Candidate Tumor Suppressor Research Articles

Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis

PurposeUveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. MethodsFFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976–1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as “Early metastasis” (metastases and death within 2–5 years after enucleation, n = 8), “Late metastasis” (metastases and death within 9–21 years after enucleation, n = 7) and “No metastasis” (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). ResultsDNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups (“Early”, “Late”, “No”) nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in “Subset Early metastasis” vs “Subset No metastasis” and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in “Subset Early metastasis vs “Subset No metastasis” and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. ConclusionsDNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

Long non-coding RNA TUSC7 suppressed colorectal cancer progression via regulation of miR-23b/PDE7A Axis.

Despite advances in our understanding of the roles of the long noncoding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) in cancer biology, which has been identified to act as a tumor suppressor by regulating cell proliferation, apoptosis, migration, invasion, cell cycle and tumor growth, its function in colorectal cancer remains unknown. The expression levels of TUSC7 in colorectal cancer tissues and cell lines were determined, and the biological functions of TUSC7 to cancer progression in colorectal cancer were investigated via correlation analysis of clinical samples, cell viability assay, transwell assay and apoptosis analysis. Further, the molecular regulatory mechanisms of TUSC7 were demonstrated by luciferase reporter assay and western blotting. We observed that the expression of TUSC7 was markedly decreased in colorectal cancer cell lines. Moreover, the lower expression of TUSC7 was correlated with advanced clinical grades and poorer survival and may be an independent risk factor for colorectal cancer. Moreover, the expression of TUSC7 inhibited cell proliferation, invasion and epithelial-to-mesenchymal transition (EMT), while it facilitated apoptosis through competitively binding miR-23b. We also found that TUSC7 decreased the expression of phosphodiesterase 7A (PDE7A), a downstream target of miR-23b, through the TUSC7/miR-23b/PDE7A axis. We demonstrated the expression of TUSC7 suppressed colorectal cancer progression through the TUSC7/miR-23b/PDE7A axis, suggesting that TUSC is a potential target for therapeutic intervention in colorectal cancer.

LncRNA TUSC7 inhibits osteosarcoma progression through the miR‑181a/RASSF6 axis.

Osteosarcoma (OS) is one of the most aggressive malignancies, accompanied by an elevated incidence and a decreased rate of healing. Recently, several long non-coding RNAs (lncRNAs) have been reported to be involved in OS progression. Although tumor suppressor candidate 7 (TUSC7) was reported as a novel lncRNA, little is known about its biological functions in OS. The present study was designed to explore whether TUSC7 was involved in the pathological development of OS using various methods, including hematoxylin and eosin staining, Cell Counting Kit-8 assay, colony formation assay and Transwell assay. The present study revealed that TUSC7 expression was downregulated in OS tissues and cell lines compared with in normal tissues and cell lines. Functionally, the current results revealed that overexpression of TUSC7 inhibited OS cell proliferation, migration and invasion, while promoting apoptosis in vitro and in vivo. Next, the subcellular distribution of TUSC7 was examined by nuclear/cytoplasmic RNA fractionation and reverse transcription-quantitative PCR. Mechanistic studies revealed that TUSC7 exerted its role by sponging microRNA (miR)-181a in OS cell lines. Ras association domain family member 6 (RASSF6) was confirmed as a target gene of miR-181a, and the expression levels of RASSF6 were negatively regulated by miR-181a. Additionally, the results of rescue experiments suggested that overexpression of miR-181a neutralized the inhibitory effects of TUSC7 overexpression on OS cells. Overall, the present study demonstrated that the tumor suppressor role of TUSC7 in OS progression was mediated through the miR-181a/RASSF6 axis, which may represent a new therapeutic target for OS.

Loss of p73 Gene Expression in Leukemias/Lymphomas Due to Hypermethylation

The p73 gene, a member of the p53 family, is a new candidate tumor suppressor gene. To investigate the possibility of genetic alteration of p73 in leukemia and lymphoma, we examined 55 cell lines and 39 patient samples together with 17 nonhematopoietic cancer cell lines. Gene expression of p73 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cell lines (5 of 7 pre B/B-acute lymphoblastic leukemia [ALL], 13 of 21 T-ALL/lymphoblastic lymphomas [LBL], 9 of 10 B-non-Hodgkin's lymphomas [B-NHL], 8 of 9 acute myelogenous leukemias [AML], 2 of 2 T-NHL, 3 of 3 multiple myeloma), and in patient samples (16 of 23 pre B-ALL, 5 of 8 T-ALL/LBL, 5 of 8 B-NHL). PCR-single-strand conformation polymorphism (SSCP) of cDNAs showed no mutation in 43 p73-expressing cell lines within the regions that corresponded to the 5 mutational hotspots of the p53 gene. Neither homologous deletion nor rearrangement of the p73 gene were found by Southern blot analysis in any of the cell lines that lack expression of p73. In contrast to prior published data, analysis of a polymorphic site showed that the p73 gene was expressed biallelically in cell lines and normal peripheral blood. Notably, the p73-negative cell lines were hypermethylated at a CpG island in the 5' untranslated region of the p73 mRNA, and treatment of these cell lines with 5-azacytidine (5-AC), a demethylation reagent, induced p73 expression. Taken together, we found that a sizable proportion (32%) of ALL/B-NHL cell lines and primary tumors had negligible or limited expression of the p73 gene associated with hypermethylation of the gene. These findings suggest that silencing of the p73 gene by hypermethylation may contribute to development and/or progression of lymphoid neoplasms.

Pathophysiological significance of N-myc downstream-regulated gene 2 in cancer development through protein phosphatase 2A phosphorylation regulation.

N‐myc downstream‐regulated gene 2 (NDRG2) is a candidate tumor suppressor in various cancers, including adult T‐cell leukemia/lymphoma (ATLL). NDRG2, as a stress‐responsive protein, is induced by several stress‐related signaling pathways and NDRG2 negatively regulates various signal transduction pathways. Although it has not been found to function alone, NDRG2 binds serine/threonine protein phosphatase 2A (PP2A), generating a complex that is involved in the regulation of various target proteins. The main function of NDRG2 is to maintain cell homeostasis by suppressing stress‐induced signal transduction; however, in cancer, genomic deletions and/or promoter methylation may inhibit the expression of NDRG2, resulting in enhanced tumor development through overactivated signal transduction pathways. A wide variety of tumors develop in Ndrg2‐deficient mice, including T‐cell lymphoma, liver, lung and other tumors, the characteristics of which are similar to those in Pten‐deficient mice. In particular, PTEN is a target molecule of the NDRG2/PP2A complex, which enhances PTEN phosphatase activity by dephosphorylating residues in the PTEN C‐terminal region. In ATLL cells, loss of NDRG2 expression leads to the failed recruitment of PP2A to PTEN, resulting in the inactivation of PTEN phosphatase with phosphorylation, ultimately leading to the activation of PI3K/AKT. Thus, NDRG2, as a PP2A adaptor, regulates the global phosphorylation of important signaling molecules. Moreover, the downregulation of NDRG2 expression by long‐term stress‐induced methylation is directly correlated with the development of ATLL and other cancers. Thus, NDRG2 might be important for the development of stress‐induced leukemia and other cancers and has become an important target for novel molecular therapies.

Intracellular hyaluronic acid-binding protein 4 (HABP4): a candidate tumor suppressor in colorectal cancer.

Hyaluronic Acid-binding protein 4 (HABP4) is a regulatory protein of 57 kDa that is functionally involved in transcription regulation and RNA metabolism and shows several characteristics common to oncoproteins or tumor suppressors, including altered expression in cancer tissues, nucleus/cytoplasm shuttling, intrinsic lack of protein structure, complex interactomes and post translational modifications. Its gene has been found in a region on chromosome 9q22.3-31, which contains SNP haplotypes occurring in individuals with a high risk for familial colon cancer. To test a possible role of HABP4 in tumorigenesis we generated knockout mice by the CRISPR/Cas9 method and treated the animals with azoxymethane (AOM)/dextran sodium sulfate (DSS) for induction of colon tumors. HABP4–/– mice, compared to wild type mice, had more and larger tumors, and expressed more of the proliferation marker proteins Cyclin-D1, CDK4 and PCNA. Furthermore, the cells of the bottom of the colon crypts in the HABP4–/– mice divided more rapidly. Next, we generated also HABP4–/– HCT 116 cells, in cell culture and found again an increased proliferation in clonogenic assays in comparison to wild-type cells. Our study of the protein expression levels of HABP4 in human colon cancer samples, through immunohistochemistry assays, showed, that 30% of the tumors analyzed had low expression of HABP4. Our data suggest that HABP4 is involved in proliferation regulation of colon cells in vitro and in vivo and that it is a promising new candidate for a tumor suppressor protein that can be explored both in the diagnosis and possibly therapy of colon cancer.

Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Abstract PO-129: Using the castration resistant prostate cancer patient-derived xenograft (PDX) tumor model to identify mRNA/miRNA biomarkers which distinguish abiraterone treatment responders and non-responders

Abstract Prostate cancer (PCa) is the most frequent cancer occurring in men in the United States and is the second leading cause of cancer deaths in men. Median survival for patients with metastatic hormone refractory PCa (HRPC/CRPC) is around 18 months. Abiraterone (Abi) is a next-generation androgen receptor targeting agent which has significant effects on PCa patients' survival, and has been approved for treatment of metastatic and CRPC. However, it is clear that there is development of Abi resistance (Abi-R). Abi inhibits the steroidogenic enzyme CYP17A1, a critical enzyme in androgen biosynthesis in the adrenals, testis as well as tumor tissues. To screen and identify biomarkers (genes and microRNA (miRNA)) for Abi-R in a heterogenous tumor, we used patient derived PCa xenograft animal model (PCa-PDX mice). Recent advances in the development of PDX models and their increasing sophistication have led to their escalating use for anticancer drug research and development, and as predictive clinical models. Hence, for the correct identification of Abi-R markers, we proposed using PCa-PDX animal model (PDX mice). We hypothesized that changes in miRNA expression underlie Abi resistance (Abi-R) mechanisms. Each tumor bearing mouse was castrated resulting in tumor regression followed by regrowth/relapse phase. Mice were then treated with Abi. The tumors were collected either once they regressed (called CRABS), or reinoculated into castrated host if they showed continued growth. The new host was treated with Abi once tumors reached 50-300 mm3. Tumors were harvested when they reached a volume of ~600-800 mm3 (CRABR2). The RNA was extracted from tumors using the miRvana kits, and RNA/miRNA-seq performed. We compared the Abi sensitive vs the Abi resistant (CRABR2) groups, to identify 316 genes and 55 miRNA whose expression was significantly changed between these two groups. We used Venn diagrams to overlap the validated gene targets for the miRNA (5,557 genes) with our 316 gene dataset. 85 common genes were identified, of which 63 genes were validated targets of hsa-miR-335 (1.7 fold in CRABS vs CRABR2), and 8 genes were validated targets of hsa-miR-574 (0.4 fold in CRABS vs CRABR2). Hsa-miR-335 has been previously identified to function as a candidate tumor suppressor in PCa. Reduced expression of miR-335 was found in human PCa tissues comparing with paired adjacent benign prostate tissues (P &amp;lt; 0.05). Moreover, the increased expression of miR-335 suppressed cell proliferation, invasion and migration of PCa cell lines in vitro. It has been found to suppress bone metastasis in PCa via targeting endothelial nitric oxide synthase. MicroRNA-574-5p promotes metastasis of non-small cell lung cancer, triple negative breast cancer and colorectal cancer to name a few. However, their function in Abi resistance has not been explored. In conclusion, we have identified two promising miRNA for distinguishing Abi sensitive from resistant tumors whose targeting should have profound effects on sensitizing castration resistant tumors to Abi. Citation Format: Bin Ouyang, Dan Song, Jacek Biesiada, Yan Ren, Mario Medvedovic, Pheruza Tarapore. Using the castration resistant prostate cancer patient-derived xenograft (PDX) tumor model to identify mRNA/miRNA biomarkers which distinguish abiraterone treatment responders and non-responders [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-129.

Proteogenomic Characterization of Human Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis, and the situation has not improved despite extensive clinical and scientific research. Here, we report proteogenomic analysis of PDAC. Mutation-phosphorylation correlations identified signaling pathways associated with somatic mutations in significantly mutated genes. mRNA-protein abundance correlations revealed oncogene and tumor suppressor candidates correlating with patient survival. Integrated clustering of mRNA, protein, and phosphorylation data identified six PDAC subtypes (Sub1-6), which were indistinguishable using mRNA data alone. mRNA and protein signatures defining Sub1-6 revealed that Sub1, Sub2-4, and Sub5-6 were precursor, invasive, and immunogenic tumors, respectively. In the Sub2-4 group, proliferation was highest for Sub4; Sub6 in the Sub5-6 group had an increased pancreatic secretion capacity. Orthotopic mouse PDAC models revealed higher numbers of pro-tumorigenic immune cells in Sub4 tumors, inhibiting T cell proliferation. Our proteogenomic analysis provides therapeutic targets and improves understanding of cancer biology and patient stratification in PDAC.

Theoretical and in silico Analyses Reveal MYC as a Dynamic Network Biomarker in Colon and Rectal Cancer.

In this article, we make a theoretical and in silico study for uncovering and evaluating biomarkers in colon and rectal cancer (CRC) by the dynamic network biomarker (DNB) theory. We propose a strategy to employ the theoretical concept of UICC TNM classification in CRC. To reveal the critical transition of CRC, the DNB algorithm was implemented to analyze the genome-wide dynamic network through temporal gene expression data. The relationship between gene sets and clinical features was evaluated by weighted gene co-expression network analysis. The results show that MYC was significantly associated with tumor amplification, tumor immune cells, and survival times. The candidate tumor suppressor genes were ZBTB16, MAL, LIFR, and SLIT2. Protein–protein interaction (PPI) analysis shows that these candidate tumor suppressor genes were significant in immune cells. Data from the Human Protein Atlas showed that a high expression of these candidate tumor suppressor genes was associated with favorable prognosis in TNM stages I–IV. In conclusion, this work provides significant and novel information regarding the TNM stage, cause, and consequences of elevated MYC expression in CRC. MYC expression levels had significant negative correlations with tumor suppressor genes and immune cells.

FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.

Bayesian copy number detection and association in large-scale studies

BackgroundGermline copy number variants (CNVs) increase risk for many diseases, yet detection of CNVs and quantifying their contribution to disease risk in large-scale studies is challenging due to biological and technical sources of heterogeneity that vary across the genome within and between samples.MethodsWe developed an approach called CNPBayes to identify latent batch effects in genome-wide association studies involving copy number, to provide probabilistic estimates of integer copy number across the estimated batches, and to fully integrate the copy number uncertainty in the association model for disease.ResultsApplying a hidden Markov model (HMM) to identify CNVs in a large multi-site Pancreatic Cancer Case Control study (PanC4) of 7598 participants, we found CNV inference was highly sensitive to technical noise that varied appreciably among participants. Applying CNPBayes to this dataset, we found that the major sources of technical variation were linked to sample processing by the centralized laboratory and not the individual study sites. Modeling the latent batch effects at each CNV region hierarchically, we developed probabilistic estimates of copy number that were directly incorporated in a Bayesian regression model for pancreatic cancer risk. Candidate associations aided by this approach include deletions of 8q24 near regulatory elements of the tumor oncogene MYC and of Tumor Suppressor Candidate 3 (TUSC3).ConclusionsLaboratory effects may not account for the major sources of technical variation in genome-wide association studies. This study provides a robust Bayesian inferential framework for identifying latent batch effects, estimating copy number, and evaluating the role of copy number in heritable diseases.

SMOC2, an intestinal stem cell marker, is an independent prognostic marker associated with better survival in colorectal cancers

We aimed to investigate the expression profile of SPARC-related modular calcium-binding protein 2 (SMOC2) during colorectal cancer (CRC) progression and assess its prognostic impact in CRC patients. In our study, we showed that SMOC2 transcript level was higher in CRC samples than in normal mucosa (P = 0.017); this level was not associated with candidate cancer stem cell markers (CD44, CD166, CD133, and CD24) or intestinal stem cell markers (LGR5, ASCL2, and EPHB2) except for OLFM4 (P = 0.04). Immunohistochemical analysis showed that SMOC2-positive cells were confined to the crypt bases in the normal intestinal mucosa, hyperplastic polyps, and sessile serrated adenomas, whereas traditional serrated adenomas and conventional adenomas exhibited focal or diffuse distribution patterns. In total, 28% of 591 CRCs were positive for SMOC2, but SMOC2 positivity had negative correlations with lymphatic invasion (P = 0.002), venous invasion (P = 0.002), and tumor stage (P < 0.001). However, a positive association with nuclear β-catenin expression was seen. Furthermore, while upregulated SMOC2 expression was maintained during the adenoma-carcinoma transition, it decreased in cancer cells at the invasive front but did not decline further during lymph node metastasis. SMOC2 positivity showed no correlations with molecular abnormalities, including microsatellite instability, CpG island methylator phenotype, and mutations of KRAS and BRAF. In addition, we showed comprehensively that SMOC2 positivity is an independent prognostic marker for better clinical outcomes in a large cohort of CRC patients (P = 0.006). In vitro studies also demonstrated that induced SMOC2 expression in DLD1 cells exerts a suppressive role in tumor growth as well as in migration, colony, and sphere formation abilities. Taken together, our results suggest SMOC2 as a candidate tumor suppressor in CRC progression.

11q Deletion or ALK Activity Curbs DLG2 Expression to Maintain an Undifferentiated State in Neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature" that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, highlighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas.

Downregulation of microRNA-320a inhibits proliferation and induces apoptosis of retinoblastoma cells via targeting TUSC3.

MicroRNA (miR)-320a is specific to vertebrates and has been indicated to serve a role in a number of cancer types, such as gastric, colorectal, pancreatic and ovarian cancer. miR-320a has been reported to be expressed at high levels in retinoblastoma tissues; however its role and mechanism of function in retinoblastoma remain to be elucidated. The aim of the present study was to investigate the role of miR-320a in retinoblastoma cells and the underlying mechanisms. The expression of miR-320a in retinoblastoma cell lines Y79 and WERI-Rb-1, and normal human retinal pigment epithelial cell line ARPE-19 was examined via reverse transcription-quantitative PCR (RT-qPCR). TargetScan bioinformatics analysis and dual-luciferase reporter assay were used to predict and reveal the target gene of miR-320a. Target gene expression was detected via RT-qPCR in retinoblastoma cell lines and ARPE-19 cells. Subsequently, gain- or loss-of-function experiments for miR-320a and tumor suppressor candidate 3 (TUSC3) were performed to study the role of miR-320a/TUSC3 in retinoblastoma cells. Cell viability and apoptosis were assessed via MTT and flow cytometry analysis, respectively. Compared with ARPE-19 cells, miR-320a was prominently expressed in retinoblastoma cell lines. TUSC3 was predicted to be a target gene of miR-320a. Compared with ARPE-19 cells, the expression of TUSC3 in retinoblastoma cell lines was reduced. The results of MTT and flow cytometry analysis revealed that overexpression of TUSC3 reduced the viability of retinoblastoma cells and induced apoptosis. Additional analysis indicated that miR-320a inhibitor enhanced the expression of the target gene TUSC3, thereby inhibiting retinoblastoma cell viability and inducing apoptosis. The effects of miR-320a inhibitor on retinoblastoma cells were inhibited by TUSC3-short hairpin RNA. miR-320a regulated the viability and apoptosis of retinoblastoma cells via targeting TUSC3. Therefore, the present study provided a reference for investigating a potential target for the clinical treatment of retinoblastoma.

Effects of lncRNA TUSC7 on the malignant biological behavior of osteosarcoma cells via regulation of miR-375.

The present study aimed at investigating how long-chain non-coding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) regulates the malignant biological behavior of osteosarcoma cells. Tumor tissues and adjacent tissues of 30 patients with osteosarcoma were collected, and the expression levels of lncRNA TUSC7 and miR-375 were detected by RT-qPCR. lncRNA TUSC7 mimic and miR-375 mimic transfection models were established in MG63 osteosarcoma cells, and Transwell assays were used to detect the migration ability of MG63 cells. An MTT assay was used to assess the proliferation ability of MG63 cells. lncRNA TUSC7 in osteosarcoma tissue was significantly lower than that of adjacent tissues, while miR-375 levels were significantly higher than that of adjacent tissues; the two levels have a negative correlation. lncRNA TUSC7 mimic inhibited MG63 proliferation and migration abilities. miR-375 mimic promoted MG63 proliferation and migration abilities. The lncRNA TUSC7 mimic and miR-375 mimic co-transfection system could partially rescue the inhibition of lncRNA TUSC7 mimic on MG63 cells. In conclusion, lncRNA TUSC7 inhibited the proliferation and migration of MG63 osteosarcoma cells by regulating miR-375.

Abstract 3430: The protein phosphatase 4 complex is a tumor suppressor in prostate cancer

Abstract Regions of recurring chromosomal deletions have been annotated in clinical prostate cancer (PCa), but for many of them there is an incomplete understanding of their contributions to disease progression. From an in vivo shRNA enrichment screen of the PCa deletome, we identified the serine threonine protein phosphatase 4 (PP4) regulatory subunit 2 (PPP4R2) as a candidate tumor suppressor, which undergoes copy number loss in 16% of primary PCa. Knockdown or CRISPR-Cas9 mediated knockout of PPP4R2 increased the growth rate of PCa xenograft tumors. Along with the catalytic subunit PP4C and an additional regulatory subunit (either PP4R3A or PP4R3B), PP4R2 forms two distinct heterotrimeric phosphatase holoenzymes. PP4R2 was critical for holoenzyme stability, as its deletion resulted in rapid, moderate downregulation of the other PP4 subunit proteins. The increased tumor growth rate and downregulation of PP4C resulting from loss of PP4R2 could be replicated by combined deletion of PPP4R3A and PPP4R3B, or by directly targeting PPP4C itself. Collectively, these findings implicated PP4C as the likely direct mediator of tumor suppression through the PP4 complex. This was confirmed in both PPP4R2 and PPP4R3A/PPP4R3B knockout cells in vivo by restoring PP4C to its normal physiological level through a dox-inducible cDNA, which reversed the accelerated tumor growth rate conferred by the deletion of the regulatory subunits. Phosphatase activity of PP4C was critical, as add-back of a catalytically dead PP4C mutant (D82A) was unable to reverse the growth rate of PPP4C knockout tumors. Interestingly, we found that the androgen receptor (AR), which is the principal therapeutic target for PCa, was co-immunoprecipitated by PP4C and its regulatory subunits. This suggests that AR could be a substrate for PP4C and is potentially present in a hyperphosphorylated and hyperactivated state in PCa that have decreased PP4 phosphatase activity. Our findings highlight a previously unappreciated tumor suppressor role for the PP4 complex in PCa, with the regulatory subunit PP4R2 providing a key function in stabilizing PP4C and thereby preserving its phosphatase activity. Citation Format: Philip A. Watson, Kayla E. Lawrence, Tejasveeta V. Nadkarni, Taslima F. Ishmael, Eugine Lee, Aura C. Agudelo Rivera, Jillian R. Love, John Wongvipat, Ling Cai, Abbas Nazir, Phillip J. Iaquinta, Kenneth Chang, Yupu Liang, Greg J. Hannon, Charles L. Sawyers. The protein phosphatase 4 complex is a tumor suppressor in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3430.

Abstract 17: Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients

Abstract Background: Lynch Syndrome (LS) is a hereditary condition secondary to germline mutations in DNA mismatch repair (MMR) genes, which predisposes patients to develop different tumor types but mainly colorectal and endometrial cancers. Due to the genetic predisposition, chemopreventive strategies become critical in these individuals. One such chemopreventive compound, statin, a potent pharmacologic inhibitor of cholesterol biosynthesis, is known to exert other pleotropic effects such as modulation of cell proliferation, apoptosis, and inflammation, mainly due to inhibition of the mevalonate pathway. Although, chemoprevention studies in the past were performed in cancer stem cell models the effects have not been previously assessed in the physiologic and MMR-deficient stem cell niche. Here, we have successfully established ex-vivo cultures of crypt-villus structures (termed ‘mini-guts' or organoids) in order to interrogate the chemopreventive effects of statins in Lynch Syndrome patients. Methods: Fresh endoscopic biopsy samples from a total of six LS patients followed at The University of Texas MD Anderson Cancer Center were obtained and ex-vivo organoid cultures were established. Each organoid line (passage &amp;lt; P6) was treated with two different doses of Simvastatin (5 and 10 µM) or DMSO (control) for 48 hours. Total RNA was isolated from organoids treated with control and Simvastatin 10 µM and RNASeq analysis was performed using Illumina HiSeq 4000. Validation of several critical genes was performed by qRT-PCR assay. Results: RNAseq analysis accounted for a total of 4,386 genes dysregulated in organoids treated with Simvastatin as compared to control. Stem cell markers, LGR5 and ASCL2, and differentiation markers, KRT20 (enterocytes) and MUC2 (goblet), were found to be induced by simvastatin. Gene Set Enrichment Analysis (GSEA) using Hallmark pathway gene sets showed significant enrichment for E2F target genes apart from canonical cholesterol homeostasis pathway. Ingenuity pathways analysis (IPA) revealed a list of top 10 genes which are upregulated/downregulated and implicated in cancer pathway. Validation of selected E2F Hallmark pathway genes (E2F1, TOP2A, Survivin, and KI-67) and IPA cancer pathway genes (KLF2, KLF6, EZH2, p57, and p21) showed a significant and dose dependent modulation upon treatment with Simvastatin. Conclusions: Treatment with Simvastatin exhibited a profound effect on key genes involved in maintenance of stem cell niche in patient-derived organoids from LS. Simvastatin had a significant effect on E2F target genes implicated in regulation of cell cycle and proliferation via modulation of Ki-67, TOP2A, and Survivin. Along with increase in cyclin-dependent kinase inhibitor p21, there was significant induction of p57, a candidate tumor suppressor gene. Hence, Simvastatin, while it maintains the stem niche, exerts its anti-proliferative effects by specifically modulating genes involved in cell cycle and apoptosis. Citation Format: Prashant Bommi, Wenhui Wu, Laura Reyes, Kyera Evans, Selvi Thirumurthi, Patrick Lynch, Krishna Sinha, Eduardo Vilar. Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 17.

Abstract 5973: GIPC2 is a candidate tumor suppressor gene in sporadic pheochromocytoma

Abstract Pheochromocytoma (PCC) is a rare endocrine tumor of the chromaffin cells in adrenal medulla or the paraganglia. Currently about 60% of PCCs can be explained by germline or somatic mutations in well-established susceptibility genes such as RET, VHL and SDHB, while for the remaining, mainly sporadic cases the pathogenesis is still unclear. We explored the molecular basis of these PCCs by analyzing 22 sporadic adrenal PCCs, using SNP6.0 arrays to identify minimal region of the most common chromosomal loss, and expression profiling to identify genes with suppressed expression relative to normal medulla. Here we report the identification of GIPC2, a gene located at 1p31.1 encoding a PDZ domain containing adaptor protein with preferential expression in adrenal, as a putative tumor suppressor gene for sporadic PCC. In a cohort of 55 sporadic PCCs, 38 were found with reduced genomic copy number of GIPC2, with accompanying reduced expression of mRNA. Much reduced or loss of GIPC2 protein expression in tumor tissues was confirmed with Western blot or immunostaining. No GIPC2 mutations were found in the cohort but significant promoter hypermethylation was demonstrated using MassARRAY EpiTYPER assay and methylation-specific PCR. The GIPC2 protein was expressed mostly in the nucleus. With GIPC2 overexpression or siRNA knockdown in both PCC derived cell lines (rat PC12 or hPheo, a cell line derived from human PCC) and primary rat adrenal chromaffin cells (ACC), we showed that GIPC2 suppressed cell proliferation by inducing cyclin kinases such as p27, and by suppressing the activation of MAPK/Erk pathways without affecting pAKT or mTOR pathways. PC12 cells stably overexpressing GIPC2 failed to develop tumor in nude mice. To explore the molecular mechanism of GIPC2 in cell cycle regulation, we searched for its interaction proteins in 293T cells via mass spectrometry and identified NONO, a nucleoprotein with a known role in regulating cell cycle. Reciprocal interaction between GIPC2 and NONO was demonstration in PC12 or hPheo cells in vivo using immune-IP or FRET. We showed that knockdown of NONO blocked GIPC2-mediated p27 up-regulation. Furthermore both GIPC2 and NONO regulated p27 promoter through the same DNA binding site and a GGCC box was significant for GIPC2-regulated p27 promoter activity through NONO. Finally, we found that PCC-causing mutations in RET and SDHB, but not VHL, genes could lead to ACC proliferation and p27 downregulation via down-regulating GIPC2. In summary, our study indicates that GIPC2 acts as a candidate tumor suppressor gene in sporadic pheochromocytomas and regulates cell cycle gene p27 through NONO. Our results provide a new insight into the molecular mechanism of sporadic PCCs. Citation Format: Yeqing Dong, Zhengguang Guo, Chengyan Fan, Zhi Zheng. GIPC2 is a candidate tumor suppressor gene in sporadic pheochromocytoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5973.