cDNA Array Research Articles

Effect of heparin on the biological properties and molecular signature of human mesenchymal stem cells

Chronic use of heparin as an anti-coagulant for the treatment of thrombosis or embolism invokes many adverse systemic events including thrombocytopenia, vascular reactions and osteoporosis. Here, we addressed whether adverse effects might also be directed to mesenchymal stem cells that reside in the bone marrow compartment. Harvested human bone marrow-derived mesenchymal stem cells (hMSCs) were exposed to varying doses of heparin and their responses profiled. At low doses (<200ng/ml), serial passaging with heparin exerted a variable effect on hMSC proliferation and multipotentiality across multiple donors, while at higher doses (≥100μg/ml), heparin supplementation inhibited cell growth and increased both senescence and cell size. Gene expression profiling using cDNA arrays and RNA-seq analysis revealed pleiotropic effects of low-dose heparin on signaling pathways essential to hMSC growth and differentiation (including the TGFβ/BMP superfamily, FGFs, and Wnts). Cells serially passaged in low-dose heparin possess a donor-dependent gene signature that reflects their altered phenotype. Our data indicate that heparin supplementation during the culturing of hMSCs can alter their biological properties, even at low doses. This warrants caution in the application of heparin as a culture supplement for the ex vivo expansion of hMSCs. It also highlights the need for careful evaluation of the bone marrow compartment in patients receiving chronic heparin treatment.

Quantification of insulin receptor mRNA splice variants as a diagnostic tumor marker in breast cancer.

The mature human insulin receptor (INSR) has two isoforms: The A isoform and the B isoform. INSR upregulation has been suggested to play a role in cancer. To establish quantitative PCR method for INSR transcript variants and examine their differential expression as a diagnostic tumor marker in breast cancer. The differential expression of IR-A and IR-B were evaluated by TaqMan qRT-PCR assay in the commercially available Breast Cancer Disease cDNA and Cancer Survey cDNA arrays. The mRNA expression levels of IR-A was statistically significantly higher in breast cancer when compared to normal breast tissue while IR-B mRNA expression was down regulated significantly in breast cancer. Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of IR-A mRNA in clinical stage (CS)-IV, and lower IR-B levels in CS-IIA, CS-IIIB and CS-IIIC. However, IR-A:IR-B ratio showed a statistically significant increase in all stages. Cancer Survey cDNA array demonstrated lower levels of IR-B mRNA in breast adenocarcinoma, liver carcinoma and lung carcinoma only while IR-A expression was significantly altered in kidney carcinoma without any significant differences in IR-A:IR-B ratios. The results demonstrate an increased IR-A:IR-B ratio in all clinical stages of breast cancer. Thus, IR-A:IR-B ratio may have a diagnostic biomarker utility in breast cancer.

Expression and pathological effects of periostin in human osteoarthritis cartilage.

BackgroundOsteoarthritis (OA) is one of the most common joint diseases in elderly people, however, the underlying mechanism of OA pathogenesis is not completely clear. Periostin, the extracellular protein, has been shown by cDNA array analysis to be highly expressed in OA, but its function is not fully understood. The purpose of this study was to examine the expression and function of periostin in human OA.MethodsHuman cartilage and synovia samples were used for the analysis of periostin expression and function. The human cartilage samples were obtained from the knees of patients undergoing total knee arthroplasty as OA samples and from the femoral bone head of patients with femoral neck fracture as control samples. Quantitative RT-PCR, ELISA, and immunohistochemistry were used for analysis of periostin expression in cartilage and synovia. Human primary chondrocytes isolated from control cartilage were stimulated by periostin, and the alteration of OA related gene expression was examined using quantitative RT-PCR. Immunocytochemistry of p65 was performed for the analysis of nuclear factor kappa B (NFκB) activation.ResultsThe periostin mRNA was significantly higher in OA cartilage than in control cartilage. Immunohistochemical analysis of periostin showed that the main positive signal was localized in chondrocytes and their periphery matrix near the erosive area, with less immunoreactivity in deeper zones. There was positive correlation between Mankin score and periostin immunoreactivity. The periostin expression was also detected in the fibrotic cartilage and tissue of subchondral bone. In cultured human chondrocytes, periostin induced the expression of interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, and nitric oxide synthase-2 (NOS2) in a dose- and time-dependent manner. The activation of NFκB signaling was recognized by the nuclear translocation of p65. Periostin-induced upregulation of these genes was suppressed by NFκB inactivation in chondrocytes.ConclusionPeriostin was upregulated in OA cartilage, and it may amplify inflammatory events and accelerate OA pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-015-0682-3) contains supplementary material, which is available to authorized users.

Abstract 5126: Establishment of an esophageal small cell carcinoma cell line (TYUC-1)

Abstract Background: Small cell carcinoma of the esophagus is rare and its prognosis is extremely poor. However, there is currently no in vitro model of this disease. Methods: After obtaining written informed consent, we obtained a tumor from a 56-year-old female patient with esophageal small cell carcinoma in 2009 and transplanted it into a Severe Combined Immuno Deficiency (SCID) mouse. After successful transplantation, a tumor from the back of this mouse was minced with a sharp scissors without enzymes and was cultured in Ham F12/DMDM supplemented with 5% fetal bovine serum. Results: Culture cells grew as adherent cells during the initial culture phase and gradually grew as floating aggregated cells. We carefully maintained these floating cells and transferred them without enzymes. These cells could passage to another flask with a split ratio of 1/10. We named these cells “TYUC-1”, and passaged them more than 85 times. The population doubling-time of TYUC-1 was approximately 30 hours. The G-band karyotype confirmed that these cells had a human origin. These cells could be transplanted to SCID mice with the same histological features of primary esophageal small cell carcinoma, and were shown to express TrkA, NGF, NSE, FGFRL1, p53, CD56, ALDH1A1, and CD44, but not CD133. A cDNA array also revealed that 9 genes were overexpressed while the expression of 33 genes was significantly lower than that in 28 esophageal squamous cell carcinoma (ESCC) cell lines. Whole exome sequencing revealed that TYUC-1 had TP53, PIK3CA, KMT2D, CACNA1H, NCOR1 and HRAS mutations. Furthermore, TYUC-1 had mutations in 128 genes, whereas 28 ESCC cell lines had no mutations in these genes. Conclusions: We newly established TYUC-1 cells that exhibited neuroendocrine characteristics and expressed several stem cell-related molecules. These cells may be useful for understanding the characteristics of esophageal small cell carcinoma. Citation Format: Yutaka Shimada, Tomoyuki Okumura, Yoshinori Takei, Kazuaki Watanabe, Akira Hirasawa, Arito Yamane, Masahiko Nishiyama, Takuya Nagata, Kazuhiro Tsukada, Kazuharu Shimizu. Establishment of an esophageal small cell carcinoma cell line (TYUC-1). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2015-5126

Abstract 3207: The roles of cancer-associated fibroblasts in stemness maintenance of lung cancer cells

Abstract Lung cancer is the leading cause of death worldwide including Taiwan. A lot of therapies have been developed to improve cure rate of lung cancer, however the prognosis remains dismal. Cancer cells can acquire resistance to therapies by several known or unknown mechanisms. Microenvironment is also important for acquiring resistance to corresponding treatments of cancer cells. Cancer-associated fibroblasts (CAFs) are known to support many different aspects of tumor initiation, growth and progression by secretion of growth stimulatory, pro-survival and angiogenic factors. In our studies, we compare the differences between normal fibroblasts (NFs) and CAFs which were cultured from surgical specimens of lung cancer patients. CAFs shows increasing tumorous sphere formation ability and significantly up-regulating stem cell markers such as nanog, klf-4, Sox-2, Oct4 and c-myc of cancer cells. We also found that CAFs regulate cancer stem cells (CSCs) growth through numerous paracrine factors such as IGF-II, HGF and SDF1 and by inducing expression of the corresponding receptors in CSCs, including IGF1R, IGF2R, HGFR and CXCR4. In our studies, CAFs obtain more advanced ability compared to NFs, but the mechanisms of how NFs are educated to become CAFs are still unknown, thus we trying to clarify the mechanisms by genome and epigenome-wide high-throughput analysis, including cDNA array, DNA methylation array and miRNA array. Citation Format: Chih-An Lin, Chao-Chi Ho, Jeremy J.W. Chen. The roles of cancer-associated fibroblasts in stemness maintenance of lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2015-3207

Abstract 3094: Sprouty and cell proliferation in colorectal cancer

Abstract BACKGROUND &amp; AIMS: Sprouty (SPRY) proteins are evolutionarily conserved modulators of mitogen-activated protein kinase pathway. SPRY2 appears to function as a tumor suppressor in other cancers, whereas we reported that SPRY2 functions as an oncogene in colorectal cancer (CRC). To further understand the oncogenic potential of SPRY1 and SPRY2 in CRC, we investigated the mechanisms by which these proteins regulate cell proliferation in CRC. MATERIALS &amp; METHODS: Human CRC cDNA arrays were employed to assess mRNA levels of SPRY1, SPRY2 and p21. SPRY1 and SPRY2 were knocked-down in CaCo2 colon cancer cells by siRNA. Effects of SPRY were assessed by immunoblotting, real time PCR, luciferase reporter assay, and confocal microscopy. Mouse embryonic fibroblasts (MEFs) derived from SPRY mutant mice were utilized to dissect SPRY functions in mesenchymal cells. RESULTS: In this study, surprisingly, we present evidence to suggest that concomitant activation of ERK, p-38 and AKT signaling cascades is insufficient to drive cancer cell proliferation in SPRY modulated cells. Suppression of SPRY in colon cancer cells upregulates p21, a cell cycle inhibitor. SPRY2 dependent p21 promoter activation may account for upregulation of p21 expression and inhibition of cancer cell proliferation. Underscoring the biological relevance of these findings, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression of p21. Finally, in a human CRC cDNA array we established an inverse correlation of SPRY1 and SPRY2 with p21 mRNA expression. CONCLUSIONS: Together, this study indicates that SPRY is a target of therapeutic intervention in CRC. Citation Format: Qiong Zhang, Katherine Shim, Kevin Wright, Alexander Jurkevich, Sharad Khare. Sprouty and cell proliferation in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3094. doi:10.1158/1538-7445.AM2015-3094

Abstract 5139: Cell sorting strategies for the isolation of cancer cells and associated fibroblasts from tumor biopsies, surgical resections, and patient-derived xenografts

Abstract The ability of early passage patient-derived tumor xenografts (PDXs) to recapitulate human tumor biology makes them invaluable tools for pre-clinical drug development (1, 2). Similarly, clinically-annotated human tumor cell lines of low passage have potential as scalable biosimilar reagents for in vitro study. Here, we describe an integrated platform for the purification, expansion, and quality control of tumor cells and cancer-associated fibroblasts (CAFs) from patient biopsies, surgical resections, and mouse PDX models. Following dissociation of solid tumors, cells are cultured on Matrigel® in fibroblast conditioned media containing Y-27632 (a Rho-associated kinase inhibitor). Cells are then analyzed by FACS using a panel of species- or cell type-specific antibodies. This panel (anti-mCD9, hCD9, hCD24, hCD90, MHC, HLA, and hEPCAM) permits quantitation of the percentage of mouse/human cells, and the percentages of tumor cells and CAFs in each patient specimen. From these data, a cell sorting strategy can be selected (e.g. mCD9 and hCD90 with hEpCAM or CD24) that will permit purification of both tumor cells and CAFs, along with removal of any mouse fibroblast contamination (for PDX tumors). Samples are sorted multiple times until target populations reach 99.99% purity (typically 2-3 rounds). Purified cells are then returned to culture, expanded over several weeks, and frozen at low passage. The purity and identity of these cultures is quality-assured using 1) mouse-specific QPCR, 2) a cDNA array designed to differentiate fibroblasts from tumor cells, and 3) STR genotyping. Tumor lines and CAFs are then scaled and re-qualified at passages 10 and 20, an essential step given the propensity of low-frequency mouse and human fibroblasts to ‘overtake’ impure cultures with time. Lastly, cancer lines and CAF cultures undergo genomic characterization and are evaluated for in vivo tumorigenicity (or the lack thereof for CAFs). To date, the platform has been used to generate a growing number of purified cancer and CAF pairs from patient materials. In conclusion, our goal is to provide useful reagents to the extramural community that will accompany current efforts within the Division of Cancer Treatment and Diagnosis (DCTD, NCI) aimed at generating a large and extensively characterized repository of PDX models. Funded by NCI Contract No. HHSN261200800001E.

Abstract 5014: Neutrophils protect B-cell lymphomas against chemotherapy via cell-cell interactions mediated by CD44 and ICAM1 receptors

Abstract Background: Neutrophils are innate immune cells that play an established role in the killing of microorganisms. While much less studied than adaptive immune cells, neutrophils are detected in different solid tumors. Current evidence suggests that neutrophils show high functional plasticity and can display either protumor or antitumor activity. Given the abundance of significant portion of tumor-associated neutrophils in many cancer models and due to lack of information about the role of neutrophils in lymphoma, we investigated the impact of neutrophils on the sensitivity of B cell lymphomas to chemotherapy. Methods: Freshly isolated human neutrophils were cocultured with different types of human B cell non Hodgkin lymphomas (NHL) in the presence of various chemotherapies (vincristine, doxorubicin, cisplatin, bortezomib and mafosfamide). Chemotherapeutic effect on cell proliferation and cytotoxicity was assessed by flow cytometry using CFSE and DAPI assays, respectively. Expression profiling of cells exposed to neutrophils was performed by Illumina cDNA array. Blocking antibodies were added to the coculture system. In vivo, RL cells were co-injected with neutrophils subcutaneously into SCID CB17 mice and treated with vincristine. Ex vivo, peripheral blood was obtained from chronic lymphocytic leukemia (CLL) patients. CLL B cells were cocultured with autologous neutrophils then exposed to vincristine, ibrutinib or idelalisib. CLL cells were analyzed for their content in Bcl2, BclXL and Mcl1. Results: We found that freshly isolated human neutrophils protect NHL cells against the chemotherapeutic cytotoxicity. Neutrophils protective role was not due to sequestration of chemotherapy. This effect was not due to soluble factors produced by neutrophils and required direct cell-cell interactions between neutrophils and NHL cells. Expression profiling of RL cells in the presence of neutrophils showed enhanced expression of CD44. Blocking antibodies against CD44 and ICAM1 receptors inhibit neutrophil-mediated protection of RL cells against chemotherapy in vitro. In vivo, freshly isolated human neutrophils co-injected with tumor cells reduced sensitivity of RL cells to vincristine resulting in enhanced tumor growth. Similarly autologous neutrophils protected fresh CLL cells from the cytotoxic effect of various agents. Exposure of CLL cells to neutrophils increased the content of Bcl2 and Mcl1 with no effect on BclXL. Conclusion: Neutrophils protect B cell lymphomas against chemotherapy in vitro and in vivo. This protective role is due to cell-cell interactions mediated by CD44 and ICAM1 receptors. Our results also suggest that neutrophils enhance CLL survival by inducing the expression of anti apoptotic proteins Bcl2 and Mcl1. Our findings underline the importance of neutrophils in tumor growth or chemoresistance and may contribute to therapeutic strategies to inhibit their protective effect. Citation Format: Taghreed Hirz, Justine Esmenjaud, Anne Evesque, Doriane Mathe-Poloni, Charles Dumontet. Neutrophils protect B-cell lymphomas against chemotherapy via cell-cell interactions mediated by CD44 and ICAM1 receptors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2015-5014

Abstract 4909: Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma

Abstract To evaluate the insulin receptor isoform mRNA expression status in NSCLC patients RNA-seq data from 614 NSCLC [355 adenocarcinomas(LUAD) and 259 squamous cell carcinomas (LUSC)] and 92 normal lung specimens were obtained from The Cancer Genome Atlas (TCGA) to evaluate the mRNAexpression of insulin receptor isoform A (IR-A) and insulin receptor isoform B(IR-B). The differential expression status of the insulin receptor isoforms in NSCLC patients was confirmed using qRT-PCR assays with lung cancer cDNA arrays and primary tumor samples. The mRNA expression levels of IR-B were significantly lower in some NSCLC samples compared to normal lung specimens,including both LUAD and LUSC. Notably, no IR-B transcripts were detected - only the IR-A isoform was expressed in 11% of NSCLC patients. This decrease in IR-B expression contributed to an elevated IR-A/IR-B ratio, which was also associated with lower epithelial-mesenchymal transition gene signatures in NSCLC and longer patient survival under standard of care in LUSC. In addition to NSCLC, RNA-seq data from TCGA revealed a similar increase in IR-A/IR-B ratio in many other cancer types, with high prevalence in acute myeloid leukemia,glioblastoma multiforme, and brain lower grade glioma. Our results indicate a common reduction of the mRNA expression level of IR-B and an increased IR-A/IR-B mRNA ratio in NSCLC and other tumor types. The relationship of altered IR-A/IR-B ratios with cancer progression and patient survival should be prospectively explored in future studies. Citation Format: Jiaqi Huang, Wei Zhu, Liyan Jiang, Katie Streicher, Chris Morehouse, Philip Brohawn, XiaoXiao Ge, Zhengwei Dong, Xiaolu Yin, Guanshan Zhu, Yi Gu, Parthiv Mahadevia, Brandon W. Higgs, Yihong Yao. Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2015-4909

The role of branched chain amino acid and tryptophan metabolism in rat’s behavioral diversity: Intertwined peripheral and brain effects

Previously, we showed that a transient early-in-life interference with the expression of multiple genes by mithramycin (MTR) followed by later-in-life exposure to chronic stress, leads to a “daring” and novelty seeking behavior in rats. In this study we searched for molecular changes that contribute to this behavioral alteration. We applied a non-hypothesis driven strategy using whole genome cDNA array analysis (WGA) followed by Genome Scale Metabolic modeling analysis (GSMM). Gene expression validation was performed by qRT-PCR and immunoblotting. Brain and serum amino acids levels were measured by HPLC. WGA data directed us towards metabolic pathways and GSMM pointed at branched chain amino acids (BCAA) pathway. Out of 21 amino acids analyzed in the prefrontal cortex of MTR+Stress rats only tryptophan, whose brain levels depend on serum BCAA levels, showed a significant decrease. No change was observed in serotonin or kynurenine levels. However, a significant reduction in mRNA and protein levels of the large neutral amino acid transporter (LAT1), which transports BCAA and tryptophan into the brain, as well as in serum levels of tryptophan/BCAA ratio were observed. The latter may be attributed to the failure to increase serum insulin, following stress, in rats pre-exposed to mithramycin. Finally, significant correlations were observed between the anxiety index and tryptophan and between T-maze errors and LAT1. This study shows a specific behavioral pattern, which is linked to modulations in fluxes of amino acids both peripheral and central, which converge and reciprocally interact, and may thus be equally important targets for therapeutic intervention.

LDOC1 silenced by cigarette exposure and involved in oral neoplastic transformation.

Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC through alteration of the methylome of oral cells. Here we report that cigarette smoke condensate (CSC) significantly changes the genomic 5-methyldeoxycytidine content and nuclear accumulation of DNA methyltransferase 1 (DNMT1) and DNMT3A in human untransformed oral cells. By using integrated analysis of cDNA and methylation arrays of the smoking-associated dysplastic oral cell line and OSCC tumors, respectively, we identified four epigenetic targets--UCHL1, GPX3, LXN, and LDOC1--which may be silenced by cigarette. Results of quantitative methylation-specific PCR showed that among these four genes, LDOC1 promoter was the most sensitive to CSC. LDOC1 promoter hypermethylation and gene silencing followed 3 weeks of CSC treatment. LDOC1 knockdown led to a proliferative response and acquired clonogenicity of untransformed oral cells. Immunohistochemistry showed that LDOC1 was downregulated in 53.3% (8/15) and 57.1% (20/35) of premalignant oral tissues and early stage OSCCs, respectively, whereas 76.5% (13/17) of normal oral tissues showed high LDOC1 expression. Furthermore, the microarray data showed that LDOC1 expression had decreased in the lung tissues of current smokers compared with that in those of never smokers and had significantly decreased in the lung tumors of smokers compared with that in normal lung tissues. Our data suggest that CSC-induced promoter methylation may contribute to LDOC1 downregulation, thereby conferring oncogenic features to oral cells. These findings also imply a tumor suppressor role of LDOC1 in smoking-related malignancies such as OSCC and lung cancer.

Heterogeneous Nuclear Ribonucleoprotein A1 Improves the Intestinal Injury by Regulating Apoptosis Through Trefoil Factor 2 in Mice with Anti-CD3–induced Enteritis

The role of hnRNP A1 in the onset of intestinal inflammation remains unclear. This study investigated the function of hnRNP A1 in mice enteritis models. C57Bl6/J mice were intraperitoneally injected with anti-CD3 antibodies to develop enteritis. In the DSS-induced colitis group, the mice were allowed free access to 3% DSS solution in their drinking water for 5 days. 3H-mannitol flux and complementary DNA array tests were used to assess the intestinal barrier function and messenger RNA (mRNA) expression, respectively. Real-time PCR was performed after immunoprecipitation with anti-hnRNP antibodies to determine the specific mRNA binding of hnRNP A1. The hnRNP A1 expression was increased in the intestine of the mouse at 24 hours after treatment with anti-CD3 antibodies and 5 days after starting DSS administration. Small interfering RNA (siRNA) against hnRNP A1 exacerbated the intestinal injuries in both models. According to the microarray analysis, trefoil factor 2 (TFF2) was identified as a candidate molecule targeted by hnRNP A1 in the anti-CD3 antibody-induced enteritis group. Moreover, the binding between hnRNP A1 and TFF2 mRNA significantly increased in the enteritis mice, and the administration of siRNA against either hnRNP A1 or TFF2 exacerbated the degree of intestinal injury. In the DSS-induced colitis group, treatment with the siRNA of hnRNP A1 worsened the intestinal injury, while the expression of TFF3 did not change. hnRNP A1 improves intestinal injury in anti-CD3 antibody-induced enteritis mice through the upregulation of TFF2, which regulates apoptosis and enhances epithelial restoration, whereas this molecule ameliorates DSS-induced colitis through a different pathway.

Different maturation cocktails provide dendritic cells with different chemoattractive properties.

BackgroundDendritic cells (DC) are currently implemented as immunotherapeutic strategy for the treatment of tumor patients based on their central role in the immune system. Despite good results were obtained in vitro and in animal models, their clinical use has provided limited success suggesting the requirement to optimise the protocol for their production.MethodsA cDNA array was performed on FastDC obtained from the differentiation of human peripheral blood monocytes stimulated with the clinical gold standard or with two alternative maturation cocktails combining interferon (IFN)γ and ligands for different toll like receptors (TLR).ResultsA stronger modulation of the DC transcriptome with respect to immature DC was found in alternatively stimulated DC when compared to DC stimulated with the clinical gold standard. A major class of molecules differentially expressed using distinct DC stimulation protocols were chemokines. Validation of their differential expression pattern at the mRNA and protein level confirmed the secretion of inflammatory chemokines by the alternative DC. Functional analyses of the chemotactic properties of DC “wash out” supernatants highlighted the ability of alternative, but not of gold standard DC to efficiently recruit immune cells with a prevalence of monocytes. Effector cells belonging to the innate as well as adaptive immunity were also attracted and the interaction with alternative DC resulted in enhanced secretion of IFNγ and induction of cytotoxic activity. Using leukocytes from cancer patients, it was demonstrated that the monocyte-attracting activity targeted cells with an inflammatory phenotype characterised by high levels of HLA-DR expression.ConclusionsDespite other classes of immune modulatory genes differently expressed in the alternative DC require to be investigated and characterised regarding their functional consequences, the reduced maturation state and chemoattractive properties of the gold standard versus alternative DC clearly promote the necessity to change the clinically used maturation cocktail of DC in order to improve the outcome of patients treated with DC-based vaccines.

Identification of defense-related genes in banana roots infected by Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium oxysporum f. sp. cubense tropical race 4 is a lethal pathogen, affecting banana production by causing vascular wilt. To date, the molecular basis of resistant cultivars is largely unknown. In this study, suppression subtractive hybridization (SSH) libraries were constructed for the roots of two iso-line cultivars—a Foc-susceptible cultivar and its tolerant mutant—using both uninfected controls and plants artificially challenged with pathogen conidiospores. Unigenes from SSH libraries were used to develop a cDNA array, and then microarray analysis was carried out to verify those genes exhibiting differential expression. A total of 257 unigenes was obtained from the SSH libraries and microarray detection confirmed that most of them were differentially expressed. Of these, 116 unigenes had matched proteins in gene ontology and were classified into 38 different functional groups. The transcript levels of up to 19 putative defense genes were followed for a whole infection period in four cultivars differing in resistance level. At the early stages of infection (within 48 h), nineteen genes varied by a factor of more than three, subsequently only four genes (Pectinesterase inhibitor, PEI; Peroxidase, POD; Pathogenesis-related protein 1, PR1; and Pathogenesis-related protein 3, PR3) maintained their up-regulation. This indicates that anti-oxidation, cell wall modification and synthesis of anti-fungal proteins are the most important aspects to study if we are to understand the Foc resistance mechanism in commercial banana plants.

Abstract A36: Unveiling biomarker and mechanism of drug resistance to molecular targeted therapeutics in renal cell carcinoma

Abstract Introduction and Objective: Renal cell carcinoma (RCC) is the most lethal of the urologic malignancies and it is largely incurable when it becomes metastatic. Recent development of molecular targeted agents such as tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors have provided some survival benefit for a subset of RCC patients. Overall, only 30% patients exhibit a response to these agents, however, almost all patients eventually develop drug resistance. Currently, there is no reliable biomarker(s) for predicting drug response for individual patients and the mechanism(s) of drug resistance is largely unknown. It is known that DAB2IP, a novel member of the Ras GTPase-activating protein gene family, is associated with cell proliferation, apoptosis, metastasis and chemo- or radio-resistance in several cancer types. In the present study, we decided to investigate the association between DAB2IP expression and therapeutic response to molecular targeted agents in RCC patients and explored the possible mechanism of DAB2IP in regulating drug response of RCC cells. Methods: DAB2IP expression was evaluated by immunohistochemistry (IHC) in a cohort of RCC patients who had received targeted therapies. Correlation between DAB2IP expression and patients' survival was analyzed. The effect of DAB2IP on RCC cells was examined using a panel of knockdown (KD) or overexpression cells generated by gene transfection and also determined for their in vitro and in vivo (xenograft model) responses to targeted agents treatment. The mechanism of drug resistance was delineated by signaling cascade profiling with a variety of molecular biologic techniques such as cDNA array, ChIP, reporter gene assay, real-time PCR and Western blot. Results: The levels of DAB2IP expression in RCC specimens determined by IHC positively correlate with the survival of patients receiving targeted therapies. DAB2IP KD RCC cells exhibit drug resistance to mTOR inhibitor Temsirolimus (Tem) treatment compared with the control from both in vitro and in vivo experiments. In contrast, increased DAB2IP in RCC cells by gene transfection can sensitize their response to Tem. Mechanistically, loss of DAB2IP was able to activate both the PI3K/mTOR pathway and the ERK/RSK1 pathway that further led to increased expression of hypoxia-inducible factor (HIF)-2α protein. Inhibitor(s) of either the mTOR pathway or the ERK pathway only partially suppressed growth of DAB2IP KD cells. However, the combination of both inhibitors achieved a synergistic growth inhibition in resistant cells. In this event, HIF-2α, but not HIF-1α, appeared to be a key converging factor and we further demonstrated that silencing of HIF-2α expression with specific shRNA was able to reverse the resistant phenotype in DAB2IP KD cells. Conclusions: DAB2IP represents a new predictive biomarker for the therapeutic response of RCC patients to targeted therapy. Most importantly, its functional role in RCC provides new therapeutic strategies in combating this lethal disease. Citation Format: Jiancheng Zhou, Kaijie Wu, Eunjin Yun, Payal Kapur, Rey-Chen Pong, Dalin He, Jer-Tsong Hsieh. Unveiling biomarker and mechanism of drug resistance to molecular targeted therapeutics in renal cell carcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A36.

Elevated NIBP/TRAPPC9 mediates tumorigenesis of cancer cells through NFκB signaling.

Regulatory mechanisms underlying constitutive and inducible NFκB activation in cancer remain largely unknown. Here we investigated whether a novel NIK- and IKK2-binding protein (NIBP) is required for maintaining malignancy of cancer cells in an NFκB-dependent manner. Real-time polymerase chain reaction analysis of a human cancer survey tissue-scan cDNA array, immunostaining of a human frozen tumor tissue array and immunoblotting of a high-density reverse-phase cancer protein lysate array showed that NIBP is extensively expressed in most tumor tissues, particularly in breast and colon cancer. Lentivirus-mediated NIBP shRNA knockdown significantly inhibited the growth/proliferation, invasion/migration, colony formation and xenograft tumorigenesis of breast (MDA-MB-231) or colon (HCT116) cancer cells. NIBP overexpression in HCT116 cells promoted cell proliferation, migration and colony formation. Mechanistically, NIBP knockdown in cancer cells inhibited cytokine-induced activation of NFκB luciferase reporter, thus sensitizing the cells to TNFα-induced apoptosis. Endogenous NIBP bound specifically to the phosphorylated IKK2 in a TNFα-dependent manner. NIBP knockdown transiently attenuated TNFα-stimulated phosphorylation of IKK2/p65 and degradation of IκBα. In contrast, NIBP overexpression enhanced TNFα-induced NFκB activation, thus inhibiting constitutive and TNFα-induced apoptosis. Collectively, our data identified important roles of NIBP in promoting tumorigenesis via NFκΒ signaling, spotlighting NIBP as a promising target in cancer therapeutic intervention.

Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

Altered Sirtuin 7 Expression is Associated with Early Stage Breast Cancer.

BACKGROUNDTo evaluate sirtuin-7 (SirT7) mRNA expression status in breast cancer patients with different metastatic stages and survey SirT7 mRNA expression status in eight different types of cancer.METHODSThe expression of SirT7 in the commercially available TissueScan qPCR Breast Cancer Disease cDNA arrays containing 16 normal, 23 Stage I, 36 IIA, 22 IIB, 8 IIIA, 23 IIIA, 6 IIIB, 13 IIIC, and 5 IV were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Similar analysis was performed in TissueScan qPCR Cancer Survey cDNA array, which includes breast, colon, kidney, liver, lung, ovarian, prostate, and thyroid specimens.RESULTSThe mRNA expression levels of SirT7 were significantly higher in breast cancer samples compared to normal breast specimens (P < 0.001). Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of SirT7 mRNA in CS-I, CS-II, and CS-III when compared to normal breast tissue (P < 0.05). Notably, SirT7 mRNA levels were higher in CS-I, CS-IIA, CS-IIB, and CS-IIIA (P < 0.05). Additionally, there were significantly lower SirT7 mRNA levels in thyroid carcinoma when compared to their corresponding normal tissue (P < 0.05).CONCLUSIONSOur results indicate an increase in the mRNA expression level of SirT7 in breast cancer, particularly in CS-I, CS-IIA, CS-IIB, and CS-IIIA. The relationship of altered SirT7 with breast cancer progression and patient survival should be prospectively explored in future studies.

Gene Expression Study on Similarity Effect of Thymoquinone on Epithelial Breast Cancer and Colorectal Cancer Cell Lines

Adenocarcinoma is known as a common type of cancer which includes 85% of breast carcinoma and 95% of colorectal carcinoma. Up until now, the cytotoxic effect of Thymoquinone on different types of tumor cells has been reported. It was hypothesized that Thymoquinone has similar effect on cancer arising from epithelial cells through gene expression analysis in MCF7 breast cancer and HT-29 colon cancer cell lines. The quantity and quality of RNA samples were identified using RNeasyPlus Mini kit and RNA 6000 Nano LabChip kit, respectively. The purified RNA samples of MCF7 cells were used in two-color 8×60K cDNA array platform with SurePrint Agilent technology. The hybridized cRNA/cDNA probes were identified due to labeling with red and green cyanine dyes using GEX hybridization buffer HI-RPM. LOWESS normalization reduced the dye bias on the array slide using feature extraction software. Gene ontology analysis was done after performing different steps of filtering to reduce not satisfied genes using Gene Spring software. Two-step RT-qPCR assay using Taq Man fast advanced master mix analyzed the most up and down regulated genes in MCF7 compared to HT-29 cancer cell lines. The 10 different cancer cell lines in a form of universal reference RNA was used as standard data set comparison and as a positive control RNA in cDNA array and RT-qPCR assay, respectively. The T-test statistical analysis of independent samples showed that there is no significant difference between two types of cell lines due to Thymoquinone treatment with p-value 0.844. Among the selected genes; CARD16, UGT1A8, SLC7A11, IFIT1, IF16 and IFIT3 were expressed significantly (0.009, 0.0001, 0.037, 0.098, 0.0001 and 0.033, respectively) in breast cancer compare to colon cancer cells. The findings indicated similar effect of Thymoquinone on cancer arising from epithelial cells.

UV-protective Effects of Phytoecdysteroids from Microsorum grossum Extracts on Human Dermal Fibroblasts

Microsorum grossum (Polypodiaceae), locally called metuapua'a, is one of the most frequently used fern species in Polynesian traditional medicine. Fronds or rhizomes of this species are common ingredients of popular medicine recipes to cure various ailments. M. grossum frond and rhizome extracts contain, as their main bioactive components, phytoecdysteroids such as 20-hydroxyecdysone, known to have many interesting biological activities and considered to be adaptogenic compounds [1]. The skin-active effect of M. grossum extract was investigated in two ways on human dermal fibroblasts: a transcriptomic study with c-DNA array for gene expression modulation and a Stress Induced Premature Senescence (SIPS) test. The total extract of M. grossum up-regulates Heme Oxygenase 1 (HO1), an enzyme which protects cells from oxidative stress and which is responsible for skin photoimmunoprotection. The present paper also reports that premature senescence of human skin induced by repeated UV irradiations can be prevented by an ecdysteroid fraction of M. grossum. Our data indicate that extracts of M. grossum could protect skin against oxidative stresses and suggest that they could be used as innovative active cosmetic ingredients.