Pathogenesis Of Multiple Cancers Research Articles

Circ_0070203 Promotes Epithelial-mesenchymal Transition in Ovarian Serous Cystadenocarcinoma through miR-370-3p/TGFβR2 Axis.

Circular RNAs (circRNAs) are important biological molecules associated with the pathogenesis of multiple cancers. This work aimed to investigate the function and molecular mechanism of circ_0070203 in high-grade serous ovarian cystadenocarcinoma (HGSOC). circRNA microarray was conducted to detect the circ_0070203 expression in HGSOC tissues. Bioinformatics analysis was used to find the binding sites between circ_0070203, miR- 370-3p and TGFβR2. Real-time quantitative reverse transcription PCR (RT-qPCR) was executed to detect the expressions of circ_0070203, miR-370-3p and TGFβR2 in HGSOC tissues and SKOV3 cells. Dual-luciferase reporter gene assay was used to validate the relationships between miR-370-3p and circ_0070203 or TGFβR2. Besides, transwell assays were conducted to assess the migrative, invasive abilities of ovarian cancer (OC) cells. Western blotting was adopted to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins. The related patents were also studied during the research. Circ_0070203 and TGFβR2 were upregulated, while miR-370-3p was downregulated in FIGO stage III-IV HGSOC tissues and SKOV-3 cell lines. circ_0070203 overexpression changed the expression of other EMT-related proteins and enhanced the migrative, invasive abilities of OC cells, while silencing circ_0070203 worked oppositely. Mechanistically, circ_0070203 could upregulate TGFβR2 expression in OC cells via sponging miR-370-3p. Circ_0070203 could promote the epithelial-mesenchymal transition, invasion, and metastasis of HGSOC via regulating the miR-370-3p/TGFβR2 axis. Our findings provided a potential biomarker for HGSOC therapy.

IGF2BP3 loss inhibits cell progression by upregulating has_circRNA_103820, and hsa_circRNA_103820-encoded peptide inhibits cell progression by inactivating the AKT pathway in lung cancer.

N6-methyladenosine (m6A) modification and m6A-related RNA-binding proteins (RBPs) play vital roles in various aspects of circRNA metabolism. Hsa_circRNA_103820 is implicated in the pathogenesis of multiple cancers, including lung cancer (LC). Moreover, bioinformatics analysis has suggested that hsa_circRNA_103820 possesses potential peptide-coding ability. Thus, we aimed to investigate the function and peptide-coding potential of hsa_circRNA_103820 in this study. Cell viability, apoptosis rate, and migratory and invasive abilities were assessed using CCK-8, flow cytometry, and transwell assays, respectively. Hsa_circRNA_103820 level was measured using RT-qPCR assay, and the interaction between hsa_circRNA_103820 and IGF2BP3 was examined through RIP and RT-qPCR assays. The coding ability of hsa_circRNA_103820 and protein levels were determined through western blot assay. The results showed that hsa_circRNA_103820 reduced cell viability, attenuated cell migratory and invasive abilities, and promoted cell apoptosis in LC. IGF2BP3 negatively regulated hsa_circRNA_103820 expression and interacted with it. Hsa_circRNA_103820 knockdown alleviated si-IGF2BP3-mediated anti-viability, anti-migration, anti-invasion, and pro-apoptosis effects in LC cells. Moreover, a 188-amino acid (aa) peptide encoded by hsa_circRNA_103820 decreased cell viability, facilitated cell apoptosis, and inhibited cell migration and invasion in LC. Collectively, hsa_circRNA_103820, regulated by IGF2BP3, encodes a 188-aa peptide and inhibits the malignant progression of LC cells by inhibiting the AKT pathway.

Super Enhancer Regulatory Gene FYB1 Promotes the Progression of T Cell Acute Lymphoblastic Leukemia by Activating IGLL1.

Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL. In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated in vitro and/or in vivo. As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database. In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells. Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients.

Targeting Pokemon is a novel strategy to suppress cancer aggressiveness of non-small cell lung cancer: Identification of Pokemon as ideal target for developing anti-NSCLC drugs

Although it is widely reported that Pokemon acts as an oncogene in the pathogenesis of multiple cancers, but its role and detailed molecular mechanisms in regulating non-small cell lung cancer (NSCLC) progression have not been fully delineated. Here, by performing Real-Time qPCR analysis, we verified that Pokemon was high-expressed in NSCLC tissues and cells, compared to the corresponding normal lung tissues and epithelial cells. Then, the small interfering RNA (siRNA) for Pokemon was transfected into the NSCLC cells to verify its biological functions, and our results suggested that silencing of Pokemon suppressed the malignant phenotypes, including cell viability, mitosis, colony formation, epithelial-mesenchymal transition (EMT), mobility and cancer stem cell (CSC) properties in NSCLC cells. Mechanistically, we confirmed that knockdown of Pokemon decreased the expression levels of phosphorylated Akt (p-Akt), phosphorylated GSK-3β (p-GSK-3β) and Snail to inactivate the oncogenic Akt/GSK-3β/Snail signal pathway, and deletion of Snail also had similar effects to hamper the development of NSCLC. Next, our rescuing experiments validated that Pokemon ablation-induced suppressing effects on NSCLC cell malignancy were all abrogated by overexpressing Snail. Finally, the in vivo experiments confirmed that silencing of Pokemon downregulated Snail to hamper tumorigenesis of NSCLC cells in xenograft tumor-bearing mice models. Taken together, we firstly uncovered the underlying mechanisms by which the Pokemon/Akt/GSK-3β/Snail signal pathway contributed to the development of NSCLC, and this signal pathway could be potentially used as therapeutic targets for the development of personalized anti-NSCLC drugs.

KIAA1429 Promotes Nasopharyngeal Carcinoma Progression by Mediating m6A Modification of PTGS2.

Emerging evidence suggests that dysregulation of a N6-methyladenosine (m6A) methyltransferase KIAA1429 participates in the pathogenesis of multiple cancers except for nasopharyngeal carcinoma (NPC). This study is aimed to explore the function of KIAA1429 in NPC progression. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were used to confirm the mRNA expression in NPC by bioinformatic analysis. The levels of KIAA1429 and PTGS2 was detected by quantitative reverse transcription polymerase chain reaction and Western blotting. To investigate the effects of KIAA1429/PTGS2 knockdown or overexpression vectors on NPC cell malignancy, cell and animal experiments were performed. Finally, MeRIP and mRNA stability assays were used to verify the m6A modification and mRNA stability, respectively. KIAA1429 was upregulated in NPC tissues and cells. After transfecting KIAA1429 knockdown or overexpression vectors in NPC cells, we proved that KIAA1429 overexpression promoted proliferation, migration, invasion, and tumor growth, whereas KIAA1429 knockdown showed the opposite effect. Our results also indicated that KIAA1429 mediated m6A modification of PTGS2, enhancing PTGS2 mRNA stability in NPC cells. In addition, PTGS2 could also regulate the effects of KIAA1429 on NPC cell malignancy. This study confirmed the oncogenic function of KIAA1429 in NPC through m6A-modification of PTGS2, suggesting that targeting KIAA1429-mediated m6A modification of PTGS2 might provide a new therapeutic strategy for NPC.

KIAA1429 regulates alternative splicing events of cancer-related genes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains one of the most fatal malignancies with high morbidity and mortality rates in the world, whose molecular pathogenesis is incompletely understood. As an RNA-binding protein participating in the processing and modification of RNA, KIAA1429 has been proved to be implicated in the pathogenesis of multiple cancers. However, how KIAA1429 functions in alternative splicing is not fully reported. In the current study, multi-omics sequencing data were used to analyze and decipher the molecular functions and the underlying mechanisms of KIAA1429 in HCC samples. RNA sequencing data (RNA-seq) analysis demonstrated that in HCCLM3 cells, alternative splicing (AS) profiles were mediated by KIAA1429. Regulated AS genes (RASGs) by KIAA1429 were enriched in cell cycle and apoptosis-associated pathways. Furthermore, by integrating the RNA immunoprecipitation and sequencing data (RIP-seq) of KIAA1429, we found that KIAA1429-bound transcripts were highly overlapping with RASGs, indicating that KIAA1429 could globally regulate the alternative splicing perhaps by binding to their transcripts in HCCLM3 cells. The overlapping RASGs were also clustered in cell cycle and apoptosis-associated pathways. In particular, we validated the regulated AS events of three genes using clinical specimens from HCC patients, including the exon 6 of BPTF gene and a marker gene of HCC. In summary, our results shed light on the regulatory functions of KIAA1429 in the splicing process of pre-mRNA and provide theoretical basis for the targeted therapy of HCC.

Periodontitis as a Risk Factor for Breast Cancer - What We Know Till Date?

A healthy microbiome is important for human health because it exhibits a variety of functions in the human body wherein the microbiome dysbiosis can lead to a variety of diseases, including cancer. Recent advances in technology and cost reduction of sequencing have made it possible and much easier for researchers to investigate the role of the microbiome in carcinogenesis. Furthermore, modulation of microflora may serve as an effective adjunct to conventional anticancer therapy that is very important to improve the patient's quality of life. Additionally, microbiome biomarkers can also be used as a diagnostic tool for cancer. So far the association between oral microbial consortia and their interactions with the host in maintaining the human health and the pathogenesis of multiple cancers has gained much popularity in the scientific research community. While the interactions of oral microflora are better established in cancer- like gastric cancer, it is far less understood in others like breast cancer. Therefore, this review briefly outlines the current information on the role of oral microbiota in breast cancer with emphasis on the mechanisms of oral microflora induced carcinogenesis and discusses the emerging role of periodontitis as a risk factor for breast cancer. Clinical relevance; Periodontitis is a very common disease that is characterized by chronic polymicrobial infection and inflammation of gingiva. It might be associated as a risk factor for breast cancer. If this association is validated in large cohort studies, it would serve as a non-invasive biomarker for breast cancer.

MiR-365 secreted from M2 Macrophage-derived extracellular vesicles promotes pancreatic ductal adenocarcinoma progression through the BTG2/FAK/AKT axis.

Clinical and experimental evidence indicates that tumour‐associated macrophages support cancer progression. Moreover, macrophage‐derived extracellular vesicles (EVs) are involved in pathogenesis of multiple cancers, yet the functions of molecular determinants in which have not been fully understood. Herein, we aim to understand whether macrophage modulates pancreatic ductal adenocarcinoma (PDAC) progression in an EV‐dependent manner and the underlying mechanisms. microRNA (miR)‐365 was experimentally determined to be enriched in the EVs from M2 macrophages (M2‐EVs), which could be transferred into PDAC cells. Using a co‐culture system, M2‐EVs could enhance the proliferating, migrating and invading potentials of PDAC cells, while inhibition of miR‐365 in M2‐EVs could repress these malignant functions. B‐cell translocation gene 2 (BTG2) was identified to be a direct target of miR‐365, while the focal adhesion kinase (F/ATP)‐dependent tyrosine kinase (AKT) pathway was activated by miR‐365. We further demonstrated that overexpression of BTG2 could delay the progression of PDAC in vitro, whereas by impairing BTG2‐mediated anti‐tumour effect, M2‐EV‐miR‐365 promoted PDAC progression. For validation, a nude mouse model of tumorigenesis was established, in which we found that targeting M2‐EV‐miR‐365 contributed to suppression of tumour growth. Collectively, M2‐EVs carry miR‐365 to suppress BTG2 expression, which activated FAK/AKT pathway, thus promoting PDAC development.

NGS-based accurate and efficient detection of circulating cell-free mitochondrial DNA in cancer patients

Mitochondrial DNA (mtDNA) mutations are closely implicated in the pathogenesis of multiple cancers, making circulating cell-free mtDNA (ccf-mtDNA) as a potential non-invasive tumor biomarker. However, an effective approach to comprehensively profile ccf-mtDNA mutations is still lacking. In this study, we first characterized ccf-mtDNA by low-depth whole-genome sequencing (WGS) and found that plasma DNA samples exhibited a dramatic decrease in mtDNA copy number when compared with fresh tumor tissues. Further analysis revealed that plasma ccf-mtDNA had a biased distribution of fragment size with a peak around 90 bp. Based on these insights, we developed a robust captured-based mtDNA deep-sequencing approach that enables accurate and efficient detection of plasma ccf-mtDNA mutations by systematic optimization of probe quantity and length, hybridization temperature, and PCR amplification cycles. Moreover, we found that placement of isolated plasma for 6 h at both 4°C and room temperature (RT) led to a dramatic decrease of ccf-mtDNA stability, highlighting the importance of proper plasma sample processing. We further showed that the optimized approach can successfully detect a substantial fraction of tumor-specific mtDNA mutations in plasma ccf-mtDNA specifically from hepatocellular carcinoma (HCC) patients but not from colorectal cancer (CRC) patients, suggesting the presence of a potential cancer-specific difference in the abundance of tumor-derived mtDNA in plasma.

Identification of small molecule drugs and development of a novel autophagy-related prognostic signature for kidney renal clear cell carcinoma.

Abnormal autophagic levels have been implicated in the pathogenesis of multiple cancers, however, its role in tumors is complex and has not yet been explored clearly. Hence, we aimed to explore the prognostic values of autophagy‐related genes (ARGs) for kidney renal clear cell carcinoma (KIRC). Differentially expressed ARGs and transcription factors (TFs) were identified in KIRC patients obtaining from the The Cancer Genome Atlas (TCGA) database. Then, networks between TFs and ARGs, gene ontology functional annotations and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were conducted. Next, we performed consensus clustering, COX regression analysis and Lasso regression analysis to identify the prognostic ARGs. Finally, an individual prognostic index (PI, riskScore) was established. Based on TCGA cohort and ArrayExpress cohort, Survival analysis, ROC curve, independent prognostic analysis, and clinical correlation analysis were also performed to evaluate this PI. Based on differentially expressed ARGs, KIRC patients were successfully divided into two clusters (P = 5.916e‐04). AS for PI, it was constructed based on 11 ARGs and significantly classified KIRC patients into high‐risk group and low‐risk group in terms of OS (P = 4.885e‐15 for TCGA cohort, P = 6.366e‐03 for ArrayExpress cohort). AUC of its ROC curve reached 0.747 for TCGA cohort and 0.779 for ArrayExpress cohort. What's more, this PI was proven to be a valuable independent prognostic factor in both univariate and multivariate COX regression analysis (P < .001). Prognostic nomograms were also performed to visualize the relationship between individual predictors and survival rates in patients with KIRC. By means of connectivity map database, emetine, cephaeline and co‐dergocrine mesilate related to ARGs were found to be negatively correlated with KIRC. This study provided an effective PI for KIRC and also displayed networks between TFs and ARGs. KIRC patients were successfully divided into two clusters based on differentially expressed ARGs. Besides, small molecule drugs related to ARGs were also identified for KIRC.

Germline APOBEC3B deletion influences clinicopathological parameters in luminal-A breast cancer: evidences from a southern Brazilian cohort.

APOBEC3A and APOBEC3B cytidine deaminases have been implicated in the pathogenesis of multiple cancers, including breast cancer (BC). A germline deletion linking APOBEC3A and APOBEC3B loci (A3A/B) has been associated with higher APOBEC-mediated mutational burden, but its association with BC risk have been controversial. Therefore, this study investigated the association between A3A/B and BC susceptibility and clinical presentation in a Brazilian cohort. A3A/B deletion was evaluated through allele-specific PCR in 341 BC patients and 397 women without familial or personal history of neoplasia from Brazil and associations with susceptibility to BC subtypes were tested through age-adjusted logistic models while correlations with clinicopathological parameters were tested using Kendall's tests. No association was found between A3A/B and BC susceptibility; however, in Luminal-A BCs, it was positively correlated with tumor size (Tau-c = 0.125) and Ki67 (Tau-c = 0.116) and negatively correlated with lymph node metastasis (LNM) (Tau-c = -0.162). The negative association between A3A/B with LNM in Luminal-A BCs remained significant even after adjusting for tumor size and Ki67 in logistic models (OR = 0.22; p = 0.008). These results show that although A3A/B may not modify BC susceptibility in Brazilian population, it may affect clinicopathological features in BC subtypes, promoting tumor cell proliferation while being negatively associated with LNM in Luminal-A BCs.

Discovery of a Synergistic Inhibitor of cAMP-Response Element Binding Protein (CREB)-Mediated Gene Transcription with 666-15.

CREB is a transcription factor implicated in the pathogenesis of multiple cancers. Targeting CREB is a promising strategy to develop potential cancer therapeutics. Previously, we identified 666-15 as a potent CREB inhibitor. Herein, we designed an ester prodrug of 666-15 through a long-range O,N-acyl transfer reaction for improved aqueous solubility. Unexpectedly, we discovered a small molecule 11 (653-47) that can potentiate the CREB inhibitory activity of 666-15 although 653-47 alone does not inhibit CREB.

Knockdown of NSD2 Suppresses Renal Cell Carcinoma Metastasis by Inhibiting Epithelial-Mesenchymal Transition.

Background: Renal cell carcinoma (RCC) accounts for around 85% of all primary kidney neoplasms, which is one of top 10 common cancers worldwide. Nuclear receptor suppressor of variegation, enhancer of zeste, and trithorax (SET) domain-containing 2 (NSD2), belonging to NSD protein family, functions as an oncogene in the pathogenesis of multiple cancers.Methods: GEO database was used to analyze the expression of NSD2 mRNA in renal cancer. Furthermore, NSD2 protein level in clear cell RCC (ccRCC) tissues was detected by immunohistochemistry (IHC). Knockdown efficiency of different siRNAs was evaluated by quantitative real-time PCR (qRT-PCR) and western blot analysis. The biological role and molecular mechanism of NSD2 in RCC metastasis were investigated via a series of functional experiments.Results: NSD2 mRNA was massively amplified in several types of renal cancer, especially in metastatic ccRCC. The expression level of NSD2 protein was elevated in ccRCC tissues, but not correlated with pathological grading. The migratory and invasive properties were significantly repressed in NSD2-silenced RCC cells, concurrent with an increase of E-cadherin expression and a decrease of N-cadherin and Vimentin expression.Conclusion: Down-regulation of NSD2 could potently suppress cell migration and invasion through inhibiting epithelial-mesenchymal transition (EMT), indicating that NSD2 may be a potential therapeutic target for metastatic RCC.

Long noncoding RNA LINC00152 promotes cell proliferation through competitively binding endogenous miR-125b with MCL-1 by regulating mitochondrial apoptosis pathways in ovarian cancer.

Recently, an increasing number of studies have focused on the key function of long noncoding RNAs (lncRNAs) in biological activity. Abnormal lncRNA expression was found to relate to the development and pathogenesis of multiple cancers. LncRNA LINC00152 served as an oncogene in multiple cancers; however, its role in ovarian cancer remains unknown. In our research study, LINC00152 was upregulated in ovarian cancer tissues and cell lines. An increasing LINC00152 level was positively correlated with the histological grade, clinical stage, and poor prognosis of ovarian cancer patients. In addition, knockdown of LINC00152 reduced cell growth, induced cell apoptosis, and suppressed tumor growth. Moreover, we revealed that LINC00152 and Myeloid cell leukemia‐1 (MCL‐1) were targeted by miR‐125b and had the same miR‐125b combining site. The miR‐125b level was negatively correlated with the expression of LINC00152, while MCL‐1 was positively related to the LINC00152 level. MiR‐125b could affect LINC00152 levels as evaluated by qRT‐PCR. Finally, we affirmed that LINC00152 mediated cell proliferation by affecting MCL‐1 expression and MCL‐1‐mediated mitochondrial apoptosis pathways and by working as a competitive endogenous RNA (ceRNA) of miR‐125b. In summary, based on ceRNA theory, the combined research on miR‐125b and MCL‐1, and taking LINC00152 as a new study point, we provide new insight into the molecular mechanism of reversing cell proliferation in ovarian cancer.

TRIM50 suppressed hepatocarcinoma progression through directly targeting SNAIL for ubiquitous degradation

Tripartite motif-containing 50 (TRIM50) belongs to the tripartite motif (TRIM) protein family, which has been implicated in the pathogenesis of multiple cancers. However, the role of TRIM50 in hepatocellular carcinoma (HCC) remains to be clarified. Here we showed that TRIM50 expression was significantly decreased in liver cancer tissues compared with corresponding non-cancerous liver tissues, and its decreased expression was significantly correlated with advanced disease progression. Gain-of-function assay by exogenous overexpression of TRIM50 in HCC cells showed that proliferation, colony formation, migration and invasion of HCC cells were significantly inhibited, whereas loss-of-function assay by TRIM50 knockdown showed that these malignant behaviors of HCC cells were significantly increased. Further investigation showed that TRIM50 could directly bind with SNAIL and induced K-48 linked poly-ubiquitous degradation of SNAIL protein, which further reversed SNAIL-mediated epithelial-to-mesenchymal transition (EMT) process of HCC cells. In vivo assay by xenograft tumor model verified the antitumor effect of TRIM50 on HCC. Taken together, these results showed that TRIM50 acted as a tumor suppressor in HCC cells by directly targeting SNAIL and reversing EMT, which further indicated that positive modulation of TRIM50 might be a novel therapeutic strategy for SNAIL overexpressed HCC cells.

Incidence of pneumonitis in patients with solid tumors treated with everolimus: A systematic review and meta- analysis of randomized controlled trials.

e22220Background: Aberrant signaling through the PI3K/AKT/mTOR signaling pathway has been implicated in the pathogenesis of multiple cancers. Everolimus (E), an mTOR inhibitor, has been employed in...

The analysis of lncRNA HOTAIR rs12826786 C>T polymorphism and gastric cancer susceptibility in a Turkish population: lack of any association in a hospital-based case-control study.

The HOX transcript antisense intergenic RNA (HOTAIR), a well-known long noncoding RNA (lncRNA), has been widely identified to participate in pathogenesis of multiple cancers. An aberrant up-regulation and biological functions have been observed in gastric cancer (GC). A common single nucleotide polymorphism (SNP) (rs12826786 C>T) at the HOTAIR has been reported to influence HOTAIR expression, but its association with GC has yet to be investigated in Turkish population. The aim of the present study was to investigate whether HOTAIR rs12826786 C>T polymorphism could be involved in the risk of GC susceptibility in Turkish population. We genotyped HOTAIR rs12826786 C>T polymorphism in 312 Turkish individuals including 105 GC patients and 207 healthy controls matched on age and gender by a Real-Time Polymerase Chain Reaction (PCR) with the TaqMan assay. No statistically significant differences were found in the allele or genotype distributions of the HOTAIR rs12826786 C>T polymorphism among GC and healthy control subjects (P>0.05). Our results demonstrate that the HOTAIR rs12826786 C>T polymorphism has not been in any major role in genetic susceptibility to gastric carcinogenesis, at least in the population studied here. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins.

Abstract 2997: Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis

Abstract Introduction: Aberrant hedgehog (Hh) pathway activation contributes to the pathogenesis of multiple cancers. Currently available Hh pathway inhibitors target Smoothened (Smo) which can acquire mutations causing drug resistance. Therefore, a major challenge is to identify compounds that inhibit Hh signaling downstream of Smoothened. Methods: We conducted a high-throughput screening (HTS) campaign of 337,000 small molecules using a Hh-dependent differentiation assay of C3H10T1/2 cells into osteoclasts. Primary active screening hits were validated by a cell-based assay cascade including motor neuron differentiation from mouse embryonic stem cells, Gli reporter activation in ShhLight2 cells and proliferation of heterozygous Patched (PTCH+/-) medulloblastoma (MB) cells. Smo binding was monitored in membrane-based Cyclopamine competition assays. To identify the underlying target identification of promising hits, affinity chromatography was performed followed by label-free quantitative (LFQ) tandem mass spectrometry. Effects on cytoplasmic dynein were examined in Smo trafficking and in vitro microtubules motility assays as well as by live cell imaging of labeled endosome movement. Results: We identified dynarrestin, a novel aminothiazole that potently blocks Hh signaling and the proliferation of Hh-dependent PTCH+/- MB tumor cells (IC50∼70nM). Dynarrestin does not bind to Smo and is able to suppress the Hh pathway in the presence of the Smo agonist SAG and following the knockdown of the Hh pathway suppressor SUFU indicating that dynarrestin inhibits Hh signaling downstream of both Smo and SUFU. Chemical LFQ proteomics identified cytoplasmic dynein as the target of dynarrestin. Dyneins convert energy from ATP hydrolysis into motor activity and are essential for many cellular processes, including Hh signaling. Unlike other dynein inhibitory molecules, e.g. Ciliobrevins, dynarrestin inhibits Smoothened trafficking but not ciliogenesis. We further demonstrate that dynarrestin reversibly interferes with endosome movement, proper mitotic spindle orientation, and dynein-based microtubule translocation in vitro without blocking ATP hydrolysis. Conclusions: Dynarrestin provides a novel dynein inhibitor with distinct specificity that uniquely facilitates probing dynein function. Given its multiple contributions to Hh signaling, mitosis and other cellular events, dynein remains a highly attractive target for medicinal chemistry programs aimed at exploiting and modulating its complex, poorly understood chemical cycle to interfere with different aspects of activity and function. We believe that unraveling dynein-dependent cellular processes by dynarrestin has great potential for development into novel anti-cancer drugs controlling Hh signaling downstream of Smoothened. Citation Format: Matthias Baumann, Susanne Höing, Ting-Yu Yeh, Nancy Martinez, Peter Habenberger, Lea Kremer, Hannes CA. Drexler, Philipp Küchler, Peter Reinhardt, Axel Choidas, Mia-Lisa Zischinsky, Gunther Zischinsky, Stephanie A. Ketcham, Lydia Wagner, Peter Nussbaumer, Slava Ziegler, Bert Klebl, Trina Schroer, Hans R. Schöler, Herbert Waldmann, Jared L. Sterneckert. Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis. [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 2997.

High TDP43 expression is required for TRIM16-induced inhibition of cancer cell growth and correlated with good prognosis of neuroblastoma and breast cancer patients

Tripartite Motif-containing protein 16 (TRIM16) is a member of a large family of tripartite motif (TRIM) proteins, that has been implicated in the pathogenesis of multiple cancers. However, the mechanism by which TRIM16 acts as a tumour suppressor is currently unknown. We used the versatile yeast two-hybrid assay on a cDNA library from human testes, which has relative high TRIM16 expression, to identify potential TRIM16-binding proteins. We identified transactive response DNA-binding protein 43 (TDP43) as a novel TRIM16 binding protein. Co-immunoprecipitation assay demonstrated that TDP43 bound TRIM16 in neuroblastoma and breast cancer cells. Enforced over-expression of TRIM16 increased the protein half-life of TDP43, through the inhibition of the proteosomal degradation pathway. High levels of TRIM16 and TDP43 are associated with good prognosis in both human neuroblastoma and breast cancer tissues. Importantly, we found TDP43 expression was required for TRIM16-induced inhibition of neuroblastoma and breast cancer cell growth and the repressive effect of TRIM16 on cell cycle regulatory proteins, E2F1 and pRb. Taken together, our data suggest that TRIM16 and TDP43 are both good prognosis indicators; also we showed that TRIM16 inhibits cancer cell viability by a novel mechanism involving interaction and stabilisation of TDP43 with consequent effects on E2F1 and pRb proteins.

Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood.A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases.Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases.On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance.Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made.At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample.Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential.We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. [Display omitted] DisclosuresTownsley:Novartis: Research Funding; GSK: Research Funding.