection fraction decrease, peripheral oedema and syncope in randomized placebo-controlled clinical trials. Real-life data confirm these findings, and suggested additional risks of torsade de pointes/QT prolongation and supraventricular arrhythmias with BRAF/MEK inhibitors.

Rapid, complete and sustained tumour response to the TRK inhibitor larotrectinib in an infant with recurrent, chemotherapy-refractory infantile fibrosarcoma carrying the characteristic ETV6-NTRK3 gene fusion.

BACKGROUND: The ETV6-NTRK3 gene fusion is present in the majority of cases of infantile fibrosarcoma (IFS) and acts as a potent oncogenic driver. We report the very rapid, complete, and sustained response of an advanced, chemotherapy-refractory, recurrent IFS to targeted treatment with the oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib. PATIENT AND METHODS: A male infant born with a large congenital IFS of the tongue had the tumour surgically resected at age 4 days. Within 2 months, he developed extensive lymph node recurrence that progressed during two cycles of vincristine-doxorubicin-cyclophosphamide chemotherapy. At screening, a large right cervical mass was clinically visible. Magnetic resonance imaging (MRI) revealed bilateral cervical and axillary lymph node involvement as well as infiltration of the floor of the mouth. The largest lesion measured 5.5×4.5×4.4 cm (ca. 55 cm3). The patient started outpatient oral larotrectinib at 20 mg/kg twice daily at age 3.5 months. RESULTS: After 4 days on treatment, the parents noted that the index tumour was visibly smaller and softer. The rapid tumour regression continued over the following weeks. On day 56 of treatment, the first scheduled control MRI showed the target lesion had shrunk to 1.2×1.2×0.8 cm (ca. 0.6 cm3), corresponding to a complete response according to the Response Evaluation Criteria In Solid Tumors version 1.1. This response was maintained over subsequent follow-up visits, and on day 112 at the second control MRI the target lymph node was completely normal. At last follow-up, the disease remained in complete remission after 16 months on larotrectinib, with negligible toxicity and no safety concerns. CONCLUSION(S): Selective TRK inhibition by larotrectinib offers a novel, highly specific and highly effective therapeutic option for IFS carrying the characteristic ETV6-NTRK3 gene fusion. Its use should be considered when surgery is not feasible. (NCT02637687).

The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner.

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

Controlled Nano-Bio Interface of Functional Nanoprobes for in Vivo Monitoring Enzyme Activity in Tumors.

Engineering inorganic nanoparticles with a biocompatible shell to improve their physicochemical properties is a vital step in taking advantage of their superior magnetic, optical, and photothermal properties as multifunctional molecular imaging probes for disease diagnosis and treatment. The grafting/peeling-off strategy we developed for nanoparticle surface coating can fully control the targeting capability of functional nanoprobes by changing their colloidal behaviors such as diffusion and sedimentation rates at the desired sites. We demonstrated that a cleavable coating layer initially immobilized on the surface of magnetic resonance imaging probes not only makes the nanoparticles water-soluble but also can be selectively removed by specific enzymes, thereby resulting in a significant decrease of their water solubility in an enzyme-rich environment. Upon removal of surface coating, the changes in hydrodynamic size and surface charges of nanoprobes as a result of interacting with biomolecules and proteins lead to dramatic changes in their in vivo colloidal behaviors ( i. e., slow diffusion rates, tendency to aggregate and precipitate), which were quantitatively evaluated by examining changes in their hydrodynamic sizes, magnetic properties, and count rates during the size measurement. Because the retention time of nanoprobes within the tumor tissues depends on the uptake and excretion rate of the nanoprobes through the tumors, selective activation of nanoprobes by a specific enzyme resulted in much higher tumor accumulation and longer retention time within the tumors than that of the inactive nanoprobes, which passively passed through the tumors. The imaging contrast effect of tumors using activatable nanoprobes was significantly improved over using inactive probes. Therefore, the grafting/peeling-off strategy, as a general design approach for surface modification of nanoprobes, offers a promising and highly efficient way to render the nanoparticles suitable for targeted imaging of tumors.

Density-dependent ERK MAPK expression regulates MMP-9 and influences growth.

Previous work has shown that expression of the extracellular signal-regulated kinase (ERK) is decreased by high density in normal fibroblast cells, and this was correlated with increased expression of mitogen-activated protein kinase phosphatases. Because of these differences in ERK regulation upon contact inhibition, it is likely that other cellular responses may be influenced by the attainment of a contact-inhibited state. Expression of matrix metalloproteinase-9 and cadherin cleavage were both found to be decreased upon reaching high culture density. Inhibition of ERK activity with the MEK inhibitor PD98059 resulted in increased expression of cadherins, while constitutive activation of ERK through the use of expression of an ERK construct with a D319N sevenmaker mutation resulted in decreased expression of cadherins and enhanced colony formation of HT-1080 fibrosarcoma cells. Taken together, these results corroborate a role for the regulation of ERK upon the attainment of a contact-inhibited state with increased expression of cadherins.

Oncogenic R132 IDH1 Mutations Limit NADPH for De Novo Lipogenesis through (D)2-Hydroxyglutarate Production in Fibrosarcoma Sells.

Neomorphic mutations in NADP-dependent isocitrate dehydrogenases (IDH1 and IDH2) contribute to tumorigenesis in several cancers. Although significant research has focused on the hypermethylation phenotypes associated with (D)2-hydroxyglutarate (D2HG) accumulation, the metabolic consequences of these mutations may also provide therapeutic opportunities. Here we apply flux-based approaches to genetically engineered cell lines with an endogenous IDH1 mutation to examine the metabolic impacts of increased D2HG production and altered IDH flux as a function of IDH1 mutation or expression. D2HG synthesis in IDH1-mutant cells consumes NADPH at rates similar to de novo lipogenesis. IDH1-mutant cells exhibit increased dependence on exogenous lipid sources for in vitro growth, as removal of medium lipids slows growth more dramatically in IDH1-mutant cells compared with those expressing wild-type or enzymatically inactive alleles. NADPH regeneration may be limiting for lipogenesis and potentially redox homeostasis in IDH1-mutant cells, highlighting critical links between cellular biosynthesis and redox metabolism.

Use of RIP1 Kinase Small-Molecule Inhibitors in Studying Necroptosis.

RIP1 kinase plays a key role in regulating signaling pathways downstream of a number of innate immune receptors such as TNFRI and TLRs. The discovery of Necrostatin-1 (Nec-1) as a small-molecule inhibitor of RIP1 kinase has been very instrumental in defining the necroptotic and other signalling pathways regulated by RIP1, but certain characteristics of Nec-1 limits its utility in experimental systems. Next generation RIP1 kinase inhibitors have been identified and the use of these tool inhibitors along with Nec-1 has revealed that RIP1 is emerging as a key driver of inflammation and tissue injury in the pathogenesis of various diseases. Further studying the role of RIP1 to carefully unravel the complex biology requires the selection of the correct tool small-molecule inhibitors. In addition, it is important to consider the proper application of current tool inhibitors and understand the current limitiations. Here we will discuss key parameters that need to be considered when selecting and applying tool inhibitors to novel biological assays and systems. General protocols to explore the in vitro and in vivo potency, cellular selectivity, and pharmacokinetic properties of current small-molecule inhibitors of RIP1 kinase are provided.

Pan-Trk Immunohistochemistry Identifies NTRK Rearrangements in Pediatric Mesenchymal Tumors.

Activating neurotrophic receptor kinase (NTRK) fusions define certain pediatric mesenchymal tumors, including infantile fibrosarcoma and cellular mesoblastic nephroma. Traditionally, molecular confirmation of these fusions has included either fluorescent in situ hybridization for ETV6 rearrangements or reverse-transcriptase polymerase chain reaction for the classic ETV6-NTRK3 fusion. However, these methods overlook variant NTRK rearrangements, which are increasingly appreciated as recurrent events in a subset of pediatric mesenchymal tumors. New therapeutic agents successfully target these fusions and may prevent morbid surgeries in very young children, making recognition of tumors harboring NTRK rearrangements of increasing importance. We evaluated the performance of immunohistochemical (IHC) staining using pan-Trk and TrkA antibodies in 79 pediatric mesenchymal tumors. Negative controls included pediatric mesenchymal tumors not harboring (n=28) or not expected to harbor (n=22) NTRK fusions. NTRK rearrangements were detected predominantly by DNA-based next-generation sequencing assays, specifically UW OncoPlex and UCSF500 Cancer Gene Panel. Pan-Trk IHC (EPR17341) was 97% sensitive and 98% specific for the presence of an NTRK rearrangement, and TrkA IHC (EP1058Y) was 100% sensitive and 63% specific for the presence of an NTRK rearrangement. Tumors with NTRK1 or NTRK2 rearrangements showed cytoplasmic staining, whereas tumors with NTRK3 rearrangements showed nuclear +/- cytoplasmic staining. We conclude that pan-Trk IHC is a highly sensitive and specific marker for NTRK rearrangements in pediatric mesenchymal tumors.

The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial ß-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an  endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.

A new gallium complex inhibits tumor cell invasion and matrix metalloproteinase MMP-14 expression and activity.

In this study, we investigated the effect of [N-(5-chloro-2-hydroxyphenyl)-l-aspartato] chlorogallate (GS2), a new water soluble gallium complex, on cell invasion and on the expression and activity of matrix metalloproteinases (MMPs) in human metastatic HT-1080 fibrosarcoma and MDA-MB 231 breast carcinoma cells. The effect on cell invasion was studied using a modified Boyden chamber coated with a type-I collagen. We analyzed the effect of GS2 on MMP-2, MMP-9, and MMP-14 via zymography and enzymatic assay using high affinity fluorogenic substrates. The expression of MMP mRNA was analyzed via qRT-PCR. GS2 induced a decrease in cell invasion. A dose-dependent inhibition effect was observed on the activities of MMP-2, MMP-9, and MMP-14 with the IC50 values of 168, 82, and 20 µM, respectively. A decrease in the expression of MMP-14 mRNA was observed in both cell lines, whereas the expression of MMP-2 and MMP-9 mRNA was decreased only in the MDA-MB231 cells. Data obtained for the expression of MMP-14 mRNA were confirmed via Western blotting. In fact, MMP-14 expression was decreased in the presence of GS2. Overall, these data show that GS2 is a promising compound for anti-invasive and anticancer therapy.

Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.

Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)-lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex-binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules.

Recurrent BRAF Gene Rearrangements in Myxoinflammatory Fibroblastic Sarcomas, but Not Hemosiderotic Fibrolipomatous Tumors.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a low grade soft tissue sarcoma with a predilection for acral sites, being associated with a high rate of local recurrence but very infrequent distant metastases. Although a t(1;10) translocation resulting in TGFBR3-MGEA5 fusion has been reported as a recurrent genetic event in MIFS, this abnormality is seen only in a subset of cases. As no studies to date have investigated the spectrum of alternative genetic alterations in TGFBR3-MGEA5 fusion negative MIFS, we undertook a genetic analysis of this particular cohort for further molecular classification. Triggered by an index case occurring in the finger of a 37-year-old female and harboring a novel TOM1L2-BRAF fusion by targeted RNA sequencing we investigated potential recurrent BRAF abnormalities by screening a large group of 19 TGFBR3-MGEA5 fusion negative MIFS by fluorescence in situ hybridization. There were 6 (32%) additional MIFS with BRAF genetic abnormalities, including 5 gene rearrangements and one showing BRAF amplification. Interestingly, VGLL3 amplification, a recurrent genetic abnormality coexisting with t(1;10) in some MIFS, was also detected by fluorescence in situ hybridization in 4/6 (67%) BRAF-rearranged MIFS, but not in the BRAF-amplified case. Up-regulated VGLL3 mRNA expression was also demonstrated in the index case by RNA sequencing. The 7 BRAF-rearranged/amplified MIFS arose in the fingers (n=3), and 1 each in wrist, forearm, foot, and knee, of adult patients (36 to 74 y; M:F=4:3). The histologic spectrum ranged from predominantly solid growth of plump histiocytoid to epithelioid tumor cells with focal myxoid change to a predominantly myxoid background with scattered tumor cells. Varying degree of inflammatory infiltrates and large tumor cells with virocyte-like macronucleoli were observed in most cases. Immunohistochemical stains of phosphorylated ERK, a downstream effector of BRAF activation, were positive in all 4 cases tested (2 diffuse strong, 2 focal strong). Unlike t(1;10), BRAF rearrangements were only found in MIFS but not in 6 hemosiderotic fibrolipomatous tumor (HFLT) lacking TGFBR3-MGEA5 fusions (including 2 pure HFLT, 2 hybrid HFLT-MIFS, and 2 associated with pleomorphic hyalinizing angiectatic tumors).

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function.

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

Inhibitory effect of blue light emitting diode on migration and invasion of cancer cells.

The aim of this study was to determine the effects and molecular mechanism of blue light emitting diode (LED) in tumor cells. A migration and invasion assay for the metastatic behavior of mouse colon cancer CT-26 and human fibrosarcoma HT-1080 cells was performed. Cancer cell migration-related proteins were identified by obtaining a 2-dimensional gel electrophoresis (2-DE) in total cellular protein profile of blue LED-irradiated cancer cells, followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. Protein levels were examined by immunoblotting. Irradiation with blue LED inhibited CT-26 and HT-1080 cell migration and invasion. The anti-metastatic effects of blue LED irradiation were associated with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression. P38 MAPK phosphorylation was increased in blue LED-irradiated CT-26 and HT-1080 cells, but was inhibited after pretreatment with SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK phosphorylation by SB203580 treatment increased number of migratory cancer cells in CT-26 and HT-1080 cells, indicating that blue LED irradiation inhibited cancer cell migration via phosphorylation of p38 MAPK. Additionally blue LED irradiation of mice injected with CT-26 cells expressing luciferase decreased early stage lung metastasis compared to untreated control mice. These results indicate that blue LED irradiation inhibits cancer cell migration and invasion in vitro and in vivo.

A modified gelatin zymography technique incorporating total protein normalization.

Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.

Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death.

The nuclear export protein, exportin-1 (XPO1/CRM1), is overexpressed in many cancers and correlates with poor prognosis. Selinexor, a first-in-class Selective Inhibitor of Nuclear Export (SINE) compound, binds covalently to XPO1 and blocks its function. Treatment of cancer cells with selinexor results in nuclear retention of major tumor suppressor proteins and cell cycle regulators, leading to growth arrest and apoptosis. Recently, we described the selection of SINE compound resistant cells and reported elevated expression of inflammation-related genes in these cells. Here, we demonstrated that NF-κB transcriptional activity is up-regulated in cells that are naturally resistant or have acquired resistance to SINE compounds. Resistance to SINE compounds was created by knockdown of the cellular NF-κB inhibitor, IκB-α. Combination treatment of selinexor with proteasome inhibitors decreased NF-κB activity, sensitized SINE compound resistant cells and showed synergistic cytotoxicity in vitro and in vivo. Furthermore, we showed that selinexor inhibited NF-κB activity by blocking phosphorylation of the IκB-α and the NF-κB p65 subunits, protecting IκB-α from proteasome degradation and trapping IκB-α in the nucleus to suppress NF-κB activity. Therefore, combination treatment of selinexor with a proteasome inhibitor may be beneficial to patients with resistance to either single-agent.