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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.

Many studies have reported a positive association between lower socioeconomic status (SES) and higher head and neck cancer (HNC) risk. Fewer studies have examined the impact of SES on the association between alcohol or cigarette use and HNC risk. The current case‐control study (1104 HNC cases and 1363 controls) investigated the influence of education, a SES indicator, on the association between HNC and the use of alcohol, cigarettes, or betel quids in Taiwan, a country with universal health care. Our results showed a larger increase in HNC risk associated with alcohol among those with lower educational level (odds ratio [OR] = 2.07; 95% confidence interval [CI], 1.53‐2.80) than those with higher educational level (OR = 1.38; 95% CI, 1.04‐1.85) (heterogeneity‐P = .03). Educational level had an influence on the association between alcohol use and HNC risk among those with genetic susceptibility (ALDH2‐deficient) to the carcinogenic effect of alcohol. The association between cigarette or betel quid use and HNC risk was similar between the high and low educational groups. National policies and social interventions have led to the decline in the prevalence of cigarette and betel quid users in Taiwan. In contrast, due to the lack of adequate alcohol control policies, alcohol consumption in Taiwan has continued to rise. A higher impact of alcohol on HNC risk among lower SES individuals even with universal health care could be the result of insufficient alcohol control policies in Taiwan.

Simple Summary Head and neck squamous cell carcinoma (HNSCC), which arises from the oral epithelium, is one of the most common cancers worldwide. Despite excellent diagnosis and treatment improvements, the mortality rate associated with HNSCC is still extremely high. Current data suggest that dysregulation of exosomes and metabolic abnormalities are involved in the initiation and progression of HNSCC. Thus, approaches for targeting exosomes in the tumor microenvironment and metabolic reprogramming pathways represent potential therapeutic strategies. Moreover, some miRNAs are thought to have significant functions in regulating the progression of HNSCC. The present article aims to summarize the current knowledge concerning the important miRNAs in both exosomes and cancer metabolism, as well as discuss future perspectives regarding their future diagnostic potential and treatment recommendations. Abstract MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that negatively regulate gene expression by binding to target mRNAs. Deregulated miRNAs can act as either oncogenic miRNAs or tumor suppressor miRNAs in controlling proliferation, differentiation, apoptosis, metastasis, epithelial–mesenchymal transition, and immune responses, which are all involved in the carcinogenesis process of HNSCC. Recent findings have shown that metabolic reprogramming is an important hallmark of cancer, which is necessary for malignant transformation and tumor development. Some reprogrammed metabolisms are believed to be required for HNSCC against an unfavorable tumor microenvironment (TME). The TME is composed of various cell types embedded in the altered extracellular matrix, among which exosomes, secreted by cancer cells, are one of the most important factors. Tumor-derived exosomes reshape the tumor microenvironment and play a crucial role in cell-to-cell communication during HNSCC development. Exosomes encapsulate many biomolecules, including miRNAs, circulate in body fluids, and can transmit intercellular regulatory messages to nearby and distant sites, which indicates that exosomal miRNAs have the potential to become non-invasive biomarkers. This review aims to clarify the functions of diverse miRNAs in HNSCC metabolic reprogramming and tumor-derived exosomes. In addition, it also emphasizes the potential role of miRNA as a biomarker in the diagnosis, prognosis, and treatment of HNSCC cancer.

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Human papillomavirus (HPV) is recognized as a major cause of oropharyngeal cancer (OPC) in Western countries. Less is known regarding its contribution to the OPC occurring in Asia. The current study aimed to investigate the association between antibody responses to HPV16 E7 and head and neck cancer (HNC) risk in a hospital‐based case–control study conducted in Taiwan with 693 HNC cases and 1,035 controls. A positive association was observed between seropositivity to HPV16 E7 and OPC risk, whereas no significant association was found in the non‐OPC cases. The increased OPC risk associated with seropositivity to HPV16 E7 was more significant among nonbetel quid or noncigarette users. Seropositivity to HPV16 E7 showed moderate agreement with P16 expression in OPC. OPC patients that were seropositive to HPV16 E7 or p16 positive were more highly educated and less likely to use alcohol, betel quids, and cigarettes compared to HPV16 E7 seronegative or p16 negative OPC patients. Furthermore, patients with p16 positive OPC were more likely to be women compared to patients with p16 negative OPC, likely owing to the low prevalence of alcohol, betel quid, and cigarette users among women. Overall, this study suggested that similar to Western countries, HPV may also be an important risk factor of OPC in Taiwan. With the declining consumption of betel quids and cigarettes in Taiwan, a higher percentage of OPC cases in Taiwan will be attributed to HPV in the future. Public health measures, including HPV vaccination, need to be implemented to prevent the occurrence of HPV‐positive OPC., The current study found a a positive association between seropositivity to HPV16 E7 and oropharyngeal cancer risk. This study suggested that similar to Western countries, HPV may also be an important risk factor of OPC in Taiwan.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear. Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2. Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.

Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography-tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, β3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not β1, β4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.

We report structure-activity relationships of 1-arylsulfonyl indoline based benzamides. The benzamide (9) exhibits striking tubulin inhibition with an IC50 value of 1.1 μM, better than that of combretastain A-4 (3), and substantial antiproliferative activity against a variety of cancer cells, including MDR-positive cell lines with an IC50 value of 49 nM (KB), 79 nM (A549), 63 nM (MKN45), 64 nM (KB-VIN10), 43 nM (KB-S15), and 46 nM (KB-7D). Dual inhibitory potential of compound 9 was found as it demonstrated significant inhibitory potential against HDAC1, 2 and 6 in comparison to MS-275 (6). Some key interactions of 9 with the amino acid residues of the active site of tubulin and with amino acid residues of HDAC 1 isoform have been figured out by molecular modeling. Compound 9 also demonstrated significant in vivo efficacy in the human non-small cell lung cancer A549 xenograft model as well as B-cell lymphoma BJAB xenograft tumor model.

Background Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. Methods Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. Results We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. Conclusions Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.

The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes the alkyl groups from the O6 position of guanine and is then degraded via ubiquitin-mediated degradation. Previous studies indicated that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) facilitates the ubiquitination and degradation of MGMT in several types of cancer cells. However, the underlying mechanism of MGMT ubiquitination remains unclear. In this study, we demonstrated for the first time that ubiquitin-conjugating enzyme E2 B (UBE2B) is a novel regulator of MGMT ubiquitination mediated by BCNU in nasopharyngeal carcinoma (NPC) cells. The E3 ubiquitin ligase RAD18, a partner of UBE2B, is also involved in BCNU-mediated MGMT ubiquitination. Overexpression/knockdown of UBE2B enhanced/reduced BCNU-mediated MGMT ubiquitination. Surprisingly, UBE2B knockdown significantly increased BCNU cytotoxicity in NPC cells. Therefore, loss of UBE2B seems to disrupt ubiquitin-mediated degradation of alkylated MGMT. We found that UBE2B knockdown reduced MGMT activity, suggesting that loss of UBE2B leads to the accumulation of deactivated MGMT and suppresses MGMT protein turnover in BCNU-treated cells. These findings indicate that UBE2B modulates sensitivity to BCNU in NPC cells by regulating MGMT ubiquitination.

Homoharringtonine (HHT), an inhibitor of protein synthesis, has been used to treat leukemia. Its therapeutic effects on non-small cell lung adenocarcinoma carrying KRAS mutation and their immune system are less understood. The present study examined the therapeutic efficacy and the immune effects of HHT in two murine lung tumor models, xenograft and transgenic, carrying the Kras mutation G12D and G12C respectively. HHT exhibited efficient anticancer activity, significantly suppressing lung tumor growth in vitro and in vivo. The levels of 22 cytokines and chemokines in splenocytes of tumor-bearing mice were examined. Interleukin-12 expression was lower in splenocytes of HHT-treated mice when compared to the controls as demonstrated by a cytokine array and an enzyme-linked immunosorbent assay. The expression levels of CD80, CD86, and CD69 in B220+ B cells from splenocytes of HHT-treated mice were higher than that of control mice in two mouse tumor models. Furthermore, antitumor effect of HHT was attenuated with depletion of B cells. Increased numbers of CD80+ and CD86+ B cells were observed in the mice treated with narciclasine, another translation inhibitor. In conclusion, HHT changed the features of immune cells, and exhibited efficient anti-tumor activity against lung tumor carrying mutant Kras expression.

Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical.

Clec4a has been reported to be an immune suppressor of dendritic cells (DCs), but its potential role in cancer therapy remains to be elucidated. The present study investigated whether downregulating the expression of Clec4a via skin delivery of small hairpin RNA (shRNA) using a gene gun produced stronger host immunity and inhibited tumor progression in animal models. Administration of Clec4a2 shRNA delayed tumor growth in both mouse bladder and lung tumor-bearing mouse models. The result was further confirmed with a compensation experiment showing that the antitumor effects induced by Clec4a2 shRNA were restored by co-injection of a plasmid expressing exogenous Clec4a2. Increased numbers of infiltrating CD4+ and CD8+ T cells at tumor sites were observed in mice treated with Clec4a2 shRNA. Splenocytes from mice with Clec4a2 shRNA administration exhibited stronger cytotoxic activity compared with splenocytes from control mice. CD8-deletion in vivo abrogated the antitumor effects elicited by Clec4a2 shRNA. Additionally, shClec4a enhanced the antitumor effects of the Neu DNA vaccine in the MBT-2 tumor model. In summary, the findings provide evidence that silencing of Clec4a2 expression via skin delivery of shRNA produces an effective antitumor response and that Clec4a2 shRNA may have therapeutic potential as an adjuvant for cancer immunotherapy.

Purpose: Targeting tumor-associated macrophages with colony-stimulating factor 1 receptor (CSF-1R) inhibition reveals a strategy for cancer therapy. Here, we studied the impact of CSF1R germline genetic variant on CSF-1R signaling and the susceptibility to CSF-1R inhibitors. Experimental designs: CSF1R germline genetic variants were studied in 140 cancer patients. CSF-1R phosphorylation, endocytosis, and macrophage polarization were measured as the response to CSF-1 stimulation. Tumor-associated macrophages in surgical specimens and sensitivity to CSF-1R inhibitors were used to determine macrophage function. Results: A CSF1R c.1085A>G genetic variant causing the change of histidine to arginine in the domain of receptor dimerization was identified as a high allele frequency in Eastern Asian population. Cancer patients with this variant allele had less M2-like tumor-associated macrophages accompanied by low VEGF expression in tumor tissues. Importantly, CSF1R genetic variant was significantly associated with disease-free survival in colorectal, endometrial, and ovarian cancer. In terms of differentiation, macrophages with CSF1R c.1085A>G genetic variant displayed a refractory response to CSF-1 stimulation and macrophage survival was sensitive to CSF-1R inhibitors with IC50 of 0.1 to 1 nmol/L range. On contrast, CSF-1 induced a prominent phosphorylation and rapid endocytosis of CSF-1R, leading to an M2-like dominant polarization in macrophages with CSF1R c.1085 genotype A_A, in which CSF-1R inhibitors of PLX3397, BLZ945, and GW2580 inhibited macrophage survival with IC50 of 10 to 100 nmol/L range. Conclusions: The CSF1R c.1085A>G genetic variant regulates tumor immunity by altering the polarization and function of macrophages. This genetic variant confers the sensitivity to CSF-1R inhibitors, implying as a biomarker in targeting CSF-1R signaling for cancer treatment. Clin Cancer Res; 23(20); 6021–30. ©2017 AACR.

Cancer stem-like cells (CSC) evolve to overcome the pressures of reduced oxygen, nutrients or chemically induced cell death, but the mechanisms driving this evolution are incompletely understood. Here, we report that hypoxia-mediated downregulation of the dual specificity phosphatase 2 (DUSP2) is critical for the accumulation of CSC in colorectal cancer. Reduced expression of DUSP2 led to overproduction of COX-2–derived prostaglandin E2, which promoted cancer stemness via the EP2/EP4 signaling pathways. Genetic and pharmacological inhibition of PGE2 biosynthesis or signal transduction ameliorated loss-of-DUSP2–induced tumor growth and cancer stemness. Genome-wide profile analysis revealed that genes regulated by DUSP2 were similar to those controlled by histone deacetylase. Indeed, treatment with novel histone deacetylase inhibitors abolished hypoxia-induced DUSP2 downregulation, COX-2 overexpression, cancer stemness, tumor growth, and drug resistance. Our findings illuminate mechanisms of cancer stemness and suggest new cancer therapy regimens. Cancer Res; 77(16); 4305–16. ©2017 AACR.

Metastasis of the cervical lymph nodes frequently leads to poor survival of patients with oral squamous cell carcinoma (OSCC). The underlying mechanisms of lymph node metastasis are unclear. Wingless-type MMTV integration site family, member 5B (WNT5B), one component of the WNT signal pathway, was markedly up-regulated in OSCC sublines with high potential of lymphatic metastasis compared to that in OSCC cells with low nodal metastasis. Increased WNT5B mRNA was demonstrated in human OSCC tissues in comparison with adjacent non-tumorous tissues. Interestingly, the high level of WNT5B protein in serum was associated with lymph node metastasis in OSCC patients. Knockdown of WNT5B expression in OSCC sublines did not affect tumour growth but impaired lymph node metastasis and tumour lymphangiogenesis of orthotopic transplantation. Conditioned medium from WNT5B knockdown cells reduced the tube formation of lymphatic endothelial cells (LECs). In contrast, recombinant WNT5B enhanced the tube formation, permeability and migration of LECs. In LECs stained with phalloidin, the morphology of those treated with recombinant WNT5B changed from flat to spindle-like. Recombinant WNT5B also increased α-smooth muscle actin and inhibited the expression of vascular endothelial-cadherin but retained characteristics of endothelial cells. The results suggest that WNT5B functions in the partial endothelial-mesenchymal transition (EndoMT). Furthermore, WNT5B-induced tube formation was impaired in the LECs following the knockdown of EndoMT-related transcription factor, SNAIL or SLUG. The WNT5B-induced expression of Snail or Slug was abolished by IWR-1-endo and Rac1 inhibitors, which are involved in the WNT/β-catenin and planar cell polarity pathways, respectively. Collectively, the data suggest that WNT5B induces tube formation by regulating the expression of Snail and Slug proteins through activation of canonical and non-canonical WNT signalling pathways.

MicroRNAs (miRNAs) are involved in the tumourigenesis of various cancers by regulating their downstream targets. To identify the changes of miRNAs in oral squamous cell carcinoma (OSCC), we investigated the expression profiles of miRNAs in 40 pairs of OSCC specimens and their matched non-tumour epithelial tissues. Our data revealed higher miR-455-5p expression in the tumour tissues than in the normal tissues; the expression was also higher in oral cancer cell lines than in normal keratinocyte cell lines. MiR-455-5p knockdown reduced both the anchorage-independent growth and the proliferative ability of oral cancer cells, and these factors increased in miR-455-5p-overexpressing cells. Furthermore, by analysing the array data of patients with cancer and cell lines, we identified ubiquitin-conjugating enzyme E2B (UBE2B) as a target of miR-455-5p, and further validated this using 3'-untranslated region luciferase reporter assays and western blot analysis. We also demonstrated that UBE2B suppression rescued the impaired growth ability of miR-455-5p-knockdown cells. Furthermore, we observed that miR-455-5p expression was regulated, at least in part, by the transforming growth factor-β (TGF-β) pathway through the binding of SMAD3 to specific promoter regions. Notably, miR-455-5p expression was associated with the nodal status, stage, and overall survival in our patients, suggesting that miR-455-5p is a potential marker for predicting the prognosis of patients with oral cancer. In conclusion, we reveal that miR-455-5p expression is regulated by the TGF-β-dependent pathway, which subsequently leads to UBE2B down-regulation and contributes to oral cancer tumourigenesis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR‐424‐5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR‐424‐5p. The miR‐424‐5p‐induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR‐424‐5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR‐424‐5p expression could be induced by the cytokine IL‐8 primarily through enhancing STAT5 transcriptional activity rather than NF‐κB signaling. Antagomir‐mediated inactivation of miR‐424‐5p prevented the IL‐8‐induced cell migration and invasion, indicating that miR‐424‐5p is required for IL‐8‐induced cellular invasiveness. Taken together, these data indicate that STAT5‐dependent expression of miR‐424‐5p plays an important role in mediating IL‐8/STAT5/SOCS2 feedback loop, and scavenging miR‐424‐5p function using antagomir may have therapeutic potential for the treatment of OSCC., Highlights miR‐424‐5p is overexpressed in OSCC.miR‐424‐5p directly targets SOCS2, leading to increased cell migration and invasion.STAT5 activation is required for IL‐8‐mediated miR‐424‐5p transcription.miR‐424‐5p plays an important role in mediating IL‐8/STAT5/SOCS2 feedback loop.

In many solid tumors, tissue of the mesenchymal subtype is frequently associated with epithelial&ndash, mesenchymal transition (EMT), strong stromal infiltration, and poor prognosis. Emerging evidence from tumor ecosystem studies has revealed that the two main components of tumor stroma, namely, infiltrated immune cells and cancer-associated fibroblasts (CAFs), also express certain typical EMT genes and are not distinguishable from intrinsic tumor EMT, where bulk tissue is concerned. Transcriptomic analysis of xenograft tissues provides a unique advantage in dissecting genes of tumor (human) or stroma (murine) origins. By transcriptomic analysis of xenograft tissues, we found that oral squamous cell carcinoma (OSCC) tumor cells with a high EMT score, the computed mesenchymal likelihood based on the expression signature of canonical EMT markers, are associated with elevated stromal contents featured with fibronectin 1 (Fn1) and transforming growth factor-&beta, (Tgf&beta, ) axis gene expression. In conjugation with meta-analysis of these genes in clinical OSCC datasets, we further extracted a four-gene index, comprising FN1, TGFB2, TGFBR2, and TGFBI, as an indicator of CAF abundance. The CAF index is more powerful than the EMT score in predicting survival outcomes, not only for oral cancer but also for the cancer genome atlas (TCGA) pan-cancer cohort comprising 9356 patients from 32 cancer subtypes. Collectively, our results suggest that a further distinction and integration of the EMT score with the CAF index will enhance prognosis prediction, thus paving the way for curative medicine in clinical oncology.

Background Tumor hypoxia-induced epithelial–mesenchymal transition (EMT) is critical in promoting cancer metastasis. We recently discovered a novel microtubule inhibitor, MPT0B098, that employs a novel antitumor mechanism. It destabilizes hypoxia-inducible factor (HIF)-1α mRNA by blocking the function of human antigen R. Thus, we proposed that MPT0B098 modulates hypoxia-induced EMT. Methods In vitro IC50 values were determined through the methylene blue dye assay. To investigate molecular events, reverse transcriptase-polymerase chain reaction, Western blotting, immunofluorescence staining, and wound healing assay were employed. Results MPT0B098 significantly inhibited HIF-1α expression, epithelial-to-mesenchymal morphology changes, and migratory ability in the human head and neck squamous cell carcinoma cell line OEC-M1. Furthermore, after MPT0B098 treatment, the expression of two mesenchymal markers, vimentin and N-cadherin, was downregulated under hypoxic conditions. Moreover, MPT0B098 suppressed hypoxia-induced EMT in part by inhibiting EMT-activating transcription factors, Twist and SNAI2/Slug. In addition, the inhibition of hypoxia-induced F-actin rearrangement and focal adhesion kinase phosphorylation may have contributed to suppression of EMT by MPT0B098in OEC-M1 cells. MPT0B098 significantly inhibited transforming growth factor(TGF)-β-induced phosphorylation of receptor-associated Smad2/3 by downregulating TGF-β mRNA and protein expression. Conclusions Taken together, this study provides a novel insight into the role of MPT0B098 in inhibiting hypoxia-induced EMT, suggesting its potential use for treating head and neck cancers. Electronic supplementary material The online version of this article (10.1186/s12929-018-0432-6) contains supplementary material, which is available to authorized users.

This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11-23) on the basis of scaffold hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with various amines, and simplifies the structural identification process. Among the synthetic compounds, 6-dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (12) and 7-pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (23) exhibit remarkable anti-proliferative activity against the cancer cell lines tested with mean IC50 values of 0.14 and 0.27 μM, respectively. Compound 23 showed moderate inhibitory activity against tubulin polymerization with an IC50 value of 5.9 μM. In a western blot analysis, 23 caused induction of HSP70 and degradation of Akt, revealing that it possesses HSP90 inhibitory activity.

Aberrant Wnt signaling pathway is a common feature of tumors and also plays important roles in tumor progression and metastasis of many cancer types. Various lines of evidence suggest that genetic defects affect Wnt pathway components, as well as epigenetic mechanisms that modulate the suppressors of Wnt pathway in oral squamous cell carcinoma. Recently, the newly discovered microRNAs are important molecular regulators in gene expression through transcription and translation repression. They play fundamental roles in a wide spectrum of biological functions, including cancer. In this review, we aim to accumulate recent research findings on the roles of Wnt/β-catenin signaling and discuss how microRNAs affect Wnt/β-catenin signaling in oral squamous cell carcinoma tumorigenesis. Apparently, investigations into the role of microRNAs with regard to the Wnt pathway in oral squamous cell carcinoma may help in the development of better strategies for tumor treatment.

// Yi-Chen Yen 1, * , Jenn-Ren Hsiao 2, * , Shih Sheng Jiang 1 , Jeffrey S. Chang 3 , Ssu-Han Wang 1 , Ying-Ying Shen 4 , Chung-Hsing Chen 5 , I-Shou Chang 1, 5 , Jang-Yang Chang 6 , Ya-Wen Chen 1, 7 1 National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan 2 Department of Otolaryngology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan 3 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 4 Pathology Core Laboratory, National Health Research Institutes, Miaoli, Taiwan 5 Institute of Population Health Sciences, National Health Research Institutes, Miaoli, Taiwan 6 Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, Tainan, Taiwan 7 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Ya-Wen Chen, e-mail: ywc@nhri.org.tw Keywords: oral squamous cell carcinoma, lymph node metastasis, migration, insulin-like growth factor binding protein 3, integrin β1 Received: August 11, 2015 Accepted: October 05, 2015 Published: October 19, 2015 ABSTRACT Frequent metastasis to the cervical lymph nodes leads to poor survival of patients with oral squamous cell carcinoma (OSCC). To understand the underlying mechanisms of lymph node metastasis, two sublines were successfully isolated from cervical lymph nodes of nude mice through in vivo selection, and identified as originating from poorly metastatic parental cells. These two sublines specifically metastasized to cervical lymph nodes in 83% of mice, whereas OEC-M1 cells did not metastasize after injection into the oral cavity. After gene expression analysis, we identified insulin-like growth factor binding protein 3 (IGFBP3) as one of the significantly up-regulated genes in the sublines in comparison with their parental cells. Consistently, meta-analysis of the public microarray datasets and IGFBP3 immunohistochemical analysis revealed increased both levels of IGFBP3 mRNA and protein in human OSCC tissues when compared to normal oral or adjacent nontumorous tissues. Interestingly, the up-regulated IGFBP3 mRNA expression was significantly associated with OSCC patients with lymph node metastasis. IGFBP3 knockdown in the sublines impaired and ectopic IGFBP3 expression in the parental cells promoted migration, transendothelial migration and lymph node metastasis of orthotopic transplantation. Additionally, ectopic expression of IGFBP3 with an IGF-binding defect sustained the IGFBP3-enhanced biological functions. Results indicated that IGFBP3 regulates metastasis-related functions of OSCC cells through an IGF-independent mechanism. Furthermore, exogenous IGFBP3 was sufficient to induce cell motility and extracellular signal-regulated kinase (ERK) activation. The silencing of integrin β1 was able to impair exogenous IGFBP3-mediated migration and ERK phosphorylation, suggesting a critical role of integrin β1 in IGFBP3-enchanced functions.

MicroRNAs (miRNAs) are small RNAs that suppress gene expression by their interaction with 3'untranslated region of specific target mRNAs. Although the dysregulation of miRNAs has been identified in human cancer, only a few of these miRNAs have been functionally documented in breast cancer. Thus, defining the important miRNA and functional target involved in chemoresistance is an urgent need for human breast cancer treatment. In this study, we, for the first time, identified a key role of miRNA 520h (miR-520h) in drug resistance. Through protecting cells from paclitaxel-induced apoptosis, expression of miR-520h promoted the drug resistance of human breast cancer cells. Bioinformatics prediction, compensatory mutation and functional validation further confirmed the essential role of miR-520h-suppressed Death-associated protein kinase 2 (DAPK2) expression, as restoring DAPK2 abolished miR-520h-promoted drug resistance, and knockdown of DAPK2 mitigated cell death caused by the depletion of miR-520h. Furthermore, we observed that higher level of miR-520h is associated with poor prognosis and lymph node metastasis in human breast cancer patients. These results show that miR-520h is not only an independent prognostic factor, but is also a potential functional target for future applications in cancer therapeutics.

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.

Growth differentiation factor-10 (GDF10), commonly referred as BMP3b, is a member of the transforming growth factor-β (TGF-β) superfamily. GDF10/BMP3b has been considered as a tumor suppressor, however, little is known about the molecular mechanism of its roles in tumor suppression in oral cancer. Clinical significance of GDF10 downregulation in oral squamous cell carcinoma (OSCC) was evaluated using three independent cohorts of OSCC patients. The molecular mechanisms of GDF10 in the suppression of cell survival, cell migration/invasion and epithelial-mesenchymal transition (EMT) were investigated by using oral cancer cell lines. The present study shows that GDF10 is downregulated during oral carcinogenesis, and GDF10 expression is also an independent risk factor for overall survival of OSCC patients. Overexpression of GDF10 attenuates cell proliferation, transformation, migration/invasion, and EMT. GDF10-inhibited EMT is mediated by ERK signaling but not by typical TGF-β signaling. In addition, overexpression of GDF10 promotes DNA damage-induced apoptosis and sensitizes the response to all-trans retinoic acid (ATRA) and camptothecin (CPT). Intriguingly, the expression of GDF10 is induced by type III TGF-β receptor (TGFBR3) through TGF-β-SMAD2/3 signaling. Our findings suggest that TGFBR3 is an upstream activator of GDF10 expression and they share the same signaling to inhibit EMT and migration/invasion. These results support that GDF10 acts as a hinge to collaborate with TGFBR3 in the transition of EMT-MET program. Taken together, we illustrated the clinical significance and the molecular mechanisms of tumor-suppressive GDF10 in OSCC.

Poor oral hygiene may lead to overgrowth of pathogenic oral bacteria, which may induce chronic inflammation to promote the oncogenesis of oral squamous cell carcinoma (OSCC). This study investigated the association between oral bacterial profile and OSCC risk in a case-control study of 138 OSCC cases and 151 controls (88 cases and 90 controls for the discovery group and 50 cases and 61 controls for the validation group). Oral bacterial profiles were characterized by targeted sequencing of the 16S rRNA gene. Three species of periodontopathogenic bacteria, Prevotella tannerae, Fusobacterium nucleatum, and Prevotella intermedia, were associated with an increased OSCC risk. This association was modified by the genetic polymorphisms of TLR2 and TLR4. Use of alcohol, betel quids and cigarettes and poor oral hygiene were associated with a higher percentage of oral periodontopathogenic bacteria. The association between alcohol and periodontopathogenic bacteria was modified by the genetic polymorphism of ALDH2, with a stronger positive association observed among the ALDH2-deficient individuals. The percentage of periodontopathogenic bacteria was positively correlated with the level of salivary IL1β, an inflammatory cytokine. Overall, our results showed a positive association between periodontopathogenic bacteria and OSCC risk and this relationship may be influenced by lifestyle and genetic factors. Our results provided further biological support for the established association between poor oral hygiene and OSCC risk. This suggested that improving oral hygiene may reduce OSCC risk and should be part of a public health campaign to prevent the occurrence of OSCC.

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Background and Purpose The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER+) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer. Experimental Approach The potency of YM155 in SK-BR-3, MDA-MB-231, MCF7 and its tamoxifen-resistant sublines, TamR6, TamR7, TamR8, TamC3 and TamC6, were determined by MTT assay. Western blot analysis, flow cytometric analysis, reverse transcription-PCR, fluorescent microscopy and comet assay were used to determine the molecular mechanism of action of YM155 in different breast cancer cell lines. Key Results YM155 was equally potent towards the parental ER+/caspase-3-deficient MCF7 breast cancer cells and its tamoxifen-resistant sublines in vitro. The ER−/HER2+ SK-BR-3 breast cancer cells and the triple-negative/caspase-3-expressing metastatic aggressive MDA-MB-231 breast cancer cells were also sensitive to YM155 with IC50 values in the low nanomolar range. Targeting survivin by YM155 modulated autophagy, induced autophagy-dependent caspase-7 activation and autophagy-dependent DNA damage in breast cancer cells. Interestingly, YM155 also induced XIAP degradation and the degradation of XIAP might play an important role in YM155-induced autophagy in breast cancer cells. Conclusions and Implications YM155 is a potent survivin inhibitor that has potential for the management of various breast cancer subtypes regardless of the expression of ER, HER2 and caspase-3. Importantly, this study provides new insights into YM155's molecular mechanism of action and therapeutic potential in the treatment of tamoxifen-resistant breast cancer.

Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

Cisplatin can cause intrastrand and interstrand crosslinks between purine bases and is a chemotherapeutic drug widely used to treat cancer. However, the major barrier to the efficacy of the treatment is drug resistance. Homologous recombination (HR) plays a central role in restoring stalled forks caused by DNA lesions. Here, we report that chronic treatment with cisplatin induces HR to confer cisplatin resistance in nasopharyngeal carcinoma (NPC) cells. A high frequency of sister chromatid exchanges (SCE) occurs in the cisplatin-resistant NPC cells. In addition, several genes in the Fanconi anemia (FA) and template switching (TS) pathways show elevated expression. Significantly, depletion of HR gene BRCA1, TS gene UBC13, or FA gene FANCD2 suppresses SCE and causes cells to accumulate in the S phase, concomitantly with high γH2AX foci formation in the presence of low-dose cisplatin. Consistent with this result, depletion of several genes in the HR, TS, or FA pathway sensitizes the cisplatin-resistant NPC cells to cisplatin. Our results suggest that the enhanced HR, in coordination with the FA and TS pathways, underlies the cisplatin resistance. Targeting the HR, TS, or FA pathways could be a potential therapeutic strategy for treating cisplatin-resistant cancer.

2-Hydroxy-3,4,5-trimethoxybenzophenones (8–16) manifest pseudo-ring formation involving intramolecular hydrogen bonding of the 2-OH and the carbonyl group. Among the synthetic products described in this report, (3-hydroxy-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxyphenyl)-methanone (14) and (3-amino-4-methoxyphenyl)(2-hydroxy-3,4,5-trimethoxy-phenyl)methanone (16) exhibit significant antiproliferative activity against KB cells with IC50 values of 11.1 and 11.3 nM, respectively. These two compounds also displayed tubulin affinity comparable to that of combretastatin A-4. In studies with human umbilical vein endothelial cells, compounds 14 and 16 revealed concentration-dependent vascular-disrupting properties. The results support the rationale of the pseudo-ring concept and suggest further investigation of A-ring modification in these benzophenones.

SOX2 is a transcription factor essential for self-renewal and pluripotency of embryonic stem cells. Recently, SOX2 was found overexpressed in the majority of the lung squamous cell carcinoma (SQC), in which it acts as a lineage-survival oncogene. However, downstream targets/pathways of SOX2 in lung SQC cells remain to be identified. Here, we show that BMP4 is a downstream target of SOX2 in lung SQC. We found that SOX2-silencing-mediated inhibition of cell growth was accompanied by upregulation of BMP4 mRNA and its protein expression. Meta-analysis with 293 samples and qRT-PCR validation with 73 clinical samples revealed an inversely correlated relationship between levels of SOX2 and BMP4 mRNA, and significantly lower mRNA levels in tumor than in adjacent normal tissues. This was corroborated by immunohistochemistry analysis of 35 lung SQC samples showing lower BMP4 protein expression in tumor tissues. Cell-based experiments including siRNA transfection, growth assay and flow cytometry assay, further combined with a xenograft tumor model in mice, revealed that reactivation of BMP4 signaling could partially account for growth inhibition and cell cycle arrest in lung SQC cells upon silencing SOX2. Finally, chromatin immunoprecipitation analysis and luciferase reporter assay revealed that SOX2 could negatively regulate BMP4 promoter activity, possibly through binding to the promoter located in the first intron region of BMP4. Collectively, our findings suggest that BMP4 could act as a tumor suppressor and its downregulation by elevated SOX2 resulting in enhanced growth of lung SQC cells.

Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of ∼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 μM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.

Cathepsin S plays multiple roles in MHC class II antigen presentation, extracellular matrix degradation, angiogenesis, and tumorogenesis. Our previous study revealed that targeting cathepsin S could induce cellular cytotoxicity and reduce cell viability. For the current study, we further investigated the molecular mechanism responsible for targeting cathepsin S-induced cell death and its association with autophagy. Distinct from regulation of the classic autophagy pathway by reactive oxygen species (ROS), we demonstrated that autophagy is the genuine regulator of early ROS production. The molecular silencing of autophagy-dependent ATG genes (ATG5, ATG7, and LC3) and the pharmacologic inhibition of autophagy with 3-MA and wortmannin reduced ROS production significantly. In addition, xanthine oxidase (XO), which is upregulated by autophagy, is required for early ROS production, oxidative DNA damage, and consequent cell death. Autophagy inhibition suppresses the upregulation of XO, which is induced by cathepsin S inhibition, resulting in reduced ROS generation, DNA damage, and cell death. Collectively, our study reveals a noncanonical molecular pathway in which, after the inhibition of cathepsin S, autophagy induces early ROS production for oxidative DNA damage and cell death through XO.

Preliminary biological data on 7-anilino-6-azaindoles (8-11) suggested that hydrophobic substituents at C7 contribute to enhancement of antiproliferative activity. A novel series of 7-aryl-6-azaindole-1-benzenesulfonamides (12-22) were developed and showed improved cytotoxicity compared to ABT751 (5). The conversion of C7 phenyl rings into C7 heterocycles led to a remarkable improvement of antiproliferative activity. Among all the synthetic products, 7-(2-furanyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (21) exhibited the most potent anticancer activity against KB, HT29, MKN45, and H460 cancer cell lines with IC50 values of 21.1, 32.0, 27.5, and 40.0 nM, respectively. Bioassays indicated that 21 not only inhibits tubulin polymerization by binding to tubulin at the colchicine binding site but also arrests the cell cycle at the G2/M phase with slight arrest at the sub-G1 phase. Compound 21 also functions as a vascular disrupting agent and dose-dependently inhibits tumor growth without significant change of body weight in an HT29 xenograft mouse model. Taken together, compound 21 has potential for further development as a novel class of anticancer agents.

Oxaliplatin treatment is a mainstay of treatment for advanced gastrointestinal tract cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain largely obscured. We previously demonstrated that increased DNA repair capacity and copper-transporting ATPase 1 (ATP7A) level contributed to oxaliplatin resistance in the human gastric carcinoma cell line TSGH-S3 (S3). In the present study, we applied gene array technology to identify additional resistance factors in S3 cells. We found that interleukin-6 (IL-6), aldo-keto reductase 1C1 (AKR1C1), and AKR1C3 are the top 3 upregulated genes in S3 cells when compared with parent TSGH cells. Despite a higher level of endogenous IL-6 in S3, IL-6 receptor (IR-6R, gp-80, and gp-130) levels were similar between TSGH and S3 cells. The addition of exogenous IL-6, IL-6 targeted siRNA, or neutralizing antibodies neither affected Stat3 activation, a downstream target of IL-6, nor changed oxaliplatin sensitivity in S3 cells. However, manipulation of AKR1C activity with siRNA or AKR1C inhibitors significantly reversed oxaliplatin resistance. AKR1Cs are classical antioxidant response element (ARE) genes that can be transcriptionally upregulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown of Nrf2 not only decreased the levels of AKR1C1, AKR1C2, and AKR1C3 mRNA and protein but also reversed oxaliplatin resistance in S3 cells. Taken together, these results suggest that activation of the Nrf2/AKR1C axis may contribute to oxaliplatin resistance in S3 cells but that the IL-6 signaling pathway did not contribute to resistance. Manipulation of Nrf2/AKR1Cs activity may be useful for management of oxaliplatin-refractory gastric cancers.

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.

The strong associations between oral squamous cell carcinoma (OSCC) and dietary habits such as alcohol consumption (A), betel quid chewing (B) and cigarette smoking (C) and its predominance in men have been well documented; however, systemic analysis of OSCC is limited. Our study applied high-throughput screening methods to identify causative epigenetic targets in a cohort of men with ABC-associated OSCC. We identified BEX1 and LDOC1 as two epigenetically silenced X-linked tumour suppressors and demonstrated a functional link between the transcription of BEX1 and LDOC1 and promoter hypermethylation. Methylation of the BEX1 and LDOC1 promoters was associated significantly (p

Parathyroid Hormone-Like Hormone (PTHLH) is an autocrine/paracrine ligand that is up-regulated in head and neck squamous cell carcinoma (HNSCC). However, the cellular function and regulatory mechanism in HNSCC remains obscure. We investigated the clinical significance of PTHLH in HNSCC patients, and verified the role of RUNX2/PTHLH axis, which is stimulated HNSCC cell growth. In patients, PTHLH is a poor prognosis marker. PTHLH expression lead to increasing the cell proliferation potential through an autocrine/paracrine role and elevating blood calcium level in Nod-SCID mice. In public HNSCC microarray cohorts, PTHLH is found to be co-expressed with RUNX2. Physiologically, PTHLH is regulated by RUNX2 and also acting as key calcium regulator. However, elevations of calcium concentration also increased the RUNX2 expression. PTHLH, calcium, and RUNX2 form a positive feedback loop in HNSCC. Furthermore, ectopic RUNX2 expression also increased PTHLH expression and promoted proliferation potential through PTHLH expression. Using cDNA microarray analysis, we found PTHLH also stimulated expression of cell cycle regulators, namely CCNA2, CCNE2, and CDC25A in HNSCC cells, and these genes are also up-regulated in HNSCC patients. In summary, our results reveal that PTHLH expression is a poor prognosis marker in HNSCC patients, and RUNX2-PTHLH axis contributes to HNSCC tumor growth.

The polymerization of tubulin molecules forms microtubules which are considered an attractive target for cancer treatment. Herein, we synthesized a new tubulin inhibitor, MPT0B169 (2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,​3-dihydro-1H-indol-7-yl]-acetamide) and demonstrated its action in leukemia cell lines HL60 and NB4 and lymphoma cell line U937. We found that MPT0B169 prevented tubulin assembly by binding the colchicine-binding site of tubulin in vitro. MPT0B169 also induced tubulin depolymerization in vivo. MPT0B169 inhibited the growth of HL60, NB4, and U937 cells in dose- and time-dependent manners. It also inhibited the growth of paclitaxel-resistant cancer cells. In addition, MPT0B169 caused G2/M cell cycle arrest in nonresistant and paclitaxel-resistant cancer cells, with a concomitant increase in cyclin B1 levels and cyclin-dependent kinase 1 (CDK1) phosphorylation. These results suggest that MPT0B169, a tubulin inhibitor, inhibits cell growth and induces G2/M cell cycle arrest of cancer cells through the disruption of tubulin polymerization.

Background To better evaluate and improve the efficacy of dendritic cell (DC)-based cancer immunotherapy, we conducted a clinical study of patients with advanced colorectal cancer using carcinoembryonic antigen (CEA)-pulsed DCs mixed with tetanus toxoid and subsequent interleukin-2 treatment. The tetanus toxoid in the vaccine preparation serves as an adjuvant and provides a non-tumor specific immune response to enhance vaccine efficacy. The aims of this study were to (1) evaluate the toxicity of this treatment, (2) observe the clinical responses of vaccinated patients, and (3) investigate the immune responses of patients against CEA before and after treatment. Methods Twelve patients were recruited and treated in this phase I clinical study. These patients all had metastatic colorectal cancer and failed standard chemotherapy. We first subcutaneously immunized patients with metastatic colorectal cancer with 1 × 106 CEA-pulsed DCs mixed with tetanus toxoid as an adjuvant. Patients received 3 successive injections with 1 × 106 CEA-pulsed DCs alone. Low-dose interleukin-2 was administered subcutaneously following the final DC vaccination to boost the growth of T cells. Patients were evaluated for adverse event and clinical status. Blood samples collected before, during, and after treatment were analyzed for T cell proliferation responses against CEA. Results No severe treatment-related side effects or toxicity was observed in patients who received the regular 4 DC vaccine injections. Two patients had stable disease and 10 patients showed disease progression. A statistically significant increase in proliferation against CEA by T cells collected after vaccination was observed in 2 of 9 patients. Conclusions The results of this study indicate that it is feasible and safe to treat colorectal cancer patients using this protocol. An increase in the anti-CEA immune response and a clinical benefit was observed in a small fraction of patients. This treatment protocol should be further evaluated in additional colorectal cancer patients with modifications to enhance T cell responses. Trial registration ClinicalTrials.gov (identifier NCT00154713), September 8, 2005 Electronic supplementary material The online version of this article (doi:10.1186/s12929-016-0279-7) contains supplementary material, which is available to authorized users.

EGF-mediated EGFR endocytosis plays a crucial role in the attenuation of EGFR activation by sorting from early endosomes to late endosomes and transporting them into lysosomes for the final proteolytic degradation. We previously observed that cathepsin S (CTSS) inhibition induces tumour cell autophagy through the EGFR-mediated signalling pathway. In this study, we further clarified the relationship between CTSS activities and EGFR signalling regulation. Our results revealed that CTSS can regulate EGFR signalling by facilitating EGF-mediated EGFR degradation. CTSS inhibition delayed the EGFR degradation process and caused EGFR accumulation in the late endosomes at the perinuclear region, which provides spatial compartments for prolonged EGFR and sustained downstream signal transducer and activator of transcription 3 and AKT signalling. Notably, cellular apoptosis was markedly enhanced by combining treatment with the EGFR inhibitor Iressa and CTSS inhibitor 6r. The data not only reveal a biological role of CTSS in EGFR signalling regulation but also evidence a rationale for its clinical evaluation in the combination of CTSS and EGFR tyrosine kinase inhibitors.

Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

Epigenetic correlates of the head and neck cancer may illuminate its pathogenic roots. Through a gene set enrichment analysis, we found that the oncogenic transcription factor RUNX2 is widely upregulated in the head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis, where it also predicts poor prognosis in patients with HNSCC. Enforced expression of ectopic RUNX2 promoted the metastatic capabilities of HNSCC, whereas RUNX2 silencing inhibited these features. Mechanistic investigations showed that manipulating levels of activin A (INHBA) could rescue or compromise the RUNX2-mediated metastatic capabilities of HNSCC cells. Furthermore, we found that miR-376c-3p encoded within the 3′-untranslated region of RUNX2 played a pivotal role in regulating RUNX2 expression in highly metastatic HNSCC cells, where it was downregulated commonly. Restoring miR-376c expression in this setting suppressed expression of RUNX2/INHBA axis along with metastatic capability. Clinically, we observed an inverse relationship between miR-376c-3p expression and the RUNX2/INHBA axis in HNSCC specimens. In summary, our results defined a novel pathway in which dysregulation of the RUNX2/INHBA axis due to miR-376c downregulation fosters lymph node metastasis in HNSCC. Cancer Res; 76(24); 7140–50. ©2016 AACR.

Summary Objectives Arginine is a nonessential amino acid which can regulate tumor growth. Argininosuccinate synthetase (ASS) is the rate-limiting enzyme for de novo arginine production. The expression pattern of ASS and the feasibility of arginine deprivation therapy in head and neck cancer have not been investigated. Materials and methods The growth-inhibitory effect of arginine deprivation therapy was assessed either by proliferation assay with head and neck cancer cells cultured in arginine-free medium, or by tetrazolium/formazan dye assay with cells treated with an arginine-depleting drug (arginine deiminase, ADI). The tumor ASS status of 73 oral squamous carcinoma (OSCC) patients was then evaluated immunohistochemically and subsequently correlated with the corresponding clinicopathological parameters. Results Head and neck cancer cells cultured in arginine-free medium either completely stopped proliferating, or proliferated minimally. In addition, ADI treatment inhibited the growth of all 8 head and neck cancer cell lines to different degrees. Although cellular ASS level did not correlate well with ADI-sensitivity among these cell lines, knockdown of endogenous ASS potentiated the growth-inhibitory effect of ADI in each individual cell line (FaDu and OEC-M1). In multivariable analysis, high tumor ASS level independently predicted an unfavorable disease-free survival in OSCC patients. Conclusion High tumor ASS status is an independent variable predicting a poor disease-free survival in OSCC patients. Arginine deprivation therapy may potentially be used as a new approach to treat head and neck cancer.

Summary Background Deregulation of mammalian target of rapamycin (mTOR) signalling is common in human hepatocellular carcinoma (HCC). Aim To determine the maximum tolerated dose (MTD) of the oral mTOR inhibitor everolimus in advanced HCC patients. Methods Patients with locally advanced or metastatic HCC (Child-Pugh class A or B) were enrolled in an open-label phase 1 study and randomly assigned to daily (2.5–10 mg) or weekly (20–70 mg) everolimus in a standard 3 + 3 dose-escalation design. MTD was based on the rate of dose-limiting toxicities (DLTs). Secondary endpoints included safety, pharmacokinetics and tumour response. In a post hoc analysis, serum hepatitis B virus (HBV) DNA levels were quantified. Results Thirty-nine patients were enrolled. DLTs occurred in five of 21 patients in the daily and two of 19 patients in the weekly cohort. Daily and weekly MTDs were 7.5 mg and 70 mg respectively. Grade 3/4 adverse events with a ≥10% incidence were thrombocytopenia, hypophosphataemia and alanine transaminase (ALT) elevation. In four hepatitis B surface antigen (HBsAg)-seropositive patients, grade 3/4 ALT elevations were accompanied by significant (>1 log) increases in serum HBV levels. The incidence of hepatitis flare (defined as ALT increase >100 IU/mL from baseline) in HBsAg-seropositive patients with and without detectable serum HBV DNA before treatment was 46.2% and 7.1% respectively (P

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDI(TM) Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)-DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.