Alpha-Ketoglutarate Regulates Tnfrsf12a/Fn14 Expression via Histone Modification and Prevents Cancer-Induced Cachexia.

Previous studies have shown that inhibition of TNF family member FN14 (gene: TNFRSF12A) in colon tumors decreases inflammatory cytokine expression and mitigates cancer-induced cachexia. However, the molecular mechanisms underlying the regulation of FN14 expression remain unclear. Tumor microenvironments are often devoid of nutrients and oxygen, yet how the cachexic response relates to the tumor microenvironment and, importantly, nutrient stress is unknown. Here, we looked at the connections between metabolic stress and FN14 expression. We found that TNFRSF12A expression was transcriptionally induced during glutamine deprivation in cancer cell lines. We also show that the downstream glutaminolysis metabolite, alpha-ketoglutarate (aKG), is sufficient to rescue glutamine-deprivation-promoted TNFRSF12A induction. As aKG is a co-factor for histone de-methylase, we looked at histone methylation and found that histone H3K4me3 at the Tnfrsf12a promoter is increased under glutamine-deprived conditions and rescued via DM-aKG supplementation. Finally, expression of Tnfrsf12a and cachexia-induced weight loss can be inhibited in vivo by DM-aKG in a mouse cancer cachexia model. These findings highlight a connection between metabolic stress and cancer cachexia development.

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines.

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.

Is CCL2 an Important Mediator of Mast Cell-Tumor Cell Interactions in Oral Squamous Cell Carcinoma?

In this study, we aimed to evaluate the influence of interactions between mast cells (MCs) and oral squamous cell carcinoma (OSCC) tumor cells on tumor proliferation and invasion rates and identify soluble factors mediating this crosstalk. To this end, MC/OSCC interactions were characterized using the human MC cell line LUVA and the human OSCC cell line PCI-13. The influence of an MC-conditioned (MCM) medium and MC/OSCC co-cultures on the proliferative and invasive properties of the tumor cells was investigated, and the most interesting soluble factors were identified by multiplex ELISA analysis. LUVA/PCI-13 co-cultures increased tumor cell proliferation significantly (p = 0.0164). MCM reduced PCI-13 cell invasion significantly (p = 0.0010). CC chemokine ligand 2 (CCL2) secretion could be detected in PCI-13 monocultures and be significantly (p = 0.0161) increased by LUVA/PCI-13 co-cultures. In summary, the MC/OSCC interaction influences tumor cell characteristics, and CCL2 could be identified as a possible mediator.

The effect of miR-372-5p regulation on CDX1 and CDX2 in the gastric cancer cell line.

OBJECTIVES: MicroRNA expression disruptions play an important function in the expansion of gastric cancer. Previous investigation has indicated that miR-372-5p doing as an oncogene in several malignancies. CDX1 and CDX2, as target genes of miR-372-5p, play the role of tumor suppressors and oncogenes in gastric cancer cells, respectively. The current investigation explored the effects of miR-372-5p regulation on CDX2 and CDX1 in AGS cell lines and studied their molecular mechanism. METHODS: hsa-miR-372-5p miRCURY LNA miRNA Inhibitors and Mimic were transfected into AGS cell line. The cell viability and cell cycle calculation were defined by MTT assay and flow cytometry, respectively. The Expression levels of miR-372-5p, CDX1, CDX2 and transfection efficiency were measured using Real-time PCR. Statistical investigation p values <0.05 were considered to be meaningful. RESULTS: miR-372-5p particularly was upregulated in control cells and also after transfection by mimic. While its expression was reduced by the inhibitor. Upregulation of miR-372-5p remarkably increased cell growth and led to accumulation in the G2/M phase, although the inhibitor decreased cell growth and accumulation in the S phase. Accordingly, upregulation of miR-372-5p increased CDX2 and decreased CDX1 expression. By inhibition of miR-372-5p, expression of CDX2 was decreased and expression of CDX1 was increased. CONCLUSIONS: Up and down-regulation of miR-372-5P has a potential effect on the expression levels of its target genes, CDX1 and CDX22. Accordingly, the downregulation of miR-372-5p may be assumed as a possible therapeutic target in treating gastric cancer.

A radioiodinated rucaparib analogue as an Auger electron emitter for cancer therapy.

INTRODUCTION: Radioligand therapy (RLT) is an expanding field that has shown great potential in the fight against cancer. Radionuclides that can be carried by selective ligands such as antibodies, peptides, and small molecules targeting cancerous cells have demonstrated a clear improvement in the move towards precision medicine. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage repair signalling pathway, with PARP inhibitors olaparib, talazoparib, niraparib, veliparib, and rucaparib having FDA approval for cancer therapy in routine clinical use. Based on our previous work with the radiolabelled PARP inhibitor [18F]rucaparib, we replaced the fluorine-18 moiety, used for PET imaging, with iodine-123, a radionuclide used for SPECT imaging and Auger electron therapy, resulting in 8-[123I]iodo-5-(4-((methylamino)methyl)phenyl)-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-1-one, ([123I]GD1), as a potential radiopharmaceutical for RLT. METHODS: [123I]GD1 was synthesized via copper-mediated radioiodination from a selected boronic esters precursor. In vitro uptake, retention, blocking, and effects on clonogenic survival with [123I]GD1 treatment were tested in a panel of cancer cell lines. Enzymatic inhibition of PARP by GD1 was also tested in a cell-free system. The biodistribution of [123I]GD1 was investigated by SPECT/CT in mice following intravenous administration. RESULTS: Cell-free enzymatic inhibition and in vitro blocking experiments confirmed a modest ability of GD1 to inhibit PARP-1, IC50 = 239 nM. In vitro uptake of [123I]GD1 in different cell lines was dose dependent, and radiolabelled compound was retained in cells for >2 h. Significantly reduced clonogenic survival was observed in vitro after exposure of cells for 1 h with as low as 50 kBq of [123I]GD1. The biodistribution of [123I]GD1 was further characterized in vivo showing both renal and hepatobiliary clearance pathways with a biphasic blood clearance. CONCLUSION: We present the development of a new theragnostic agent based on the rucaparib scaffold and its evaluation in in vitro and in vivo models. The data reported show that [123I]GD1 may have potential to be used as a theragnostic agent.

Overexpression of synaptic vesicle protein Rab GTPase 3C promotes vesicular exocytosis and drug resistance in colorectal cancer cells.

Rab GTPase 3C (RAB3C) is a peripheral membrane protein that is involved in membrane trafficking (vesicle formation) and cell movement. Recently, researchers have noted the exocytosis of RAB proteins, and their dysregulation is correlated with drug resistance and the altered tumor microenvironment in tumorigenesis. However, the molecular mechanisms of exocytotic RABs in the carcinogenicity of colorectal cancer (CRC) remain unknown. Researchers have used various in silico datasets to evaluate the expression profiles of RAB family members. We confirmed that RAB3C plays a key role in CRC progression. Its overexpression promotes exocytosis and is related to the resistance to several chemotherapeutic drugs. We established a proteomic dataset based on RAB3C, and found that dystrophin is one of the proteins that is upregulated with the overexpression of RAB3C. According to our results, RAB3C-induced dystrophin expression promotes vesicle formation and packaging. A connectivity map predicted that the cannabinoid receptor 2 (CB2) agonists reverse RAB3C-associated drug resistance, and that these agonists have synergistic effects when combined with standard chemotherapy regimens. Moreover, we found high dystrophin expression levels in CRC patients with poor survival outcomes. A combination of the dystrophin and RAB3C expression profiles can serve as an independent prognostic factor in CRC and is associated with several clinicopathological parameters. In addition, the RAB3C-dystrophin axis is positively correlated with the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) genetic alterations in CRC patients. These findings can be used to provide novel combined therapeutic options for the treatment of CRC.

Dipyridamole interacts with the N-terminal domain of HSP90 and antagonizes the function of the chaperone in multiple cancer cell lines.

Molecular chaperone HSP90 has been considered as a promising target for anti-cancer drug development for years. However, due to the heat shock response induced by the ATP competitive inhibitors against HSP90, the therapeutic efficacies of the compounds are compromised, which consequently restricts the clinical use of HSP90-targeted inhibitors. Therefore, there is a need to discover novel HSP90-targeted modulators which exhibit acceptable inhibition activity against the chaperone and do not induce significant heat shock response in the meantime. Here in this study, we firstly developed a tip-based affinity selection-mass spectrometry platform with optimized experimental conditions/parameters for HSP90-targeted active compound screening, and then applied it to fish out inhibitors against HSP90 from a collection of 2,395 compounds composed of FDA-approved drugs and drug candidates. Dipyridamole, which acts as an anti-thrombotic agent by modulating multiple targets and has a long history of safe use, was identified to interact with HSP90's N-terminal domain. The following conducted biophysical and biochemical experiments demonstrated that Dipyridamole could bind to HSP90's ATP binding pocket and function as an ATP competitive inhibitor of the chaperone. Finally, cellular-based assays including CESTA, cell viability assessment and proteomic analysis etc. were performed to evaluate whether the interaction between HSP90 and Dipyridamole contributes to the anti-tumor effects of the compound. We then found that Dipyridamole inhibits the growth and proliferation of human cancer cells by downregulating cell cycle regulators and upregulating apoptotic cell signaling, which are potentially mediated by the binding of Dipyridamole to HSP90 and to PDEs (phosphodiesterases), respectively.

Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

Acquired chemoresistance to proteasome inhibitors (PIs), such as bortezomib (BTZ), becomes an intractable obstacle in the management of multiple myeloma (MM) in the clinic, but the underlying mechanisms are still not well elucidated. In the current study, we established bortezomib-resistant (BR) myeloma cells and performed stable isotope labeling by amino acids in cell culture (SILAC) assay to screen profiled protein expression. The level of deoxyuridine triphosphatase (DUT), an important enzyme of nucleotide metabolism, increased in the BR MM cells. Retrospective analysis indicated patients with higher DUT expression had poorer responses to PI-based treatment and clinical outcomes. DUT knockdown by RNAi effectively minimized BTZ resistance in MM cells. Moreover, DUT knockdown was accompanied with the downregulation of proliferating cell nuclear antigen (PCNA), contributing to decelerating cell growth, as well as augmented apoptosis due to bortezomib treatment. In contrast, DUT overexpression in parental MM.1S and LP-1 cells enhanced BTZ resistance. Furthermore, acquired resistance to BTZ could trigger the modulation of mitochondrial metabolism and function, as evidenced by elevated expression of genes associated with mitochondrial metabolism, as well as altered oxygen consumption rate and adenosine triphosphate (ATP) production in BR MM cells. DUT inhibition partially attenuated mitochondrial modulation, and instead favored an early impairment of mitochondrial integrity upon BTZ exposure so as to restrict MM progression and overcome drug resistance to BTZ treatment both in vitro and in vivo. In conclusion, we unveiled previously unrecognized effects of DUT on acquired drug resistance of MM, thus manipulating DUT may be efficacious for sensitizing MM cells to PIs.

Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis.

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers. Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors. Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice. Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity.

In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.

ATF2 loss promotes tumor invasion in colorectal cancer cells via upregulation of cancer driver TROP2.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Modulating Nucleus Oxygen Concentration by Altering Intramembrane Cholesterol Levels: Creating Hypoxic Nucleus in Oxic Conditions.

We propose a novel mechanism by which cancer cells can modulate the oxygen concentration within the nucleus, potentially creating low nuclear oxygen conditions without the need of an hypoxic micro-environment and suited for allowing cancer cells to resist chemo- and radio-therapy. The cells ability to alter intra-cellular oxygen conditions depends on the amount of cholesterol present within the cellular membranes, where high levels of cholesterol can yield rigid membranes that slow oxygen diffusion. The proposed mechanism centers on the competition between (1) the diffusion of oxygen within the cell and across cellular membranes that replenishes any consumed oxygen and (2) the consumption of oxygen in the mitochondria, peroxisomes, endoplasmic reticulum (ER), etc. The novelty of our work centers around the assumption that the cholesterol content of a membrane can affect the oxygen diffusion across the membrane, reducing the cell ability to replenish the oxygen consumed within the cell. For these conditions, the effective diffusion rate of oxygen becomes of the same order as the oxygen consumption rate, allowing the cell to reduce the oxygen concentration of the nucleus, with implications to the Warburg Effect. The cellular and nucleus oxygen content is indirectly evaluated experimentally for bladder (T24) cancer cells and during the cell cycle, where the cells are initially synchronized using hydroxeaurea (HU) at the late G1-phase/early S-phase. The analysis of cellular and nucleus oxygen concentration during cell cycle is performed via (i) RT-qPCR gene analysis of hypoxia inducible transcription factors (HIF) and prolyl hydroxylases (PHD) and (ii) radiation clonogenic assay every 2 h, after release from synchronization. The HIF/PHD genes allowed us to correlate cellular oxygen with oxygen concentration in the nucleus that is obtained from the cells radiation response, where the amount DNA damage due to radiation is directly related to the amount of oxygen present in the nucleus. We demonstrate that during the S-phase cells can become hypoxic in the late S-phase/early G2-phase and therefore the radiation resistance increases 2- to 3-fold.

Mutational signatures are markers of drug sensitivity of cancer cells.

Genomic analyses have revealed mutational footprints associated with DNA maintenance gone awry, or with mutagen exposures. Because cancer therapeutics often target DNA synthesis or repair, we asked if mutational signatures make useful markers of drug sensitivity. We detect mutational signatures in cancer cell line exomes (where matched healthy tissues are not available) by adjusting for the confounding germline mutation spectra across ancestries. We identify robust associations between various mutational signatures and drug activity across cancer cell lines; these are as numerous as associations with established genetic markers such as driver gene alterations. Signatures of prior exposures to DNA damaging agents - including chemotherapy - tend to associate with drug resistance, while signatures of deficiencies in DNA repair tend to predict sensitivity towards particular therapeutics. Replication analyses across independent drug and CRISPR genetic screening data sets reveal hundreds of robust associations, which are provided as a resource for drug repurposing guided by mutational signature markers.

MiR-26b-5p in small extracellular vesicles derived from dying tumor cells after irradiation enhances the metastasis promoting microenvironment in esophageal squamous cell carcinoma.

Radiation therapy is effective in achieving local control in esophageal squamous cell carcinoma; however, changes in the tumor microenvironment induced by radiation can also promote metastasis. Dying tumor cells play vital roles in promoting the survival of living tumor cells; however, few studies have investigated the effects of dying tumor cells on the tumor microenvironment. Since myeloid-derived suppressor cells (MDSCs) and macrophages constitute the pre-metastatic niche (PMN), we used a 4-nitroquinoline-1-oxide induced in situ tumor model to investigate the effects of irradiation on MDSCs and macrophages in esophageal squamous cell carcinoma (ESCC). When primary tumor sites were irradiated, we observed an increase in MDSCs in the spleen and the deposition of PMN components in lung and liver. Enhanced MDSC accumulation and function were induced by small extracellular vesicles (sEVs) isolated from irradiated tumor-bearing mice. The MDSC induction function of sEVs after irradiation was reaffirmed using sEVs derived from ESCC cell lines. The irradiation-induced upregulation of miR-26b-5p in sEVs enhanced MDSC expansion and activation by targeting phosphatase and tensin homolog. Our results first elucidated a mechanism by which dying tumor cells enhanced the deposition of PMN components and potentiated MDSCs in ESCC after irradiation. sEVs played a vital role in mediating signals between the primary tumor and the microenvironment to form a metastasis-promoting microenvironment after irradiation. Furthermore, miR-26b-5p or PI3K/AKT signaling pathway inhibitors should be evaluated in clinical trials in combination with radiotherapy as a strategy to improve outcomes.

Inhibition of the CDK2 and Cyclin A complex leads to autophagic degradation of CDK2 in cancer cells.

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

miRNA profiling of human nasopharyngeal carcinoma cell lines HONE1 and CNE2 after X-ray therapy.

BACKGROUND: Radiotherapy is the main treatment for nasopharyngeal carcinoma. The radioresistance mechanism of cells is related to miRNAs. OBJECTIVES: To investigate the miRNA profiling of HONE1 and CNE2 after X-ray therapy. MATERIAL AND METHODS: The HONE1 and CNE2 cells were treated with X-ray at 4 Gy, 8 Gy, 16 Gy, and 20 Gy doses. The cell lines CNE2 with the best therapy effects and HONE1 with the worst therapy effects were screened out. Apoptosis and cell viability were detected with flow cytometry and Cell Counting Kit-8 (CCK-8). High-throughput sequencing was performed. A miRNA library was constructed. The miRNA annotation expression distribution, family prediction and target gene interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. RESULTS: The 24-hour 20 Gy dose X-rays were selected as the optimal therapy conditions. The CNE2_C, CNE2_M, HONE1_C and HONE1_M miRNAs accounted for 26.5%, 31.7%, 21.3%, and 22.9% of the Cleandata reads count, respectively, and the contents of rRNAs accounted for 24.9%, 14.7%, 25.1%, and 25.1% of the Cleandata reads count, respectively. The miRNAs with differential expression between the HONE1 and CNE2 cell lines including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-125a-5p, hsa-miR-26a-5p, hsa-let-7f-5p, hsa-miR-20a-5p, and hsa-miR-24a-3p. There were also differentially expressed miRNAs in HONE1_C vs. HONE1_M, such as hsa-miR-21-5p and hsa-let-7i-5p. The differentially expressed miRNA in CNE2_C vs. CNE2_M was hsa-miR-148b-3p. The Gene Ontology analysis showed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were mainly enriched in biological process such as negative and positive regulation of transcription from RNA polymerase II promoter, cellular component such as cytosol and molecular function such as protein binding factor. The KEGG pathway analysis revealed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were enriched in the cancer-related pathways, such as pathways in cancer, MAPK signaling pathway and Wnt signaling pathway. CONCLUSIONS: Twelve miRNAs and 9 genes which contribute to X-ray radiation resistance were identified. Among those with differential expression between the HONE1 and CNE2 cell lines, which played a regulatory role in multiple pathways, were hsa-miR-20a-5p, hsa-let-7a-5p, hsa-let-7f5p, hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-148b-3p, and hsa-miR-200c-3p. The corresponding genes were MAPK1, SOS1, TGFBR1, TGFBR2, TP53, CASP3, CCNE2, PTEN, and CDK2.

Antagonism of the transient receptor potential melastatin­2 channel leads to targeted antitumor effects in primary human malignant melanoma cells.

Melanoma continues to be the most aggressive and devastating form of skin cancer for which the development of novel therapies is required. The present study aimed to determine the effects of antagonism of the transient receptor potential melastatin­2 (TRPM2) ion channel in primary human malignant melanoma cells. TRPM2 antagonism via use of the antifungal agent, clotrimazole, led to decreases in cell proliferation, as well as dose­dependent increases in cell death in all melanoma cell lines investigated. The targeting of TRPM2 channels was verified using TRPM2 knockdown, where treatment with TRPM2 small­interfering RNA led to similar levels of cell death in all melanoma cell lines when compared with clotrimazole treatment. Minimal effects on proliferation and cell death were observed following antagonism or knockdown of TRPM2 in non­cancerous human keratinocytes. Moreover, characteristics of TRPM2 were explored in these melanoma cells and the results demonstrated that TRPM2, localized to the plasma membrane as a non­specific ion channel in non­cancerous cells, displayed a nuclear localization in all human melanoma cell lines analyzed. Additional characterization of these melanoma cell lines confirmed that each expressed one or more established multidrug resistance genes. Results of the present study therefore indicated that antagonism of the TRPM2 channel led to antitumor effects in human melanoma cells, including those that are potentially unresponsive to current treatments due to the expression of drug resistance genes. The unique cellular localization of TRPM2 and the specificity of the antitumor effects elicited by TRPM2 antagonism suggested that TRPM2 possesses a unique role in melanoma cells. Collectively, the targeting of TRPM2 represents a potentially novel, efficacious and readily accessible treatment option for patients with melanoma.