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Lung adenocarcinoma (LUAD) causes severe cancer death worldwide. E2F2 is a canonical transcription factor implicated in transcription regulation, cell cycle and tumorigenesis. The role of E2F2 as well as its transcription regulatory network in LUAD remains obscure. In this study, we constructed a weighted gene co-expression network and identified several key modules and networks overrepresented in LUAD, including the E2F2-centered transcription regulatory network. Function analysis revealed that E2F2 overexpression accelerated cell growth, cell cycle progression and cell motility in LUAD cells whereas E2F2 knockdown inhibited these malignant phenotypes. Mechanistic investigations uncovered various E2F2-regulated downstream genes and oncogenic signaling pathways. Notably, three core transcription factors of E2F2, B-Myb and FOXM1 from the LUAD transcription regulatory network exhibited positive expression correlation, associated with each other, mutually transactivated each other, and regulated similar downstream gene cascades, hence constituting a consolidated core transcription regulatory circuitry. Moreover, E2F2 could promote and was essentially required for LUAD growth in orthotopic mouse models. Prognosis modeling revealed that a two-gene signature of E2F2 and PLK1 from the transcription regulatory circuitry remarkably stratified patients into low- and high-risk groups. Collectively, our results clarified the critical roles of E2F2 and the exquisite core transcription regulatory circuitry of E2F2/B-Myb/FOXM1 in LUAD progression.

Filopodia, a finger-like structure and actin-rich plasma-membrane protrusion at the leading edge of the cell, has important roles in cell motility. However, the mechanisms of filopodia generation are not well-understood via the actin-related protein 2/3 (ARP2/3) complex in Non-Small Cell Lung Cancer (NSCLC) cells. We previously have demonstrated that PRR11 associates with the ARP2/3 complex to regulate cytoskeleton-nucleoskeleton assembly and chromatin remodeling. In this study, we further demonstrate that PRR11 involves in filopodia formation, focal adhesion turnover and cell motility through ARP2/3 complex. Cell phenotype assays revealed that the silencing of PRR11 increased cellular size and inhibited cell motility in NSCLC cells. Mechanistically, PRR11 recruited and co-localized with Arp2 at the membrane protrusion to promote filopodia formation but not lamellipodia formation. Notably, PRR11 mutant deletion of the proline-rich region 2 (amino acid residues 185–200) abrogated the effect of filopodia formation. In addition, PRR11-depletion inhibited filopodial actin filaments assembly and increased the level of active integrin β1 in the cell surface, whereas reduced the phosphorylation level of focal adhesion kinase (FAKY397) to repress focal adhesion turnover and cell motility in NSCLC cells. Taken together, our findings indicate that PRR11 has critical roles in controlling filopodia formation, focal adhesion turnover and cell motility by recruiting ARP2/3 complex, thus dysregualted expression of PRR11 potentially facilitates tumor metastasis in NSCLC cells.

B-Myb is an important transcription factor that plays a critical role in gene expression regulation and tumorigenesis. However, its functional implication in colorectal cancer remains elusive. In this study, we found that B-Myb was significantly upregulated at both mRNA and protein levels in colorectal cancer samples compared to non-tumor counterparts. B-Myb overexpression accelerated cell proliferation, cell cycle progression and cell motility in colorectal cancer cells, and promoted tumor growth in orthotopic nude mouse models in vivo. In contrast, B-Myb depletion inhibited these malignant phenotypes. Mechanistic investigations revealed that E2F2 was a novel transcriptional target of B-Myb and is essential to B-Myb-induced malignant phenotypes. Notably, B-Myb and E2F2 exhibited positive expression correlation, and interacted with each other in colorectal cancer cells. In addition to their autoregulatory mechanisms, B-Myb and E2F2 can also directly transactivate each other, thus constituting consolidated reciprocal feed-forward transactivation loops. Moreover, both B-Myb and E2F2 are required for the activation of ERK and AKT signaling pathways in colorectal cancer cells. Taken together, our data clarified a critical role for B-Myb in colorectal cancer and unraveled an exquisite mutual collaboration and reciprocal cross regulation between B-Myb and E2F2 that contribute to the malignant progression of human colorectal cancer.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Radiotherapy plays a major role in the management of nasopharyngeal carcinoma (NPC). However, the radioresistant cells limit its efficiency and drive recurrence inside the irradiated tumor volume leading to poor outcome for patients. To illuminate the signal pathway involved in the radioresistance and evaluate the potential for predicting NPC response to radiotherapy, we established the radioresistant NPC cell line (CNE2-RR) derived from NPC cell line CNE2 by gradually increased the radiation dose (total 60 Gy), and the radioresistance of CNE2-RR cells was evaluated by the colony formation, FCM and comet assays. Furthermore, comparison of established CNE2-RR cell line to parental cell line found the homologous recombination repair (HRR) proteins differences involved in NPC radioresistance. In addition, the differentially expressed proteins were further validated by western blotting, immunofluorescence and IHC in tumor xenografs and radioresistant NPC tissues. Furthermore, the correlation of HRR proteins expression levels with NPC radioresistance were evaluated. The results showed that the upregulation of HRR proteins were significantly correlated with NPC radioresistance. In addition, using the Youden Index cutoff value, a panel of the HRR proteins analyses revealed a sensitivity of 70%, specificity of 72%. Furthermore, silencing NFBD1 enhanced the radiosensitivity of CNE2-RR cells by impairing IR-inducing γ-H2AX and HR proteins foci formation. These results suggest that controlling the HRR signaling pathway may hold promise to overcome NPC radioresistance.

Nasopharyngeal carcinoma (NPC) is a common malignant carcinoma of the head and neck, and the biological mechanisms underlying the pathogenesis of NPC remain not fully understood. In the present study, we systematically analyzed four independent NPC transcriptomic datasets and focused on identifying the critical molecular networks and novel key hub genes implicated in NPC. We found totally 170 common overlapping differentially expressed genes (DEGs) in the four NPC datasets. GO and KEGG pathway analysis revealed that cell cycle dysregulation is a critical event in NPC. Protein–protein interaction (PPI) network analysis identified a 15 hub-gene core network with overexpressed kinesin family member 2C (KIF2C) as a central regulator. Loss-of-function study demonstrated that knockdown of KIF2C significantly inhibited cell growth and cell motility, and delayed cell cycle progression, accompanied with dramatic mitotic defects in spindle formation in NPC cells. RNA-seq analysis revealed that KIF2C knockdown led to deregulation of various downstream genes. KIF2C could also regulate the AKT/mTOR pathways, and enhance paclitaxel sensitivity in NPC cells. Taken together, our results suggest that cell cycle dysregulation is a critical event during NPC pathogenesis and KIF2C is a novel key mitotic hub gene with therapeutic potential in NPC.

Rationale: Colorectal cancer (CRC) is one of the most common cancers worldwide. Ciclopirox olamine (CPX) has recently been identified to be a promising anticancer candidate; however, novel activities and detailed mechanisms remain to be uncovered. Methods: The cytotoxic potential of CPX towards CRC cells was examined in vitro and in vivo. The global gene expression pattern, ROS levels, mitochondrial function, autophagy, apoptosis, etc. were determined between control and CPX-treated CRC cells. Results: We found that CPX inhibited CRC growth by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. The anti-cancer effects of CPX involved the downregulation of DJ-1, and overexpression of DJ-1 could reverse the cytotoxic effect of CPX on CRC cells. The loss of DJ-1 resulted in mitochondrial dysfunction and ROS accumulation, thus leading to CRC growth inhibition. The cytoprotective autophagy was provoked simultaneously, and blocking autophagy pharmacologically or genetically could further enhance the anti-cancer efficacy of CPX. Conclusion: Our study demonstrates that DJ-1 loss-induced ROS accumulation plays a pivotal role in CPX-mediated CRC inhibition, providing a further understanding for CRC treatment via modulating compensatory protective autophagy.

Data in this article are associated with the research article "Ampholytic ion-exchange materials coated with small zwitterion for high-efficacy purification of ionizable soluble biomacromolecules" (Rao et al., 2018) [1]. This article provided data on how to design ampholytic ion-exchange material (AIEM) for the purification of ionizable soluble biomacromolecules for both high activity yields and favorable homogeneity, with two uricases as protein models and a plasmid as DNA model. Data were made publicly available for further analyses.

For the purification of soluble proteins and nucleic acids through ion-exchange, the ampholytic ion-exchange materials (AIEMs) were designed, which possessed both short aliphatic carboxyl and short aliphatic amines/imidazole at optimized ratios on solid supports coated with high density of small zwitterions; under optimized conditions, the soluble ionizable biomacromolecules were adsorbed on those AIEMs via electrostatic attractions and eluted effectively through electrostatic repulsions. As the proof-of-concept, magnetic submicron particles bearing short aliphatic carboxyl and the coats of small zwitterion served as the starting solid supports, which were conjugated with lysine alone, or with lysine plus glycine or N,N‑dimethylethylenediamine, to yield magnetic AIEMs whose surfaces possessed zero net charges at different pH. Such magnetic AIEMs exhibited ideal efficacy to release acid red 13 as an anion at the elution pH optimized for strong electrostatic repulsions; those magnetic AIEMs were proven absorbing under optimized conditions for the purification of soluble proteins stable at pH close to their isoelectric points and solid-phase extraction of nucleic acids in applicable biological mixtures. Therefore, the designed AIEMs are promising for the high-efficacy purification of ionizable soluble biomacromolecules.

BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

Our previous studies have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene and implicates in regulating the proliferation in lung cancer. However, its precise role in cell cycle progression remains unclear. Our recent evidences show that PRR11 silencing has an effect on autophagy in non-small-cell lung cancer (NSCLC) cells. Two human NSCLC cell lines, H1299 and A549 were transiently transfected with against PRR11 siRNA. The Cell Counting Kit-8 and plate clone formation assay showed that downregulation of PRR11 inhibited the cell proliferation associated with cell cycle related genes reduced. And our data suggested that PRR11 depletion expression enhanced the autophagosomes formation, followed with downregulation of P62 and upregulation of LC3-II protein. Furthermore, the immunoblotting results indicated that silencing of PRR11 inactivated the Akt/mTOR signaling pathway. Collectively, these results demonstrated PRR11 had an effective role in autophagy in NSCLC cells through Akt/mTOR autophagy signaling pathways. Therefore, it is helpful to clarify the function of PRR11 in tumorigenesis of NSCLC. Keywords: Akt/mTOR, Autophagy, Cell proliferation, Non-small-cell lung cancer, PRR11

// Xuefei Feng 1, * , Chundong Zhang 2, * , Ling Zhu 1 , Lian Zhang 2 , Hongxia Li 1 , Longxia He 1 , Yan Mi 1 , Yitao Wang 2 , Jiang Zhu 1 and Youquan Bu 2 1 Department of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China 2 Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China * These authors have contributed equally to this work Correspondence to: Jiang Zhu, email: zhujiang163mail@163.com Youquan Bu, email: buyqcn@cqmu.edu.cn , buyqcn@aliyun.com Keywords: DEPDC1, cell cycle, mitosis, nasopharyngeal carcinoma Received: January 09, 2017 Accepted: June 02, 2017 Published: June 29, 2017 ABSTRACT DEP domain containing 1 (DEPDC1) is a newly identified cancer-related and cell cycle related gene and has been demonstrated as a novel therapeutic target for bladder cancer. However, the functional involvement and therapeutic potential of DEPDC1 in nasopharyngeal carcinoma (NPC) remains unclear. Our results showed that DEPDC1 was overexpressed at both mRNA and protein levels in NPC tissues compared with normal or non-tumor tissues. The siRNA-mediated DEPDC1 depletion resulted in significant inhibition of proliferation and delay in cell cycle progression in both NPC cell lines, CNE-1 and HNE-1. Detailed analysis with indirect immunofluorescence assays revealed that DEPDC1 depletion caused significant mitotic arrest accompanied with mitotic defects such as multipolar spindles and multiple nuclei followed by apoptotic cell death. Notably, DEPDC1 depletion also reduces migration and invasion ability in both cell lines. Consistent with its regulatory role in NF-κB pathway, knockdown of DEPDC1 caused significant upregulation of A20 and downregulation of mutiple NF-κB downstream target genes implicated in proliferation and tumorigenesis (c-Myc, BCL2, CCND1, CCNB1 and CCNB2), and metastasis (MMP2, MMP9, ICAM1, vimentin, Twist1). Moreover, in vivo study demonstrated that DEPDC1 knockdown also caused significant inhibition of tumor growth in the NPC xenograft nude mouse model. Taken together, our present study demonstrated that DEPDC1 is essentially required for the accelerated cell cycle progression and motility in NPC cells, and strongly suggested that DEPDC1 may serve as a novel therapeutic target in NPC.

Redox imbalance is resulted from the destruction of balance between oxidants and antioxidants. The dominant oxidants are reactive oxygen species (ROS), which are involved in multiple cellular processes by physiologically transporting signal as a second messenger or pathologically oxidizing DNA, lipids, and proteins. Generally speaking, low concentration of ROS is indispensable for cell survival and proliferation. However, high concentration of ROS is cytotoxic. Additionally, ROS are now known to induce the oxidative modification of macromolecules especially proteins. The redox modification of proteins is involved in numerous biological processes related to diseases including CRC. Herein, we attempt to afford an overview that highlights the crosstalk between redox imbalance and CRC.

We previously demonstrated that proline-rich protein 11 (PRR11) and spindle and kinetochore associated 2 (SKA2) constituted a head-to-head gene pair driven by a prototypical bidirectional promoter. This gene pair synergistically promoted the development of non-small cell lung cancer. However, the signaling pathways leading to the ectopic expression of this gene pair remains obscure. In the present study, we first analyzed the lung squamous cell carcinoma (LSCC) relevant RNA sequencing data from The Cancer Genome Atlas (TCGA) database using the correlation analysis of gene expression and gene set enrichment analysis (GSEA), which revealed that the PRR11-SKA2 correlated gene list highly resembled the Hedgehog (Hh) pathway activation-related gene set. Subsequently, GLI1/2 inhibitor GANT-61 or GLI1/2-siRNA inhibited the Hh pathway of LSCC cells, concomitantly decreasing the expression levels of PRR11 and SKA2. Furthermore, the mRNA expression profile of LSCC cells treated with GANT-61 was detected using RNA sequencing, displaying 397 differentially expressed genes (203 upregulated genes and 194 downregulated genes). Out of them, one gene set, including BIRC5, NCAPG, CCNB2, and BUB1, was involved in cell division and interacted with both PRR11 and SKA2. These genes were verified as the downregulated genes via RT-PCR and their high expression significantly correlated with the shorter overall survival of LSCC patients. Taken together, our results indicate that GLI1/2 mediates the expression of the PRR11-SKA2-centric gene set that serves as an unfavorable prognostic indicator for LSCC patients, potentializing new combinatorial diagnostic and therapeutic strategies in LSCC.

Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer that is prevalent among people of southern Chinese ancestry in southern China and Southeast Asia. Radiotherapy and cisplatin (CDDP)-based chemotherapy are the main treatment options. Unfortunately, disease response to concurrent chemoradiotherapy varies among patients with NPC, and many cases are resistant to CDDP and radiotherapy. NFBD1 functions in cell cycle checkpoint activation and DNA repair following DNA damage. In this study, we identified the NFBD1 as a tractable molecular target to chemosensitize NPC cells. NFBD1 expression in NPC CNE1 cell lines was depleted using lentivirus-mediated short hairpin RNA, and the elevated sensitivity of these NFBD1-inhibited NPC cells to therapeutic reagent CDDP and 5-fluorouracil (5-FU) was evaluated using MTS assays. Flow cytometry analysis also showed that NFBD1 knockdown led to an obvious induction of apoptosis in CDDP- or 5-FU-treated CNE1 cells. Furthermore, we implicated the involvement of NFBD1 in Rad51 and DNA-PKcs foci formation following CDDP or 5-FU chemotherapy. In conclusion, NFBD1 knockdown improves the chemosensitivity of NPC cells by inhibiting cell growth and promoting apoptosis through the impairment of DNA damage repair, suggesting NFBD1 as a novel therapeutic target for NPC.

Lysyl oxidase like 2, LOXL2, as a member of the lysyl oxidase (LOX) family, has been shown to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 is also engaged to transcription regulation, cell signaling transduction and cell adhesion regulation. It has been reported that LOXL2 is highly expressed in several types of tumors and promotes cell proliferation and migration in various cancer cells. However, the regulatory mechanism of LOXL2 expression remains largely unknown. To further investigate its transcriptional regulatory mechanism, LOXL2 promoter region has been cloned and identified in the present study. Chromatin state analysis revealed that LOXL2 gene locus contained an active promoter near its first exon. We then constructed five different LOXL2 gene promoter luciferase reporter constructs covering 1.7 kb upstream of LOXL2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all the five constructs showed notable promoter activity, and LOXL2 core promoter was located in a region of 185 bp near the transcription initiation site. Transcriptional factor binding analysis indicated that, LOXL2 promoter lacked classical TATA box, but contained putative binding sites for classic transcriptional factors such as Sp1 and NF-κB. Ectopic overexpression of Sp1 significantly enhanced LOXL2 promoter activity as well as its endogenous expression in cells. In contrast, mithramycin A (a selective Sp1 inhibitor) treatment repressed LOXL2 promoter as well as its endogenous transcription. Site directed mutagenesis assay further confirmed that the Sp1 binding sites were essential for proximal prompter activity of LOXL2 gene. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 bound LOXL2 promoter in vivo. Of note, the expression of Sp1 and LOXL2 are positively correlated, and the higher expression of LOXL2 is associated with poor prognosis in colorectal cancer, strongly suggesting the implication of Sp1-mediated LOXL2 transactivation in the pathogenesis of colorectal cancer.

Our previous study found that two novel cancer-related genes, PRR11 and SKA2, constituted a classic gene pair that was regulated by p53 and NF-Y in lung cancer. However, their role and regulatory mechanism in breast cancer remain elusive. In this study, we found that the expression levels of PRR11 and SKA2 were upregulated and have a negative prognotic value in breast cancer. Loss-of-function experiments showed that RNAi-mediated knockdown of PRR11 and/or SKA2 inhibited proliferation, migration, and invasion of breast cancer cells. Mechanistic experiments revealed that knockdown of PRR11 and/or SKA2 caused dysregulation of several downstream genes, including CDK6, TPM3, and USP12, etc. Luciferase reporter assays demonstrated that wild type p53 significantly repressed the PRR11-SKA2 bidirectional promoter activity, but not NF-Y. Interestingly, NF-Y was only essential for and correlated with the expression of PRR11, but not SKA2. Consistently, adriamycin-induced (ADR) activation of endogenous p53 also caused significant repression of the PRR11 and SKA2 gene pair expression. Notably, breast cancer patients with lower expression levels of either PRR11 or SKA2, along with wild type p53, exhibited better disease-free survival compared to others with p53 mutations and/or higher expression levels of either PRR11 or SKA2. Collectively, our study indicates that the PRR11 and SKA2 transcription unit might be an oncogenic contributor and might serve as a novel diagnostic and therapeutic target in breast cancer. [BMB Reports 2019; 52(2): 157-162].

Metastasis remains a big barrier for the clinical treatment of colorectal cancer (CRC). Our previous proteomics analysis identified DJ-1 as a potential metastasis biomarker of CRC. In this study, we found that DJ-1 was upregulated in CRC. The levels of DJ-1 were closely correlated with the depths of invasion and predicted patient outcome. Enforced expression of DJ-1 could enhance CRC proliferation and metastasis in vitro and in vivo by stimulating Wnt-β-catenin signaling. Specifically, DJ-1-induced β-catenin nuclear translocation stimulated TCF transcription activity, which promoted BMP4 expression for CRC cell migration and invasion, and elevated CCND1 expression for CRC cell proliferation, respectively. Furthermore, DJ-1-induced Wnt signaling activation was dependent on PLAGL2 expression. In conclusion, our study demonstrates that DJ-1 can promote CRC metastasis by activating PLAGL2–Wnt–BMP4 axis, suggesting novel therapeutic opportunities for postoperative adjuvant therapy in CRC patients.

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.

Hyaluronan (HA) is a key component of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the hyaluronan synthases including HAS1, HAS2 and HAS3. The expression and regulation of HAS1-3 are implicated in numerous physiological and pathological processes. The promoters of human HAS1 and HAS2 genes have been identified previously whereas HAS3 promoter remains unclear. In the present study, we have for the first time identified and characterized the human HAS3 gene promoter region. 5' RACE assay revealed two novel transcriptional variants of HAS3 gene with distinct transcription start sites. Progressive deletion analysis of the 5'-flanking region of HAS3 gene demonstrated that HAS3 proximal promoter is mainly restricted to a 450-bp region (i.e. -761 to -305 bp upstream of the major HAS3 transcription start site), whereas its core promoter is located to a minimal 129-bp region (i.e. -433 to -305 bp upstream of the major HAS3 transcription start site). Transcriptional factor binding analysis indicated that HAS3 gene promoter lacks of canonical TATA box, but contains classical GC box as well as other putative binding sites for transcriptional factors such as C/EBP and NFκB. In addition, site-directed mutagenesis assay demonstrated that the proximal Sp1 binding site is essential for the robust proximal promoter activity of HAS3 gene whereas the core MTE (core promoter motif ten elements) motif is required for the basic core promoter activity of HAS3 gene. Our present study should facilitate further studies on the mechanism regulating the expression of this important gene.

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]

// Ya Zhou 1, 2, * , Yuanyuan Zhu 1, 2, * , Xiaoyan Fan 1, 2, * , Chundong Zhang 1, 2 , Yitao Wang 1, 2 , Lian Zhang 1, 2 , Huan Zhang 3 , Tao Wen 4 , Kaina Zhang 3 , Xiao Huo 5 , Xue Jiang 1, 2 , Youquan Bu 1, 2 and Ying Zhang 1, 2 1 Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China 2 Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China 3 College of Pharmacy, Chongqing Medical University, Chongqing 400016, China 4 First Clinical College, Chongqing Medical University, Chongqing 400016, China 5 College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016, China * These authors contributed equally to this work Correspondence to: Ying Zhang, email: zhangying078@aliyun.com Keywords: ovarian cancer, NID1, EMT, metastasis, chemoresistance Abbreviations: ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; FAK: focal adhesion kinase; MET: mesenchymal-epithelial transition; NID1: nidogen-1 Received: January 09, 2017 Accepted: February 08, 2017 Published: March 13, 2017 ABSTRACT Nidogen-1 (NID1) has been identified as a novel candidate diagnostic biomarker of ovarian cancer in our previous study. Nevertheless, the role of NID1 in the pathogenesis of ovarian cancer is unclear. In the present study, we demonstrated that NID1 was a mesenchymal associated gene and its high expression was significantly correlated with shorter overall survival of ovarian cancer patients. The ectopic expression of NID1 in OVCAR-3 cells revealed a epithelial-mesenchymal transition (EMT) phenotype accompanied by enhancement of motility, invasiveness and cisplatin resistance, whereas the knockdown of NID1 was sufficient to convert HEY cells into epithelial phenotype with decreased capability of motility, invasiveness and cisplatin resistance. Mechanistic studies disclosed that NID1 activated ERK/MAPK signaling pathway to promote EMT. Collectively, our findings have uncovered the molecular mechanisms of NID1 in promoting ovarian cancer metastasis and chemoresistance, and provide a rationale for the therapeutic potential of NID1 suppression in ovarian cancer.

We previously identified proline-rich protein 11 (PRR11) as a novel cancer-related gene that is implicated in the regulation of cell cycle and tumorigenesis. Our recent study demonstrated that PRR11 and its adjacent gene, kinetochore associated 2 (SKA2), constitute a classic head-to-head gene pair that is coordinately regulated by nuclear factor Y (NF-Y). In the present study, we further show that the PRR11-SKA2 bidirectional transcription unit is an indirect target of the tumor suppressor p53. A luciferase reporter assay revealed that overexpression of wild type p53, but not mutant p53, significantly represses the basal activity and NF-Y mediated transactivation of the PRR11-SKA2 bidirectional promoter. Deletion and mutation analysis of the PRR11-SKA2 promoter revealed that p53-mediated PRR11-SKA2 repression is dependent on the presence of functional NF-Y binding sites. Furthermore, a co-immunoprecipitation assay revealed that p53 associates with NF-Y in lung cancer cells, and a chromatin immunoprecipitation assay showed that p53 represses PRR11-SKA2 transcription by reducing the binding amount of NF-Y in the PRR11-SKA2 promoter region. Consistently, the ability of p53 to downregulate PRR11-SKA2 transcription was significantly attenuated upon siRNA-mediated depletion of nuclear factor Y subunit beta (NF-YB). Notably, lung cancer patients with lower expression of either PRR11 or SKA2 along with wild type p53 exhibited the best overall survival compared with others with p53 mutation and/or higher expression of either PRR11 or SKA2. Taken together, our results demonstrate that p53 negatively regulates the expression of the PRR11-SKA2 bidirectional transcription unit through NF-Y, suggesting that the inability to repress the PRR11-SKA2 bidirectional transcription unit after loss of p53 might contribute to tumorigenesis.

MCPH1, initially identified as an hTERT repressor, has recently been implicated in mediating DNA damage response and maintaining chromosome integrity. This study is to investigate its potential role in the onset of cervical cancer. In the study, decreased expression of MCPH1 was observed in 19 of 31 cases (61.3 %) at mRNA level and 44 of 63 cases (69.8 %) at protein level of cervical tumor tissues compared with the paired nontumor tissues. Reduced MCPH1 protein expression was significantly associated with high-tumor grade (1 vs. 3 P = 0.013; 2 vs. 3 P = 0.047). In addition to inhibit SiHa cell migration and invasion, the overexpression of MCPH1 inhibited cervical cancer cells growth through inducing S phase arrest and mitochondrial apoptosis. Further analysis demonstrated cyclinA2/CDK2, CDC25C-cyclinB/CDC2, and p53/p21 pathways were involved in the MCPH1 overexpression-induced S phase arrest. Moreover, the overexpression of MCPH1 activated mitochondrial apoptosis through regulating several apoptosis-related proteins such as p53, Bcl-2, Bax, cytochrome c, caspase-3, and PARP-1. Our findings indicate that downregulated MCPH1 correlates with tumor progression in cervical cancer, and MCPH1 has an important role in regulating cell growth through regulating the cell cycle and apoptosis. Thus, it may be a crucial tumor suppressor gene and a novel candidate therapeutic target for cervical cancer.

Fangchinoline (Fan) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of Fan on cell growth and proliferation in breast cancer cells remain to be elucidated. Here, we show that Fan inhibited cell proliferation in the MDA-MB-231 breast cancer cell line through suppression of the AKT/Gsk- 3beta/cyclin D1 signaling pathway. Furthermore, Fan induced apoptosis by increasing the expression of Bax (relative to Bcl-2), active caspase 3 and cytochrome-c. Fan significantly inhibited cell proliferation of MDA- MB-231 cells in a concentration and time dependent manner as determined by MTT assay. Flow cytometry analysis demonstrated that Fan treatment of MDA-MB-231 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. Further analysis demonstrated that Fan decreased the phosphorylation of AKT and GSK-3beta. In addition, Fan up-regulated active caspase3, cytochrome-c protein levels and the ratio of Bax/Bcl-2, accompanied by apoptosis. Taken together, these results suggest that Fan is a potential natural product for the treatment of breast cancer.

Glioblastoma multiforme (GBM) is the most malignant glioma. In the current study, 149 astrocytoma gene expression datasets were classified by prediction analysis of microarray. Strikingly, disks large homolog 3 (DLG3), a membrane-associated guanylate kinase-family gene, had the highest score in the GBM subset. DLG3 mRNA expression is significantly down-regulated in GBM relative to normal tissue and grade II or grade III astrocytoma according to the results of real-time polymerase chain reaction, and its protein expression shows an obvious difference by immunohistochemistry. Further assays show that DLG3 over-expression induces mitotic cell cycle arrest and apoptosis, and it inhibits proliferation and migration. However, DLG3 over-expression has almost no affect on invasion. The DLG3 protein expression in human brain GBM tissue and its effects on GBM cell invasion were not expected. Our data suggest that DLG3 is down-regulated in this cancer type. To our knowledge, this is the first report to clearly demonstrate the possible involvement of DLG3 in GBM.

ACER2 is a critical sphingolipid metabolizing enzyme, and has been shown to be remarkably up-regulated following various stimuli such as DNA damage. However, the transcriptional regulatory mechanism of ACER2 gene and its potential role in the regulation of autophagy remain unknown. In this study, we have for the first time identified the human ACER2 gene promoter, and found that human ACER2 transcription is directly regulated by p53 and ACER2 is implicated in the induction of autophagy as well as apoptosis. A series of luciferase reporter assay demonstrated that ACER2 major promoter is located within its first intron where the consensus p53-binding sites exist. Consistently, forced expression of p53 significantly stimulated ACER2 transcription. Notably, p53-mediated autophagy and apoptosis were markedly enhanced by ACER2. Depletion of the essential autophagy gene ATG5 revealed that ACER2-induced autophagy facilitates its effect on apoptosis. Further studies clearly showed that ACER2-mediated autophagy and apoptosis are accompanied by ROS generation. In summary, our present study strongly suggests that ACER2 plays a pivotal role in p53-induced autophagy and apoptosis, and thus might serve as a novel and attractive molecular target for cancer treatment.

Lung cancer is the most common cause of cancer-related death in the world. The main types are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), the latter including squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma. NSCLCs account for about 80% of all lung cancer cases. Microcephalin (MCPH1), also called BRIT1 (BRCT-repeat inhibitor of hTERT expression), plays an important role in the maintenance of genomic stability. Recently, several studies have provided evidence that the expression of MCPH1 gene is decreased in several different types of human cancers. We evaluated the expression of protein MCPH1 in 188 lung cancer and 20 normal lung tissues by immunohistochemistry. Positive MCPH1 staining was found in all normal lung samples and only some cancerous tissues. MCPH1-positive cells were significantly lower in lung carcinoma compared with normal tissues. Furthermore, we firstly found that MCPH1 expression in lung adenocarcinoma is higher than its expression in squamous cell carcinoma. Change in MCPH1 protein expression may be associated with lung tumorigenesis and may be a useful biomarker for identification of pathological types of lung cancer.

Mouse ovarian surface epithelium (OSE) is a single layer of cubodial epithelial cells that covers the ovary surface and is involved in regulating the secretion and transport of 17β-hydroxysteroid dehydrogenase. Recently, OSE cells have attracted particular interest as a major source of ovarian cancer. Death-associated protein DAXX along with PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) reportedly play roles in transcriptional regulation and apoptosis. However, little is known regarding a role for DAXX in mOSE cells. In this study, we both over-expressed DAXX and depleted DAXX in primary mOSE cells. We found that Daxx deletion accelerated senescence in a p53/p21-dependent manner and promoted DNA damage by interacting with PML bodies without affecting cell cycle progression. These results suggest that DAXX may transform mOSE cells to an ovarian oncogenic phenotype and may be an anti-cancer target.

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.

This study was undertaken to determine the effects of Pyracantha fortuneana (Maxim.) Li polysaccharides (PFP) on antioxidant and immune functions in mice. Results from this study showed that PFP administration significantly increased thymus and spleen indices, promoted splenocyte proliferation and natural killer (NK) cell activity, and elevated CD4 T cell numbers as well as CD4(+)/CD8(+) ratios. PFP also increased interleukin-2 (IL-2) levels, and decreased the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in splenocytes. In addition, PFP treatment led to remarkable increases in glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities, and dramatic decreases in malondialdehyde (MDA) levels in splenocytes. Moreover, PFP increased mRNA and protein expression of nuclear factor erythroid 2-related factor (Nrf2) in splenocytes. Taken together, these results suggest that PFP treatment enhances the immune function and decreases the oxidative stress in mice.

After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).

NFBD1/MDC1 is a large nuclear protein mainly participating in DNA damage response, indicating its therapeutic potential as a radio-/chemosensitizer target in cancer field. Esophageal cancer ranks among one of the most frequent cause of cancer death in the world. In this study, we used three representative esophageal cancer cell lines to investigate the effects of NFBD1 silencing on cell proliferation, cell morphology, and cell cycle distribution. Synthetic small interfering RNA (siRNA) duplexes against NFBD1 were introduced into three esophageal cancer cell lines, which subsequently resulted in a significant inhibition in NFBD1 expression in the cells. Our results have shown that a targeted siRNA depletion of NFBD1 resulted in a significant growth inhibition, morphology change, and cell cycle alterations in esophageal cancer cells. Furthermore, NFBD1 depletion also sensitized all the three esophageal cancer cell lines to chemotherapeutic agents including adriamycin and cisplatin. Taken together, our study strongly suggested that NFBD1 may serve as a potential therapeutic target in human esophageal cancer.