IFIT2 Depletion Promotes Cancer Stem Cell-like Phenotypes in Oral Cancer.

(1) Background: Cancer stem cells (CSCs) are a small cell population associated with chemoresistance, metastasis and increased mortality rate in oral cancer. Interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) depletion results in epithelial to mesenchymal transition, invasion, metastasis, and chemoresistance in oral cancer. To date, no study has demonstrated the effect of IFIT2 depletion on the CSC-like phenotype in oral cancer cells. (2) Methods: Q-PCR, sphere formation, Hoechst 33,342 dye exclusion, immunofluorescence staining, and flow cytometry assays were performed to evaluate the expression of the CSC markers in IFIT2-depleted cells. A tumorigenicity assay was adopted to assess the tumor formation ability. Immunohistochemical staining was used to examine the protein levels of IFIT2 and CD24 in oral cancer patients. (3) Results: The cultured IFIT2 knockdown cells exhibited an overexpression of ABCG2 and CD44 and a downregulation of CD24 and gave rise to CSC-like phenotypes. Clinically, there was a positive correlation between IFIT2 and CD24 in the patients. IFIT2high/CD24high/CD44low expression profiles predicted a better prognosis in HNC, including oral cancer. The TNF-α blockade abolished the IFIT2 depletion-induced sphere formation, indicating that TNF-α may be involved in the CSC-like phenotypes in oral cancer. (4) Conclusions: The present study demonstrates that IFIT2 depletion promotes CSC-like phenotypes in oral cancer.

TIMP3 Gene Polymorphisms of -1296 T > C and -915 A > G Increase the Susceptibility to Arsenic-Induced Skin Cancer: A Cohort Study and In Silico Analysis of Mutation Impacts

Long-term exposure to arsenic may induce several human cancers, including non-melanoma skin cancer. The tissue inhibitor of metalloproteinase (TIMP)-3, encoded by the TIMP3 gene, may inhibit tumor growth, invasion, and metastasis of several cancer types. In this study, we aimed to investigate effects of the TIMP3 -1296 T > C (rs9619311) and -915 A > G (rs2234921) single-nucleotide polymorphisms (SNPs) on skin cancer risk in an arsenic-exposed population, and to evaluate the influence of allele-specific changes by an in silico analysis. In total, 1078 study participants were followed up for a median of 15 years for newly diagnosed skin cancer. New cases were identified through linkage to the National Cancer Registry of Taiwan. A Cox regression analysis was used to evaluate the effects of TIMP3 variants. Transcription factor (TF) profiling of binding sites of allele-specific changes in SNPs was conducted using the JASPAR scan tool. We observed borderline associations between TIMP3 genotypes and skin cancer risk. However, when combined with high arsenic exposure levels, the rs9619311 C allele, rs2234921 G allele, or C-G haplotype groups exhibited a greater risk of developing skin cancer compared to the respective common homozygous genotype group. The in silico analysis revealed several TF motifs located at or flanking the two SNP sites. We validated that the C allele of rs9619311 attenuated the binding affinity of BACH2, MEIS2, NFE2L2, and PBX2 to the TIMP3 promoter, and that the G allele of rs2234921 reduced the affinity of E2F8 and RUNX1 to bind to the promoter. Our findings suggest significant modifications of the effect of the association between arsenic exposure and skin cancer risk by the TIMP3 rs9619311 and rs2234921 variants. The predicted TFs and their differential binding affinities to the TIMP3 promoter provide insights into how TIMP3 interacts with arsenic through TFs in skin cancer formation.

Discovery of Oral Anticancer 1,2-Bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine Hybrids That Inhibit Angiogenesis and Induce DNA Cross-Links.

Designing hybrid molecules with dual functions is one approach to improve the therapeutic efficacy of combination treatment. We have previously conjugated phthalazine and bis(hydroxymethyl)pyrrole pharmacophores to form hybrids bearing antiangiogenesis and DNA interstrand cross-linking activities. To improve the bioavailability, we adopted a benzology approach to design and synthesize a new series of 1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazines. These new hybrids retained the dual functions and could be formulated into vehicles for intravenous and oral administration. Among them, we demonstrated that compound 19a with dimethylamine at the C6 position markedly suppressed the tumor growth of human small cell lung cancer cell line H526, squamous lung cancer cell line H520, and renal cancer cell line 786-O in nude mice, implying that compound 19a is a broad-spectrum anticancer agent. Our results implicated that the conjugation of antiangiogenic and DNA cross-linking is likely to be a helpful approach to improving the efficacy of combination therapy.

GT-Repeat Polymorphism in the HO-1 Gene Promoter Is Associated with Risk of Liver Cancer: A Follow-Up Study from Arseniasis-Endemic Areas in Taiwan.

The induction of heme oxygenase-1 (HO-1) has been shown to have therapeutic potential in experimental models of hepatitis and liver fibrosis, which are closely related to liver cancer. In humans, HO-1 induction is transcriptionally modulated by the length of a GT-repeat [(GT)n] in the promoter region. We aimed to investigate the effect of HO-1 (GT)n variants on liver cancer in a human population. We determined the HO-1 genotype in 1153 study subjects and examined their association with liver cancer risk during a 15.9-year follow-up. Allelic polymorphisms were classified as short [S, <27 (GT)n] or long [L, ≥27 (GT)n]. Newly developed cancer cases were identified through linkage to the National Cancer Registry of Taiwan. Multivariate Cox regression analysis was used to evaluate the effect of the HO-1 (GT)n variants. Alpha-fetoprotein (AFP) and cirrhosis history were also examined. The S/S genotype was found to be significantly associated with liver cancer risk, compared to the L/S and L/L genotypes. The S/S genotype group also had a higher percentage of subjects with abnormal AFP levels than other groups. There were significant percentages of cirrhosis among groups who carried S-alleles. Our findings indicate that short (GT)n variants in the HO-1 gene may confer susceptibility to rather than protection from liver cirrhosis/cancer.

Genetic profiles of 103,106 individuals in the Taiwan Biobank provide insights into the health and history of Han Chinese.

Personalized medical care focuses on prediction of disease risk and response to medications. To build the risk models, access to both large-scale genomic resources and human genetic studies is required. The Taiwan Biobank (TWB) has generated high-coverage, whole-genome sequencing data from 1492 individuals and genome-wide SNP data from 103,106 individuals of Han Chinese ancestry using custom SNP arrays. Principal components analysis of the genotyping data showed that the full range of Han Chinese genetic variation was found in the cohort. The arrays also include thousands of known functional variants, allowing for simultaneous ascertainment of Mendelian disease-causing mutations and variants that affect drug metabolism. We found that 21.2% of the population are mutation carriers of autosomal recessive diseases, 3.1% have mutations in cancer-predisposing genes, and 87.3% carry variants that affect drug response. We highlight how TWB data provide insight into both population history and disease burden, while showing how widespread genetic testing can be used to improve clinical care.

SAHA Overcomes 5-FU Resistance in IFIT2-Depleted Oral Squamous Cell Carcinoma Cells.

Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is a member of the interferon-stimulated gene family that contains tetratricopeptide repeats (TPRs), which mediate protein-protein interactions in various biological systems. We previously showed the depletion of IFIT2 enhanced cell migration and metastatic activity in oral squamous cell carcinoma (OSCC) cells via the activation of atypical PKC signaling. In this study, we found that IFIT2-knockdown cells displayed higher resistance to 5-fluorouracil (5-FU) than control cells. The comet assay and annexin V analysis showed decreased DNA damage and cell death in IFIT2-knockdown cells compared to control cells treated with 5-FU. Cell cycle progression was also perturbed by 5-FU treatment, with the accumulation of IFIT2-depleted cells in S phase in a time-dependent manner. We further observed the overexpression of thymidylate synthase (TS) and thymidine kinase (TK) in IFIT2-knockdown cells. Inhibition of TS alone or double inhibition of TS and TK1 using the siRNA technique increased susceptibility to 5-FU in IFIT2-knockdown cells. We further identified that suberanilohydroxamic acid (SAHA) treatment decreased the expression of TS in IFIT2-knockdown cells and demonstrated that pretreatment with SAHA sensitized IFIT2-knockdown cells to 5-FU in vitro and in vivo. In conclusion, IFIT2 knockdown enhances TS expression, which mediates 5-FU resistance, and SAHA pretreatment suppresses TS expression and hence sensitizes cells to 5-FU. SAHA will be an effective strategy for the treatment of OSCC patients with 5-FU resistance.

Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives.

A series of 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives and their alkyl (ethyl and isopropyl) carbamates and 12,13-bis(hydroxymethyl)-9,14-dihydro-dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives were synthesized for antiproliferative evaluation. The preliminary antitumour studies revealed that these two types of bis(hydroxymethyl) derivatives showed significant antitumour activities and were able to inhibit the growth of various human tumour cell lines in vitro. Several of the derivatives were demonstrated to cause DNA interstrand cross-links by an alkaline agarose gel shifting assay. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, delaying cell cycle progression in the G2/M phase and triggering apoptosis. Compound 21a, dissolved in a vehicle suitable for intravenous administration, was selected for antitumour studies in animal models. We demonstrated that at a dose that did not cause body weight loss in mice, compound 21a could significantly suppress the growth of tumour xenografts of human lung cancer H460 and colorectal cancer HCT-116 cells in nude mice. Our present results confirm the antitumour activities of these conjugates.

Combinatorial Low Dose Arsenic Trioxide and Cisplatin Exacerbates Autophagy via AMPK/STAT3 Signaling on Targeting Head and Neck Cancer Initiating Cells.

Head and neck squamous cell carcinoma (HNSCC) is a highly lethal disease with high-level of epidemic both in the world and Taiwan. Previous studies support that head and neck cancer-initiating cells (HN-CICs), a subpopulation of cancer cells with enhanced stemness properties, contribute to therapy resistance and tumor recurrence. Arsenic trioxide (As2O3; ATO) has shown to be an effective anti-cancer drug targeting acute promyelocytic leukemia (APL). Combinatorial treatment with high dose of ATO and cisplatin (CDDP) exert synergistic apoptotic effects in cancer cell lines of various solid tumors, however, it may cause of significant side effect to the patients. Nevertheless, none has reported the anti-cancerous effect of ATO/CDDP targeting HN-CICs. In this study, we aim to evaluate the low dose combination of ATO with conventional chemo-drugs CDDP treatment on targeting HN-CICs. We first analyzed the inhibitory tumorigenicity of co-treatment with ATO and chemo-drugs on HN-CICs which are enriched from HNSCC cells. We observed that ATO/CDDP therapeutic regimen successfully synergized the cell death on HN-CICs with a Combination Index (CI) <1 by Chou-Talalay's analysis in vitro. Interestingly, the ATO/CDDP regimen also induced exaggerated autophagy on HN-CICs. Additionally, this drug combination strategy also empowered both preventive and therapeutic effect by in vivo xenograft assays. Finally, we provide the underlying molecular mechanisms of ATO-based therapeutic regimen on HN-CICs. Together, low dose of combinatorial ATO/CDDP regimen induced cell death as well as exacerbated autophagy via AMPK-STAT3 mediated pathway in HN-CICs.

4(1H)-quinolone derivatives overcome acquired resistance to anti-microtubule agents by targeting the colchicine site of ß-tubulin.

Developing new therapeutic strategies to overcome drug resistance of cancer cells is an ongoing endeavor. From among 2 million chemicals, we identified ethyl 4-oxo-2-phenyl-1,4-dihydroquinoline-6-carboxylate (AS1712) as a low-toxicity inhibitor of lung cancer cell proliferation and xenograft tumor growth. We show that AS1712 is active against broad cancer cell lines and is able to bind in the colchicine-binding pocket of ß-tubulin, thereby inhibiting microtubule assembly and, consequently, inducing mitotic arrest and apoptosis. Our cell-based structure-activity relationship study identified a new lead compound, RJ-LC-15-8, which had a greater anti-proliferative potency for H1975 cells than did AS1712, while maintaining a similar mechanism of action. Notably, AS1712 and RJ-LC-15-8 overcame P-glycoprotein efflux pump and ß-tubulin alterations that lead to acquired resistance against microtubule-targeting drugs of cancer cells. AS1712 and RJ-LC-15-8 may be lead compounds that overcome acquired resistance to microtubule-targeting agents of cancer cells.

Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine Hybrids as Potent Anticancer Agents that Inhibit Angiogenesis and Induce DNA Interstrand Cross-links.

Hybrid molecules are composed of two pharmacophores with different biological activities. Here, we conjugated phthalazine moieties (antiangiogenetic pharmacophore) and bis(hydroxymethyl)pyrrole moieties (DNA cross-linking agent) to form a series of bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine hybrids. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M phase, triggering apoptosis, and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g., 29dL) significantly suppressed the growth of small-cell lung cancer cell (H526) xenografts in mice. Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the intensity of γ-H2AX, a marker of DNA damage, increased. The present data revealed that the conjugation of antiangiogenic and DNA-damaging agents can generate potential hybrid agents for cancer treatment.

IFIT1 and IFIT3 promote oral squamous cell carcinoma metastasis and contribute to the anti-tumor effect of gefitinib via enhancing p-EGFR recycling.

IFIT1 and IFIT3 are abundant products of interferon-stimulating genes. While the importance of IFIT1 and IFIT3 in the prognosis of cancer has been reported, the molecular basis of IFIT1 and IFIT3 in cancer progression remains unexplored. In the present study, we investigated the modes of action and the clinical significance of IFIT1 and IFIT3 in oral squamous cell carcinoma (OSCC). Ectopic expression of IFIT1 or IFIT3 induced OSCC cell invasion by promoting the epithelial-mesenchymal transition, whereas IFIT1 or IFIT3 knockdown exhibited opposite effects. Overexpression of IFIT1 or IFIT3 promoted tumor growth, regional and distant metastasis in xenograft and orthotopic nude mice models. Most importantly, IFIT1 or IFIT3 overexpression increased the levels of p-EGFRY1068 and p-AKTS473 in OSCC cells and also enhanced tumor inhibitory effect of gefitinib. By immunoprecipitation and LC-MS/MS analysis, we found that IFIT1 and IFIT3 interacted with ANXA2 that enhanced p-EGFRY1068 endosomal recycling. Depletion of ANXA2 using siRNA therefore abolished p-EGFRY1068 and p-AKTS473 expression in IFIT1- or IFIT3-overexpressed cells. Furthermore, a significant positive association of increased IFIT1 and IFIT3 expression with advanced T-stage, lymph node metastasis, perineural invasion, lymphovascular invasion, extranodal extension, and poor overall survival rate was confirmed in OSCC patients. We also found a statistically positive correlation of p-EGFRY1068 expression with IFIT1 and IFIT3 in OSCC tumors and poor clinical outcome in patients. Collectively, we demonstrated a novel role of IFIT1 and IFIT3 in driving OSCC progression and metastasis by interacting with ANXA2 and hence enhancing p-EGFR recycling and its downstream signaling.

Emerging Functions of Human IFIT Proteins in Cancer.

Interferon-induced protein with tetratricopeptide repeats (IFIT) genes are prominent interferon-stimulated genes (ISGs). The human IFIT gene family consists of four genes named IFIT1, IFIT2, IFIT3, and IFIT5. The expression of IFIT genes is very low in most cell types, whereas their expression is greatly enhanced by interferon treatment, viral infection, and pathogen-associated molecular patterns (PAMPs). The proteins encoded by IFIT genes have multiple tetratricopeptide repeat (TPR) motifs. IFIT proteins do not have any known enzymatic roles. However, they execute a variety of cellular functions by mediating protein-protein interactions and forming multiprotein complexes with cellular and viral proteins through their multiple TPR motifs. The versatile tertiary structure of TPR motifs in IFIT proteins enables them to be involved in distinct biological functions, including host innate immunity, antiviral immune response, virus-induced translation initiation, replication, double-stranded RNA signaling, and PAMP recognition. The current understanding of the IFIT proteins and their role in cellular signaling mechanisms is limited to the antiviral immune response and innate immunity. However, recent studies on IFIT protein functions and their involvement in various molecular signaling mechanisms have implicated them in cancer progression and metastasis. In this article, we focused on critical molecular, biological and oncogenic functions of human IFIT proteins by reviewing their prognostic significance in health and cancer. Research suggests that IFIT proteins could be novel therapeutic targets for cancer therapy.

Prognostic Values of EPDR1 Hypermethylation and Its Inhibitory Function on Tumor Invasion in Colorectal Cancer.

Aberrant DNA methylation is a potential mechanism underlying the development of colorectal cancer (CRC). Thus, identification of prognostic DNA methylation markers and understanding the related molecular functions may offer a new perspective on CRC pathogenesis. To that end, we explored DNA methylation profile changes in CRC subtypes based on the microsatellite instability (MSI) status through genome-wide DNA methylation profiling analysis. Of 34 altered genes, three hypermethylated (epidermal growth factor, EGF; carbohydrate sulfotransferase 10, CHST10; ependymin related 1, EPDR1) and two hypomethylated (bone marrow stromal antigen 2, BST2; Rac family small GTPase 3, RAC3) candidates were further validated in CRC patients. Based on quantitative methylation-specific polymerase chain reaction (Q-MSP), EGF, CHST10 and EPDR1 showed higher hypermethylated levels in CRC tissues than those in adjacent normal tissues, whereas BST2 showed hypomethylation in CRC tissues relative to adjacent normal tissues. Additionally, among 75 CRC patients, hypermethylation of CHST10 and EPDR1 was significantly correlated with the MSI status and a better prognosis. Moreover, EPDR1 hypermethylation was significantly correlated with node negativity and a lower tumor stage as well as with mutations in B-Raf proto-oncogene serine/threonine kinase (BRAF) and human transforming growth factor beta receptor 2 (TGFßR2). Conversely, a negative correlation between the mRNA expression and methylation levels of EPDR1 in CRC tissues and cell lines was observed, revealing that DNA methylation has a crucial function in modulating EPDR1 expression in CRC cells. EPDR1 knockdown by a transient small interfering RNA significantly suppressed invasion by CRC cells, suggesting that decreased EPDR1 levels may attenuate CRC cell invasion. These results suggest that DNA methylation-mediated EPDR1 epigenetic silencing may play an important role in preventing CRC progression.

HSP40 co-chaperone protein Tid1 suppresses metastasis of head and neck cancer by inhibiting Galectin-7-TCF3-MMP9 axis signaling.

Human tumorous imaginal disc (Tid1), a DnaJ co-chaperone protein, is classified as a tumor suppressor. Previously, we demonstrated that Tid1 reduces head and neck squamous cell carcinoma (HNSCC) malignancy. However, the molecular details of Tid1-mediated anti-metastasis remain elusive. Methods: We used affinity chromatography and systemic mass spectrometry to identify Tid1-interacting client proteins. Immunohistochemical staining of Tid1 in HNSCC patient tissues was examined to evaluate the association between the expression profile of Tid1-interacting client proteins with pathologic features and prognosis. The roles of Tid1-interacting client proteins in metastasis were validated both in vitro and in vivo. The interacting partner and downstream target of Tid1-interacting client protein were determined. Results: Herein, we first revealed that Galectin-7 was one of the Tid1-interacting client proteins. An inverse association of protein expression profile between Tid1 and Galectin-7 was determined in HNSCC patients. Low Tid1 and high Galectin-7 expression predicted poor overall survival in HNSCC. Furthermore, Tid1 abolished the nuclear translocation of Galectin-7 and suppressed Galectin-7-induced tumorigenesis and metastasis. Keratinocyte-specific Tid1-deficient mice with 4-nitroquinoline-1-oxide (4NQO) treatment exhibited increased protein levels of Galectin-7 and had a poor survival rate. Tid1 interacted with Galectin-7 through its N-linked glycosylation to promote Tid1-mediated ubiquitination and proteasomal degradation of Galectin-7. Additionally, Galectin-7 played a critical role in promoting tumorigenesis and metastatic progression by enhancing the transcriptional activity of TCF3 transcription factor through elevating MMP-9 expression. Conclusions: Overall, future treatments through activating Tid1 expression or inversely repressing the oncogenic function of Galectin-7 may exhibit great potential in targeting HNSCC progression.

Upregulation of E3 Ubiquitin Ligase CBLC Enhances EGFR Dysregulation and Signaling in Lung Adenocarcinoma.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family, which has E3 ubiquitin ligase activity toward activated receptor tyrosine kinases. CBLC is frequently upregulated in non-small cell lung cancer (NSCLC), yet very little is known about the functions of CBLC in tumorigenesis. Here we show that CBLC is an epigenetically demethylated target and its expression can be upregulated in NSCLC after treatment with the DNA methylation inhibitor 5'-azacytidine. Depletion of CBLC significantly inhibited cell viability and clonogenicity in vitro and reduced tumor growth in a xenograft model. CBLC silencing further sensitized EGFR-mutated NSCLC cells to treatment with tyrosine kinase inhibitors. Conversely, ectopic expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by competing with CBL for EGFR binding. Analysis of ubiquitin linkages on activated EGFR (aEGFR) revealed that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. This CBLC-mediated polyubiquitination promoted either preferential recycling of aEGFR back to the plasma membrane or trafficking to the cell nucleus. IHC analyses revealed a positive correlation between phospho-EGFR and CBLC in lung adenocarcinoma. In summary, we demonstrate a novel mechanism by which aEGFR escapes lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy.Significance: This work demonstrates the role of CBLC expression as a diagnostic biomarker and potential therapeutic target in lung adenocarcinoma. Cancer Res; 78(17); 4984-96. ©2018 AACR.

Arsenic trioxide-mediated suppression of miR-182-5p is associated with potent anti-oxidant effects through up-regulation of SESN2.

Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 (SESN2) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3'- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

ROS-independent ER stress-mediated NRF2 activation promotes warburg effect to maintain stemness-associated properties of cancer-initiating cells.

Cancer-initiating cells (CICs) are responsible for tumor initiation, progression, and therapeutic resistance; moreover, redox homeostasis is important in regulating cancer stemness. Previously, we have identified that cancer cells containing low intracellular reactive oxygen species levels (ROSLow cells) display enhanced features of CICs. However, the specific metabolic signatures of CICs remain unclear and are required for further characterization by systemic screenings. Herein, we first showed CICs mainly relying on glycolysis that was important for the maintenance of stemness properties. Next, we revealed that NRF2, a master regulator of antioxidants, was able to maintain low intracellular ROS levels of CICs, even though in the absence of oxidative stress. We further characterized that NRF2 activation was required for the maintenance of CICs properties. Of ROSLow cells, NRF2 activation not only directly activates the transcription of genes encoding glycolytic enzymes but also inhibited the conversion of pyruvate to acetyl-CoA by directly activating pyruvate dehydrogenase kinase 1 (PDK1) to lead to inhibition of tricarboxylic acid (TCA) cycle; therefore, to promote Warburg effect. A positive regulatory ROS-independent ER stress pathway (GRP78/p-PERK/NRF2 signaling) was identified to mediate the metabolic shift (Warburg effect) and stemness of CICs. Lastly, co-expression of p-PERK and p-NRF2 was significantly associated with the clinical outcome. Our data show that NRF2 acting as a central node in the maintenance of low ROS levels and stemness associated properties of the CICs, which is significantly associated with the clinical outcome, but independent from ROS stress. Future treatments by inhibiting NRF2 activation may exhibit great potential in targeting CICs.

A Low-Toxicity DNA-Alkylating N-Mustard-Quinoline Conjugate with Preferential Sequence Specificity Exerts Potent Antitumor Activity Against Colorectal Cancer.

Efficacy and safety are fundamental prerequisites for anticancer drug development. In the present study, we explored the anti-colorectal cancer (CRC) activity of SL-1, a DNA-directed N-mustard-quinoline conjugate. The N-mustard moiety in SL-1 induced DNA strand breaks, interstrand cross-links (ICLs), G2/M arrest, and apoptosis, whereas its quinoline moiety preferentially directed SL-1 to target the selective guanine sequence 5'-G-G/C-N-G-C/T-3'. Notably, SL-1 was highly cytotoxic to various CRC cell lines. Experiments using xenograft models revealed that SL-1 was more potent than 5-fluorouracil (5-FU) and oxaliplatin for suppressing the growth of RKO and RKO-E6 (oxaliplatin-resistant subline) cells as well as metastatic SW620 cells. In addition, SL-1 combined with 5-FU was more effective than oxaliplatin and 5-FU for suppressing RKO or SW620 cell growth in mice. Significantly, compared with cisplatin, oxaliplatin, or 5-FU, SL-1 alone or in combination with 5-FU did not cause obvious kidney or liver toxicity in ICR mice. In summary, SL-1, a DNA-directed alkylating agent, is established as an anti-CRC agent with high efficacy and low toxicity and thus warrants further development for the treatment of CRC patients.

Novel N-mustard-benzimidazoles/benzothiazoles Hybrids, Synthesis and Anticancer Evaluation.

BACKGROUND: Bendamustine, an N-mustard-benzoimidazole hybrid conjugate, was recently approved for the treatment of chronic lymphocytic leukemia. However, the short half-life of bendamustine may limit its clinical applications. OBJECTIVE: The purpose of this study is to design and synthesize compounds with a more favorable pharmacokinetic profile. METHODS: We synthesized a series of hybrid molecules comprising a phenyl N-mustard moiety and benzothiazole or benzimidazole scaffold linked via a urea linker and evaluated their antitumor activity and plasma stability. RESULTS: We revealed that these agents exhibited significant cytotoxicity against a panel of human lymphoblastic leukemia and human solid tumor cells in culture. Human lymphoblastic leukemia CCRM-CEM cells were the most sensitive to the tested compounds. In general, the new hybrids were as potent as cisplatin, but significantly more cytotoxic than bendamustine. Phenyl N-mustard-benzothiazole compound 27d and phenyl N-mustardbenzimidaloe compound 32b possessed significant cytotoxicity and led to apoptotic death in the treated tumor cells. These two agents were able to induce DNA interstrand cross-linking and arrested cell cycle progression at the G2/M phase. Furthermore, we showed that these new hybrids were more chemically stable than bendamustine in rat plasma. CONCLUSION: Our results suggest that conjugation of phenyl N-mustard pharmacophore at C6 of benzimidazole or at C8 of the benzothiazole ring via a urea linker is likely an approach to increase the chemical stability and bioavailability. Highlights ⇒ Series of benzimidazoles and benzothiazoles linked to N-mustard were synthesized. ⇒ The newly synthesized derivatives induced DNA interstrand cross-links. ⇒ These derivatives induced cell cycle arrest in the G2/M phase and triggered apoptosis in H460 cells. ⇒ The new compounds are more cytotoxic than bendamustine. ⇒ The new compounds were chemically more stable than bendamustine in rat plasma.

Promoter hypermethylation of LGALS4 correlates with poor prognosis in patients with urothelial carcinoma.

Galectine-4 (gal-4), encoded by the LGALS4 gene, was recently shown to exhibit a tumor suppressive effect in colorectal carcinoma and pancreatic adenocarcinoma, although how the expression of this gene is regulated remains unknown. No reports describe the significance of gal-4 in the malignant potential of urothelial tumors. Thus, we analyzed LGALS4 methylation and gene expression and their clinical relevance and biological function in urothelial carcinoma (UC). LGALS4 methylation was initially identified as a progression biomarker for UC patients through genome-wide DNA methylation profiling of 16 tumor samples. Bisulfite sequencing PCR and immunohistochemistry were performed to validate the promoter methylation and expression of LGALS4. We used quantitative methylation-specific PCR to determine the methylation levels of LGALS4 normalized to ACTB in the tumor samples of 79 UC patients and compared the levels between patients with different clinicopathological characteristics. The association with survival probability was analyzed with the Kaplan-Meier method and Cox regression analysis. The ectopic expression of gal-4 in cancer cell lines was used to address its biological function in UC in vitro. The promoter hypermethylation of LGALS4 (>2.51, log10 scale) revealed a positive correlation with high levels of both histological grade and tumor T category and with lymph node metastasis (all P≤0.001). In addition, LGALS4 hypermethylation was an independent predictor of inferior survival in UC patients (P<0.05). The ectopic expression studies demonstrated that gal-4 suppressed urothelial cancer cell growth, migration, and invasion. Thus, LGALS4 may function as a tumor suppressor gene in UC progression. Our findings provide evidence that methylation-mediated LGALS4 gene repression may be involved in urothelial tumor progression.