Gene Summary

Gene:HBEGF; heparin binding EGF like growth factor
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:proheparin-binding EGF-like growth factor
Source:NCBIAccessed: 01 September, 2019


What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 01 September 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Glycoproteins
  • ErbB Receptors
  • Cell Proliferation
  • Sialyltransferases
  • Messenger RNA
  • Proto-Oncogene Proteins
  • Cell Movement
  • EGF Family of Proteins
  • Stomach Cancer
  • Neoplasm Metastasis
  • Ovarian Cancer
  • siRNA
  • ras Proteins
  • Up-Regulation
  • Pancreatic Cancer
  • Heparin-binding EGF-like Growth Factor
  • RNA Interference
  • Ligands
  • Breast Cancer
  • Transcription
  • Prostate Cancer
  • Cancer Gene Expression Regulation
  • Tetraspanin 29
  • Intercellular Signaling Peptides and Proteins
  • Protein Kinase Inhibitors
  • Chromosome 5
  • Transcription Factor RelA
  • Transfection
  • Receptor, erbB-2
  • Amphiregulin
  • Biomarkers, Tumor
  • Immunohistochemistry
  • Lung Cancer
  • Neoplasm Proteins
  • Tumor Burden
  • Phosphorylation
  • alpha-Fetoproteins
  • Gene Expression
  • Wound Healing
  • Gene Expression Profiling
  • Epidermal Growth Factor
  • Signal Transduction
Tag cloud generated 01 September, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: HBEGF (cancer-related)

Sethuraman A, Brown M, Krutilina R, et al.
BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion.
Breast Cancer Res. 2018; 20(1):117 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Metastasis is responsible for a significant number of breast cancer-related deaths. Hypoxia, a primary driving force of cancer metastasis, induces the expression of BHLHE40, a transcription regulator. This study aimed to elucidate the function of BHLHE40 in the metastatic process of breast cancer cells.
METHODS: To define the role of BHLHE40 in breast cancer, BHLHE40 expression was knocked down by a lentiviral construct expressing a short hairpin RNA against BHLHE40 or knocked out by the CRISPR/Cas9 editing system. Orthotopic xenograft and experimental metastasis (tail vein injection) mouse models were used to analyze the role of BHLHE40 in lung metastasis of breast cancer. Global gene expression analysis and public database mining were performed to identify signaling pathways regulated by BHLHE40 in breast cancer. The action mechanism of BHLHE40 was examined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (CoIP), exosome analysis, and cell-based assays for metastatic potential.
RESULTS: BHLHE40 knockdown significantly reduced primary tumor growth and lung metastasis in orthotopic xenograft and experimental metastasis models of breast cancer. Gene expression analysis implicated a role of BHLHE40 in transcriptional activation of heparin-binding epidermal growth factor (HBEGF). ChIP and CoIP assays revealed that BHLHE40 induces HBEGF transcription by blocking DNA binding of histone deacetylases (HDAC)1 and HDAC2. Cell-based assays showed that HBEGF is secreted through exosomes and acts to promote cell survival and migration. Public databases provided evidence linking high expression of BHLHE40 and HBEGF to poor prognosis of triple-negative breast cancer.
CONCLUSION: This study reveals a novel role of BHLHE40 in promoting tumor cell survival and migration by regulating HBEGF secretion.

Kato Y, Ninomiya K, Ohashi K, et al.
Combined effect of cabozantinib and gefitinib in crizotinib-resistant lung tumors harboring ROS1 fusions.
Cancer Sci. 2018; 109(10):3149-3158 [PubMed] Free Access to Full Article Related Publications
The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has shown dramatic effects in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusion genes. However, patients inevitably develop resistance to this agent. Therefore, a new treatment strategy is required for lung tumors with ROS1 fusion genes. In the present study, lung cancer cell lines, HCC78 harboring SLC34A2-ROS1 and ABC-20 harboring CD74-ROS1, were used as cell line-based resistance models. Crizotinib-resistant HCC78R cells were established from HCC78. We comprehensively screened the resistant cells using a phosphor-receptor tyrosine kinase array and RNA sequence analysis by next-generation sequencing. HCC78R cells showed upregulation of HB-EGF and activation of epidermal growth factor receptor (EGFR) phosphorylation and the EGFR signaling pathway. Recombinant HB-EGF or EGF rendered HCC78 cells or ABC-20 cells resistant to crizotinib. RNA sequence analysis by next-generation sequencing revealed the upregulation of AXL in HCC78R cells. HCC78R cells showed marked sensitivity to EGFR-TKI or anti-EGFR antibody treatment in vitro. Combinations of an AXL inhibitor, cabozantinib or gilteritinib, and an EGFR-TKI were more effective against HCC78R cells than monotherapy with an EGFR-TKI or AXL inhibitor. The combination of cabozantinib and gefitinib effectively inhibited the growth of HCC78R tumors in an in vivo xenograft model of NOG mice. The results of this study indicated that HB-EGF/EGFR and AXL play roles in crizotinib resistance in lung cancers harboring ROS1 fusions. The combination of cabozantinib and EGFR-TKI may represent a useful alternative treatment strategy for patients with advanced NSCLC harboring ROS1 fusion genes.

Yu OM, Benitez JA, Plouffe SW, et al.
YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.
Oncogene. 2018; 37(41):5492-5507 [PubMed] Free Access to Full Article Related Publications
The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Liao WC, Liao CK, Tsai YH, et al.
DSE promotes aggressive glioma cell phenotypes by enhancing HB-EGF/ErbB signaling.
PLoS One. 2018; 13(6):e0198364 [PubMed] Free Access to Full Article Related Publications
Remodeling of the extracellular matrix (ECM) in the tumor microenvironment promotes glioma progression. Chondroitin sulfate (CS) proteoglycans appear in the ECM and on the cell surface, and can be catalyzed by dermatan sulfate epimerase to form chondroitin sulfate/dermatan sulfate (CS/DS) hybrid chains. Dermatan sulfate epimerase 1 (DSE) is overexpressed in many types of cancer, and CS/DS chains mediate several growth factor signals. However, the role of DSE in gliomas has never been explored. In the present study, we determined the expression of DSE in gliomas by consulting a public database and conducting immunohistochemistry on a tissue array. Our investigation revealed that DSE was upregulated in gliomas compared with normal brain tissue. Furthermore, high DSE expression was associated with advanced tumor grade and poor survival. We found high DSE expression in several glioblastoma cell lines, and DSE expression directly mediated DS chain formation in glioblastoma cells. Knockdown of DSE suppressed the proliferation, migration, and invasion of glioblastoma cells. In contrast, overexpression of DSE in GL261 cells enhanced these malignant phenotypes and in vivo tumor growth. Interestingly, we found that DSE selectively regulated heparin-binding EGF-like growth factor (HB-EGF)-induced signaling in glioblastoma cells. Inhibiting epidermal growth factor receptor (EGFR) and ErbB2 with afatinib suppressed DSE-enhanced malignant phenotypes, establishing the critical role of the ErbB pathway in regulating the effects of DSE expression. This evidence indicates that upregulation of DSE in gliomas contributes to malignant behavior in cancer cells. We provide novel insight into the significance of DS chains in ErbB signaling and glioma pathogenesis.

Hotblack A, Holler A, Piapi A, et al.
Tumor-Resident Dendritic Cells and Macrophages Modulate the Accumulation of TCR-Engineered T Cells in Melanoma.
Mol Ther. 2018; 26(6):1471-1481 [PubMed] Free Access to Full Article Related Publications
Ongoing clinical trials explore T cell receptor (TCR) gene therapy as a treatment option for cancer, but responses in solid tumors are hampered by the immunosuppressive microenvironment. The production of TCR gene-engineered T cells requires full T cell activation in vitro, and it is currently unknown whether in vivo interactions with conventional dendritic cells (cDCs) regulate the accumulation and function of engineered T cells in tumors. Using the B16 melanoma model and the inducible depletion of CD11c

Zhao J, Shi L, Zeng S, et al.
Importin-11 overexpression promotes the migration, invasion, and progression of bladder cancer associated with the deregulation of CDKN1A and THBS1.
Urol Oncol. 2018; 36(6):311.e1-311.e13 [PubMed] Related Publications
OBJECTIVES: We recently determined that a novel oncogene, IPO11 from 5q12, participates in bladder cancer (BCa) progression. However, the biological function of IPO11 and the molecular mechanisms through which it contributes to BCa progression remain unclear. The aim of this study was to investigate the role of IPO11 in BCa aggressiveness and elucidate the molecular mechanisms underlying its effects in BCa.
MATERIALS AND METHODS: The mRNA expression levels of IPO11 in BIU-87, RT4, UMUC3, EJ, 5637, T24, J82, and HT-1376 cell lines were determined using quantitative real-time polymerase chain reaction. Expression of importin-11 was detected in 134 formalin-fixed and paraffin-embedded (FFPE) BCa tissues and 10 paired nonneoplastic bladder tissue specimens by immunohistochemistry. The copy number of IPO11 was examined in 25 FFPE BCa specimens using fluorescent in situ hybridization. The effects of IPO11 on migration, invasion, and cell proliferation were investigated in EJ and 5637 cell lines using RNA interference. Potential molecular mechanisms were investigated using whole transcriptome sequencing and bioinformatic approaches in EJ cells and IPO11-silenced EJ cells and verified using quantitative real-time polymerase chain reaction.
RESULTS: Endogenous IPO11 mRNA was highly expressed in 6 invasive BCa cell lines (EJ, HT-1376, UMUC3, 5637, J82, and T24) but had a low expression in the noninvasive BCa cell line BIU-87 and the papillary BCa cell line RT4. Immunohistochemical staining revealed that 87 (64.9%) of 134 FFPE BCa tissues displayed importin-11 overexpression. Moreover, importin-11 overexpression was positively associated with increased tumor stages and tumor grades, lymphatic invasion, and lymph node metastasis. Furthermore, importin-11 overexpression was detected in 100% (14/14) of BCa tissues with IPO11 amplification, and IPO11 amplification was not observed in 2 additional BCa tissues with importin-11 overexpression. Small interfering RNA-mediated knockdown of IPO11 is sufficient to inhibit the motility and invasiveness of EJ and 5637 cells. IPO11 knockdown also inhibited cell proliferation in EJ cells, whereas this was not observed in 5637 cells or the in vivo experiments. Using whole transcriptome sequencing, we found that 22 genes (including IPO11) were differentially expressed in IPO11-silenced EJ cells compared with wild-type EJ cells, 4 of which were upregulated, and 18 of which were downregulated. KEGG pathway enrichment analysis of the significantly differentially expressed genes showed that the proteoglycans in cancer pathway (pathway Id: hsa05205) was most significantly enriched among 10 genetically altered pathways and referred to 6 significantly altered genes (CDKN1A, HBEGF, PTK2, THBS1, CCNG2, and EGR1). The next 3 most significantly enriched pathways in order were the p53, ErbB, and BCa pathways. CDKN1A and THBS1 were the most 2 frequently covered genes and were involved in 9 and 6 pathways, respectively. They were also 2 key proteins in the BCa pathway (pathway Id: hsa05219) that were downregulated in IPO11-knockdown EJ cells compared with wild-type EJ cells.
CONCLUSIONS: Importin-11 overexpression can promote BCa cell invasiveness, probably associated with the deregulation of CDKN1A and THBS1 primarily through the activation of the proteoglycans in cancer pathway and the classical BCa pathway. Importin-11 may be a useful target through which the progression of noninvasive BCa to invasive BCa can be blocked.

Yun S, Kwak Y, Nam SK, et al.
Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.
Cancer Res Treat. 2018; 50(4):1351-1361 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients' survival with CRC.
Materials and Methods: The expression of EGFR ligands, including heparin binding epidermal growth factor-like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH.
RESULTS: Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS; p=0.018) and overall survival (OS; p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis.
CONCLUSION: Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Miyata K, Yotsumoto F, Fukagawa S, et al.
Serum Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) as a Biomarker for Primary Ovarian Cancer.
Anticancer Res. 2017; 37(7):3955-3960 [PubMed] Related Publications
Ovarian cancer is the most lethal malignancy among gynaecological cancers. Although many anticancer agents have been developed for the treatment of ovarian cancer, it continues to have an extremely poor prognosis. Heparin-binding epidermal growth factor-like grown factor (HB-EGF) has been reported to be a rational therapeutic target for ovarian cancer. Here, we evaluated the clinical significance of serum HB-EGF by examining the association between prognosis and serum HB-EGF levels in patients with primary ovarian cancer. We found that high serum HB-EGF concentrations were significantly associated with poor prognosis in a combined cohort of patients with all stages of ovarian cancer, as well as in a subset of patients with advanced disease. In addition, serum HB-EGF levels increased as the cancer advanced. These data suggest that serum HB-EGF may be a target for the design of novel therapies for ovarian cancer.

De Marco C, Laudanna C, Rinaldo N, et al.
Specific gene expression signatures induced by the multiple oncogenic alterations that occur within the PTEN/PI3K/AKT pathway in lung cancer.
PLoS One. 2017; 12(6):e0178865 [PubMed] Free Access to Full Article Related Publications
Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%), though only 30 differentially expressed genes (DEGs) (15 up-regulated, 12 down-regulated and 3 discordant) out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%). Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated), DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated) and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated). The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10). Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206) or PI3K (LY294002) further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively) correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6). Finally, we made use of Ingenuity Pathway Analysis (IPA) to investigate the relevant BioFunctions enriched by the costitutive activation of AKT1-, PI3K- or PTEN-dependent signalling in lung epithelial cells. Expectedly, the analysis of the DEGs common to all three alterations highlighted a group of BioFunctions that included Cell Proliferation of tumor cell lines (14 DEGs), Invasion of cells (10 DEGs) and Migration of tumour cell lines (10 DEGs), with a common core of 5 genes (ATF3, CDKN1A, GDF15, HBEGF and LCN2) that likely represent downstream effectors of the pro-oncogenic activities of PI3K/AKT signalling. Conversely, IPA analysis of exclusive DEGs led to the identification of different downstream effectors that are modulated by mutant AKT1 (TGFBR2, CTSZ, EMP1), mutant PIK3CA (CCND2, CDK2, IGFBP2, TRIB1) and PTEN loss (ASNS, FHL2). These findings not only shed light on the molecular mechanisms that are activated by aberrant signalling through the PI3K/AKT pathway in lung epithelial cells, but also contribute to the identification of previously unrecognised molecules whose regulation takes part in the development of lung cancer.

Schrevel M, Osse EM, Prins FA, et al.
Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.
Int J Oncol. 2017; 50(6):1947-1954 [PubMed] Free Access to Full Article Related Publications
In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

Cavallini A, Rotelli MT, Lippolis C, et al.
Human microRNA expression in sporadic and FAP-associated desmoid tumors and correlation with beta-catenin mutations.
Oncotarget. 2017; 8(26):41866-41875 [PubMed] Free Access to Full Article Related Publications
Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with β-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis.The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.

Byeon SJ, Lee HS, Kim MA, et al.
Expression of the ERBB Family of Ligands and Receptors in Gastric Cancer.
Pathobiology. 2017; 84(4):210-217 [PubMed] Related Publications
OBJECTIVE: Gastric cancer (GC) is the second most common cancer and the third leading cause of cancer-related death in Korea. Alterations in the ERBB (homology to the erythroblastoma viral gene product, v-erbB) receptor family and ERBB-related signaling pathways are frequently observed in GC. However, the roles of the ERBB receptors and their ligands in GC are not well established.
METHODS: We evaluated the expression levels of various ERBB receptor ligands (i.e., heparin-binding epidermal growth factor-like growth factor [HBEGF], transforming growth factor-α [TGFA], amphiregulin [AREG], epiregulin [EREG], epidermal growth factor [EGF], and betacellulin [BTC]) and 3 ERBB family receptors (i.e., epidermal growth factor receptor [EGFR], human EGFR2 [HER2], and ERBB3) in 313 cases of GC using immunohistochemistry, fluorescence in situ hybridization, and mRNA in situ hybridization.
RESULTS: A high expression of EGFR, HER2, and ERBB3 was observed in 30, 32, and 27 cases, respectively. A high expression of HBEGF, TGFA, AREG, EREG, EGF, and BTC was observed in 91, 97, 151, 74, 26, and 37 cases, respectively. A high expression of TGFA was associated with better survival, while a high expression of BTC was associated with worse survival. These results were confirmed using Cox proportional hazards analysis. HBEGF, TGFA, AREG, tumor-node-metastasis classification, Lauren's classification, and ERBB3 were significant survival parameters in multivariate analysis.
CONCLUSION: Among the ERBB family receptors and ligands examined, 3 ligands (i.e., TGFA, HBEGF, and AREG) and ERBB3 had a prognostic impact.

Cooper ML, Choi J, Karpova D, et al.
Azacitidine Mitigates Graft-versus-Host Disease via Differential Effects on the Proliferation of T Effectors and Natural Regulatory T Cells In Vivo.
J Immunol. 2017; 198(9):3746-3754 [PubMed] Free Access to Full Article Related Publications
Azacitidine (AzaC) mitigates graft-versus-host disease (GvHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust graft-versus-leukemia effect. Previous studies have failed to investigate the role of natural regulatory T cells (nTregs) on the mitigation of GvHD by AzaC, instead focusing on the generation of suppressive Tregs (CD4

Kumar SS, Tomita Y, Wrin J, et al.
High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.
Clin Transl Oncol. 2017; 19(6):718-726 [PubMed] Related Publications
PURPOSE: Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy.
METHODS: Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression.
RESULTS: Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression.
CONCLUSION: High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

Komisarof J, McCall M, Newman L, et al.
A four gene signature predictive of recurrent prostate cancer.
Oncotarget. 2017; 8(2):3430-3440 [PubMed] Free Access to Full Article Related Publications
Prostate cancer is the most common form of non-dermatological cancer among US men, with an increasing incidence due to the aging population. Patients diagnosed with clinically localized disease identified as intermediate or high-risk are often treated by radical prostatectomy. Approximately 33% of these patients will suffer recurrence after surgery. Identifying patients likely to experience recurrence after radical prostatectomy would lead to improved clinical outcomes, as these patients could receive adjuvant radiotherapy. Here, we report a new tool for prediction of prostate cancer recurrence based on the expression pattern of a small set of cooperation response genes (CRGs). CRGs are a group of genes downstream of cooperating oncogenic mutations previously identified in a colon cancer model that are critical to the cancer phenotype. We show that systemic dysregulation of CRGs is also found in prostate cancer, including a 4-gene signature (HBEGF, HOXC13, IGFBP2, and SATB1) capable of differentiating recurrent from non-recurrent prostate cancer. To develop a suitable diagnostic tool to predict disease outcomes in individual patients, multiple algorithms and data handling strategies were evaluated on a training set using leave-one-out cross-validation (LOOCV). The best-performing algorithm, when used in combination with a predictive nomogram based on clinical staging, predicted recurrent and non-recurrent disease outcomes in a blinded validation set with 83% accuracy, outperforming previous methods. Disease-free survival times between the cohort of prostate cancers predicted to recur and predicted not to recur differed significantly (p = 1.38x10-6). Therefore, this test allows us to accurately identify prostate cancer patients likely to experience future recurrent disease immediately following removal of the primary tumor.

Shetty P, Patil VS, Mohan R, et al.
Annexin A2 and its downstream IL-6 and HB-EGF as secretory biomarkers in the differential diagnosis of Her-2 negative breast cancer.
Ann Clin Biochem. 2017; 54(4):463-471 [PubMed] Related Publications
Background AnnexinA2 (AnxA2) membrane deposition has a critical role in HB-EGF shedding as well as IL-6 secretion in breast cancer cells. This autocrine cycle has a major role in cancer cell proliferation, migration and metastasis. The objective of the study is to demonstrate annexinA2-mediated autocrine regulation via HB-EGF and IL-6 in Her-2 negative breast cancer progression. Methods Secretory annexinA2, HB-EGF and IL-6 were analysed in the peripheral blood sample of Her-2 negative ( n = 20) and positive breast cancer patients ( n = 16). Simultaneously, tissue expression was analysed by immunohistochemistry. The membrane deposition of these secretory ligands and their autocrine regulation was demonstrated using triple-negative breast cancer cell line model. Results Annexina2 and HB-EGF expression are inversely correlated with Her-2, whereas IL-6 expression is seen in both Her-2 negative and positive breast cancer cells. RNA interference studies and upregulation of annexinA2 proved that annexinA2 is the upstream of this autocrine pathway. Abundant soluble serum annexinA2 is secreted in Her-2 negative breast cancer (359.28 ± 63.73 ng/mL) compared with normal (286.10 ± 70.04 ng/mL, P < 0.01) and Her-2 positive cases (217.75 ± 60.59 ng/mL, P < 0.0001). In Her-2 negative cases, the HB-EGF concentrations (179.16 ± 118.81 pg/mL) were highly significant compared with normal (14.92 ± 17.33 pg/mL, P < 0.001). IL-6 concentrations were increased significantly in both the breast cancer phenotypes as compared with normal ( P < 0.001). Conclusion The specific expression pattern of annexinA2 and HB-EGF in triple-negative breast cancer tissues, increased secretion compared with normal cells, and their major role in the regulation of EGFR downstream signalling makes these molecules as a potential tissue and serum biomarker and an excellent therapeutic target in Her-2 negative breast cancer.

Zhang Y, Velez-Delgado A, Mathew E, et al.
Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the establishment of an immunosuppressive environment in pancreatic cancer.
Gut. 2017; 66(1):124-136 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity.
OBJECTIVE: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment.
METHODS: Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b
RESULTS: Depletion of myeloid cells prevented Kras
CONCLUSION: Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8

Mullapudi N, Ye B, Suzuki M, et al.
Genome Wide Methylome Alterations in Lung Cancer.
PLoS One. 2015; 10(12):e0143826 [PubMed] Free Access to Full Article Related Publications
Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.

Wen T, Hou K, Li Z, et al.
Silencing β-linked N-acetylglucosamine transferase induces apoptosis in human gastric cancer cells through PUMA and caspase-3 pathways.
Oncol Rep. 2015; 34(6):3140-6 [PubMed] Related Publications
β-linked N-acetylglucosamine (GlcNAc) is a monosaccharide that is catalyzed by O-GlcNAcylation transferase (OGT) to bind serine or threonine hydroxyl moieties of numerous nuclear and cytoplasmic proteins. Recent studies have shown that O-GlcNAcylation is elevated in various cancer types, which is associated with oncogenesis and tumor progression. However, whether OGT is expressed and/or plays a role in gastric cancer is unknown. In the present study, we used qPCR to determine that OGT mRNA levels are significantly elevated in gastric cancer tissues compared with that in corresponding adjacent tissues. In addition, in vivo silencing of OGT in nude mice suppressed tumor proliferation and decreased tumor burden. Furthermore, in vitro OGT knockdown induced more cell apoptosis through increasing PUMA and caspase-3 expression. We used a glycan-binding protein gene microarray to identify potential downstream target genes of OGT, and found that apoptosis-related genes such as galectin and HBEGF were decreased after OGT suppression, suggesting that OGT silencing induces apoptosis in gastric cancer tissues. We concluded that OGT plays a key role in gastric cancer proliferation and survival, and could be a potential target for therapy.

Figueroa JD, Koutros S, Colt JS, et al.
Modification of Occupational Exposures on Bladder Cancer Risk by Common Genetic Polymorphisms.
J Natl Cancer Inst. 2015; 107(11) [PubMed] Free Access to Full Article Related Publications
Few studies have demonstrated gene/environment interactions in cancer research. Using data on high-risk occupations for 2258 case patients and 2410 control patients from two bladder cancer studies, we observed that three of 16 known or candidate bladder cancer susceptibility variants displayed statistically significant and consistent evidence of additive interactions; specifically, the GSTM1 deletion polymorphism (P interaction ≤ .001), rs11892031 (UGT1A, P interaction = .01), and rs798766 (TMEM129-TACC3-FGFR3, P interaction = .03). There was limited evidence for multiplicative interactions. When we examined detailed data on a prevalent occupational exposure associated with increased bladder cancer risk, straight metalworking fluids, we also observed statistically significant additive interaction for rs798766 (TMEM129-TACC3-FGFR3, P interaction = .02), with the interaction more apparent in patients with tumors positive for FGFR3 expression. All statistical tests were two-sided. The interaction we observed for rs798766 (TMEM129-TACC3-FGFR3) with specific exposure to straight metalworking fluids illustrates the value of integrating germline genetic variation, environmental exposures, and tumor marker data to provide insight into the mechanisms of bladder carcinogenesis.

Shang H, Braggio D, Lee YJ, et al.
Targeting the Notch pathway: A potential therapeutic approach for desmoid tumors.
Cancer. 2015; 121(22):4088-96 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Desmoid tumors (DTs) are rare mesenchymal lesions that can recur repeatedly. When it is feasible, DTs are surgically resected; however, this often results in high recurrence rates. Recently, treatment with PF-03084014, a potent γ-secretase inhibitor, has been shown to have antitumor activity in several tumor types by affecting the WNT/β-catenin pathway. Consequently, Notch pathway inhibition by PF-03084014 might be a promising approach for DT treatment.
METHODS: The expression of Notch pathway components was analyzed in DT tissues and cell strains with immunohistochemistry and Western blotting, respectively. A panel of DT cell strains was exposed to PF-03084014 and evaluated for cell proliferation. Antitumor effects were assessed via cell cycle, apoptosis, and migration and invasion analysis. Cells treated with PF-03084014 were characterized with a gene array analysis combined with Ingenuity Pathway Analysis.
RESULTS: The results showed that Notch pathway components were expressed at different levels in DTs. Hes1 (Hes Family BHLH Transcription Factor 1) was overexpressed in DT tumors versus dermal scar tissue, and PF-03084014 caused significant decreases in Notch intracellular domain and Hes1 expression in DT cell strains. PF-03084014 decreased DT cell migration and invasion and also caused cell growth inhibition in DT cell strains, most likely through cell cycle arrest. Gene array analysis combined with Ingenuity Pathway Analysis showed that Wnt1-inducible signaling pathway protein 2 possibly regulated Notch and WNT pathways after treatment with PF-03084014 through integrin.
CONCLUSION: Our findings suggest that the Notch pathway is an important DT therapeutic target. Furthermore, PF-03084014 has significant antitumor activity against DTs, and it may be an alternative strategy for DT treatment.

Cursons J, Leuchowius KJ, Waltham M, et al.
Stimulus-dependent differences in signalling regulate epithelial-mesenchymal plasticity and change the effects of drugs in breast cancer cell lines.
Cell Commun Signal. 2015; 13:26 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: The normal process of epithelial mesenchymal transition (EMT) is subverted by carcinoma cells to facilitate metastatic spread. Cancer cells rarely undergo a full conversion to the mesenchymal phenotype, and instead adopt positions along the epithelial-mesenchymal axis, a propensity we refer to as epithelial mesenchymal plasticity (EMP). EMP is associated with increased risk of metastasis in breast cancer and consequent poor prognosis. Drivers towards the mesenchymal state in malignant cells include growth factor stimulation or exposure to hypoxic conditions.
METHODS: We have examined EMP in two cell line models of breast cancer: the PMC42 system (PMC42-ET and PMC42-LA sublines) and MDA-MB-468 cells. Transition to a mesenchymal phenotype was induced across all three cell lines using epidermal growth factor (EGF) stimulation, and in MDA-MB-468 cells by hypoxia. We used RNA sequencing to identify gene expression changes that occur as cells transition to a more-mesenchymal phenotype, and identified the cell signalling pathways regulated across these experimental systems. We then used inhibitors to modulate signalling through these pathways, verifying the conclusions of our transcriptomic analysis.
RESULTS: We found that EGF and hypoxia both drive MDA-MB-468 cells to phenotypically similar mesenchymal states. Comparing the transcriptional response to EGF and hypoxia, we have identified differences in the cellular signalling pathways that mediate, and are influenced by, EMT. Significant differences were observed for a number of important cellular signalling components previously implicated in EMT, such as HBEGF and VEGFA. We have shown that EGF- and hypoxia-induced transitions respond differently to treatment with chemical inhibitors (presented individually and in combinations) in these breast cancer cells. Unexpectedly, MDA-MB-468 cells grown under hypoxic growth conditions became even more mesenchymal following exposure to certain kinase inhibitors that prevent growth-factor induced EMT, including the mTOR inhibitor everolimus and the AKT1/2/3 inhibitor AZD5363.
CONCLUSIONS: While resulting in a common phenotype, EGF and hypoxia induced subtly different signalling systems in breast cancer cells. Our findings have important implications for the use of kinase inhibitor-based therapeutic interventions in breast cancers, where these heterogeneous signalling landscapes will influence the therapeutic response.

Schneider MA, Granzow M, Warth A, et al.
Glycodelin: A New Biomarker with Immunomodulatory Functions in Non-Small Cell Lung Cancer.
Clin Cancer Res. 2015; 21(15):3529-40 [PubMed] Related Publications
PURPOSE: In recent years, immune therapeutic strategies against non-small cell lung cancer (NSCLC) based on tissue-derived biomarkers, for example PD1/PD-L1 (CD274), have evolved as novel and promising treatment options. However, the crosstalk between tumor and immune cells is poorly understood. Glycodelin (gene name PAEP), initially described in the context of pregnancy and trophoblastic implantation, is a secreted immunosuppressive glycoprotein with an as-of-yet largely unknown function in lung cancer.
EXPERIMENTAL DESIGN: In this study, we characterized the expression and role of glycodelin in NSCLC through mRNA and protein expression analyses, functional knockdown experiments, and correlations with clinicopathologic parameters.
RESULTS: Glycodelin mRNA expression was significantly elevated in tumors (n = 336) compared with matched normal tissue (P < 0.0001). Overall survival (OS) was significantly reduced in NSCLC with high glycodelin mRNA levels in women but not in men. Glycodelin was detected in the sera of patients, and the levels correlated with recurrence and metastatic disease. Knockdown of glycodelin with siRNAs in NSCLC cell lines resulted in significant upregulation of immune system modulatory factors such as PDL1, CXCL5, CXCL16, MICA/B, and CD83 as well as proliferation stimulators EDN1 and HBEGF. Furthermore, decreased migration of tumor cells was observed.
CONCLUSIONS: Altogether, the comprehensive characterization of glycodelin in NSCLC provides strong support for its use as a biomarker with immune modulatory function.

Cushman SM, Jiang C, Hatch AJ, et al.
Gene expression markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).
Clin Cancer Res. 2015; 21(5):1078-86 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Formalin-fixed, paraffin-embedded tumor samples from CALGB 80203 were analyzed for expression of EGFR axis-related genes to identify prognostic or predictive biomarkers for cetuximab treatment.
PATIENTS AND METHODS: Patients (238 total) with first-line metastatic colorectal cancer (mCRC) were randomized to FOLFOX or FOLFIRI chemotherapy ± cetuximab. qRT-PCR analyses were conducted on tissues from 103 patients at baseline to measure gene expression levels of HER-related genes, including amphiregulin (AREG), betacellulin (BTC), NT5E (CD73), DUSP4, EGF, EGFR, epigen (EPGN), epiregulin (EREG), HBEGF, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), PHLDA1, and TGFA. The interactions between expression levels and treatment with respect to progression-free survival (PFS) and overall survival (OS) were modeled using multiplicative Cox proportional hazards models.
RESULTS: High tumor mRNA levels of HER2 [hazard ratio (HR), 0.64; P = 0.002] and EREG (HR, 0.89; P = 0.016) were prognostic markers associated with longer PFS across all patients. HER3 and CD73 expression levels were identified as potential predictive markers of benefit from cetuximab. In KRAS wild-type (WT) tumors, low HER3 expression was associated with longer OS from cetuximab treatment, whereas high HER3 expression was associated with shorter OS from cetuximab treatment (chemo + cetuximab: HR, 1.15; chemo-only: HR, 0.48; Pinteraction = 0.029). High CD73 expression was associated with longer PFS from cetuximab treatment in patients with KRAS-WT (chemo + cetuximab: HR, 0.91; chemo-only: HR, 1.57; Pinteraction = 0.026) and KRAS-mutant (Mut) tumors (chemo + cetuximab: HR, 0.80; chemo-only: HR, 1.29; P = 0.025).
CONCLUSIONS: Gene expression of HER3 and CD73 was identified as a potential predictive marker for cetuximab. These data implicate HER axis signaling and immune modulation as potential mechanisms of cetuximab action and sensitivity.

Wei LQ, Liang HT, Qin DC, et al.
MiR-212 exerts suppressive effect on SKOV3 ovarian cancer cells through targeting HBEGF.
Tumour Biol. 2014; 35(12):12427-34 [PubMed] Related Publications
MicroRNAs (miRNAs) play critical roles in the development and progression of ovarian cancer. We found that miR-212 was significantly downregulated in serum and tissues from epithelial ovarian cancer (EOC) patients. Overexpression of miR-212 in ovarian cancer cells inhibited cell proliferation, migration, and invasion. Luciferase reporter assay confirmed HBEGF as a direct target of miR-212. Overexpression of miR-212 decreased HBEGF expression at both the protein and messenger RNA (mRNA) levels. Knockdown of HBEGF expression in SKOV3 cell line significantly inhibited cell growth, migration, and invasion. HBEGF mRNA level was upregulated in EOC tissues and inversely correlated with miR-212 expression in tissues. Upregulation of HBEGF could attenuate the effect induced by miR-212. These findings indicate that miR-212 displays a tumor-suppressive effect in human ovarian cancer. And miR-212 suppresses cell proliferation, migration, and invasion by targeting the HBEGF transcript, highlighting the therapeutic potential of miR-212 and HBEGF in epithelial ovarian cancer treatment.

Olbryt M, Habryka A, Student S, et al.
Global gene expression profiling in three tumor cell lines subjected to experimental cycling and chronic hypoxia.
PLoS One. 2014; 9(8):e105104 [PubMed] Free Access to Full Article Related Publications
Hypoxia is one of the most important features of the tumor microenvironment, exerting an adverse effect on tumor aggressiveness and patient prognosis. Two types of hypoxia may occur within the tumor mass, chronic (prolonged) and cycling (transient, intermittent) hypoxia. Cycling hypoxia has been shown to induce aggressive tumor cell phenotype and radioresistance more significantly than chronic hypoxia, though little is known about the molecular mechanisms underlying this phenomenon. The aim of this study was to delineate the molecular response to both types of hypoxia induced experimentally in tumor cells, with a focus on cycling hypoxia. We analyzed in vitro gene expression profile in three human cancer cell lines (melanoma, ovarian cancer, and prostate cancer) exposed to experimental chronic or transient hypoxia conditions. As expected, the cell-type specific variability in response to hypoxia was significant. However, the expression of 240 probe sets was altered in all 3 cell lines. We found that gene expression profiles induced by both types of hypoxia were qualitatively similar and strongly depend on the cell type. Cycling hypoxia altered the expression of fewer genes than chronic hypoxia (6,132 vs. 8,635 probe sets, FDR adjusted p<0.05), and with lower fold changes. However, the expression of some of these genes was significantly more affected by cycling hypoxia than by prolonged hypoxia, such as IL8, PLAU, and epidermal growth factor (EGF) pathway-related genes (AREG, HBEGF, and EPHA2). These transcripts were, in most cases, validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our results indicate that experimental cycling hypoxia exerts similar, although less intense effects, on the examined cancer cell lines than its chronic counterpart. Nonetheless, we identified genes and molecular pathways that seem to be preferentially regulated by cyclic hypoxia.

Sheng Z, Murphy SF, Guo S, Green MR
A diphtheria toxin negative selection in RNA interference screening.
Methods Mol Biol. 2014; 1176:59-72 [PubMed] Related Publications
RNA interference (RNAi) screening is a powerful technique for understanding the molecular biology of cancer and searching drug targets. Genes and their upstream activators that are essential for the survival of cancer cells often dictate cancer formation/progression. Hence, they are preferable therapeutic targets. Identifying these genes using RNAi is, however, problematic because knocking them down leads to cell death. Here we describe a diphtheria toxin (DT) negative selection method to circumvent the problem of cell death in RNAi screening. DT fails to kill mouse cells due to the lack of functional DT receptor (DTR). Thus, we first prepare a construct encoding a human functional DTR driven by the promoter of mouse Atf5, a gene essential for the survival of malignant glioma. Then a DT-sensitive mouse malignant glioma cell line is established by over-expressing this DTR. Finally, an RNAi screen is performed in this cell line and genes that activate Atf5 expression are identified. The negative selection approach described here allows RNAi screening to be used for identifying genes controlling cell survival in cancers or perhaps other human diseases with potential in therapeutic intervention.

Okamoto A, Asai T, Kato H, et al.
Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF.
Biochem Biophys Res Commun. 2014; 449(4):460-5 [PubMed] Related Publications
An Fab' antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab' fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.

Cervigne NK, Machado J, Goswami RS, et al.
Recurrent genomic alterations in sequential progressive leukoplakia and oral cancer: drivers of oral tumorigenesis?
Hum Mol Genet. 2014; 23(10):2618-28 [PubMed] Free Access to Full Article Related Publications
A significant proportion (up to 62%) of oral squamous cell carcinomas (OSCCs) may arise from oral potential malignant lesions (OPMLs), such as leukoplakia. Patient outcomes may thus be improved through detection of lesions at a risk for malignant transformation, by identifying and categorizing genetic changes in sequential, progressive OPMLs. We conducted array comparative genomic hybridization analysis of 25 sequential, progressive OPMLs and same-site OSCCs from five patients. Recurrent DNA copy number gains were identified on 1p in 20/25 cases (80%) with minimal, high-level amplification regions on 1p35 and 1p36. Other regions of gains were frequently observed: 11q13.4 (68%), 9q34.13 (64%), 21q22.3 (60%), 6p21 and 6q25 (56%) and 10q24, 19q13.2, 22q12, 5q31.2, 7p13, 10q24 and 14q22 (48%). DNA losses were observed in >20% of samples and mainly detected on 5q31.2 (35%), 16p13.2 (30%), 9q33.1 and 9q33.29 (25%) and 17q11.2, 3p26.2, 18q21.1, 4q34.1 and 8p23.2 (20%). Such copy number alterations (CNAs) were mapped in all grades of dysplasia that progressed, and their corresponding OSCCs, in 70% of patients, indicating that these CNAs may be associated with disease progression. Amplified genes mapping within recurrent CNAs (KHDRBS1, PARP1, RAB1A, HBEGF, PAIP2, BTBD7) were selected for validation, by quantitative real-time PCR, in an independent set of 32 progressive leukoplakia, 32 OSSCs and 21 non-progressive leukoplakia samples. Amplification of BTBD7, KHDRBS1, PARP1 and RAB1A was exclusively detected in progressive leukoplakia and corresponding OSCC. BTBD7, KHDRBS1, PARP1 and RAB1A may be associated with OSCC progression. Protein-protein interaction networks were created to identify possible pathways associated with OSCC progression.

LaPorta E, Welsh J
Modeling vitamin D actions in triple negative/basal-like breast cancer.
J Steroid Biochem Mol Biol. 2014; 144 Pt A:65-73 [PubMed] Free Access to Full Article Related Publications
Breast cancer is a heterogeneous disease with six molecularly defined subtypes, the most aggressive of which are triple negative breast cancers that lack expression of estrogen receptor (ER) and progesterone receptor (PR) and do not exhibit amplification of the growth factor receptor HER2. Triple negative breast cancers often exhibit basal-like gene signatures and are enriched for CD44+ cancer stem cells. In this report we have characterized the molecular actions of the VDR in a model of triple negative breast cancer. Estrogen independent, invasive mammary tumor cell lines established from wild-type (WT) and VDR knockout (VDRKO) mice were used to demonstrate that VDR is necessary for 1,25-dihydroxyvitamin D3 (1,25D) mediated anti-cancer actions in vitro and to identify novel targets of this receptor. Western blotting confirmed differential VDR expression and demonstrated the lack of ER, PR and Her2 in these cell lines. Re-introduction of human VDR (hVDR) into VDRKO cells restored the anti-proliferative actions of 1,25D. Genomic profiling demonstrated that 1,25D failed to alter gene expression in KO240 cells whereas major changes were observed in WT145 cells and in KO clones stably expressing hVDR (KO(hVDR) cells). With a 2-fold cutoff, 117 transcripts in WT145 cells and 197 transcripts in the KO(hVDR) clones were significantly altered by 1,25D. Thirty-five genes were found to be commonly regulated by 1,25D in all VDR-positive cell lines. Of these, we identified a cohort of four genes (Plau, Hbegf, Postn, Has2) that are known to drive breast cancer invasion and metastasis whose expression was markedly down regulated by 1,25D. These data support a model whereby 1,25D coordinately suppresses multiple proteins that are required for survival of triple-negative/basal-like breast cancer cells. Since studies have demonstrated a high prevalence of vitamin D deficiency in women with basal-like breast cancer, correction of vitamin D deficiency in these women represents a reasonable, but as yet untested, strategy to delay recurrence and extend survival. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

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