Tumor Suppressor Genes RB1 Research Articles

Radiation compromised survival of patients with heritable retinoblastoma (H1): what will be the long-term consequences of current eye salvage therapies?

Temming and colleagues (1) present important data from the German national retinoblastoma reference center (University Hospital Essen) confirming the previously observed (2) long-term very serious consequence of radiation treatment to save vision for children with heritable retinoblastoma: second cancers. The long-term overall survival of 633 patients with heritable retinoblastoma diagnosed between 1940 and 2008 was studied. Heritable retinoblastoma is initiated by constitutional mutation in the RB1 tumor suppressor gene, indicated as “H1” in the 8 th edition of the TNMH cancer staging for retinoblastoma (3). Most of the children (93%) had bilateral disease, while 7% were unilaterally affected and shown to be H1 by a close relative or genetic testing. No child with heritable unilateral retinoblastoma died as a result of a second cancer, compared to 9% of bilaterally affected patients. This may suggest that carriers of a reduced expressivity/penetrance RB1 mutation also have fewer second cancers.

Promoter methylation of RB1, P15, P16, and MGMT and their impact on the clinical course of pilocytic astrocytomas.

Promoter methylation of P15, P16, RB transcriptional corepressor 1 (RB1) and O-6-methylguanine-DNA methyltransferase (MGMT) impacts the prognosis of numerous glioma subtypes. However, whether promoter methylation of these genes also has an impact on the clinical course of pilocytic astrocytoma remains unclear. Using methylation-specific polymerase chain reaction, the methylation status of the tumor suppressor genes P15, P16, RB1, and MGMT in pilocytic astrocytomas (n=18) was analyzed. Immunohistochemical staining for the R132H mutation of the isocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1) gene was performed. Clinical data including age, gender, localization of tumor, extent of resection, treatment modality, progression-free survival and overall survival were collected. The methylation index for P15, P16, RB1 and MGMT was 0.0, 0.0, 5.6% (1/18) and 44.5% (8/18), respectively. If the MGMT promoter was methylated, the probability of relapse and second subsequent therapy was significantly increased (P=0.019). The one patient with methylation of P15 demonstrated a poor clinical course. The pilocytic astrocytomas of all 18 patients revealed wild-type IDH1. Clinically, there was a significant correlation of subtotal resection with the occurrence of relapse (P=0.005) and of the localization of the tumor with the extent of resection (P=0.031). Gross total resection was achieved significantly more often in pediatric patients than in adult patients (P=0.003). Adult patients demonstrated more relapses following the first tumor resection (P=0.001). The present study indicates that methylation of MGMT is associated with a poor clinical course and represents an age-independent risk factor for an unfavorable outcome. Other influential factors of outcome were the age of the patient and extent of resection.

Preliminary results: Comprehensive national retinoblastoma cohort in Finland ‐ RB1 mutation spectrum

AbstractPurposeRetinoblastoma (Rb), a malignancy of developing retina, emerges almost always after biallelic mutation and inactivation of tumor suppressor gene RB1. In heritable Rb, a child carries germline RB1 mutation. Identifying this will help in future risk management and family planning. Recently, we have made an effort to create a comprehensive national register for Rb. DNA sequencing for mutations in RB1 is being offered to every patient not previously tested and the results are included in the register.MethodsOf the 226 Rb patients registered in the Finnish Retinoblastoma Register until January 2016, 72 have so far underwent germline DNA testing and 40 patients from 32 unrelated families tested positive for mutation. RB1 mutation detection methods varied depending on the time period. Mutations were checked for pathogenicity from the Leiden Open Variation Database (http://rb1-lsdb.d-lohmann.de).ResultsRB1 mutations comprised 90% of heritable (bilateral, familial or both) Rb and 10% were sporadic unilateral tumors (unifocal or too advanced to assess focality). The distribution of mutation types was 46% nonsense, 15% frameshift, 15% splice site, 3% large indel, 8% missense, 13% chromosomal deletions and none promoter. Two novel mutations (c.1A>C and c.2042_2203+?del) were detected. Five mutations (13%) had been reported only once and nine mutations (23%) three times or less.ConclusionsThe frequencies of the type of mutation in the RB1 gene in the preliminary results of our national cohort expectedly follow the mutation spectrum described worldwide.

Development of zebrafish medulloblastoma-like PNET model by TALEN-mediated somatic gene inactivation.

Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b-TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Injection of rb1-TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3′ mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.

Clonal composition of human ovarian cancer based on copy number analysis reveals a reciprocal relation with oncogenic mutation status

Intratumoral heterogeneity of cancer cells remains largely unexplored. Here we investigated the composition of ovarian cancer and its biological relevance. A whole-genome single nucleotide polymorphism array was applied to detect the clonal composition of 24 formalin-fixed, paraffin-embedded samples of human ovarian cancer. Genome-wide segmentation data consisting of the log2 ratio (log2R) and B allele frequency (BAF) were used to calculate an estimate of the clonal composition number (CC number) for each tumor. Somatic mutation profiles of cancer-related genes were also determined for the same 24 samples by next-generation sequencing. The CC number was estimated successfully for 23 of the 24 cancer samples. The mean ± SD value for the CC number was 1.7 ± 1.1 (range of 0–4). A somatic mutation in at least one gene was identified in 22 of the 24 ovarian cancer samples, with the mutations including those in the oncogenes KRAS (29.2%), PIK3CA (12.5%), BRAF (8.3%), FGFR2 (4.2%), and JAK2 (4.2%) as well as those in the tumor suppressor genes TP53 (54.2%), FBXW7 (8.3%), PTEN (4.2%), and RB1 (4.2%). Tumors with one or more oncogenic mutations had a significantly lower CC number than did those without such a mutation (1.0 ± 0.8 versus 2.3 ± 0.9, P = 0.0027), suggesting that cancers with driver oncogene mutations are less heterogeneous than those with other mutations. Our results thus reveal a reciprocal relation between oncogenic mutation status and clonal composition in ovarian cancer using the established method for the estimation of the CC number.

Inhibition of pRB Pathway Differentially Modulates Apoptosis in Esophageal Cancer Cells

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Current chemotherapy regimens include a combination of 5-fluorouracil (5-FU) and cisplatin, but more efficient therapy strategies are needed to increase 5-year survival. Alterations in the signaling pathway of the tumor suppressor gene Rb-1, which encodes a phosphoprotein (pRB) that negatively regulates the G1/S transition of the cell cycle, are present in 70% of all tumors, but its role in esophageal cancer is still unclear. Most of these are alterations leading to up-regulation of the activity of cyclin-dependent kinases (CDKs) to phosphorylate pRB, which suggests that keeping the wild type pRB phosphorylated might be advantageous. Besides proliferation, pRB also regulates apoptosis induced by tumor necrosis factor-alpha (TNF-α) and DNA-damage. We investigated the status of phosphorylation of pRB along esophageal tumorigenesis stages, as well as whether hyperphosphorylation of pRB could suppress apoptosis induced by cisplatin, 5-FU, or TNF-α in esophageal cancer cells. pRB phosphorylation increased progressively from normal esophageal tissue to metaplasia and adenocarcinoma, suggesting that pRB phosphorylation increases along esophageal tumor stages. When RB-1 was knocked down or CDK inhibitors reduced the levels of phosphorylated pRB, opposite apoptotic effects were observed, depending on the combination of drugs tested: whereas TNF-α- and cisplatin-induced apoptosis increased, 5-FU-induced apoptosis decreased. Taken together, these data suggest that pRB plays a role in esophageal adenocarcinoma and that, depending on the type of anti-cancer treatment, combining CDK inhibitors and chemotherapy has the potential to increase the sensitivity of esophageal cancer cells to cell death.

Generation of two H1 hESC sublines carrying a heterozygous and homozygous knock-out of RB1

Retinoblastoma is a childhood cancer of the retina caused by biallelic inactivation of the tumor suppressor gene RB1. In heritable retinoblastoma, one allele is inherited in mutant form via one of the parental germ cells. To study molecular mechanisms in retinoblastoma, two sublines of H1 hESCs were generated, carrying a knock-out allele of RB1 in the heterozygous or homozygous state. Exon 3 of RB1 was targeted and modified by nucleotide deletions using the CRISPR/Cas9 nuclease system. Based on a nearby single nucleotide polymorphism, the modification could be assigned to one allele.

Genomic analysis of follicular dendritic cell sarcoma by molecular inversion probe array reveals tumor suppressor-driven biology.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

In Vitro Differentiation of Preosteoblast-Like Cells, MC3T3-E1, to Adipocytes Is Enhanced by 1,25(OH)2 Vitamin D3.

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and are controlled by specific transcription factors. While 1,25-dihydroxyvitamin D3 (vitamin D) is known to be an important factor for osteoblast differentiation, there are conflicting reports regarding its effect on adipogenesis. In this study, we attempted to understand the effect of exposure of preosteoblasts (MC3T3-E1) to adipogenic media with and without vitamin D and determined the expression of adipogenic genes during this process. Our studies show that while transdifferentiation of preosteoblasts occurred on exposure to adipogenic media, the effect of vitamin D treatment was synergistic resulting in several hundred fold increase in adipocyte transcription factors C/EBPα and peroxisome proliferator-activated receptor-gamma (P < 0.001) along with an increase in markers of adipogenesis and accumulation of lipid droplets in cells. Vitamin D treatment was also accompanied by 100-fold to 700-fold increases in vitamin D receptor expression during the treatment period (P < 0.001). To determine how the effect of vitamin D might compare to other genetic manipulations that promote adipogenic differentiation, we stably knocked down retinoblastoma expression in MC3T3-E1 cells. Recent studies have suggested retinoblastoma (Rb1) tumor suppressor gene function to be critical to maintain osteoblasts function and inhibit adipocyte differentiation. We exposed MC3T3-E1 cells with reduced Rb1 expression to adipogenic media and found an increase in adipogenic differentiation when compared to cells with a full complement of Rb dosage. However, the extent of the change was not as dramatic as seen with vitamin D. These studies show that preosteoblasts are sensitive and respond to these manipulations that favor the adipocytic phenotype. While vitamin D is not known to directly affect targets in adipogenesis, our observations may have resulted from the malleability of preosteoblast genome in MC3T3-E1 cells, which allowed adipocyte specific gene expression under appropriate stimuli. Why this pathway is influenced and subverted by an anabolic bone factor such as vitamin D remains to be determined.

Retinoblastoma (Rb) protein expression in triple-negative breast cancer.

1097 Background: Expression of Rb, the protein product of the RB tumor suppressor gene, is required for cyclin D kinase (CDK) 4/6 inhibition in luminal breast cancers. Triple negative breast cancers (TNBC) frequently exhibit Rb loss and are thus believed to be poor candidates for CDK4/6 inhibition. However, the features associated with Rb loss in TNBC are poorly-defined. We aimed to assess whether TNBC that express the androgen receptor (AR) and resemble a more luminal subtype retain Rb more often than other TNBC. Methods: To assess the frequency and correlates of Rb expression in TNBC, we stained tissue microarrays (TMAs) containing 180 Stage I-III TNBC, 71 with germline BRCA1 mutations ( BRCA1+) and 109 without BRCA1 mutation (sporadic), using a mouse monoclonal antibody to human Rb (clone G3-245, BD Biosciences). Assessment of tumor size, histologic type, grade, lymphovascular invasion and lymph node status were assessed on histologic review. The TMAs, containing three 0.6mm cores/tumor, had been previously stained for AR, CK5/6, CK14 and EGFR. Log-binomial regression was used to calculate risk ratios (RR). Results: Fifty percent of TNBC were Rb-positive (Rb+; ≥10% nuclei staining) of which 84.4% had &gt; 50% nuclei staining. Among TNBC that were Rb-negative, 76.6% had &lt; 1% nuclei staining for Rb. AR expression (≥10% nuclei staining) was more common in Rb+ than Rb-negative TNBC (16.7% vs 4.5%; p = 0.01). In addition, Rb expression was significantly more common among sporadic than BRCA1+ TNBC (59.6% vs 35.2%, p = 0.001). Compared with Rb-negative TNBC, Rb+ tumors were associated with older age (mean 48.7 vs 45.7 years; p = 0.06) and lower histologic grade (grade 1 or 2: 9.2% vs 2.2%; p = 0.05). No other clinical or pathologic features were significantly associated with Rb status. In a multivariable model, both AR expression (RR, 1.65; 95% CI, 1.31-2.08) and lack of BRCA1 mutation (RR, 1.63; 95% CI, 1.17-2.29) significantly predicted for Rb expression among TNBC. Conclusions: Sporadic TNBC is significantly more likely to be Rb+ than BRCA1+ TNBC. Among TNBC, AR expression significantly predicts for Rb expression. These results suggest clinically relevant biomarkers that may predict for Rb expression and potential targeted therapies in TNBC.

The human retinoblastoma susceptibility gene (RB1): an evolutionary story in primates.

The tumor suppressor gene RB1 (Human Retinoblastoma Susceptibility Gene) plays a prominent role in normal development, gene transcription, DNA replication, repair, and mitosis. Its complete biallelic dysfunction in retinoblasts is the main cause of retinoblastoma in the human. Although this gene has been evolutionary conserved, comparisons between the reference and human RB1 coding region with its counterparts in 19 non-human primates showed 359 sites where nucleotide replacements took place during the radiation of these species. These resulted in missense substitutions in 97 codons, 91 of which by amino acids with radically different physicochemical properties. Several in frame deletions and two insertions were also observed in the N-terminal region of the pRB protein where the highest number of amino acid substitutions and radical amino changes were found. Fifty-six codons were inferred to be under negative selection and five under positive selection. Differences in codon usage showed evident phylogenetic signals, with hominids generally presenting higher indices of codon bias than other catarrhines. The lineage leading to platyrrhines and, within platyrrhines, the lineage leading to Saimiri boliviensis showed a high rate of nucleotide substitutions and amino acids. Finally, several RB1 alterations associated to retinoblastoma in the human were present in several non-human primates without an apparent pathological effect.

The Rb1 tumour suppressor gene modifies telomeric chromatin architecture by regulating TERRA expression

The tumour suppressor gene (Rb1) is necessary for the maintenance of telomere integrity in osteoblastic cells. We now show that the compaction of telomeric chromatin and the appropriate histone modifications of telomeric DNA are both dependent upon Rb1-mediated transcription of the telomere-derived long non-coding RNA TERRA. Expression of TERRA was reduced in Rb1 haploinsufficient cells, and further decreased by shRNA-mediated reduction of residual Rb1 expression. Restoration of Rb1 levels through lentiviral transduction was sufficient to reestablish both transcription of TERRA and condensation of telomeric chromatin. The human chromosome 15q TERRA promoter contains predicted retinoblastoma control elements, and was able to confer Rb1-dependent transcription upon a promoterless reporter gene. Chromatin immunoprecipitation revealed preferential binding of phosphorylated over non-phosphorylated Rb1 at the TERRA promoter. As Rb1-deficient cells show increased genomic instability we suggest that this novel non-canonical action of Rb1 may contribute to the tumour suppressive actions of Rb1.

Abstract A27: A linked data approach to discover HPV oncoprotiens and RB1 induced mutation associations for the retinoblastoma research

Abstract Background: LOSS or GAIN in tumor suppressor gene RB1 play a significant role as in case of loss low penetrance where only 39% of eye at risk develops in retinoblastoma. This research covers the multiple mutation types and its effects and identification of major type of mutation involved in retinoblastoma because of HPV and RB1. Methods: First, we focus on exploring gene expression (GE) patterns for RB1 and HPV associated genes from TCGA. Second, identification of validated and non-validated standard CNV ensured using the COSMIC. Finally, the clinical profiles of filtered mutations have been validated based on ICGC pathological profiling data to infer the prognostic behavior from RB1 and HPV associated genes. In order to link and retrieve patterns of a gene from TCGA, COSMIC, and ICGC repositories, we performed following steps: transform heterogeneous data repositories and their storage formats into standard Resource Description Framework (RDF) format; to discover associations by finding specific patterns (i.e. correlations) in the GE data sets; scalable querying the large volume and frequently updating datasets covering the GE data from different repositories. Results: HPV mutations indicated in more than 127 cancer studies shows deletion and amplifications are rare mutations. Retinoblastoma: Expression profile of RB1 shows mutations such as nonsense, Missense or splice events and in GBM and gliomas the expression values in splice mutations (1500-200), nonsense mutations (200-600). In principal HPV associated retinoblastoma the higher expression of HPV genes results in splice junctions and lower in nonsense mutations. Other tissues: Pattern of RB1 where the results coming from more 123 studies show the pattern of mutations similar to the results obtained from HPV associated genes. Alteration with HPV genes study based on the alteration in Altered in 90% samples of 61 cases where TP53 is holding 90% occurrence majorly as normal mutations and SNRNP70 and BRCA1 is majorly responsible truncating mutation other highly mutated genes are AP3D1, BRD4, CCHCR1, CPSF4, CREBBP, CUL3, DDX11, EP300, EP400, FRZ1, GNB2L1, GTF2B, KDM5C, NR4A1, PRP1, PLK1, SF1, SRSF1, SRSF7, SMARCB1, SNRNP70, TAF1, TBP, TMF1, TOPBP1. Whereas RB1 is associated with 11% cased of deep deletion where the V654L is the normal mutation and all others are highly truncating mutation. Survival graph for HPV and RB1 associated genes median months(m) of survival with alteration in these query genes are 103m whereas in RB1 the median month of survival are 7.63m however the disease free survival in RB1 cases are 4.50m. The p-value are 0.76, 0.67, 0.38 respectively. To demonstrate pattern of survival gene set enrichment have been performed on both gene lists where in case of RB1 genes with highest interactions are MDM2, CDK4 and TP53. In HPV genes interacting hubs are TOP1, PARP1, TP53 and ODF2. Higher interacting genes are associated with drugs. RB1 corresponded to Insulin, p a non-cancer FDA approved drug whereas HPV genes and especially TOP1 is associated with Lucanthone, Innotecan, BTBD1 and Topotecan in case of FDA approved drugs category. Cancer drugs with HPV genes are majorly associated with TOP1, PARP1 and PLK1 namely BTBD1, AZD 2281, AG14361, BI2536 and GW84382X. In ICGC effect of RB1 is on cancers e.g. melanoma 51.91% Esophageal 45.38% ovarian 31.18% liver 27.96% Pancreatic 22.55%. Associations of RB1 with other cancer mutations are either splice as in TCGA and other associated mutation with RB1 is LPAR6 majorly these mutations are in exon-region and further understand the other mutations in TCGA ICGC reveals most of these are SNPs at chromosome-13 which defines the locus of RB1 and for HPV. Higher interacting hubs from TCGA TOP1, PAPR1 and ODF2. TOP1 is associated with melanoma two donor hubs of 42.08% and 40.91% liver cancer Hepatocellular carcinoma (Virus) with 23.08 % Esophageal (15.05%) and Ovarian (11.36%). Mutations types are SNP and Splice junctions. Citation Format: Alokkumar Jha, Yasar Khan, Dietrich Rebholz-Schumann, Ratnesh Sahay. A linked data approach to discover HPV oncoprotiens and RB1 induced mutation associations for the retinoblastoma research. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A27.

Bigh3 silencing increases retinoblastoma tumor growth in the murine SV40-TAg-Rb model.

BIGH3, a secreted protein of the extracellular matrix interacts with collagen and integrins on the cell surface. BIGH3 can have opposing functions in cancer, acting either as tumor suppressor or promoter by enhancing tumor progression and angiogenesis. In the eye, BIGH3 is expressed in the cornea and the retinal pigment epithelium and could impact on the development of retinoblastoma, the most common paediatric intraocular neoplasm. Retinoblastoma initiation requires the inactivation of both alleles of the RB1 tumor suppressor gene in the developing retina and tumor progression involves additional genomic changes. To determine whether BIGH3 affects retinoblastoma development, we generated a retinoblastoma mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing in these mice resulted in enhanced tumor development in the retina. A decrease in apoptosis is involved in the initial events of tumorigenesis, followed by an increased activity of the pro-survival ERK pathway as well as an upregulation of cyclin-dependent kinases (CDKs). Taken together, these data suggest that BIGH3 acts as a tumor suppressor in the retina.

Retinoblastoma for Pediatric Ophthalmologists.

Retinoblastoma can present in 1 or both eyes and is the most common intraocular malignancy in childhood. It is typically initiated by biallelic mutation of the RB1 tumor suppressor gene, leading to malignant transformation of primitive retinal cells. The most common presentation is leukocoria, followed by strabismus. Heritable retinoblastoma accounts for 45% of all cases, with 80% being bilateral. Treatment and prognosis of retinoblastoma is dictated by the disease stage at initial presentation. The 8th Edition American Joint Committee on Cancer (AJCC) TNMH (tumor, node, metastasis, heritable trait) staging system defines evidence-based clinical and pathological staging for overall prognosis for eye(s) and child. Multiple treatment options are available in 2018 for retinoblastoma management with a multidisciplinary team, including pediatric ocular oncology, medical oncology, radiation oncology, genetics, nursing, and social work. Survival exceeds 95% when disease is diagnosed early and treated in centers specializing in retinoblastoma. However, survival rates are less than 50% with extraocular tumor dissemination. We summarize the epidemiology, genetics, prenatal screening, diagnosis, classification, investigations, and current therapeutic options in the management of retinoblastoma.

MiR-1297 promotes cell proliferation by inhibiting RB1 in liver cancer.

Liver cancer is the one of the most common causes of cancer-associated mortality worldwide. MicroRNAs (miRNAs or miRs) are important in various types of cancer, including liver cancer. In the present study, with miRNA expression profile data obtained from the Gene Expression Omnibus database, three independent methods were used to investigate the miRNAs that are involved in liver carcinogenesis, including Fisher's exact test, t-test and Wilcoxon test. Five differentially expressed miRNAs were identified. Among them, miR-1297 drew specific attention. Target gene analysis and Ingenuity Pathway Analysis revealed its potential impact on cell death and cell cycle. Cell Counting Kit-8 proliferation assay indicated that the HepG2 cell proliferation was promoted by miR-1297, while miR-1297 inhibitor could significantly inhibit the proliferation of HepG2 cells. Luciferase assays confirmed that miR-1297 directly bound to the 3'-untranslated region of retinoblastoma (RB)1, and western blotting demonstrated that miR-1297 suppressed the expression of RB1 at the protein level. RB1 is involved in the regulation of the human cell cycle pathway. It is possible that miR-1297 contributes to the carcinogenesis of liver cancer via downregulation of the tumor-suppressor gene RB1. Our results suggest that miR-1297 may serve as a potential therapeutic target of liver cancer.

The genetic evolution of skin squamous cell carcinoma: tumor suppressor identity matters.

The genetic evolution of skin squamous cell carcinoma: tumor suppressor identity matters.

Abstract 4494: Whole genome sequencing reveals different patterns of mutational mechanisms in breast tumors between women of African and European descent

Abstract Across race/ethnicities, breast cancer incidence and mortality rates markedly differ. Numerous studies have demonstrated that individuals of African ancestry acquire aggressive, early-onset breast cancers more frequently than other populations for reasons that remain unexplained. The sources of these disparities are complex, and a comprehensive characterization of mutation landscapes amongst indigenous Africans, African Americans (AA), and Caucasian breast tumors has not been performed. We generated high-depth whole genome sequencing on 31 Nigerian breast cancers (90x) along with matched blood (30x). Breast cancer whole genomes (tumor-normal pairs) from The Cancer Genome Atlas were harmonized with our samples, resulting in a cohort of 31 Nigerians (17 estrogen receptor negative, ER-), 31 AA (22 ER-), and 43 Caucasians (19 ER-). High confidence somatic mutations (substitutions and insertions/deletions) were obtained by using multiple variant callers. Regardless of race, ER- tumors carried similar numbers of mutations than their estrogen receptor positive (ER+) counterparts (Welch t-test p = 0.57 - 0.82). TP53 (64%) was the most frequently mutated gene in ER- disease, while canonical PIK3CA activating mutations were prevalent in ER+ cases (33.3%). Additionally, tumor suppressor genes RB1, NF1, and PTEN were disrupted via structural rearrangements in ∼6 to 15% of samples. Rearrangements in the H3K27 methylation regulator EZH1 were identified in six Caucasians but only one individual with African _ancest. Notably, within coding regions, no striking mutation rate differences amongst races were identified. However, global substitution patterns in ER+ and ER- cancers varied widely by race/ethnicity. In ER- cases, Nigerians carried the highest proportion of canonical APOBEC-associated substitutions, particularly C&amp;gt;T transitions. Conversely, Caucasians with ER+ disease showed a higher proportion of C&amp;gt;T than both Nigerians (Welch t-test p = 0.044) and AA (Welch t-test p = 0.011). Kataegis, or clustered mutations, was most prevalent in Nigerian samples, regardless of ER status. Evidence for kataegis was often corroborated by structural variant breakpoints and aberrant copy number states at the hypermutated locus. Mutation signature analyses highlighted multiple APOBEC signatures, with moderate contribution differences across race and ER status. Overall, our data suggests potential mutation spectra differences in Caucasian, African American and indigenous African breast tumors. Identification of these molecular characteristics by ancestry and geography may help understand race-associated phenotypes and exposures that drive outcomes in breast cancer. Citation Format: Jason J. Pitt, Toshio F. Yoshimatsu, Yonglan Zheng, Jason Grundstad, Jigyasa Tuteja, Jiebiao Wang, Abayomi Odetunde, Galina Khramtsova, Wendy Clayton, Adeyinka Ademola, Temidayo O. Ogundiran, Adenike T. Adeniji-Sofoluwe, Millicent Obajimi, Adewunmi Adeoye, Chinedum Babalola, Oladosu A. Ojengbede, Christopher O. Olopade, Oluwasola A. Olayiwola, Lin Chen, Dezheng Huo, Kevin P. White, Olufunmilayo I. Olopade. Whole genome sequencing reveals different patterns of mutational mechanisms in breast tumors between women of African and European descent. [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 4494.

Adriamycin resistance-associated prohibitin gene inhibits proliferation of human osteosarcoma MG63 cells by interacting with oncogenes and tumor suppressor genes.

The resistance of cancer cells to chemotherapeutic agents is a major obstacle for successful chemotherapy, and the mechanism of chemoresistance remains unclear. The present study developed an adriamycin-resistant human osteosarcoma MG-63 sub-line (MG-63/ADR), and identified differentially expressed proteins that may be associated with adriamycin resistance. Two dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis and a protein identification assay were performed. Western blot analysis was used to examine the prohibitin (PHB) levels in the MG-63/ADR cells. Quantitative polymerase chain reaction was utilized to detect adriamycin resistant-associated genes. Laser-scanning confocal microscope was employed to examine the colocalization of PHB with v-myc avian myelocytomatosis viral oncogene homolog (c-myc), FBJ murine osteosarcoma viral oncogene homolog (c-fos), tumor protein p53 and retinoblastoma 1 (Rb). In addition, the full length of the open reading frame of human PHB was subcloned into a lentiviral vector pLVX-puro. The proliferative rate of MG-63 cells was also investigated. The overall protein expression in MG-63/ADR cells was clearly suppressed. Three notable protein regions, representing high mobility group box 1, Ras homolog gene family, member A, and PHB, were identified to be significantly altered in MG-63/ADR cells when compared with its parental cells. Therefore, PHB modulated the chemoresistance of MG-63/ADR cells by interacting with multiple oncogenes or tumor suppressor genes (c-myc, c-fos, p53 and Rb). In addition, overexpression of PHB decreases the proliferative rate of MG-63 cells. In conclusion, PHB is an adriamycin resistance-associated gene, which may inhibit the proliferation of human osteosarcoma MG-63 cells by interacting with the oncogenes or tumor suppressor genes, c-myc, c-fos, p53 and Rb.

72. A Phase l Study of RB94 in Genitourinary Cancers Using a Tumor-Targeted Systemic Nanodelivery System

RB-94 is a truncated form of the RB tumor suppressor gene which has shown marked cytotoxicity in every human tumor type tested to date without affecting normal cells. A systemically administered, tumor-targeted nanocomplex for systemic gene delivery (termed SGT) has been developed. In the SGT nanocomplex, the payload is encapsulated within a cationic liposome. The surface of the liposome is decorated with an anti-transferrin receptor (TfR) single chain antibody fragment (scFv) designed to target cancer cells by binding to the TfR which is highly expressed on tumor cells. Here we describe the results of a DNA dose escalating first-in-man Phase l study of SGT encapsulated RB-94 (SGT-94) for the treatment of metastatic genitourinary tumors. The majority of the cancers treated in this study were bladder cancers. Treatment with SGT-94 was well tolerated with minimal side effects observed. Moreover, among the 11 evaluable patients of 13 treated, a complete remission (CR) in a lung metastasis, two partial remissions (PRs) and three incidences of stable disease (SD) were observed. The CR and PRs occurred at the highest dose (2.4 mg DNA) tested. In addition, there is strong evidence for tumor-specific targeting including data from two resected lung metastases. In these metastatic lesions, the presence of RB94 was documented by both PCR and Western blotting in the tumors. However, RB94 was absent in contiguous normal lung tissue. Therefore, this Phase I study demonstrates not only the safety, but also the anti-cancer potential of the SGT-94 nanocomplex and suggests that additional studies using SGT-94 alone or in combination with chemotherapy, radiation or other modalities are warranted.