Expression Levels In Tumor Cells Research Articles

PB1696 EVALUATION OF TUMOR CELL SENSITIVITY TO DAUNORUBICIN USING WST1 TEST IN ACUTE MYELOBLASTIC LEUKEMIA PATIENTS

Background:In recent years the MTT test for the assessment of the sensitivity of tumor cells to cytotoxic drugs in leukemia patients were used. In present study, the possibility of the WST1 test as a screening method of drug sensitivity of tumor cells of leukemia patients using peripheral blood and bone marrow samples was evaluated.Aims:The aim of this work was to study the effectiveness of the WST1 test for the initial estimation of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the treatment response of patients with acute myeloblastic leukemia.Methods:The study included 42 patients with acute myeloblastic leukemia (AML). The average age of the patients was 53.3 ± 12.7 years. Leukemia cells were obtained from peripheral blood and bone marrow of patients. The sensitivity of tumor cells of patients with AML to daunorubicin in vitro using the WST1 test was made prior to initiating of cytotoxic therapy. Confirmation of multidrug resistance by evaluation of MDR1 mRNA expression levels in tumor cells of AML patients was performed on bone marrow and/or peripheral blood samples using quantitative real‐time reverse transcription‐polymerase chain reaction (real‐time qPCR).In all patients, the diagnosis was confirmed by the study of myelogram, flow cytofluorimetry, immunocytochemistry, cytogenetic analysis, as well as standard laboratory and instrumental studies that are included in the standard examination of patients with leukemia. Based on immunopenotyping and cytogenetic analysis, each patient was assigned to a risk group and prognosis. The obtained results were compared with the clinical and hematological response of these patients to cytotoxic therapy, as well as with the MDR1 mRNA expression level.Also we evaluated the survival of leukemia patients depending on the sensitivity to daunorubicin and MDR1 mRNA expression levels in tumor cells of AML patients.All statistical analyses were done with computer software MS Excel, OriginPro 7.5, Statistica 10.0.Results:Regardless of the class of chemotherapeutic drugs administered to the patients, the survival rate was higher in the group of patients with high sensitivity to daunorubicin and low expression levels of the MDR1 mRNA in tumor cells. Patients received daunorubicin within the “7 + 3” regimen (7 days of cytarabine and 3 days of daunorubicin) showed a high correlations (r = 0.8, p <0.05) between the expression levels of MDR1 mRNA and sensitivity to daunorubicin, moderate correlations (r = 0.63, p < 0.05) between the response to chemotherapy and sensitivity to daunorubicin, and a moderate correlations between the prediction (r = 0.48, p < 0.05) and the risk group (r = 0.49, p < 0.05) and sensitivity to daunorubicin. It is important to note that a high correlations (r = 0.86, p < 0.05) was found between the expression levels of MDR1 mRNA and the response to therapy.Summary/Conclusion:The obtained data demonstrate that the WST1 test can be effectively used as a screening method for the initial assessment of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the response to treatment of AML patients.

A Feedback Loop Formed by ATG7/Autophagy, FOXO3a/miR-145 and PD-L1 Regulates Stem-Like Properties and Invasion in Human Bladder Cancer.

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 blockade have been identified to target immune checkpoints to treat human cancers with durable clinical benefit. Several studies reveal that the response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumor cells. However, the mechanistic pathways that regulate PD-L1 protein expression are not understood. Here, we reported that PD-L1 protein is regulated by ATG7-autophagy with an ATG7-initiated positive feedback loop in bladder cancer (BC). Mechanistic studies revealed that ATG7 overexpression elevates PD-L1 protein level mainly through promoting autophagy-mediated degradation of FOXO3a, thereby inhibiting its initiated miR-145 transcription. The lower expression of miR-145 increases pd-l1 mRNA stability due to the reduction of its direct binding to 3′-UTR of pd-l1 mRNA, in turn leading to increasing in pd-l1 mRNA stability and expression, and finally enhancing stem-like property and invasion of BC cells. Notably, overexpression of PD-L1 in ATG7 knockdown cells can reverse the defect of autophagy activation, FOXO3A degradation, and miR-145 transcription attenuation. Collectively, our results revealed a positive feedback loop to promoting PD-L1 expression in human BC cells. Our study uncovers a novel molecular mechanism for regulating pd-l1 mRNA stability and expression via ATG7/autophagy/FOXO3A/miR-145 axis and reveals the potential for using combination treatment with autophagy inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human BCs.

Prognostic Value of PRAME Activity in Tumor Cells of Follicular Lymphoma

Aim. To set survival parameters for follicular lymphoma (FL) patients with different PRAME expression levels in tumor cells. Materials & Methods. The study was conducted on samples of lymph nodes, blood, and bone marrow of 34 patients with newly diagnosed FL. PRAME expression levels were measured in tumor cells (centrocytes and centroblasts) by quantitative real-time PCR. The impact of different PRAME expression levels on survival parameters was studied with median follow-up of 29 months. Clinical and laboratory characteristics used for FLIPI-1 and FLIPI-2 calculations in different patient groups were compared. Results. A high (> 5 % against ABL control gene) PRAME expression level correlates with higher Ki-67 activity (p = 0.043) and larger tumor mass (p = 0.04). Survival parameters were worse with high PRAME expression level in FL cells. Combination of both high FLIPI-1/FLIPI-2 risk and high PRAME expression level determines extremely unfavorable prognosis and may result in death. Conclusion. In FL patients high PRAME expression level in tumor cells has negative prognostic value, but only in the presence of parameters determining high FLIPI-1 and FLIPI-2 risk. Juxtaposition of PRAME expression level and FLIPI-1/ FLIPI-2 values enables most reliable prediction of early mortality in FL patients.

Vliyanie na prognoz estrogenovykh retseptorov a v opukholevykh kletkakh u patsientov, radikal'no operirovannykh po povodu nemelkokletochnogo raka legkogo

Aim. Evaluation of prognostic role of estrogen receptors a (ERa) expression level at patients with non-small cell lung cancer (NSLC) after surgical treatment. Methods and materials. 115 clinical cases of patients who underwent treatment in N.N.Blokhin Russian Cancer Research Center fr om 2009 to 2016 were studied. There were 95 males and 20 females. All patients had NSLC confirmed by histological analysis and followed-up for long term results. Results. There was no statistical evidence that the level of estrogen a expression in tumor cells has an impact on relapse free survival among men with both adenocarcinoma and squamous cell carcinoma of the lung. Similar results were obtained for women group. Also, no correlations between overall survival and the level of estrogen a expression were noticed among men and women. Thus ERa can’t be used as a predictor marker for patients with NSLC. Conclusion. The level of ERa expression in tumor cells among male patients does not influence on disease prognosis regardless stage of the disease and tumor morphology and can’t be used as a prognostic factor. The impact of the ERa expression level in tumor cells among female patients on disease prognosis is questionable.

Au-CGKRK Nanoconjugates for Combating Cancer through T-Cell-Driven Therapeutic RNA Interference.

Numerous prior studies on fighting cancer have been based on using inhibitors of JAK-STAT pathway (signal transducer and activator of transcription 3 (STAT3) inhibitor in particular), a signaling pathway responsible for progression of many types of cancer cells. However, recent studies have shown that STAT3 activation leads to upregulation of program death receptor-ligand 1 (PD-L1, an immune checkpoint protein that plays a major role behind evasion of immune systems by growing tumors) expression levels in tumor cells, leading to enhanced immune suppression. This is why global efforts are being witnessed in combating cancer through use of immune checkpoint inhibitors. Herein, we report on the design, synthesis, physicochemical characterizations, and bioactivity evaluation of novel tumor- and tumor-vasculature-targeting noncytotoxic Au-CGKRK nanoconjugates (17–80 nm) for combating tumor. Using a syngeneic mouse tumor model, we show that intraperitoneal (i.p.) administration of the Au-CGKRK nanoparticles (NPs) complexed with both PD-L1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant (>70%) enhancement in overall survivability (OS) in melanoma-bearing mice (n = 5) when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western blotting) are consistent with the observed OS enhancement being a T-cell-driven process. Biodistribution study using near-infrared dye-loaded Au-CGKRK nanoconjugates revealed selective accumulation of the dye in mouse tumor. Notably, the overall survival benefits were significantly less (∼35%) when melanoma-bearing mice were treated (i.p.) with Au-CGKRK NPs complexed with only PD-L1siRNA or with STAT3siRNA alone. The presently described Au-CGKRK nanoconjugates are expected to find future use in therapeutic RNA-interference-based cancer immunotherapy.

STAT5 expression correlates with recurrence and survival in melanoma patients treated with interferon-α.

Interferons (IFN) have a direct growth-inhibiting effect on tumor cells through Janus kinase-dependent activation of the transcription factor signal transducer and activator of transcription (STAT1). In vitro, signaling through STAT5 has been demonstrated to counteract this effect and lead to IFN resistance of melanoma cell lines. In 32 patients treated with IFN-α in an adjuvant setting, we investigated paraffin-embedded tumor tissue from primary melanomas and melanoma metastases for expression of STAT3 and STAT5, by immunohistochemistry, and for expression of phosphorylated signaling transduction activating transcription factor (pSTAT)3 and pSTAT5, by immunofluorescence. Tumor cell expression levels of these proteins were correlated with patient characteristics and clinical outcomes. The patient cohort consisted of 12 (37.5%) patients at AJCC stage I/II (primary melanoma) and 20 (62.5%) at stage III/IV (metastatic melanoma). Recurrence was observed for 25 (78.1%) either during or after IFN-α therapy. χ Correlation of staining intensities with clinical data revealed association of pSTAT3 and STAT5 expression with sex (P=0.003 and 0.016, respectively) and of STAT3 with tumor stage (P=0.019). Recurrence of melanoma was found to be associated with high STAT5 expression (P=0.017). Multivariable regression analysis revealed STAT5 expression as an independent factor for predicting progression-free survival (P<0.0001) and overall survival (P=0.022). In summary, high expression of STAT5 correlated with melanoma recurrence and survival of patients treated with IFN-α in the adjuvant setting. Recently, it has been suggested that mutations of Janus kinases are involved in resistance to immune checkpoint blocker treatments implying a possible role of STAT5 for immune checkpoint resistance.

MicroRNA miR-147b promotes tumor growth via targeting UBE2N in hepatocellular carcinoma.

As the subfamily of noncoding RNA, microRNAs (miRNAs) broadly regulate the development of cancers, while their dysregulation and function in human hepatocellular carcinoma (HCC) remains largely unclear. Here, we found the expression level of microRNA-147b (miR-147b) is increased aberrantly in HCC tumor tissues, and its expression positively correlates to the tumor severity. In both MTT and colony formation assay, knockdown of miR-147b dramatically inhibits in vitro proliferation of HCC cell lines. More interestingly, we also performed in vivo tumorigenesis assay and found that miR-147b can regulate in vivo tumorigenesis in nude mice xenograft models. The ubiquitin-conjugating enzyme E2N (UBE2N) was identified directly and functionally targeted by miR-147b. The mRNA level of UBE2N is increased in HCC tumors or cell lines. Restoring UBE2N expression level in tumor cells leads to inhibition of cell proliferation, which mimics the effect upon miR-147b knockdown in the same cells. These data elucidated the oncogenic role of miR-147b in HCC development and progression with therapeutic target potentials.

Simultaneous Imaging of Endogenous Survivin mRNA and On-Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher.

The design of multifunctional drug delivery systems capable of simultaneous target detection, imaging, and therapeutics in live mammalian cells is critical for biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs) chemically modified with a small-molecule dark quencher, followed by sequential drug encapsulation, MSN capping with a dye-labeled antisense oligonucleotide, and bioorthogonal surface modification with cell-penetrating poly(disulfide)s, the authors have successfully developed the first mesoporous silica nanoquencher (qMSN), characterized by high drug-loading and endocytosis-independent cell uptake, which is able to quantitatively image endogenous survivin mRNA and release the loaded drug in a manner that depends on the survivin expression level in tumor cells. The authors further show that this novel drug delivery system may be used to minimize potential cytotoxicity encountered by many existing small-molecule drugs in cancer therapy.

β-catenin induces expression of prohibitin gene in acute leukemic cells

Prohibitin (PHB) is a multifunctional protein conserved in eukaryotic systems and shows various expression levels in tumor cells. However, regulation of PHB is not clearly understood. Here, we focused on the regulation of PHB expression by Wnt signaling, one of dominant regulatory signals in various leukemic cells. High mRNA levels of PHB were found in half of clinical leukemia samples. PHB expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. Although cell proliferating signals downregulated the transcription of PHB, treatment with lithium chloride, an analog of the Wnt signal, induced PHB level in various cell types. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This β-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicate that PHB is a direct target of β-catenin and the increased level of PHB in leukemia can be regulated by Wnt signaling.

Expression of exportin 1 (XPO1/CRM1) in pancreatic adenocarcinoma.

327 Background: Exportin 1 (XPO1/CRM1) is a nuclear export chaperone that mediates the nuclear export of several proteins that are essential to growth regulation and tumor suppression. It was found to be overexpressed in several types of human malignancies and overexpression was found to be associated with poor prognosis. Pancreatic adenocarcinoma (PAC) is associated with very poor survival and currently has no effective treatment. Selective inhibitors of nuclear export are currently undergoing phase I and II clinical trials in hematologic malignancies and solid tumors. The aim of this study was to determine the expression of XPO1/CRM1 in pancreatic adenocarcinoma (PAC) and its relation to survivin expression and proliferative activity as determined by the S-phase fraction (SPF). Methods: Sections of tissue microarray containing 77 formalin fixed and paraffin embedded human PAC were stained by immunohistochemistry (IHC) using monoclonal antibodies to XPO1/CRM1, survivin, and for Cyclin A. The levels of XPO1/CRM1 and survivin expression in tumor cells and the S-phase fraction (SPF) were determined using a quantitative digital image analysis solution (OTMIAS). Results: Sixty-six of the 77 (86%) cases were positive for XPO1/CRM1, with the mean expression value ranging from 0.3 to 53 units and the median ranging from 0.3 to 45 units. There was significant correlation between the mean and median expression values of XPO1/CRM1 in tumor cells with the mean and median values of expression of survivin (p &lt; 0.001). Moreover, the mean and median XPO1/CRM1 expression levels in tumor cells correlated significantly with SPF (p = 0.005). Conclusions: XPO1/CRM1 is expressed in a significant proportion of PAC, and increased expression correlates with both survivin expression and increased proliferative activity, suggesting that selective inhibitors of nuclear export may be effective against PAC.

The epigenetic regulation of CXCL14 plays a role in the pathobiology of oral cancers.

Background: Chemokines selectively attract and activate leukocytes and play roles in a variety of homeostatic and disease processes. Explore the biological properties of CXCL14 seems complicated due to unknown functional characteristics of CXCL14 in cancer.Methods: To study the multistep process of oral cancer development, we analyzed oral samples spanning normalcy, dysplasia and cancer from multiple perspectives, revealing a cascade of progressive changes.Results: CXCL14 protein was expressed in the cytoplasm adjacent to tumors. T classification (P<0.001), clinical stage (P=0.0013) and nodal metastasis (P=0.0035) were significantly associated with CXCL14 in relationships between CXCL14 expression levels and tumor and patient characteristics. Compared with non-tumor tissue, expression of the epidermal growth factor receptor (EGFR) gene was increased in dysplasia and was further sustained in cancer. Our data show an inverse relationship between CXCL14 and EGFR expression levels in tumor cells indicating that CXCL14 expression is beneficial for tumor suppression. To explore epigenetic regulation and the impact of CXCL14 on oral cancer, analysis of CpG islands methylation in the CXCL14 promoter region indicated that the abnormal hypermethylation of that promoter region in tumor cells and tissues is one of the mechanisms causing the reduced expression. Restoration of CXCL14 expression was induced by treatment with 5-aza-2′-deoxycytidine. Using in vivo mouse models, we demonstrate that the restoration of CXCL14 expression in irradiation-induced oral carcinoma cells induces the expression of Late Cornified Envelope (LCE) genes.Conclusions: Our data suggest that LCE genes are a novel target of CXCL14 and are likely to have a tumor suppressor function through the modulation of CXCL14 expression. In conclusion, CXCL14 might play a pivotal role in the pathobiology of oral cancer, probably by regulating DNA methylation and leukocyte migration. The level of CXCL14 expression may be a valuable adjuvant parameter to predict the prognosis of patients with oral carcinoma and may be a potential therapeutic target.

High cytotoxic T-lymphocyte-associated antigen 4 and phospho-Akt expression in tumor samples predicts poor clinical outcomes in ipilimumab-treated melanoma patients.

Ipilimumab, a fully human monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), is the first immune checkpoint inhibitor approved for the treatment of unresectable melanoma on the basis of its overall survival (OS) benefit. However, ipilimumab is associated with significant immune-related adverse events. We hypothesized that biomarker exploration of pretreatment tumor samples and correlation with clinical outcome would enable patient selection with an increased benefit/risk ratio for ipilimumab therapy. At the University of Texas MD Anderson Cancer Center, a total of 81 advanced melanoma patients were treated on the Ipilimumab Expanded Access Program from 2007 to 2008. Using immunohistochemistry, we analyzed the expression of immune checkpoint (CTLA-4, PD-1, PD-L1) and Akt-pathway proteins in formalin-fixed tumor tissue. Associations between these biomarkers and progression-free survival (PFS) and OS were analyzed with univariate and multivariate Cox proportional-hazards models. There was a significant correlation between high CTLA-4 protein expression levels in tumor cells and risk of death (P=0.02) and decreased PFS (P=0.023). In addition, high expression of CTLA-4 in peritumoral lymphocytes correlated with poor OS (P=0.023). In multivariate analysis, patients with high CTLA-4 and phospho-Akt (p-Akt) expression correlated with poor OS (log-rank test, P=0.039) and PFS (log-rank test, P=0.014). High levels of CTLA-4 and p-Akt expression in pretreatment tumor cells in melanoma patients were associated with poor clinical outcomes. Immunohistochemistry analysis of CTLA-4 and p-Akt in pretreatment tumor samples provides useful biomarkers that may enable improved patient selection for ipilimumab therapy. Prospective clinical studies are warranted to investigate the predictive value of these biomarkers.

Abstract LB-315: Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a

Abstract Bone is a preferential site of metastasis from prostate cancer (PCa). Age and obesity, conditions that increase adipocyte numbers in bone marrow, are risk factors for skeletal metastases from PCa. Research in our laboratory focuses on understanding the interactions between adipocytes and tumor cells that have infiltrated the bone marrow. Specifically, we are examining how the secretion, transport, and uptake of adipocyte-supplied factors promote metastatic progression in bone. We have previously shown that bone marrow adipocytes enhance a glycolytic phenotype in PCa cells through paracrine activation of glycolytic enzymes GLUT1, HK2, PDK1, ENO2, and LDHa at both the mRNA and protein levels. This correlated with an increase in glycolytic activity as measured by higher levels of lactate secretion and decreases in oxidative phosphorylation, indicative of an enhanced Warburg-like metabolic signature. We then assessed HIF-1a activity through mRNA analysis of HIF-1a target genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF). We found that CA9 and VEGF had elevated expression levels in tumor cells exposed to adipocytes, indicative of enhanced HIF-1a activity. Notably, PCa cells exposed to bone marrow adipocytes had significantly enhanced presence of HIF-1a in the nucleus, result further confirming HIF-1α, activation in tumor cells exposed to marrow adipocytes. Hypoxia chamber experiments revealed that these effects were observable regardless of the presence or absence of oxygen, indicating the presence of oxygen-independent HIF-1a signaling or a state of pseudohypoxia in tumor cells. These results were further validated in multiple in vitro cell culture and in vivo mouse models, where an increased production of both glycolytic genes and HIF-1α target genes was shown to correlate with marrow adiposity. Our overall hypothesis is that bone marrow adipocyte-supplied lipids within the bone microenvironment cause HIF-1a activation and metabolic switch to glycolysis in metastatic PCa cells in bone, leading to their increased aggressiveness and resistance to therapy. Citation Format: Jonathan Diedrich, Erandi Rajagurubandara, Mackenzie Herroon, Izabela Podgorski. Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-315.

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

CXCL12 and CXCR4 as predictive biomarkers of glioma recurrence pattern after total resection

Purpose of the studyPrevious studies have shown that the pattern of recurrence for glioma is related to the direction of glioma cell invasion. Recent studies demonstrated that the CXCL12/CXCR4 signaling pathway mediates cellular invasion in glioma. Therefore, in this study, we investigated the possible relationship between CXCL12/CXCR4 expression and recurrence pattern in glioma. Patients and methodsImmunohistochemical techniques were used to assess CXCL12/CXCR4 expression in 42 glioma tissues following total resection. According to magnetic resonance imaging (MRI) of gliomas, the recurrence pattern was classified as close or distant pattern. The relationship between recurrence pattern and CXCL12/CXCR4 expression were initially examined by Chi-squared analysis. The prognostic significance of CXCL12 and CXCR4 was determined by log-rank tests and COX proportional hazards model. ResultsCXCL12 was expressed mainly in vascular endothelial cells and CXCR4 was expressed mainly in tumor cells. The recurrence pattern was significantly related to the expression level of CXCL12 in vascular endothelial cells (P=0.002) and CXCR4 in tumor cells (P=0.004). However, CXCL12 and CXCR4 were not independent prognostic factors for progression-free survival or overall survival in glioma patients. ConclusionThe glioma recurrence pattern is related to CXCL12 expression levels in vascular endothelial cells and CXCR4 expression levels in tumor cells; thus, implicating the CXCL12/CXCR4 signaling pathway as a potential target for glioma therapy.

Abstract 1491: Targeting radiation-inducible cell surface TIP-1 using HVGGSSV peptide as a novel imaging and therapeutic strategy for tumors

Abstract The greatest challenge in radiotherapy is to maximize radiation doses to tumors while sparing the normal tissues. Ionizing radiation has been shown to induce expression of proteins on surface of tumor cells. Using this strategy, drug delivery can be targeted specifically to cancer and guided by use of a beam of radiation. We used phage-displayed peptide libraries to discover radiation-inducible antigens in different cancer models. One such inducible-antigen is Tax interacting protein-1 (Tip-1) which was shown to bind the hexapeptide HVGGSSV. TIP-1 is an atypical PDZ protein consisting of only a single PDZ domain that is predominantly cytosolic protein and expressed in all types of cells. Overall TIP-1 expression levels in tumor cells and non-tumor cells are similar; however it is over-expressed on the tumor cell surface following irradiation. In this study, we evaluated the affinity of binding of HVGGSSV and a scrambled peptide to TIP-1 using surface plasmon resonance (SPR) technology. A KD (dissociation constant) of 3.3e-8M for HVGGSSV and 1.37e-3M for the scrambled peptide was obtained. We next determined the specificity of HVGGSSV to bind tumors in vivo using nano SPECT technology. HVGGSSV was conjugated to a 40KDa PEG (for longer circulation time) and radiolabelled with 111Indium (111In) using diethylene triamine pentaacetic acid (DTPA) as the chelator. The A549 tumor bearing nude mice were irradiated (3Gy x 3 times) or sham irradiated prior to tail vein injections of 111In labeled HVGGSSV. The mice were imaged 48h and 96h post injection using nano CT-SPECT imager. The SPECT images revealed that HVGGSSV specifically bound to the irradiated A549 tumors while little or no binding was seen in the sham irradiated tumors. The post-SPECT imaging bio-distrubution also revealed maximum uptake in irradiated tumors. We further evaluated tumor binding of HVGGSSV in Glioma (D54), esophageal cancer (OE33), cervical cancer (HT3), and pancreatic cancer (BxPC3). The nano-SPECT imaging showed that HVGGSSV specifically bound to all the irradiated tumors tested. Phosphor images of the tumor sections showed that HVGGSSV specifically bound to the tumors and not to normal tissues. In conclusion, HVGGSSV peptide has high affinity to TIP-1 in vitro and in vivo. Radiolabeled HVGGSSV is a novel tool for imaging tumors and may be developed further as a therapeutic agent to deliver cancer specific drugs or therapeutic radioisotopes. Citation Format: Vaishali Kapoor, David Dadey, Kim Nguyen, Hua Li, Buck Rogers, Dinesh Thotala, Dennis Hallahan. Targeting radiation-inducible cell surface TIP-1 using HVGGSSV peptide as a novel imaging and therapeutic strategy for tumors. [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 1491. doi:10.1158/1538-7445.AM2015-1491

Abstract 2775: Prognostic significance of triple miRNAs, miR-9, miR-221, and miR-200c in breast cancer

Abstract MicroRNAs (miRNA) mediated epithelial-mesenchymal transition have been considered as a major event in cancer aggression and accounted for cancer progression. However, clinical association between synergistic effect of deregulated expression levels of the EMT-related miRNAs, including miR-9, miR-221, and miR-200c, and breast cancer prognoses remains underestimated. Initially, tissue samples were from 184 enrolled female patients, with invasive ductal carcinoma, followed up for more than ten years. miRNA expression levels between paired laser capture microdissected tumor and non-tumor cells from the primary tumor site was analyzed by quantitative real-time PCR (qRT-PCR). Differentially expressed status of the target miRNA in tumors relative to non-tumor parts (T/N ratio) was assessed their relationship with pathological characteristics and clinical outcomes of breast cancer. While miR-9 or miR-221 showed significantly higher expression level in tumor cells compared to the non-tumor ones, miR-200c exhibited the opposite (p&amp;lt;0.05). Based on the ROC curve, T/N ratio of 2.80- and 1.96-fold increase for miR-9 and miR-221, whereas, 1.80-fold decrease for miR-200c as risk genotypes, respectively. According to these cutoffs, high miR-9 expression in tumors was associated with advanced tumor stages (p&amp;lt;0.05); significant risk associations were shown between LNM-positive tumors and miR-9 (OR = 3.12) or miR-221 (OR = 1.88) (p&amp;lt;0.05). A joint effect on risk elevation of tumor invasion and nodal metastasis was seen in tumors having miR-9 overexpression alongside increased miR-221 or reduced miR-200c expression (p-trend&amp;lt;0.05). Considering the combined effect of these three miRNAs, patients harboring greater numbers of deregulated expression levels in tumors were found to have a higher risk of poor prognoses (high-stage (p-trend = 0.003) and LNM (p-trend = 0.028)) and reduced probability of 10-year overall survival, showing hazard ratio of 2.39 (p&amp;lt;0001 for trend test). In conclusion, deregulated levels of miR-9, miR-221, and miR-200c, may serve as prognostic biomarkers to predict disease progression and shorter OS in breast cancer. Citation Format: Chun-Wen Cheng, Jyh-Cherng Yu, Yi-Hsien Hsieh, Po-Ming Chen, Hsiao-Wei Wang, Hui-Chun Wang, Chen-Yang Shen. Prognostic significance of triple miRNAs, miR-9, miR-221, and miR-200c in breast 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 2775. doi:10.1158/1538-7445.AM2015-2775

OSTEOPROTEGERIN AS A PREDICTIVE MARKER OF THE COURSE OF BREAST CANCER

Despit e th e considerable progress in research of breast cancer (BC), taking into consideration the division into molecular subtypes, the mechanics of metastasis development in BC patients are not definitely investigated, especially at favorable prognosis. Research of new informative diagnostic and prognostic markers represents the modern problems of BC pathology in a new way. Thi s paper is devoted to study of osteoprotegerin (OPG) expression levels in tumor cells in BC patients. The study covered 83 female patients with regional BC (T2–4N0–3M0), treated from 2003 to 2010. The inclusion criteria were histologically proved diagnosis of invasive BC, age over 18 y. o., ECOG 0 or 1. In order to study OPG levels, we carried out immune histochemical test, which was carried out according to standard protocol on the sections of biopsy material. The OPG antibody from GeneTex, used in the study, was rabbit polyclonal, dilution 1:1000, incubation time – 30 minutes. Vizualization system was Real EnVision, anti-rabbit (by DAKO, Denmark). Immunohistochemical staining was studied by counting of number of positive cells in reference to total number of malignant cells. Expression over 50 % of cells was regarded as high, less than 50 % – as low. Average value of OPG expression level was 49 %, median – 50 %. Minimal value – 0 %, maximum value – 90 %. The patients were divided into 2 groups according their expression levels: Group 1 – OPG (high) expression (more than median), Group 2 – OPG (low) expression (less than median). OPG (high) Group was made by 47 patients, OPG (low) Group – by 36. When analyzing clinical and pathological characteristics of BC patients, taking into consideration OPG expression, there were noticed no reliable differences with respect to availability o r absence o f affected regional lymph nodes, category T and Ki-67 indice. The group with positive estogen-progesteron receptors showed reliably more often the high values of OPG than the group with negative receptors (p < 0,05). When analyzing the duration till the progression and overall survival, a difference between the survival and time to progression (rise of metastases) in patients with low OPG levels and the same exponents in patients with high OPG levels was established.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Identification and characterization of human Rad51 inhibitors by screening of an existing drug library

Homologous Recombination (HR) plays an essential role in cellular proliferation and in maintaining genomic stability by repairing DNA double-stranded breaks that appear during replication. Rad51, a key protein of HR in eukaryotes, can have an elevated expression level in tumor cells, which correlates with their resistance to anticancer therapies. Therefore, targeted inhibition of Rad51 through inhibitor may improve the tumor response to these therapies. In order to identify small molecules that inhibit Rad51 activity, we screened the Prestwick Library (1120 molecules) for their effect on the strand exchange reaction catalyzed by Rad51. We found that Chicago Sky Blue (CSB) is a potent inhibitor of Rad51, showing IC50 values in the low nanomolar range (400nM). Biochemical analysis demonstrated that the inhibitory mechanism probably occurs by disrupting the Rad51 association with the single-stranded DNA, which prevents the nucleoprotein filament formation, the first step of the protein activity. Structure Activity Relationship analysis with a number of compounds that shared structure homology with CSB was also performed. The sensitivity of Rad51 inhibition to CSB modifications suggests specific interactions between the molecule and Rad51 nucleofilament. CSB and some of its analogs open up new perspectives in the search for agents capable of potentiating chemo- and radio-therapy treatments for cancer. Moreover, these compounds may be excellent tools to analyze Rad51 cellular functions. Our study also highlights how CSB and its analogs, which are frequently used in colorants, stains and markers, could be responsible of unwanted side effects by perturbing the DNA repair process.