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PDF file - 515KB, Body weight change of the mice model. The body weight was recorded before treatment for the baseline, and throughout the period of experiment. The plots represent the curve of (A) the dose-dependent BGT226 study, and (B) the comparison study of individual PI3K and mTOR inhibitors.

PDF file - 757KB, Effect of the cell growth and the intracellular signals by BGT226. (A) Growth inhibitory effect of BGT226, rapamycin and LY294002. FaDu and OECM1 cells were treated with each compound in the identical concentration-dependent manner. The result was plotted (X axis, logarithm scale of concentration; y axis, ratio). (B) Effect of BGT226 and the other PI3K/mTOR targeting agents in OECM1. In the left panel, cells were treated with BGT226 120 nM or LY294002 2μM in a time-dependent manner. In the right panel, cells were treated with various PI3K/mTOR targeting agents for 30 min before whole cell lysate collection (G, BGT226 120 nM; K, BKM120 2μM; Rad, RAD001, 0.5 or 2 nM; L, LY294002 2μM; Rapa, rapamycin 100 or 200 nM; C, control).

PDF file - 468KB, (A) The inhibitory effect of BGT226 in cetuximab-resistant Hep3B cell line. Growth inhibition assay was used in this study. After subculture, cells were treated with cetuximab or BGT226 at least three generation time. The results of cetuximab were represented as curve plot in the left panel, and the IC50 with BGT226 were shown in the right table. (B) Basal expression of PTEN in all tested cell lines. All cell lysate was extracted at the time in steady growth. (C) Combination treatment of BGT226 with radiation in FaDu cells. Clonogenic assay was applied for the cell survival study, with 400 cells implanted into each of 6-well plate. After attachment, BGT226 was administrated 2 h prior to the radiation, and then replaced with drug-free medium after 24 h. The result was then assessed after incubation for 10 days.

PDF file - 1659KB, Apoptosis and autophagy-associated reaction by BGT226. (A) Apoptotic effect of BGT226. HSC3 cells incubated with either BGT226 or cisplatin for 24 h were analyzed for apoptosis-associated protein expression. Arrows indicated the cleavage-form of protein. (B) Effect of AVOs formation in FaDu cells treated with BGT226. Cells were stained with acridine orange. The extent of staining intensity in tested cells was analyzed and plotted by flow cytometry. The left rows represented cells incubated in DMSO or 3MA. The right row showed cells that were treated with BGT226 120 nM with or without the co-treatment of 3MA. The percentage of detected cells in each quadrant is marked on the panel. (C) Evaluation of cathepsin B/D/L (Cat B/D/L) activity in FaDu cells treated with BGT226. The activity of each factor was assessed by its individual activity kit (BioVisiion, CA). For the preparation, we followed manifacturer's protocol after BGT226 treatment of the cells. The samples were then transferred to 96-well plate, with the result read using fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. The data was then compared to the control, with the ratio ploted and shown in the bar chart.

Lessons Learned SCB01A is a novel microtubule inhibitor with vascular disrupting activity. This first-in-human study demonstrated SCB01A safety, pharmacokinetics, and preliminary antitumor activity. SCB01A is safe and well tolerated in patients with advanced solid malignancies with manageable neurotoxicity. Background SCB01A, a novel microtubule inhibitor, has vascular disrupting activity. Methods In this phase I dose-escalation and extension study, patients with advanced solid tumors were administered intravenous SCB01A infusions for 3 hours once every 21 days. Rapid titration and a 3 + 3 design escalated the dose from 2 mg/m2 to the maximum tolerated dose (MTD) based on dose-limiting toxicity (DLT). SCB01A-induced cellular neurotoxicity was evaluated in dorsal root ganglion cells. The primary endpoint was MTD. Safety, pharmacokinetics (PK), and tumor response were secondary endpoints. Results Treatment-related adverse events included anemia, nausea, vomiting, fatigue, fever, and peripheral sensorimotor neuropathy. DLTs included grade 4 elevated creatine phosphokinase (CPK) in the 4 mg/m2 cohort; grade 3 gastric hemorrhage in the 6.5 mg/m2 cohort; grade 2 thromboembolic event in the 24 mg/m2 cohort; and grade 3 peripheral sensorimotor neuropathy, grade 3 elevated aspartate aminotransferase, and grade 3 hypertension in the 32 mg/m2 cohort. The MTD was 24 mg/m2, and average half-life was ~2.5 hours. The area under the curve-dose response relationship was linear. Nineteen subjects were stable after two cycles. The longest treatment lasted 24 cycles. SCB01A-induced neurotoxicity was reversible in vitro. Conclusion The MTD of SCB01A was 24 mg/m2 every 21 days; it is safe and tolerable in patients with solid tumors.

Background Glioblastoma is associated with poor prognosis and high mortality. Although the use of first-line temozolomide can reduce tumor growth, therapy-induced stress drives stem cells out of quiescence, leading to chemoresistance and glioblastoma recurrence. The specificity protein 1 (Sp1) transcription factor is known to protect glioblastoma cells against temozolomide; however, how tumor cells hijack this factor to gain resistance to therapy is not known. Methods Sp1 acetylation in temozolomide-resistant cells and stemlike tumorspheres was analyzed by immunoprecipitation and immunoblotting experiments. Effects of the histone deacetylase (HDAC)/Sp1 axis on malignant growth were examined using cell proliferation–related assays and in vivo experiments. Furthermore, integrative analysis of gene expression with chromatin immunoprecipitation sequencing and the recurrent glioblastoma omics data were also used to further determine the target genes of the HDAC/Sp1 axis. Results We identified Sp1 as a novel substrate of HDAC6, and observed that the HDAC1/2/6/Sp1 pathway promotes self-renewal of malignancy by upregulating B cell-specific Mo-MLV integration site 1 (BMI1) and human telomerase reverse transcriptase (hTERT), as well as by regulating G2/M progression and DNA repair via alteration of the transcription of various genes. Importantly, HDAC1/2/6/Sp1 activation is associated with poor clinical outcome in both glioblastoma and low-grade gliomas. However, treatment with azaindolyl sulfonamide, a potent HDAC6 inhibitor with partial efficacy against HDAC1/2, induced G2/M arrest and senescence in both temozolomide-resistant cells and stemlike tumorspheres. Conclusion Our study uncovers a previously unknown regulatory mechanism in which the HDAC6/Sp1 axis induces cell division and maintains the stem cell population to fuel tumor growth and therapeutic resistance.

Glioblastoma (GBM), a WHO Grade IV glioma, is the most common and aggressive primary brain tumor. Despite the standard of care consisting of surgery removal following radio- and chemo- therapy, the prognosis of GBM patients remain dismal. The interplay between GBM cells and its microenvironment contributes to maintaining the cancer stemness, developing resistance, and forming immunosuppression. GBM-associated macrophages (GAMs) are recruited to the GBM microenvironment by cytokines/chemokines. Conversely, GAMs may promote GBM progression. Therefore, we hypothesized that GBM cells modulated immune microenvironment via macrophage activation. We integrated the bulk RNA sequencing (seq.) of the paired primary-recurrent GBM specimens, single-cell RNA seq., and in vitro validation to investigate the hypothesis. The bulk RNA seq. of the paired primary-recurrent GBM specimens indicated that recurrent GBM enhanced the neuroinflammation pathway. Among the components, we found that CXCL12, also known as stromal cell-derived factor 1 was accumulated during GBM progression and up-regulated in temozolomide (TMZ)-resistant GBM cells. On the other hand, the newly CXCL12 receptor CXCR7 may act as a scavenger for CXCL12 during developing TMZ resistance in GBM. The bulk RNA sequencing seq. from primary-recurrent GBM specimens showed the decrement of CXCR7. Single-cell RNA seq. of GBM patients’ specimens indicated CXCR7 dominantly expressed in GBM cells other than normal neural and immune cells. Developing TMZ resistance in GBM cell lines down-regulated CXCR7 expression. Furthermore, silencing CXCR7 attenuated TMZ cytotoxicity while combining CXCR7 agonists (Plerixafor, VUF11207, and TC14012) and TMZ enhanced TMZ cytotoxicity. Otherwise, the GBM-associated CXCL12 activated M0 macrophages into GAMs by facilitating macrophage proliferation and inducing pro-tumor factors, including interleukine (IL)-1β and its receptors, IL-6, MMP9, and immune checkpoint PD-L1. Mechanistically, GBM-associated CXCL12 upregulated PD-L1 in GAMs via NF-κB. Accordingly, by multimodal analysis, CXCL12 was identified with the additional role in regulating resistance in GBM cells and GBM microenvironment. Also, re-activating CXCR7 may overcome the resistance to TMZ and immune checkpoint blockade therapy. Combination of CXCR7 activation with current therapy and immunotherapy may be an effective strategy for improving the prognosis of primary GBM patients. Citation Format: Chan-Chuan Liu, Chia-Hung Chien, Wen-Bin Yang, Jian-Ying Chuang, Kwang-Yu Chang. Multimodal analysis of glioblastoma identifies the additional function of CXCL12 for modulating GBM resistance and immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5867.

Cathepsin S (CTSS) is a lysosomal enzyme that belongs to cysteine proteases. It is associated with progression of different types of cancer, mediating the antigen presentation, extracellular matrix degradation, angiogenesis, and metastasis. In recent studies, CTSS was suggested as a therapeutic target for cancer treatment. We found that, with a CTSS inhibitor RJW-58 treatment over tumor-bearing mouse, the serum showed an enhanced anti-tumor cytokine releasing of interleukin 7 (IL-7). In the mRNA array results of 40 pair head and neck patients’ sample, we found the negative correlation between CTSS and IL-7 comparing to the normal head and neck tissue. Moreover, we also found that the level of CD8 T cell infiltration is negatively correlated with the expression of CTSS in 20 head and neck cancer patients’ slides. In cell model, targeting CTSS by siRNA or RJW-58 promoted IL-7 secretion in different head and neck cancer cell lines. We demonstrated that cathepsin S decreased the secretion level of IL-7 through downregulating IL-7 receptor, a receptor that mediating IL-7 secretion, protein expression level resulting in activating lysosomal degradation pathway. Promoting cell proliferation of activated CD8 T cells was proved when CD8 T cells were treated with the condition medium from CTSS-knockdown head and neck cancer cells. In conclusion, this is the first study demonstrated that CTSS acted as a negative regulator in mediating IL-7 resulting in inducing an immune cell-suppressed microenvironment in head and neck cancer cells. (This work is supported by the NSTC grand number: MOST 110-2314-B-400-035) Citation Format: Yung-Chieh Chang, Shan-Hung Chen, Kwang-Yu Chang, Jang-Yang Chang. Cathepsin S mediates interleukin 7 to sculpture the tumor immunity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5966.

BackgroundThe mechanism by which glioblastoma evades temozolomide (TMZ)-induced cytotoxicity is largely unknown. We hypothesized that mitochondria plays a role in this process.MethodsRNA transcriptomes were obtained from tumor samples and online databases. Expression of different proteins was manipulated using RNA interference or gene amplification. Autophagic activity and mitochondrial metabolism was assessed in vitro using the respective cellular and molecular assays. In vivo analysis were also carried out in this study.ResultsHigh SH3GLB1 gene expression was found to be associated with higher disease grading and worse survival profiles. Single-cell transcriptome analysis of clinical samples suggested that SH3GLB1 and the altered gene levels of oxidative phosphorylation (OXPHOS) were related to subsets expressing a tumor-initiating cell signature. The SH3GLB1 protein was regulated by promoter binding with Sp1, a factor associated with TMZ resistance. Downregulation of SH3GLB1 resulted in retention of TMZ susceptibility, upregulated p62, and reduced LC3B-II. Autophagy inhibition by SH3GLB1 deficiency and chloroquine resulted in attenuated OXPHOS expression. Inhibition of SH3GLB1 in resistant cells resulted in alleviation of TMZ-enhanced mitochondrial metabolic function, such as mitochondrial membrane potential, mitochondrial respiration, and ATP production. SH3GLB1 modulation could determine tumor susceptibility to TMZ. Finally, in animal models, resistant tumor cells with SH3GLB1 knockdown became resensitized to the anti-tumor effect of TMZ, including the suppression of TMZ-induced autophagy and OXPHOS.ConclusionsSH3GLB1 promotes TMZ resistance via autophagy to alter mitochondrial function. Characterizing SH3GLB1 in glioblastoma may help develop new therapeutic strategies against this disease in the future.

Background Sp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown. Methods RNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo. Results Among the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid β-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ. Conclusion Sp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

Background Sp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown. Methods RNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo. Results Among the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid β-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ. Conclusion Sp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

Background: Colorectal cancer (CRC) progression and related mortality are highly associated with metabolic disorders. However, the molecular mechanism involved in the regulation of hyperlipidemia-associated CRC metastasis remains unclear. This study aimed to investigate the effects of angiopoietin-like 4 (ANGPTL4) on NADPH oxidase 4 (NOX4) expression and reactive oxygen species (ROS) production, which might provide new targets for improving outcomes in patients with hyperlipidemia-associated CRC metastasis. Methods: The clinical relevance of relationship between NOX4 expression and ANGPTL4 was examined in CRC patients by the Oncomine and TCGA data set. Expressions of NOX4, epithelial-mesenchymal transition (EMT) markers, and gene regulation of NOX4 in free fatty acids (FFAs)-treated CRC cells were determined. The FFAs-triggered metastatic ability of CRC cells under treatments of antioxidants or knockdown of NOX4, ANGPTL4, and MMPs was evaluated in vitro and in vivo. In addition, effects of antioxidants and depletion of metastasis-associated molecules on the correlation between ROS production and FFAs-promoted CRC metastasis were also clarified. Results: In this study, we found that the induction of NOX4, followed by the increased ROS was essential for oleic acid (OA)-promoted CRC cell metastasis. The depletion of ANGPTL4 significantly inhibited c-Jun-mediated transactivation of NOX4 expression, accompanied with reduced levels of ROS, MMP-1, and MMP-9, resulting in the disruption of OA-promoted CRC cell metastasis. Moreover, knockdown of ANGPTL4, NOX4, MMP-1, and MMP-9 or the treatment of antioxidants dramatically inhibited circulating OA-enhanced tumor cell extravasation and metastatic seeding of tumor cells in lungs, indicating that the ANGPTL4/NOX4 axis was critical for dyslipidemia-associated tumor metastasis. Conclusion: The coincident expression of NOX4 and ANGPTL4 in CRC tumor specimens provides the insight into the potential therapeutic targets for the treatment of dyslipidemia-associated CRC metastasis.

DNA repair promotes the progression and recurrence of glioblastoma (GBM). However, there remain no effective therapies for targeting the DNA damage response and repair (DDR) pathway in the clinical setting. Thus, we aimed to conduct a comprehensive analysis of DDR genes in GBM specimens to understand the molecular mechanisms underlying treatment resistance. Herein, transcriptomic analysis of 177 well-defined DDR genes was performed with normal and GBM specimens (n = 137) from The Cancer Genome Atlas and further integrated with the expression profiling of histone deacetylase 6 (HDAC6) inhibition in temozolomide (TMZ)-resistant GBM cells and patient-derived tumor cells. The effects of HDAC6 inhibition on DDR signaling were examined both in vitro and intracranial mouse models. We found that the expression of DDR genes, involved in repair pathways for DNA double-strand breaks, was upregulated in highly malignant primary and recurrent brain tumors, and their expression was related to abnormal clinical features. However, a potent HDAC6 inhibitor, MPT0B291, attenuated the expression of these genes, including RAD51 and CHEK1, and was more effective in blocking homologous recombination repair in GBM cells. Interestingly, it resulted in lower cytotoxicity in primary glial cells than other HDAC6 inhibitors. MPT0B291 reduced the growth of both TMZ-sensitive and TMZ-resistant tumor cells and prolonged survival in mouse models of GBM. We verified that HDAC6 regulated DDR genes by affecting Sp1 expression, which abolished MPT0B291-induced DNA damage. Our findings uncover a regulatory network among HDAC6, Sp1, and DDR genes for drug resistance and survival of GBM cells. Furthermore, MPT0B291 may serve as a potential lead compound for GBM therapy.

Background Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1–3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.

Gastric cancer (GC) is the third leading cause of cancer mortality globally and a molecularly heterogeneous disease. Identifying the driver pathways in GC progression is crucial to improving the clinical outcome. Recent studies identified ASPM (abnormal spindle-like microcephaly-associated) and FOXM1 (Forkhead box protein M1) as novel Wnt and cancer stem cell (CSC) regulators; their pathogenetic roles and potential crosstalks in GC remain unclarified. The expression patterns of ASPM isoforms and FOXM1 were profiled in normal gastric epithelial and GC tissues. The functional roles of ASPM and FOXM1 in Wnt activity, cancer stemness and GC progression, and the underlying signaling processes were investigated. Approximately one third of GC cells upregulate the expression of ASPM isoform I (ASPMiI) in their cytoplasm; the tumors with a high ASPMiI positive score (≥ 10%) are associated with a poor prognosis of the patients. Mechanistically, the molecular interplay among FOXM1, ASPMiI and DVL3 was found to converge on β-catenin to control the Wnt activity and the stemness property of GC cells. This multi-mode Wnt-regulatory module serves to reinforce Wnt signals in CSCs by transcriptional regulation (FOXM1–ASPM), protein–protein interactions (ASPMiI–DVL3–β-catenin), and nuclear translocation (FOXM1–β-catenin). This study illuminates a novel Wnt- and stemness-regulatory mechanism in GC cells and identifies a novel subset of FOXM1highASPMiIhigh GC with potential to guide Wnt- and stemness-related diagnostics and therapies.

Background The prognosis of advanced oral squamous cell carcinoma is poor. We investigated the effect of cetuximab-based sequential therapy as a primary treatment. Methods Forty-seven treatment-naive patients with advanced tumors originating from the oral cavity or oropharynx were enrolled. Neoadjuvant cetuximab, paclitaxel, and cisplatin were administered, followed by cetuximab-based radiotherapy. Immunohistochemical staining was applied to study the tissues. Results The best overall response rate was 70.2%, including 4 patients with a complete response and 29 with a partial response. The median progression-free and overall survival rates were 10.3 and 15.2 months, respectively. Patients with more than 50% tumor reduction with neoadjuvant therapy had better survival outcomes. Twenty-two patients had severe adverse events with mostly dermatological complications. Of the 16 patients who received operations, 9 had increased PD-L1 staining compared to pretreatment biopsy in the post hoc study. Conclusion The regimen was effective in selected patients. Increased PD-L1 suggested altered tumor features.

T cell activation and tolerance are tightly regulated to ensure effective elimination of foreign antigen while maintaining immune tolerance to self-antigens. Development of T cell anergy and regulatory T cell (Treg) mediated suppression both contribute to the establishment of immune tolerance. Here, we show that neuritin (Nrn1), a conserved GPI-anchored surface molecule important for the development, survival and function of neurons, is highly expressed in anergic and Treg cells. Nrn1 deficient CD4 cells are resistant to Treg cell mediated suppression, display defective anergy induction, and have reduced peripheral Treg generation. Nrn1 deficient Foxp3+ Treg cells exhibit reduced control of inflammatory colitis. Moreover, upon induction of experimental autoimmune encephalomyelitis (EAE), Nrn1 deficient mice develop non-remitting disease and have increased spinal cord inflammatory infiltrates. These in vivo findings underscore the importance of Nrn1 in immune tolerance. Recently, Nrn1 was identified as an accessory component of the ionotropic AMPA receptor (AMPAR) complex in neurons. AMPARs mediate glutamate dependent cation flux and regulate cell membrane potential. Cell membrane potential can impact nutrient uptake, calcium influx, cell size, proliferation and survival. In vitro analysis reveals that Nrn1 deficient Treg cells exhibit reduced proliferation and survival, associated with higher membrane potential, reduced nutrient sensitivity, reduced glycolysis and mTOR activation. AMPAR blockade can correct proliferation defect in Nrn1 deficient Treg cells. These findings reveal Nrn1 as an important regulator of immune tolerance functioning through the modulation of glutamate activated AMPAR.

660 Background: Nal-IRI is irinotecan (as known as CPT-11) encapsulated in a nanoliposome drug delivery system (nanoliposomal irinotecan; nal-IRI). Nal-IRI + 5-FU/LV has been approved in patients with metastatic pancreatic cancer after gemcitabine-based therapy through the NAPOLI-1 study. TAS-102 is a combination drug of trifluridine (FTD), an antineoplastic thymidine analog, and tipiracil hydrochloride (TPI), a thymidine phosphorylase inhibitor. TAS-102 has been approved in metastatic colorectal cancer and gastric or gastroesophageal junction adenocarcinoma. Given both drugs show antitumor activity in various cancers, this study is to identify the MTD (maximum tolerated dose) and recommended phase II dose (RP2D), and to explore the preliminary efficacy of nal-IRI and TAS-102 combination in solid tumors. Methods: This multi-center phase I study consists of a classical 3 + 3 dose finding portion and an expansion portion. Eligible patients have pathologically confirmed solid malignancies with no standard treatment available. Patients will be treated with nal-IRI at dose of 60, 70, 80 mg/m2 over 90 minutes on day 1, and oral TAS-102 25, 30, 35 mg/m2 twice daily on day 1-5, every 14 days. Patients who have homozygous for the UGT1A1*28 allele ( TA7/TA7) or UGT1A1*6 allele ( A/A), or double heterozygous for both UGT1A1*28 allele ( TA6/TA7) and UGT1A1*6 allele ( G/A) will be excluded from the dose finding portion. This trial is registered with ClinicalTrials.gov, NCT 03810742. Results: From February 2019 to May 2021, a total of 43 (male/female: 27/16; median age 56, range 25-69) patients (pts) were treated, including 28 in the dose finding portion in which 3, 3, 3, 3, 7, 3 and 6 pts were at levels 1, 2A, 2B, 3, 4A, 4B and 5, respectively, and 15 pts in the expansion portion. The results here are reported from the dose finding portion. The most common cancer types were cholangiocarcinoma (N = 6), neuroendocrine tumor (N = 3), and ampulla of Vater (N = 3). DLTs were noted at one patient who had G4 sepsis and G3 diarrhea at level 4A (80/30), and two subjects who had G3 diarrhea and G3 dehydration at level 5 (80/35). The dosage of the level 4A is determined to be the MTD and it is also the RP2D for the expansion phase. Most common treatment related toxicities during the DLT observation period (the first 2 cycles) are neutropenia, thrombocytopenia, diarrhea, fatigue, and vomiting. In this dose finding portion, 4 pts had partial response and 17 pts had stable disease as the best response. Conclusions: The MTD of Nal-IRI in combination with TAS-102 every 2 weeks is 80/30 mg/m2, which is also the RP2D for expansion study. The clinical benefit was observed in pts at different dose levels. The expansion portion of the study is still ongoing and pts will be followed until disease progression or consent withdrawal. Clinical trial information: NCT03810742.

The phosphoinositide-3-kinase (PI3K) pathway has widely been considered as a potential therapeutic target for head and neck cancer (HNC), however, the application of PI3K inhibitors is often overshadowed by the induction of drug resistance with unknown mechanisms. In this study, PII3K inhibitor resistant cancer cells were developed by prolonged culturing of cell lines with BEZ235, a dual PI3K and mammalian target of rapamycin (mTOR) inhibitor. The drug resistant HNC cells showed higher IC50 of the proliferation to inhibitors specifically targeting PI3K and/or mTOR, as compared to their parental cells. These cells also showed profound resistance to drugs of other classes. Molecular analysis revealed persistent activation of phosphorylated AKT at threonine 308 in the drug resistant cells and increased expression of markers for tumor-initiating cells. Interestingly, increased intra-cellular ROS levels were observed in the drug resistant cells. Among anti-oxidant molecules, the expression of SOD2 was increased and was associated with the ALDH-positive tumor-initiating cell features. Co-incubation of SOD inhibitors and BEZ235 decreased the stemness feature of the cells in vitro, as shown by results of the spheroid formation assay. In conclusion, dysregulation of SOD2 might contribute to the profound resistance to PI3K inhibitors and the other drugs in HNC cells.

It has been suggested that stress stimuli from the microenvironment maintain a subset of tumor cells with stem-like properties, including drug resistance. Here, we investigate whether Sp1, a stress-responsive factor, regulates stemness gene expression and if its inhibition sensitizes cancer cells to chemotherapy. Hydrogen peroxide- and serum deprivation-induced stresses were performed in glioblastoma (GBM) cells and patient-derived cells, and the effect of the Sp1 inhibitor mithramycin A (MA) on these stress-induced stem cells and temozolomide (TMZ)-resistant cells was evaluated. Sp1 and stemness genes were not commonly overexpressed in clinical GBM samples. However, their expression was highly induced by stress stimuli. Using MA, we demonstrated Sp1 as a critical stemness-related transcriptional factor protecting GBM cells against stress- and TMZ-induced death. Thus, Sp1 inhibition may prevent recurrence of malignant cells persisting after primary therapy.

Background Intratumor subsets with tumor-initiating features in glioblastoma are likely to survive treatment. Our goal is to identify the key factor in the process by which cells develop temozolomide (TMZ) resistance. Methods Resistant cell lines derived from U87MG and A172 were established through long-term co-incubation of TMZ. Primary tumors obtained from patients were maintained as patient-derived xenograft for studies of tumor-initating cell (TIC) features. The cell manifestations were assessed in the gene modulated cells for relevance to drug resistance. Results Among the mitochondria-related genes in the gene expression databases, superoxide dismutase 2 (SOD2) was a significant factor in resistance and patient survival. SOD2 in the resistant cells functionally determined the cell fate by limiting TMZ-stimulated superoxide reaction and cleavage of caspase-3. Genetic inhibition of the protein led to retrieval of drug effect in mouse study. SOD2 was also associated with the TIC features, which enriched in the resistant cells. The CD133+ specific subsets in the resistant cells exhibited superior superoxide regulation and the SOD2-related caspase-3 reaction. Experiments applying SOD2 modulation showed a positive correlation between the TIC features and the protein expression. Finally, co-treatment with TMZ and the SOD inhibitor sodium diethyldithiocarbamate trihydrate in xenograft mouse models with the TMZ-resistant primary tumor resulted in lower tumor proliferation, longer survival, and less CD133, Bmi-1, and SOD2 expression. Conclusion SOD2 plays crucial roles in the tumor-initiating features that are related to TMZ resistance. Inhibition of the protein is a potential therapeutic strategy that can be used to enhance the effects of chemotherapy. Graphical abstract

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Oral squamous cell carcinoma (SCC) is a prevalent malignant disease worldwide, especially so in Taiwan. Early- or even preclinical-stage detection is critical for reducing morbidity and mortality from oral SCC. Epidemiological and genome association studies are useful for identifying clinicopathological risk factors for preventive, diagnostic, and therapeutic approaches of oral SCC. For advanced oral SCC, effective treatments are critical to prolonging survival and enhancing quality of life. As oral SCC is characteristic of regional invasion with lymph node metastases, understanding the aggressive features of oral SCC, particularly in lymphangiogenesis, is essential for determining effective treatments. Emerging evidence has demonstrated that the tumor microenvironment (TME) plays a pivotal role in tumor growth, invasion, and metastases. Recent clinical successes in immune checkpoint inhibitors either alone or combined with chemotherapy have also supported the therapeutic value of immunotherapy in oral SCC. This review summarizes critical advances in basic knowledge of oral SCC from the perspective of clinicopathological risk factors, molecular tumorigenesis, and the TME. We also highlight our recent investigations on the microbiome, genome association studies, lymphangiogenesis, and immunomodulation in oral SCC. This review may provide new insights for oral SCC treatment by systematically interpreting emerging evidence from various preclinical and clinical studies.

Additional file 5: Figure S5. The assays of tumor-initiating cell (TIC) properties. (A) Tumor formation analysis with limiting dilution (50~1000 cells per injection) of U87-parental and TMZ-resistant (r#10) cells in subcutaneous flank area of NOD-SCID mice was performed and recorded. Tumor formation was defined as the measurement to reach 0.1â cm3 or larger. The number of tumor formation in total implanted mice was shown in the brackets. (B) The frequency of the TIC-featured population of GBM#2 (derived from another recurrent GBM patient) with or without TMZ treatment was estimated using the in vitro extreme limiting dilution assay.

Additional file 3: Figure S3. The mitochondrial ROS were detected with MitoSox. (A) The TMZ-induced mitochondrial ROS were in parental and resistant U87MG cells. (B) The MitoSox results of CD133+ cells from U87MG parental and resistant cells were presented. The black curves represented the unstained control, the green curves represented the untreated group, and the pink curves represented the treated group. The brackets were the mean fluorescent intensity. These plots were representative ones of the triplicate experiments.

Additional file 2: Figure S2. Kaplan-Meier curves of an array database from SurvExpress ( http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp ) [18]. The original data included samples of (A) GBM from Joo KM, et al., 2013 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42669 ), (B) low-grade glioma and GBM from Phillips HS, et al., 2006 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE4271 ), and (C) low-grade glioma and GBM from TCGA. Each line refers to cases in which SOD2 gene expression was higher or lower than the median.

Additional file 6: Figure S6. DETC inhibitor used in vitro and in vivo. (A) (Left) U87MG spheres were treated with different doses of DETC as indicated. The cell lysates were analyzed via western blot. The statistic results (Right) were shown. (B) Mice that received U87MG-r#10 cells were randomly treated with TMZ or TMZ/DETC for 5 consecutive days. Representative images of IHC staining in which the specific protein levels of the resistant xenografts were analyzed. *Pâ

Additional file 1: Figure S1. The comparison between the parental and resistant cells. (A) The western blotting results of the parental cells and single clones of the resistant cells of U87MG (left) and A172 (right) were shown. (B) SOD2 levels were detected by Western Blot assay in the parental and resistant cells of U87MG (left) and A172 (right) with or without infection of SOD2 shRNA. The long- and the short-exposure of SOD2 plots were shown. (C) Levels of ROS scavengers in U87MG (left) or A172 (right) were compared between the parental and their resistant cells (r#10 or r#6, respectively). The detection was through qPCR for triplicated experiment with standard error shown in the bar graph.

Additional file 4: Figure S4. The resistant primary cells (GBM#1) were pretreated with siRNA for SOD2 knockdown. The cells were then incubated in low serum (2%) cultures medium with or without TMZ. The western blotting result of cleaved caspase 3 after TMZ treatment was shown. (n = 3 for each group, Data are presented as mean ± standard error, *P

Additional file 7: Figure S7. The analysis from clinical samples. (A) The IHC image represented a GBM tissue section which was stained with anti-rabbit IgG as a negative control. (B) The IHC staining in the left was a representative result of SOD2 and Bmi1 from a same patient. The study was done in total of the clinical specimens from 10 patients with recurrent GBM. The area of the staining was assessed and recorded in the dotted graph at right, showing statistical correlation of SOD2 and Bmi1.

Cisplatin (CDDP) is an important anti-cancer drug commonly used in various human cancers, including nasopharyngeal carcinoma (NPC). How to overcome the drug resistance of CDDP provides opportunities to improve clinical outcomes of NPC. O(6) -methylguanine-DNA methyltransferase (MGMT) has been well-characterized to be a therapeutic determinant of O(6) -alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we showed that MGMT-proficient cells were highly resistant to the cytotoxic effects of CDDP as compared to MGMT-deficient cells. Further studies showed that the platinum level of DNA after CDDP exposure was significantly lower in MGMT-proficient cells than in MGMT-deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP-induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP-induced DNA damages. Subsequently, CDDP-bound MGMT protein became ubiquitinated and was degraded through ubiquitin-mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP-based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression-free survival (PFS; p = 0.022) and overall survival (OS; p = 0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (p = 0.01, hazard ratio 2.23) and OS (p = 0.018, hazard ratio 2.14). Our findings suggest that MGMT protein is important to determine the efficacy of CDDP in NPC.

Acquisition of temozolomide (TMZ) resistance is a major factor leading to the failure of glioblastoma (GBM) treatment. The exact mechanism by which GBM evades TMZ toxicity is not always related to the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), and so remains unclear. In this study, TMZ-resistant variants derived from MGMT-negative GBM clinical samples and cell lines were studied, revealing there to be increased specificity protein 1 (Sp1) expression associated with reduced reactive oxygen species (ROS) accumulation following TMZ treatment. Analysis of gene expression databases along with cell studies identified the ROS scavenger superoxide dismutase 2 (SOD2) as being disease-related. SOD2 expression was also increased, and it was found to be co-expressed with Sp1 in TMZ-resistant cells. Investigation of the SOD2 promoter revealed Sp1 as a critical transcriptional activator that enhances SOD2 gene expression. Co-treatment with an Sp1 inhibitor restored the inhibitory effects of TMZ, and decreased SOD2 levels in TMZ-resistant cells. This treatment strategy restored susceptibility to TMZ in xenograft animals, leading to prolonged survival in an orthotopic model. Thus, our results suggest that Sp1 modulates ROS scavengers as a novel mechanism to increase cancer malignancy and resistance to chemotherapy. Inhibition of this pathway may represent a potential therapeutic target for restoring treatment susceptibility in GBM., Graphical abstract fx1, Highlights • Sp1 levels increase in TMZ resistance of MGMT-deficient cells. • Sp1 upregulation enables GBM cells to escape TMZ-mediated toxicity. • Sp1 is a critical transcriptional activator to enhance SOD2 gene expression. • Sp1-SOD2 pathway raises GBM malignancy and chemotherapy resistance. • Inhibition of Sp1-SOD2 pathway restores treatment susceptibility in GBM.

Objective Previous studies examining the association between genetic variations in prostaglandin pathway and risk of head and neck cancer (HNC) have only included polymorphisms in the PTGS2 (COX2) gene. This study investigated the association between genetic polymorphisms of six prostaglandin pathway genes (PGDS, PTGDS, PTGES, PTGIS, PTGS1 and PTGS2), and risk of HNC. Methods Interviews regarding the consumption of alcohol, betel quid, and cigarette were conducted with 222 HNC cases and 214 controls. Genotyping was performed for 48 tag and functional single-nucleotide polymorphisms (SNPs). Results Two tag SNPs of PTGIS showed a significant association with HNC risk [rs522962: log-additive odds ratio (OR) = 1.42, 95% confidence interval (CI): 1.01–1.99 and dominant OR = 1.58, 95% CI: 1.02–2.47; rs6125671: log-additive OR = 1.49, 95% CI: 1.08–2.05 and dominant OR = 1.96, 95% CI: 1.16–3.32]. In addition, a region in PTGIS tagged by rs927068 and rs6019902 was significantly associated with risk of HNC (global P = 0.007). Finally, several SNPs interacted with betel quid and cigarette to influence the risk of HNC. Conclusions Genetic variations in prostaglandin pathway genes are associated with risk of HNC and may modify the relationship between use of betel quid or cigarette and development of HNC.

Data mining in the public domain demonstrates that cyclin-dependent kinase 4 (CDK4) is highly expressed in nasopharyngeal carcinomas (NPC). Associated with cyclin-D, CDK4 phosphorylates and inactivates retinoblastoma (Rb) protein family members and mediates progression through the G1- to the S-phase of the cell cycle. Amplification and overexpression of CDK4 has been identified in various human malignancies. However, its expression and amplification has never been systemically evaluated in NPC. This study aimed to evaluate the amplification and expression status, correlation with clinicopathological features, and prognostic implications of CDK4 based on public domain dataset and in our well-defined cohort of NPC patients. The association between CDK4 transcript level and gene dosage was explored by analysis of an independent public domain dataset. We retrospectively assessed CDK4 immunoexpression in biopsies of 124 consecutive NPC patients devoid of initial distant metastasis and treated according to consistent guidelines. The results were correlated with clinicopathological features, local recurrence-free survival (LRFS), distant metastasis-free survival (DMeFS), and disease-specific survival (DSS). High levels of CDK4 protein were positively correlated with the T 3, 4 status (p = 0.024); N 2, 3 status (p < 0.001); and the American Joint Committee on Cancer stage 3, 4 (p < 0.001). Multivariate analysis suggested high CDK4 expression was an independent prognostic indicator of worse DMeFS (p = 0.001, hazard ratio (HR) = 3.226) and DSS (p = 0.037, HR = 1.838). Although CDK4 is frequently upregulated, its gene locus is very uncommonly amplified in NPC. CDK4 overexpression is mostly independent with gene amplification and represents a potential prognostic biomarker in NPC and may indicate tumor aggressiveness through cell cycle dysregulation.

Alcohol consumption is an established risk factor for head and neck cancer (HNC). The major carcinogen from alcohol is acetaldehyde, which may be produced by humans or by oral microorganisms through the metabolism of ethanol. To account for the different sources of acetaldehyde production, the current study examined the interplay between alcohol consumption, oral hygiene (as a proxy measure for the growth of oral microorganisms), and alcohol-metabolizing genes (ADH1B and ALDH2) in the risk of HNC. We found that both the fast (*2/*2) and the slow (*1/*1 + *1/*2) ADH1B genotypes increased the risk of HNC due to alcohol consumption, and this association differed according to the slow/non-functional ALDH2 genotypes (*1/*2 + *2/*2) or poor oral hygiene. In persons with the fast ADH1B genotype, the HNC risk associated with alcohol drinking was increased for those with the slow/non-functional ALDH2 genotypes. For those with the slow ADH1B genotypes, oral hygiene appeared to play an important role; the highest magnitude of an increased HNC risk in alcohol drinkers occurred among those with the worst oral hygiene. This is the first study to show that the association between alcohol drinking and HNC risk may be modified by the interplay between genetic polymorphisms of ADH1B and ALDH2 and oral hygiene. Although it is important to promote abstinence from or reduction of alcohol drinking to decrease the occurrence of HNC, improving oral hygiene practices may provide additional benefit.

Data mining on public domain identified that stathmin 1 (STMN1) transcript was significantly higher expressed in nasopharyngeal carcinoma (NPC). Also known as the oncoprotein 18, STMN1 performs an important function in regulating rapid microtubule remodeling of the cytoskeleton in response to the cellular conditions. Immunoexpression of STMN1 was retrospectively assessed in biopsies of 124 consecutive NPC patients without initial distant metastasis and treated with consistent guidelines. The outcome was correlated with clinicopathological features and patient survivals. Results indicated that high STMN1 expressions (50 %) were correlated with advanced age (p = 0.027), higher T stage (p = 0.003), and overall clinical stage (p = 0.006) by the 7th American Joint Committee of Cancer Staging. In multivariate analyses, high STMN1 expression emerged as an independent prognosticator for worse disease-specific survival (p = 0.001), distal metastasis-free survival (p = 0.003), and local recurrence-free survival (p = 0.006). Exogenous expression of E2F transcription factor 1 (E2F1) or/and its dimeric partner, transcription factor Dp-1 (TFDP1), notably induced the STMN1 protein level in a NPC-derived cell line, TW01. Accordingly, high STMN1 protein level is commonly associated with adverse prognosticators and confers tumor aggressiveness in patients with NPC, and its upregulation might be attributed to E2F1 and/or TFDP1 transactivation.

This analysis examined the association between oral hygiene and head and neck cancer (HNC) and whether this association differed by the consumption of alcohol, betel quid, or cigarette and by the genetic polymorphisms of inflammation-related genes.Interviews regarding dental care and oral health were conducted with 317 HNC cases and 296 controls. Genotyping was performed for 6 single nucleotide polymorphisms in IL6, IL10 and PTGS2.A positive association was observed between HNC and no regular dental visits (odds ratio (OR)=2.86, 95% confidence interval (CI): 1.47-5.57), brushing teeth2times/day (OR=1.51, 95% CI: 1.02-2.23), frequent gum bleeding (OR=3.15, 95% CI: 1.36-7.28), and loss of20 teeth (OR=2.31, 95% CI: 1.05-5.07). Analysis with dental care score (range: 0-4, 4=worst dental care), which combined regular dental visits, toothbrushing, and use of dental floss and mouthwash, showed a positive trend with HNC risk, particularly among alcohol drinkers and cigarette smokers. Multifactor dimensionality reduction analysis divided the study subjects into high- and low-risk group based on combinations of dental care score and IL6 rs1800796 genotypes. Compared to the low-risk group, the high-risk group had an OR of HNC=2.16 (95% CI: 1.44-3.25).This study observed a positive association between poor oral hygiene and HNC, which appeared to differ by alcohol or cigarette consumption and the genotypes of IL6 rs1800796. Further investigations are needed to determine whether poor oral hygiene is a cause for HNC or a surrogatemarker of an unhealthy lifestyle that increases the risk of HNC.

Despite the advances in diagnostic imaging and treatment modalities, the risk stratification and final outcomes in patients with nasopharyngeal carcinomas (NPC) still remain suboptimal. Through data mining from published transcriptomic database, topoisomerase IIα (TOP2A) was first identified as a differentially upregulated gene in NPC tissues, which implicates cell division via selective cleavage, rearrangement, and re-ligation of DNA strands. Given the roles of TOP2A in prognostication and in the frontline therapeutic regimen of common carcinomas, such as breast cancer, we explored TOP2A immunoexpression status and its associations with clinicopathological variables and survival in a well-defined cohort of NPC patients. TOP2A immunohistochemistry was retrospectively performed and analyzed using H-score method for biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. Those cases with H-score larger than the median value were construed as featuring TOP2A overexpression. The findings were correlated with the clinicopathological variables, disease-specific survival (DSS) and distant metastasis-free survival (DMFS). TOP2A overexpression was significantly associated with American Joint of Cancer Committee (AJCC) stages III-IV (p = 0.019) and univariately predictive of adverse outcomes for DSS (p = 0.0078) and DMFS (p = 0.0003). In the multivariate comparison, TOP2A overexpression remained prognostically independent to portend worse DSS (p = 0.047, hazard ratio = 1.732) and DMFS (p = 0.003, hazard ratio = 2.569), together with advanced AJCC stages III-IV. TOP2A expression is upregulated in a subset of NPCs and its increased immunoexpression significantly correlated with advanced stages and tumor aggressiveness, justifying the potentiality of TOP2A as a prognostic biomarker and a novel therapeutic target of NPC.

article i nfo Complete profiling would substantially facilitate the fundamental understanding of tumor angiogenesis and of possible anti-angiogenesis cancer treatments. We developed an integrated synchrotron-based methodology withexcellent performances: detection of very small vessels by high spatialresolution (~1 μm)and nanoparticle contrast enhancement, in vivo dynamics investigations with high temporal resolution (~1 ms), and three- dimensional quantitative morphology parametrization by computer tracing. The smallest (3-10 μm) microves- sels were found to constitute >80% of the tumor vasculature and exhibit many structural anomalies. Practical applications are presented, including vessel microanalysis in xenografted tumors, monitoring the effects of anti-angiogenetic agents and in vivo detection of tumor vascular rheological properties.

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is deregulated in acute kidney injury (AKI) through an unknown mechanism. In the present study, we used a previously described mouse model of ascending urinary tract infection in which uropathogenic Escherichia coli (UPEC) were transurethrally inoculated to induce kidney infections. Here, we show that urinary MIF was upregulated during AKI while MIF was abundantly expressed in the renal cortical tubules and that UPEC infection caused a decrease in tubular MIF. Infections with UPEC in vitro caused MIF release in a cell type-dependent manner, which was independent of receptor-mediated internalization, signal transduction, and transcription. Indeed, UPEC infection-induced necrotic cell death in vitro and in vivo correlated with extracellular acidification and processed MIF secretion. These data suggest that MIF is released by necrotic renal cortical tubular cells during UPEC infection.

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in such cancers as head and neck squamous cell carcinoma (HNSCC); however, whether the metabolic enzyme, pyruvate dehydrogenase kinase 1 (PDK1), mediates EGF-enhanced HNSCC metastasis remains unclear. Of interest, we found that EGF induced PDK1 expression in HNSCC. Tumor cell transformation induced by EGF was repressed by PDK1 knockdown, and the down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells as well as the metastatic seeding of tumor cells in lungs. PDK1 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Furthermore, PDK1 overexpression induced MMP-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Of interest, depletion of fibronectin inhibited PDK1-enhanced MMP-1-3 and MMP-9 expression as well as Rac1/cdc42 activation and tumor invasion. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis

Aim To better understand the spectrum of primary lymphomas in the upper aerodigestive tract, a common site of extranodal lymphoma. Materials and methods Lymphoma cases diagnosed at an institution in southern Taiwan from 1992 to 2007 were retrospectively studied with pathology and history review, immunohistochemistry, in situ hybridisation for Epstein–Barr virus (EBER-ISH), and statistical analysis. Results 70 patients were identified. The male to female ratio was 2:1, and the median age was 61.5 years (range 8–87); 73% of cases occurred in Waldeyer9s ring or the oral cavity. Phenotypically, there were 45 (64%) B cell and 25 (36%) T cell or extranodal natural killer (NK)/T cell lymphoma (ENKL) including 42 (60%) diffuse large B cell lymphomas (DLBCLs), 22 (31%) ENKLs, three unspecified peripheral T cell lymphomas, two follicular lymphomas and one Burkitt lymphoma. EBER-ISH was positive in three (7%) of 42 DLBCLs and all 22 ENKLs. Most patients received chemotherapy with or without radiotherapy. The 5-year overall survival for all patients was 56.3% with B and T or NK/T cell lymphomas at 66.0% and 40.6%, respectively. Univariate analysis revealed that sinonasal presentation, T or NK/T cell phenotype, raised lactate dehydrogenase (LDH) activity, and Ann Arbor stage III/IV diseases were associated with prognostically significant higher hazard ratio (HR) of lymphoma-related death. However, only raised LDH remained significant on multivariate analysis. For DLBCLs, only raised LDH was prognostically significant on either univariate or multivariate analysis. Conclusions Only a limited number of lymphoma entities occurred primarily in this anatomical region. The 5-year overall survival rate was comparable to other reports, and raised LDH at diagnosis was the only significant prognostic factor identified. A relatively high incidence of EBV positivity was identified in DLBCLs in this anatomical region, and further studies are warranted to elucidate the clinicopathological significance of these tumours.

The prognosis of glioblastoma (GBM) is usually poor even following treatment with the first-line chemotherapeutic agent temozolomide (TMZ). One most known resistant mechanism is the presence of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). However, compared with MGMT-mediated innate TMZ resistance, the development of acquired resistance is considered more complex with multi-factorial involvement such as the presence of cancer stem cells (CSCs). In this study, we treated the MGMT-negative GBM cells with TMZ to investigate the acquired resistance, and found that both histone deacetylases (HDACs) and Sp1 are key factors protecting GBM against TMZ. These results include the following: (1) Stemness markers were highly increased in TMZ-resistant GBMs; (2) The activity of HDACs affected the stem-like characteristics and cell survival of GBM stem cells (GSCs); (3) An HDAC1/2/6-selective inhibitor MPT0B291 increased TMZ-sensitivity and induced senescence in TMZ-resistant cells; (4) MPT0B291 suppressed anti-senescence genes (hTERT and BMI1) expression via inhibition Sp1 transactivation; (5) Both HDACs and Sp1 were elevated and interacted with each other in GSCs and resistant GBM cells; (6) TMZ treatment induced Sp1 deacetylation, but MPT0B291 attenuated that. In summary, we verified that HDACs increases Sp1 activation via protein deacetylation and causes Sp1-downstream target upregulation, which may enrich stemness properties and protect GBM against chemotherapeutic drugs. Citation Format: Jian-Ying Chuang, Che-Chia Hsu, Kwang-Yu Chang, Wen-Chang Chang. Sp1 acetylation associates with stemness characteristics in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5905.

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin not only inhibited the expression of MMP-9 but also repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes. The inhibition of ANGPTL4 could be a potential strategy for the treatment of FFA-mediated HNSCC metastasis. Citation Format: Chih-Jie Shen, Shih-Hung Chan, Chung-Ta Lee, Wan-Chen Huang, Jhih-Peng Tsai, Kwang-Yu Chang, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid promotes head and neck squamous cell carcinoma anoikis resistance and metastasis via ANGPTL4/fibronectin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 85.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with presence of stemness-featured cells that are prone to survive from cancer treatment. We reported Sp1-induced SOD2 was associated with the process to acquire temozolomide (TMZ) resistance that the significance remained to be elucidated. Resistant models were established through long-term coincubation with TMZ in GBM cell lines, which expressed significantly higher stemness properties in extreme limiting dilution analysis (ELDA) and serial transplantation. These cells also showed increased expression of SOD2. Enrichment of stemness-featured cells by spheroid culturing or by CD133-positive cells selection resulted in increased SOD2, suggesting their associations. The impact of the protein to glioma stem cells (GSCs) was then studied through SOD2 RNA interference (RNAi), displaying downregulated stemness features in the knockdown models. Functional studies of the resistant cells showed superior scavenging ability in TMZ- or H2O2-induced ROS that would be dysfunctioned in pretreatment with SOD2 RNAi. In addition, co-treatment with the reagents over the knockdown cells resulted in enhancement of apoptosis as well as inhibition in tumor spheroid culture and cell clonogenic formation, suggesting re-sensitization to the drug. By establishing in vivo model from patient derived xenograft implantation that was obtained from clinical resistant disease, the mouse receiving combination treatment with TMZ and SOD inhibitor had lower tumor proliferation comparing to those receiving TMZ alone. These data indicate that there was an association between SOD2 and the stemness features, suggesting the protein to play important roles in regulation of GSCs behavior. Down-regulation of SOD2 levels may thus be a potential therapeutic strategy for modulating stemness properties of GSCs, making these cells more susceptive to the chemo-therapeutic agent. Citation Format: Kwang-Yu Chang, Chia-Hung Chien, Jui-Mei Chu, Pei-Hsuan Chung, Ming-Sheng Liu, Jian-Ying Chuang. Mitochondrial SOD2 is the mainstay to protect the stemness-featured glioblastoma cells against drug-induced reactive oxygen stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4887.