Tumor-relevant Genes Research Articles

Abstract 7656: Clinicogenomic anlaysis reveals a role for TP53 mutations in distant progression of high-grade glioma

Abstract Purpose: High-grade glioma (HGG), the most common form of primary brain tumor, is highly lethal and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for HGG. Here, we aimed to identify the clinicogenomic features associated with HGG prognosis and recurrence patterns. Methods: Our single-institution retrospective analysis included 220 patients initially diagnosed with adult HGG (grade 3 or 4) who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. The patients were categorized according to the 2021 WHO classification and their clinical status, survival, genomic profile, and recurrence patterns were analyzed. Results: At a median follow-up of 23 months (ranges, 2-59 months), 180 patients (82%) showed either progression or recurrence. A total of 15% of patients were reclassified under the 2021 WHO classification, revealing a clear separation of progression-free survival (PFS) and overall survival (OS) curves between grades 3 and 4 gliomas. We found that TP53 mutations were significantly associated with the occurrence of distant progression, especially in grade 4 gliomas. Additionally, TP53 mutations were more common in patients with higher Ki67 index and subependymal/leptomeningeal seeding, indicating an increased risk of distant progression in TP53-mutated grade 4 glioma patients. Following multivariate Cox regression analysis, TP53 mutations were independent predictors of OS (hazard ratio [HR]=1.47, P=0.039) and distant PFS (HR=1.71, P=0.005). Conclusion: Considering the elevated risk of distant progression in grade 4 gliomas with TP53 mutations, our results underscore the necessity for additional research into the most effective treatment approaches for these patients. Keywords: high-grade glioma, TP53, pattern of failure, distant progression, next-generation sequencing Citation Format: Byung-Hee Kang, Seungbok Lee, Joo Ho Lee. Clinicogenomic anlaysis reveals a role for TP53 mutations in distant progression of high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7656.

Identification of novel genomic hotspots and tumor-relevant genes via comprehensive analysis of HPV integration in Chinese patients of cervical cancer.

Cervical cancer accounts for 10-15% of cancer-related mortality among women globally. Infection with high-risk human papillomavirus (HPV) types constitutes a significant etiological factor in the development of cervical carcinoma. The integration of HPV DNA into the host genome is considered a pivotal event in cervical carcinogenesis. Nevertheless, the precise mechanisms underlying HPV integration and its role in promoting cancer progression remain inadequately understood. Therefore, this study aims to identify potential common denominators at HPV DNA integration sites and to analyze the adjacent cellular sequences. We conducted whole-genome sequencing on 13 primary cervical cancer samples, employing the chromosomal coordinates of 537 breakpoints to assess the statistical overrepresentation of integration sites in relation to various chromatin features. Our analysis, which encompassed all chromosomes, identified several integration hotspots within the human genome, notably at 14q32.2, 10p15, and 2q37. Additionally, our findings indicated a preferential integration of HPV DNA into intragenic and gene-dense regions of human chromosomes. A substantial number of host cellular genes impacted by the integration sites were associated with cancer, including IKZF2, IL26, AHRR, and PDCD6. Furthermore, the cellular genes targeted by integration were enriched in tumor-related terms and pathways, as demonstrated by gene ontology and KEGG analysis. In conclusion, these findings enhance our understanding of HPV integration sites and provide deeper insights into the molecular mechanisms underlying the pathogenesis of cervical carcinoma.

Abstract 936: Clinicogenomic predictors for the pattern of failure in high-grade glioma

Abstract Background: High-grade gliomas, the most common primary brain tumors, are highly lethal, and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for high-grade glioma. Here, we aimed to identify the clinicogenomic features associated with its prognosis and recurrence patterns. Materials/Methods: Our single-institution retrospective analysis included 223 patients diagnosed with high-grade gliomas who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. We categorized them according to the 2021 WHO classification, and their clinicopathological statuses, treatment characteristics, survival, and genomic profiles were analyzed. Results: The most common genomic variants were TERT promoter (51%), TP53 (39%), IDH1/2 (18%), NF1 (13%), ATRX (12%), PTEN (11%), PIK3CA (9%), SETD2 (7%), and H3F3A (6%) mutation. Regarding copy number variants, amplification of EGFR (21%), PDGFRA (11%), CDK4 (9%) and loss of CDKN2A/2B (55%), PTEN (29%), 19q (17%), NF2 (14%), and RB1 (10%) were the most common copy number aberrations. At a median follow-up of 18 months (range, 2-54 months), 174 patients (78%) had progression or recurrences. A total of 15.2% of our patients were re-classified under the 2021 WHO classification, and there was a clear separation of overall survival(OS) and progression-free survival(PFS) curves between grade 3 and 4 groups using the revised grading system. On multivariate cox regression analysis, MYCN amplification (HR 6.08, p = 0.002) and SETD2 mutation (HR 0.19, p =0.06) were independent predictors of OS and PFS. Conclusion: The assessment of genomic characteristics in conjunction with the pattern of failure for high-grade glioma aids in identifying patients who are likely to benefit from personalized medicine. The 2021 WHO grading system explains survival differences between grade 3 and 4 tumors better than the 2016 WHO classification dose. We identified SETD2 mutation and MYCN as prognostic biomarkers with potential therapeutic implications in patients with high-grade glioma. Citation Format: Joo Ho Lee, Byung-Hee Kang, Seungbok Lee. Clinicogenomic predictors for the pattern of failure in high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 936.

BIOM-18. CLINICOGENOMIC PREDICTORS FOR THE PATTERN OF FAILURE IN HIGH-GRADE GLIOMA

Abstract Background High-grade gliomas, the most common primary brain tumors, are highly lethal and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for high-grade glioma. Here, we aimed to identify the clinicogenomic features associated with its prognosis and recurrence patterns. MATERIALS/METHODS Our single-institution retrospective analysis included 182 patients diagnosed with high-grade gliomas who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. Clinicopathological status, treatment characteristics, survival, and genomic profiles were analyzed. RESULTS At a median follow-up of 23 months (range, 2-229 months), 151 patients (83%) had progression or recurrences. Local and distant recurrences were observed in 132 (72.5%) and 101 (54.9%) patients, respectively. The most common genomic variants in high-grade gliomas were TP53 (42.9%), IDH1/2 (23.1%), TERT promoter (38.5%), ATRX (13.2%), H3F3A (7.1%), and SETD2 (6.0%) mutation. Regarding copy number variants, amplification of EGFR (20.9%), PDGFRA (9.9%), MYCN (2.2%) and loss of CDKN2A/2B (49.5%), PTEN (37.9%), RB1 (17.6%), and 1p19q codeletion(9.3%) were the most common copy number aberrations. On multivariate cox regression anlalysis, MYCN amplification (HR 6.08 95% CI 1.91-19.35, p = 0.002), and SETD2 mutation (HR 0.19 95% CI 0.06-0.62, p =0.06) were independent predictors of overall survival, in relation to previously established prognostic factors including age, Eastern Cooperative Oncology Group Performance Status (ECOG PS) scale, extent of resection, MGMT promoter methylation, and IDH1/2 mutation. Interestingly, MYCN amplification (HR 5.24 95% CI 1.69-16.27, p = 0.004), SETD2 mutation (HR 0.35 95% CI 0.12-0.99, p =0.048) were independent predictors of failure from distant recurrences. CONCLUSION The assessment of genomic characteristics in conjunction with pattern of failure for high-grade glioma aids to identify patients who are likely to benefit from personalized medicine. We identified SETD2 mutation, and MYCN as a prognostic biomarkers with potential therapeutic implications in patients with high-grade glioma.

Abstract 1081: Design of combinatory RNA molecules to simultaneously activate tumor immunity and target several signaling pathways in solid tumors

Abstract Combination therapy is implicated for many oncological indications to circumvent resistance mechanisms that reduce efficacy of targeted drugs. In recent years, mRNA has been successfully utilized preclinically to deliver cytokines to tumors locally at efficacious doses. Versameb has developed a technology platform which enables combinatorial approaches for intra-tumoral application of nucleotide therapeutics that comprise an mRNA element covalently linked to up to three independent siRNA elements targeting various tumor-relevant genes. Such constructs allow a dose-synchronized treatment of different components in parallel, which opens the opportunity to address up to four cancer targets within a single RNA molecule. For proof of concept, we designed a combinatory molecule consisting of mRNA coding for human interleukin 2 (IL-2) linked to a small interfering RNA (siRNA) targeting vascular endothelial growth factor A (VEGFA). Transfection of A549 adenocarcinoma cells led to dose-dependent secretion of efficacious concentrations of IL-2 and simultaneous complete VEGFA knockdown. IL-2 protein functionality was tested in SK-OV-3 cell spheroids co-cultured with human primary peripheral blood mononuclear cells (PBMCs). After treatment, secreted IL-2 protein induced immune cell activation and consecutive spheroid killing by PBMCs from three independent donors. VEGFA functionality was tested in an in vitro vascularization assay in HUVEC cells. Cells were treated with supernatants of vehicle or RNA transfected SCC-4 head & neck cancer cells producing high endogenous VEGFA, and vascular sprouting was analyzed quantitatively. While vehicle-treated cell supernatant induced vessel sprouting, supernatant from RNA treated cells showed much reduced sprouting in vitro consistent with the lower VEGFA levels measured, confirming functionality of the siRNA effect on VEGFA protein expression. We further provide examples of combinatory molecules targeting up to 4 independent key cancer hallmarks and their functional outcomes in various cancer cell types. In conclusion, mRNA combination constructs can be developed into cancer therapeutics that target several pro-tumorigenic pathways simultaneously with a single molecule overcoming hurdles of delivery, toxicity, and regulatory requirements for combination therapies. Citation Format: Petra Hillmann, Justin A. Selvaraj, Sina E. Zimmerli, Pascale G. Birrer, Claudia C. Bohnert, Klaas P. Zuideveld, Stefan J. Scherer, Friedrich Metzger. Design of combinatory RNA molecules to simultaneously activate tumor immunity and target several signaling pathways in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1081.

MiR-129-5p exerts Wnt signaling-dependent tumor-suppressive functions in hepatocellular carcinoma by directly targeting hepatoma-derived growth factor HDGF

BackgroundIn hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis.MethodsGlobal miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined.ResultsHDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3′UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs.ConclusionsThis study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

Educational no. 3: Cytogenetic analysis (CA)

In tumor cytogenetics, individual cancer cells are genetically analyzed, either conventionally by studying the mitotic chromosomes of dividing tumor cells from in vitro cultures (metaphase analysis; karyotyping) or molecularly by identifying specific DNA sequences or chromosomal regions in the nucleus of nondividing tumor cells in situ (interphase fluorescence in situ hybridization). In a typical cytogenetic study, 20 metaphase cells and/or 200 interphase cells are microscopically investigated for visible somatic chromosome changes that might occur in a given neoplastic clone. The detection sensitivity for aberrant cells, therefrom, ranges roughly between 1 and 10% in both, conventional and molecular cytogenetics. This and the need for a culturable testing material is why cytogenetics is completely different to the more sensitive molecular genetic methods (polymerase chain reaction, deep sequencing). The great strength of cytogenetics is the acquisition of substantial amounts of information about the whole genome of a tumor, cell per cell, in a single assay, such as detection of previously unknown chromosome anomalies, revealing of primary and secondary changes, proof of clonality and cell lineage origin, positional evidence for tumor-relevant genes, knowledge of the proliferating ability of an aberrant clone, and conclusions for staging and prognosis at diagnosis, as well as information about disease regression and clonal evolution during follow-up.

Biological characteristics and karyotiping of a new isolation method for human adipose mesenchymal stem cells in vitro.

A new method was presented to prepare clinical-grade human adipose-derived stromal stem cells (ASCs) and its safety in vitro, such as biological characteristics and genetic features alteration were investigated. The morphology of the ASCs which were cultured in vitro using serum-free medium was observed. Cell cycle and CD markers profile were tested by flow cytometry, while karyotype was analyzed by the chromosome G-banding technology. Growth factors expression was tested by ELISA and tumor-related genes were analyzed by the real-time PCR, respectively. ASCs were adult stem cells with spindle shape. The proliferation ratio of ASCs began to slow down after 10 passages, and was significant after 15 passages. Cell cycle analysis revealed that the percentage of G2 phase and S phase cells was stable. There was no obvious missing, translocation or dislocation in terms of karyotype. Expression level of tumor relevant genes and cytokines at different passages had no significant difference. The clinical-grade ASCs prepared with this new method, less than ten passages, was safe for clinical trials.

Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

Abstract 2620: 3D multicellular pancreatic cancer spheroids as drug screening tool for pharmacological evaluation of EHT 5372 and other Mirk/DYRK1B inhibitors

Abstract The dual-specificity tyrosine phosphorylated and regulated kinase Mirk/DYRK1B belongs to an evolutionary conserved family of kinases involved in the control of growth and development. DYRK1B has low level of expression in most normal cell types but is amplified or over-expressed in a number of human cancers. DYRK1B regulates the exit of cancer cells from quiescence through regulating cyclin D turnover and p27kip1 stabilization, thus participating as substantial actor in the control of cancer cell cycle progression. Diaxonhit has developed a novel class of DYRK inhibitors with potent in vitro efficacy. Among them, EHT 5372 reduces tumor growth in a PANC-1 xenograft model and reduces ascites spheroids to single cells and induces their apoptosis. Three-dimensional (3D) multicellular spheroids are symmetrical cellular aggregates that model an in vitro system of intermediate complexity between monolayer cultures and tumors in vivo. Here, we have conducted genome-wide analysis of transcriptional changes between normal (monolayer, 2D) and 3D culture conditions of pancreatic PANC-1 cells using Diaxonhit's GWSATM platform to identify tumor-relevant genes and pathways. The PANC-1 multicellular spheroid model was first characterized based on phenotypical and functional properties and we then analyzed the pharmacological response of spheroids to EHT 5372 and other DYRK inhibitors treatment. A strong up-regulation of DYRK1B was evidenced and confirmed at the protein level. Stemness-related markers were investigated and some were also found much higher in the spheroids than within the monolayer cultures and further increased in a culture time-dependent manner. Stem-like cancer cells may be the cause of therapy-resistance and relapse in patients and sphere forming ability is one of properties of this quiescent cancer-initiating cells. As DYRK1B contributes to G0 arrest to maintain the viability of quiescent cancer cells, pharmacological DYRK inhibition would reduce the capacity of cells to enter into quiescence and sensitize cancer cells to conventional chemotherapeutic agents or radiation. Molecular and physiological consequences of inhibiting DYRK1B with EHT 5372 are now being investigated along with genes expression changes in stemness-related markers and in biological pathways involved in tumor cell growth regulation. A molecular characterization of pancreatic cancer cell spheroids is currently undertaken to determine the utility of the 3D assay as a surrogate tool enriched in cancer stem cells. Spheroids also enable the study of quiescence, chemoresistance and metastasis. DYRK1B inhibitors screening in 3D models is a powerful approach that can help focus on compounds active on these crucial mechanisms of cancer cell biology. The most active compounds identified in this program are likely good candidates for in vivo xenograft studies. Citation Format: Anne-Sophie CASAGRANDE, Florence BACHELOT, Emeline THROO, Florence MAHE, Bertrand LEBLOND, Thierry BESSON, Matthew PANDO, Laurent DESIRE. 3D multicellular pancreatic cancer spheroids as drug screening tool for pharmacological evaluation of EHT 5372 and other Mirk/DYRK1B inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2014-2620

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

Metal-mediated oxidative DNA damage induced by methylene blue

BackgroundMethylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage. MethodsWe investigated the mechanism of MB-induced DNA damage using 32P-5′-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV–visible spectrometry. ResultsMB induced DNA damage at the 5′-ACG-3′ sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV–visible spectrometry revealed that the absorbance of oxidized form of MB at 668nm was decreased by NADH, and the addition of metal ions attenuated the spectral change. ConclusionsMB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis. General significanceThis study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.

Targeted CRM197-PEG-PEI/siRNA Complexes for Therapeutic RNAi in Glioblastoma.

RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.

Effect of zoledronic acid exposure on Ki-67 expression and proliferation in MCF7 cells resistant to apoptosis induction.

e21129 Background: Bisphosphonates (BP) and the RANK-ligand antibody denosumab (D) are used for the treatment of bone metastases. Low to high concentrations of zoledronic acid (ZA) can induce tumor cell apoptosis in vitro via inhibition of the mevalonate pathway and/or accumulation of the ATP analog ApppI. Clinical studies indicate a benefit in tumor relapse and survival with the supportive treatment of ER-positive breast cancer using zoledronate (ZA), but the molecular mechanisms involved are under debate. Methods: MDA-MB-231 and MCF-7 breast cancer cells were treated for 3 h (pulse treatment) and 72 h (permanent treatment) with 5 – 100 µM ibandronate (IBN), alendronate (ALN), risedronate (RIS) and ZA and 1 – 100 ng/ml D for comparison. Apoptosis and proliferation was determined after 72 h. Rescue experiments for the BP effects were done using geranylgeranyl-pyrophosphate (GGPP) and atorvastatin (Ator). Microarray hybridizations were performed to identify target genes in MCF-7. Results: Permanent and pulse exposure to ZA induced apoptosis in MDA-MB-231 and inhibited proliferation in MCF-7 without affecting apoptosis. While IBN, ALN and RIS were inferior to ZA in apoptosis induction in MDA-MB-231, they were equipotent in proliferation inhibition in MCF-7. GGPP rescued ZA effects in MDA-MB-231 but not the antiproliferative effects in MCF-7, while Ator rescued the latter. qPCR and immunocytochemistry identified KLF2, KLF4, KLF6 and Ki-67 as target genes of ZA in MCF-7. RANKL did not induce proliferation in MCF-7 cells and D did not affect the untreated or RANKL pretreated cells in terms of proliferation and apoptosis. Conclusions: In summary we show here direct effects of ZA and other BP on cell proliferation and expression of tumor relevant genes in MCF-7 cells, which are relatively resistant to ZA-induced apoptosis in comparison with MDA-MB-231. Ator but not GGPP rescued these effects, possibly indicating that these effects are rather due to the accumulation of ATP analogs than to the inhibition of protein prenylation. D had no effects under these basal conditions but future experiments should address the effects in the context of estrogens and gestagens and also in coculture with bone cells.

Downregulation of Angiogenin Transcript Levels and Inhibition of Colonic Carcinoma by Gum Arabic (Acacia senegal)

Gum Arabic (GA), a nutrient from dried exudate of Acacia senegal, is widely used as emulsifier and stabilizer. It stimulates sodium and water absorption in diarrhea. This study explored the effects of GA in colonic tissue. Mice were treated with GA (10% wt/vol) in drinking water and gene array was performed. As GA modified several tumor-relevant genes, chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by 3 cycles of 3% dextrane sodium sulphate in drinking water) was induced with or without GA treatment. Within 4 days, GA treatment decreased the colonic transcript levels of the angiogenetic factors angiogenin 1, angiogenin 3, and angiogenin 4 by 78 ± 18%, 88 ± 15%, and 92 ± 13%, respectively (n = 5 each), and of further genes including CD38 antigen, aquaporin4, interleukin18, vav-3-oncogene, γ + -amino acid transporter, sulfatase1, ubiquitinD, and chemokine ligand5. According to Western blotting, GA treatment similarly decreased angiogenin protein expression, and according to immunohistochemistry, it decreased ß-catenin expression. Chemical cancerogenesis resulted in multiple colonic tumors within 12 wk. GA treatment (10% wt/wt) in drinking water significantly decreased the number of tumors by 70%. The observations disclose a powerful anticarcinogenic effect of GA. The nutrient could thus be used for the prophylaxis against colon carcinoma particularly in individuals at enhanced risk.

Abstract 594: RNAi-mediated therapeutic knockdown of HER family members in vivo through polyethylenimine (PEI)/siRNA complexes and establishment of oligomaltose-modified PEI (OM-PEIs) as novel siRNA delivery platform

Abstract The therapeutic application of RNA interference (RNAi) for the knockdown of tumor-relevant genes critically relies on the efficient delivery of small interfering RNAs (siRNAs) or shRNA-encoding DNA plasmids in vivo. Previously, we have established polyethylenimine (PEI)/siRNA complexes as a highly efficient and ease-to-use system for gene targeting and a universally applicable platform for therapeutic RNAi. Here, we analyse the anti-tumor effects of the therapeutic knockdown of various members of the HER family, i.e. HER1 (c-erbB1, EGFR), HER2 (c-erbB2/neu) and HER3 (c-erbB3) in s.c. ovarian carcinoma xenografts in mice. While little effects are seen upon HER1 knockdown, the treatment of the mice with PEI/complexed siRNAs targeting HER2 or HER3 lead to a significant reduction in tumor growth. Notably, additive anti-tumor effects are observed upon double knockdown of HER2 and HER3. The biocompatibility of this PEI-based siRNA delivery platform for in vivo RNAi can be further increased by modifications of the polyethylenimines. To this end, we systematically analyse physicochemical and biological properties of DNA and siRNA complexes prepared from a set of maltose-, maltotriose- or maltoheptaose-modified hyperbranched PEIs (termed (oligo-)maltose-modified PEIs; OM-PEIs). While (oligo-)maltose grafting generally leads to reduced in vitro DNA transfection efficacies, this effect is less profound in OM-PEI/siRNA complexes for the induction of RNAi. This is particularly true for NH2-monosubstituted OM-PEIs. Independent of the maltose architecture, DNA and siRNA complexes based on maltose-grafted PEI show considerably lower toxicity as compared to PEI complexes. More importantly, pharmacokinetics in mice are markedly altered in OM-PEI/siRNA complexes. While their halflives in the blood circulation upon i.v. injection are generally increased as compared to PEI/siRNA complexes, the tissue distribution of radioactively labelled (OM-)PEI complexed siRNA molecules critically relies on the degree and pattern of (oligo-)maltose grafting. Marked differences in siRNA tissue distribution profiles are observed between different OM-PEIs. Enhanced tissue siRNA levels upon treatment of mice with OM-PEI/siRNA complexes as compared to PEI-based complexes are observed. Taken together, we (i) demonstrate the therapeutic anti-tumor effects of PEI/siRNA-mediated (double) knockdown of members of the HER family and (ii) establish OM-PEI complexes as novel promising tools for the in vivo application of siRNAs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 594.

Genomic and Expression Profiling of Glioblastoma Stem Cell–Like Spheroid Cultures Identifies Novel Tumor-Relevant Genes Associated with Survival

Glioblastoma spheroid cultures are enriched in tumor stem-like cells and therefore may be more representative of the respective primary tumors than conventional monolayer cultures. We exploited the glioma spheroid culture model to find novel tumor-relevant genes. We carried out array-based comparative genomic hybridization of spheroid cultures derived from 20 glioblastomas. Microarray-based gene expression analysis was applied to determine genes with differential expression compared with normal brain tissue and to nonneoplastic brain spheroids in glioma spheroid cultures. The protein expression levels of three candidates were determined by immunohistochemistry on tissue microarrays and correlated with clinical outcome. Functional analysis of PDPN was done. Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serum-grown monolayer cultures established from the same patients did not match well with the respective primary tumors. Microarray-based gene expression analysis of glioblastoma spheroid cultures identified a set of novel candidate genes being upregulated or downregulated relative to normal brain. Quantitative real-time PCR analyses of 8 selected candidate genes in 20 clinical glioblastoma samples validated the microarray findings. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. Invasive capacity and RhoA activity were decreased in PDPN-silenced spheroids. We identified a set of novel candidate genes that likely play a role in glioblastoma pathogenesis and implicate AJAP1, EMP3, and PDPN as molecular markers associated with the clinical outcome of glioma patients.

Coding microsatellite instability analysis in microsatellite unstable small intestinal adenocarcinomas identifies MARCKS as a common target of inactivation

Approximately 15% of small intestinal adenocarcinomas show inactivation of DNA-mismatch repair (MMR) and display high-level microsatellite instability (MSI-H). MSI-H tumors progress as a result of mutations affecting coding microsatellites (coding microsatellite instability, cMSI) that may result in a functional inactivation of the encoded proteins and provide a selective growth advantage for the affected cell. To investigate the cMSI selection in small intestinal carcinogenesis 56 adenocarcinomas were tested for MSI. Eleven MSI-H carcinomas (19.6%) were identified and subjected to cMSI analysis in 24 potentially tumor relevant genes. Mutation frequencies were similar to those observed in colorectal cancer (CRC). Beside high frequencies of cMSI in TGFbetaR2, ACVR2, and AIM2 we detected MARCKS mutations in 10 out of 11 (91%) tumors with a 30% share of biallelic mutations. Since little is known about MARCKS expression in the intestine, we analyzed MARCKS protein expression in 31 carcinomas. In non-neoplastic mucosa, MARCKS was found to be expressed with a concentration gradient along the crypt-villus axis. In line with cMSI induced functional inactivation of MARCKS, 8 out of 11 MSI-H adenocarcinomas showed regional or complete loss of the protein. In microsatellite stable (MSS) small bowel adenocarcinoma, loss of MARCKS expression was seen in 2 out of 20 tumors (10%). In conclusion, we herein present a cMSI profile of MSI-H small intestinal adenocarcinomas identifying MARCKS as a frequent target of mutation. Loss of MARCKS protein expression suggests a significant role of MARCKS inactivation in the pathogenesis of small intestinal adenocarcinomas.

MethyQESD, a robust and fast method for quantitative methylation analyses in HNPCC diagnostics using formalin-fixed and paraffin-embedded tissue samples

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.