bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1327: Autophagy facilitates tumor promotion in PyMT tumors through up-regulation of Pparg
Authors: Okamoto Takako , Chen Song , Yeo Syn , Guan Jun-Lin
Published: August 2018

Autophagy genes have been reported to have either tumor promoting or inhibition functions in various cancers, but it is convoluting because of autophagy-independent functions of the respective autophagy genes. We previously reported that suppression of Fip200, an essential autophagy gene, inhibited tumor growth of PyMT driven mammary tumors. To dissect the specific autophagy-dependent function of FIP200 in these tumors, we recently established Fip200 Knock-in PyMT tumor mice with MMTV-Cre (cKI), allowing for conditional inhibition of autophagy in PyMT driven tumors without perturbing the non-autophagy functions of FIP200 . Compared to wild type (WT) tumors, tumor initiation, growth and metastasis were significantly suppressed in both cKI tumors and conditional Fip200 deleted tumors (cKO). Microarray analysis (GSEA) showed that adipogenesis related genes were significantly enriched in WT tumors compared to cKO tumors. The mRNA expression and protein expression of Pparg, one of the master regulators of adipogenesis, were significantly increased with autophagy induction under starvation condition (especially Glucose starvation) in PyMT isogenic cells compared to Fip200 KO and KI cells. Many Pparg target genes were down-regulated in autophagy deficient tumors. Among these, CD36, a free fatty acid (FFA) transporter, was also decreased compared to WT tumors. This suggests that FFA may have important roles as an energy source for PyMT tumor cells to survive under starvation conditions. In conclusion, our results indicate that tumor promotion of PyMT driven tumors was dependent on canonical autophagy. Our future studies will address the detailed mechanism of autophagy dependent tumor growth through metabolic regulation by Pparg.

Citation Format: Takako Okamoto, Song Chen, Syn Yeo, Jun-Lin Guan. Autophagy facilitates tumor promotion in PyMT tumors through up-regulation of Pparg [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1327.
Abstract 1333: Inactivation of NBR1 enhances sensitivity to rapamycin on human urothelial cancer cell lines through AMPK/ULK1-mediated autophagy
Authors: Kim Myeong Joo , Cho Min Ji , Chang In Ho , Whang Young Mi
Published: August 2018

Rapamycin has been highly evaluated in clinical therapeutic intervention for cancer patients as a specific inhibitor of the mammalian target of rapamycin (mTOR) kinase. Rapamycin also stimulates autophagy and mitophagy to remove damaged cells as a regulator of general autophagy. Previously, we identified increased expression of mRNA levels of NBR1 (autophagy cargo receptor) by inhibiting mTOR signaling in human urothelial cancer cell lines. Therefore, we investigated whether loss of NBR1 sensitizes human urothelial cancer cells to autophagic stimulation and stress-induced mitochondrial insults by treatment with rapamycin. NBR1-deficient cells exhibited enhanced sensitivity to rapamycin that was associated with increased autophagy and mitochondrial defects. Loss of NBR1 expression altered the cellular response to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species. Moreover, rapamycin treatment also induced autophagy activity through AMPK activation in NBR1-deficient cells, which was involved in blocked mTOR signaling and activated unc-51 like autophagy activating kinase 1 (ULK1). NBR1-deficient cells exhibited profound mitochondrial dysfunction in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-deficient human urothelial cancer through induction of autophagy activity by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential candidate for the treatment of human urothelial cancer. (NRF-2015R1A1A1A0500110 to I.H.C., NRF-2016R1D1A1B03933826 to Y.M.W., and the Korea Health Technology R&D Project HI17C0710 to C.I.H.)

Citation Format: Myeong Joo Kim, Min Ji Cho, In Ho Chang, Young Mi Whang. Inactivation of NBR1 enhances sensitivity to rapamycin on human urothelial cancer cell lines through AMPK/ULK1-mediated autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1333.
Abstract 1347: Autophagy inhibitor, chloroquine synergize oxaliplatin by modulating activity of cytosolic HMGB1 in pancreatic cancer
Authors: Lee Seonmin , Kim Kyu-Pyo , Yoo Changhoon , Kim Danbi , Kim Chorong , Chae Heejung , Kang Jihoon , Ryoo Baek-Yeol
Published: August 2018

Autophagy is a regulated, destructive catabolic mechanism to degrade cellular organelles and macromolecules. In cancer, the role of autophagy in tumor cell survival or death is still controversial because of each tumor type has a heterogeneity on metabolic status and microenvironment. In this study, we showed that autophagy inhibitor, chloroquine (CQ), increased sensitivity to oxaliplatin in pancreatic cancer cell lines. The autophagy inhibitor, chloroquine effectively inhibited autophagic flux which is important for maintenance of pancreatic cancer cell metabolism and drug resistance. Treatment of oxaliplatin after blockade of autophagy increased chromatin instability and facilitated loss of nuclear HMGB1. In consequence the cells turned into apoptotic cell death. The drug combination effect of chloroquine and oxaliplatin shows that inhibition of autophagy by chloroquine carry on dual role in pancreatic cancer cell death. First, chloroquine occurs cytosolic redox imbalance based on increase mitochondrial ROS (mtROS) following rupture of autolysosome. Second, the cytosolic redox imbalance occurred by chloroquine induces failure of autophagy by incapacitation of cytosolic HMGB1. Here we suggest that autophagy inhibition by chloroquine synergize oxaliplatin by modulating activity of cytosolic HMGB1. Also we suggest HMGB1 as a new target for therapeutic agents against pancreatic cancer.

Citation Format: Seonmin Lee, Kyu-Pyo Kim, Changhoon Yoo, Danbi Kim, Chorong Kim, Heejung Chae, Jihoon Kang, Baek-Yeol Ryoo. Autophagy inhibitor, chloroquine synergize oxaliplatin by modulating activity of cytosolic HMGB1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1347.
Abstract 1355: Mutation signatures including APOBEC in cancer cell lines
Authors: Harris Reuben S.
Published: August 2018

Background: Multiple endogenous and exogenous sources of DNA damage contribute to the overall mutation burden in cancer, with distinct and overlapping combinations contributing to each cancer type. Many mutation sources result in diagnostic mutation signatures, which can be deduced from tumor genomic DNA sequences. Examples include spontaneous hydrolytic deamination of methyl-cytosine bases in CG motifs (AGEING signature) and C-to-T and C-to-G mutations in 5'-TC(A/T) motifs (APOBEC signature).

Methods: The deconstructSigs R package was used to analyze single base substitution mutation signatures in over 1000 cancer cell lines. Two additional approaches were used to analyze the APOBEC mutation signature.

Results: Most cell lines show evidence for multiple mutation signatures. For instance, the AGEING signature, which is the largest source of mutation in most primary tumors, predominates in the majority of cancer cell lines. The APOBEC mutation signature is enriched in cancer cell lines from breast, lung, head/neck,

bladder, and cervical cancers, where this signature also comprises a large fraction of all mutations.

Conclusions: The single base substitution mutation signatures of cancer cell lines often reflect those of the original tumors from which they are derived. Cancer cell lines with enrichments for distinct mutation signatures such as APOBEC have the potential to become model systems for fundamental research on the underlying mechanisms and for advancing clinical strategies to target these processes and, ultimately, constrain tumor evolvability.

Citation Format: Reuben S. Harris. Mutation signatures including APOBEC in cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1355.
Abstract 136: T-cell clonotype convergence in colorectal cancer driven by tumor-specific neoantigens
Authors: Machado Jose C. , Reis Joana , Fernandes Margarida , Silva Rafaela , Cirnes Luis , Carneiro Fatima , Costa Jose L.
Published: August 2018

A cornerstone of the biological process which mediates response to immunotherapy in cancer patients is the expression of neoantigens resulting from somatic mutations in cancer cells. Little attention has been paid so far to the neoantigen's immune cell counterpart, the T-cell receptor (TCR). In this study, we aimed at characterizing the tumor infiltrating T-cell repertoire in colorectal cancer (CRC), and to evaluate evidence for neoantigen driven T-cell expansion within the tumor microenvironment. To characterize the T-cell repertoire, we used the Ion AmpliSeq Immune Repertoire Assay Plus with an Ion S5 XL system. This assay works with as little as 10ng of starting total RNA, allows 400bp read length, and offers complete characterization of the CDR1, CDR2 and CDR3 regions. A cohort of 20 CRC cases was used, including microsatellite instability (MSI) positive and MSI-negative cases. Our results show a higher T-cell richness in distal CRC samples compared with proximal samples (p=0.04), and lower T-cell richness in M1 as compared with M0 cases (p=0.0001). No association was observed with MSI status, inflammatory infiltrate, tumor grade or lymph node metastasis. We observed that the T-cell repertoire in CRC samples is highly convergent compared to the peripheral blood lymphocyte repertoire of healthy donors (p<0.0001). In CRC samples we observed evidence of selection of TCR clonotypes coding for the same CDR3 aminoacid sequence, suggesting neoantigen-driven TCR convergence. TCR clone overlap was observed only between pairs of samples (4 pairs of primary tumor and metastasis samples available) from the same patient, and not between samples from different patients, suggesting high tumor specificity of T-cell clonotype selection. A validation cohort of CRC cases, including additional pairs of primary tumor and metastasis samples, is currently being analyzed to validate our findings. In this study we provide evidence of T-cell clonotype selection in CRC driven by tumor-specific neoantigens. The specificity of this process is further reinforced by the T-cell clonotype overlap within patients. It remains to be seen whether it is possible to map convergent T-cell clonotypes to its respective tumor neoantigens.

Citation Format: Jose C. Machado, Joana Reis, Margarida Fernandes, Rafaela Silva, Luis Cirnes, Fatima Carneiro, Jose L. Costa. T-cell clonotype convergence in colorectal cancer driven by tumor-specific neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 136.
Abstract 1361: Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer
Authors: Duan Wenrui , Tang Shirley , Gao Li , Dotts Kathleen , Fink Andrew , Kalvala Arjun , Aguila Brittany , Villalona-Calero Miguel A.
Published: August 2018

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings.

Citation Format: Wenrui Duan, Shirley Tang, Li Gao, Kathleen Dotts, Andrew Fink, Arjun Kalvala, Brittany Aguila, Miguel A. Villalona-Calero. Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1361.
Abstract 137: Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer
Authors: Geraghty Terese , Rajagopalan Anugraha , Khan Samia , Varner Judith , Gupta Vineet
Published: August 2018

Lung cancer is the leading cause of cancer related deaths in the United States. With a 5-year survival rate of 18%, lung cancer patients are in immediate need of new therapeutic options. A major problem facing immunotherapy for cancer is the active immune suppression by the tumor. Tumors show presence of large numbers of tumor associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization to the tumor, and promote tumor survival. Integrin CD11b is highly expressed on TAMs and is essential for their recruitment and biological functions. Reasoning that CD11b activity is important for controlling tumor growth, we developed a novel small molecule agonist called leuhadherin-1 (LA1), which activates CD11b. We found that CD11b activation by LA1 significantly reduced tumor growth in wild type mice bearing Lewis Lung Carcinoma (LLC) tumors as early as one week post treatment. Conversely, LLC tumors grew at a faster rate in CD11b-/- mice, compared to wild type mice, showing that CD11b is important for controlling tumor growth. Importantly, LA1 treatment in tumor bearing CD11b-/- mice did not show any efficacy, demonstrating the specificity of LA1 to CD11b. These data suggest that CD11b activation via LA1 modulates TAMs in tumors and is a novel therapeutic strategy against cancer.

Citation Format: Terese Geraghty, Anugraha Rajagopalan, Samia Khan, Judith Varner, Vineet Gupta. Pharmacologic activation of integrin CD11b as a novel therapeutic strategy against lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 137.
Abstract 1374: Modeling the CRISPR/Cas9 structural complex with sgRNA and DNA
Authors: Wary Anita
Published: August 2018

The ability of genome editing tool to correct genetic disease, or edit the tumor causing gene, will not only benefit human race, but also plants and animal kingdom. The CRISPR/Cas9 system can be used to target specific genomic loci by single guide RNAs (sgRNAs). Using publicly available literature, we have modeled the structure of Streptococcus pyogenes Cas9 molecule in complex with sgRNA and its target DNA at 2.5 A° resolution. The crystal structure determination revealed a two-lobed architecture made of: (a) target recognition and (b) nuclease lobes, positioning the sgRNA:DNA hetero-duplex in a positively charged groove at their interface. While the recognition lobe interacts with the sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains that are responsible for cleavage of the complementary and non-complementary target DNA strands, respectively. The nuclease lobe also harbors a carboxyl-terminal domain to provide additional specificity for interaction with the protospacer adjacent motif (PAM) DNA sequence. This structural analyses show the molecular mechanism of RNA-guided DNA targeting by Cas9, thus providing the CRIPSR/Cas9 genetic machinery its ability to edit specific DNA sequence. (Key Ref: Nishimasu H et al., Cell. 2014;156:935-49)

Citation Format: Anita Wary. Modeling the CRISPR/Cas9 structural complex with sgRNA and DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1374.
Abstract 1384: The epigenetic modifier EZH2 represses antitumor immunity in melanoma
Authors: Tiffen Jessamy , Shklovskaya Elena , Gallagher Stuart , Gunatilake Dilini , Hersey Peter
Published: August 2018

Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remains a key obstacle. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Aberrant EZH2 in cancer results in the silencing of hundreds of tumor-suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq and microarrays to reveal downstream EZH2 target genes, we identified many tumor-suppressor genes including some that play a role in the antitumor immune response. Interestingly, xenografts in immune-deficient NOD SCID mice showed no significant inhibition in tumor growth when treated with an EZH2 inhibitor. However, growth inhibition was achieved when the same experiment was performed in nude mice, which retain some mediators of innate immunity. The effect of EZH2 overexpression in melanoma on immune-mediated rejection was studied in an inducible in vivo model. Subcutaneous B16 tumors expressing model antigen were initially rejected but relapsed in the majority of immunodeficient mice transferred with CD4 T cells. Long-term (120-day) survival was reduced from 40% to 22% if the tumors also overexpressed EZH2. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the antitumor immune response. The progression of several preclinical, small-molecule inhibitors of EZH2 remains promising.

Citation Format: Jessamy Tiffen, Elena Shklovskaya, Stuart Gallagher, Dilini Gunatilake, Peter Hersey. The epigenetic modifier EZH2 represses antitumor immunity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1384.
Abstract 14: Preclinical evaluation of novel HIF-1α/P300 binding inhibitors
Authors: Strope Jonathan D. , Harris Emily M. , Beedie Shaunna L. , Chau Cindy H. , Cook Kristina M. , Schofield Christopher J. , Gustafson Kirk R. , Figg William D.
Published: August 2018

A key component of tumor progression and metastasis is the hypoxic response. The hypoxic response regulates angiogenesis, tumor invasion, and metabolism. The Hypoxia Inducible Factor (HIF) is the transcriptional system responsible for the hypoxic response. The inhibition of the hypoxic response via inhibition of the HIF-1 pathway by disrupting its association with the transcriptional coactivator p300 presents a potential therapeutic target for multiple cancers where HIF is upregulated. To that end, we describe the preclinical development of previously described novel marine pyrroloiminoquinone alkaloids found using a HIF-1α/p300 assay in a high throughput screen of extracts from the National Cancer Institute's Natural Products Repository. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein−protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription. The 2 lead candidates, discorhabdin L and H were assessed in inhibition of angiogenesis in an in vitro HUVEC assay and ex vivo rat aortic ring assay. The compounds were also evaluated for in vivo efficacy in prostate cancer cell tumor xenografts. Results show that development of these compounds for clinical use is warranted and may prevent the progression of multiple tumor types.

Citation Format: Jonathan D. Strope, Emily M. Harris, Shaunna L. Beedie, Cindy H. Chau, Kristina M. Cook, Christopher J. Schofield, Kirk R. Gustafson, William D. Figg. Preclinical evaluation of novel HIF-1α/P300 binding inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 14.
Abstract 142: cdk5 inhibition promotes neurogenesis while reducing self-renewal of brain tumor stem cells
Authors: Mukherjee Subhas , Olson Cheryl , Brat Daniel
Published: August 2018

Cancer stem cells are biologically potent and support tumor growth, in part by deregulating asymmetric stem cell division. Asymmetric cell division of stem cells is a type of mitotic division where a stem cell generates one stem and one differentiated cell. Cancer stem cells, however, tend to generate more stem-like dividing cells to promote neoplasm. To uncover mechanisms and potential therapeutic targets relevant to self-renewal in human glioma stem cells (GSCs), we performed a genetic screen for kinases that reverse the tumor phenotype of our novel adult Drosophila brain tumor model and found that knockdown of the brain-specific dCdk5 was highly effective in suppressing the self-renewing stem cell components. A highly specific CDK5 inhibitor is promoting neurogenesis among these fly brain cancer stem cells. These cancer stem cells, although they express glial markers in tumors, do not differentiate into glia upon CDK5 suppression. Additionally, while the tumor generation in adult flies completely disrupts the mushroom body, the main cognitive center in adult fly brain, the CDK5 inhibition partially rescues the mushroom body and extends life span of these flies. dCdk5 and its human ortholog CDK5 (79% identity) are aberrantly activated in some forms of cancer. We demonstrate that GBMs highly express CDK5 mRNA and protein, likely related to copy number gains of chromosome 7, with strong positive correlation with stem cell markers and marked effect on tumor-propagating properties. Using human GSCs isolated from tumor spheres and mouse xenografts, we found that CDK5 directly phosphorylates CREB1 independent of cAMP and stops stem cell renewal. These data together show that CDK5 inhibition not only can attenuate self-renewal properties of brain tumor stem cells, but also can promote neurogenesis, which could potentially become a better treatment strategy for patients and improve their cognitive function as well.

Citation Format: Subhas Mukherjee, Cheryl Olson, Daniel Brat. cdk5 inhibition promotes neurogenesis while reducing self-renewal of brain tumor stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 142.
Abstract 1424: Elucidating the microRNA-203 specific biological processes in glioblastoma cells from high-throughput RNA-sequencing
Authors: Ahir Bhavesh K. , Engelhard Herbert H. , Lakka Sajani S.
Published: August 2018

Glioblastoma (GBM) is the most common primary malignant intracranial adult brain tumor. Allelic deletion on chromosome 14q play an essential role in GBM pathogenesis, and this chromosome 14q site was thought to harbor multiple tumor suppressor gene associated with GBM, a region that also encodes microRNA-203 (miR-203). This study was conducted to identify gene expression profile changes associated with mir-203 expression by high-throughput RNA sequencing. Enrichment analyses for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that mir-203 expression had a strong, negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation. The biological processes mostly influenced were p53 signaling pathway, FoxO signaling pathway, DNA replication, cell cycle, MAPK signaling pathway and Apoptosis. In total, 847 upregulated and 345 downregulated differentially expressed genes (DEGs) were identified in control versus miR203 expressing glioma cells. After GO enrichment, the downregulated DEGs and miR-203 predicted target genes such as BCL-2, SPARC, PDGFA, CREB were found to be mainly enriched in cell cycle regulation and apoptosis processes, whereas the upregulated DEGs, such as CCND1, E2F1 were involved in the DNA replication and cell cycle regulation of gene expression. We also demonstrate that mir-203 expression suppressed BCL-2 protein and mRNA by western blotting and qRT-PCR analysis. Moreover, co-transfection experiments using a luciferase-based reporter assay demonstrated that mir-203 directly regulated BCL-2 expression and BCL-2 overexpression suppressed mir-203 mediated cell death. Knowledge gained through this profiling study can facilitate targeted therapeutic interventions.

Citation Format: Bhavesh K. Ahir, Herbert H. Engelhard, Sajani S. Lakka. Elucidating the microRNA-203 specific biological processes in glioblastoma cells from high-throughput RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1424.
Abstract 1430: Improved sample indexing for high fidelity demultiplexing to increase confidence in low frequency variant calling
Authors: Irish Jonathan C. , RoseFigura Jordan , Sandhu Sukhinder K. , Carrion Bita , Kurihara Laurie , Makarov Vladimir
Published: August 2018

Due to recent scientific advances, there is a growing demand for new indices with high fidelity to eliminate errors associated with certain index combinations and sequencing chemistries. These errors are due to insufficient edit distances (the minimum number of changes required to transform one index sequence to the other) between index sequences or index hopping. Recent publications have highlighted how sequencing reads are misassigned due to "index hopping" on Illumina patterned flow cells, such as the recently launched Illumina Novaseq which can generate billions of reads (>100 exomes) in a single run. This misassignment canlead to false positives in ultra-sensitive assays where low frequency variants or nucleic acid species are monitored. We have observed misassignment due to insufficient edit distance among Illumina TruSeq HT i7 indices at a frequency up to 1.5%. Therefore, we developed 968nt i7 indices that can be paired with the existing TruSeq HT i5 indices to achieve 768 high throughput dual combinations, which were validated using a novel method on both Illumina's 2- and 4-channel technologies as both single and dual indices. This method involved thepreparation of 96 libraries with unique, non-overlapping inserts to facilitate tracking of index misassignment. This allowed us to assess not only which index has misassigned library molecules, but also pinpoint the origin and rate of misassignment within a single run. With our 96 i7 indices, misassignment was observed at rates <0.1%. For even higher fidelity de-multiplexing, we have paired our 96 indices in a non-tandem manner for single use in both the i5 and i7 positions, known as Unique Dual Indices (UDIs). The use of UDIs further eliminates index read errors that misassign reads, enabling increased confidence in calling low frequency variants. UDIs can also eliminate PCR-induced chimerisms, which can significantly improve data from a variety of assays. We are validating 96 new indices as UDIs for avoidance of index hopping and for eliminating PCR-induced chimerism during multiplexed library amplification.

Citation Format: Jonathan C. Irish, Jordan RoseFigura, Sukhinder K. Sandhu, Bita Carrion, Laurie Kurihara, Vladimir Makarov. Improved sample indexing for high fidelity demultiplexing to increase confidence in low frequency variant calling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1430.
Abstract 1432: Deranged tyrosine metabolism drives tumorigenesis in liver cancer
Authors: Tong Man , Wong Tin Lok , Luk Steve Tin-Chi , Che Noelia , Guan Xin Yuan , Yuan Yun Fei , Lee Terence Kin-Wah , Ma Stephanie
Published: August 2018

Tyrosine, like other amino acids, is the building block for proteins as well as an alternative energy source for cellular functions. Liver is the major organ where tyrosine degradation takes place to produce intermediates or precursors for gluconeogenesis and ketogenesis. In normal liver, tyrosine can be converted to fumarate and acetoacetate through multiple steps of enzymatic reactions. In patients with liver cancer, an upregulation of serum tyrosine levels has been previously reported, suggesting a deregulated tyrosine catabolism in liver cancer. In this study, we observed a significant down-regulation of the five enzymes involved in tyrosine catabolism (TAT, HPD, HGD, GSTZ1 and FAH) in tumor samples compared with normal liver samples from TCGA database. Further, downregulation of the first three enzymes significantly correlates with overall and disease-free survival in liver cancer patients. Downregulation of these enzymes is also confirmed in a separate liver cancer patient transcriptome cohort of Asian ethnicity, as well as at the proteomic level by both Western blot and immunohistochemistry assays. Our group has previously reported that downregulation of the first tyrosine catabolizing enzyme TAT (tyrosine aminotransferase) could promote liver tumorigenesis. Here, we further investigate if reprogrammed tyrosine metabolism could promote cancer development by modulating gene expression and activity of the second enzyme HPD (4-hydroxyphenylpyruvate dioxygenase) in the tyrosine catabolism pathway. We found blocking HPD gene expression or its enzymatic activity could promote cell proliferation and tumor formation in liver cancer. This could be partially attributed to enhanced mitochondrial respiratory capacity accompanied with reduction in ROS level, NADP/NADPH ratio and DNA damage. Conversely, overexpression of HPD led to an opposing effect. Taken together, this study reveals an unreported alteration of tyrosine metabolism which drives cancer development in liver cancer.

Citation Format: Man Tong, Tin Lok Wong, Steve Tin-Chi Luk, Noelia Che, Xin Yuan Guan, Yun Fei Yuan, Terence Kin-Wah Lee, Stephanie Ma. Deranged tyrosine metabolism drives tumorigenesis in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1432.
Abstract 1456: Integrin beta4-induced mitophagy promotes the lactate production of cancer-associated fibroblasts in breast cancer
Authors: Sung Jin Sol , Kang Suki , Lee Joo Hyun , Mun Seong Gyeong , Kim Baek Gil , Cho Nam Hoon
Published: August 2018

Integrin beta4 neoexpression is induced in cancer-associated fibroblasts (CAFs) by some triple negative breast cancer cells (MDA-MB-231 and BT-20) through a contact-dependent manner. Integrin beta4 expression is well known to contribute to the migration and survival of cancer cells. However, there is very little known about the role of integrin beta4 expression in CAFs, especially in the aspect of tumor progression. In our study, we found that the exogenous expression of integrin beta4 increased the lactate production of CAFs. The observation made us to hypothesize that integrin beta4 neoexpression is associated with the mitophagy, a cause for aerobic glycolysis, in CAFs. To confirm the assumption, the expression alteration of mitophagy-related genes was first screened in the CAF transfected with integrin beta4 overexpression plasmid. BCL2 interacting protein 3 like (BNIP3L) and microtubule associated protein 1 light chain 3 alpha-II (LC3-II) were significantly upregulated in the integrin beta4 overexpressing CAFs, which was reversed both in the knockdown of integrin beta4 expression and the treatment with an integrin beta4 inhibitor. Autophagosome maturation and lysosomal fusion were also increased in the integrin beta4 overexpressing CAFs compared to the control. Phosphorylation level of c-Jun was upregulated by integrin beta4 overexpression in CAFs and was able to bind to the putative promoter regions of BNIP3L and LC3-II. Taken together, these results demonstrate that integrin beta4 neoexpression induces upregulation of BNIP3L and LC3-II ultimately leading to mitophagy in CAFs, contributing to cancer metabolism via mitophagy-induced lactate production, namely reverse Warburg effect.

Citation Format: Jin Sol Sung, Suki Kang, Joo Hyun Lee, Seong Gyeong Mun, Baek Gil Kim, Nam Hoon Cho. Integrin beta4-induced mitophagy promotes the lactate production of cancer-associated fibroblasts in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1456.
Abstract 1466: DAXX localizes ATRX to suppress alternative lengthening of telomeres in osteosarcoma
Authors: Soper Sarah Faith Clatterbuck , Yost Kathryn E. , Walker Robert L. , Pineda Marbin A. , Zhu Yuelin J. , Waterfall Joshua J. , Meltzer Paul S.
Published: August 2018

To maintain genome stability, proliferating cells must add telomere sequence to counteract the chromosome end replication problem. In normal cells, telomeres are lengthened through the action of the enzyme telomerase. In about 10-15% of tumors, however, telomeres are lengthened through a telomerase-independent mechanism known as Alternative Lengthening of Telomeres or ALT. Many tumors that use ALT have poor prognoses, so ALT represents an appealing therapeutic target. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex that deposits histone variant H3.3. How these mutations facilitate the ALT pathway is not well understood. Previous work in our lab identified an ALT-positive osteosarcoma cell line, G292, in which ATRX is wild-type but DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. The DAXX-KIFC3 fusion leads to a loss of DAXX function, and inducible restoration of wild-type DAXX reversibly abrogates ALT in this cell line. We observe that expression of wild-type DAXX results in localization of ATRX to PML bodies, increased occupancy of ATRX at telomeric chromatin, and higher levels of histone H3.3 at telomeres. We conclude that full-length DAXX is required for the functional localization of ATRX to telomeres. Leveraging this our inducible system, we continue to probe the role of the ATRX/DAXX complex in suppressing ALT.

Citation Format: Sarah Faith Clatterbuck Soper, Kathryn E. Yost, Robert L. Walker, Marbin A. Pineda, Yuelin J. Zhu, Joshua J. Waterfall, Paul S. Meltzer. DAXX localizes ATRX to suppress alternative lengthening of telomeres in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1466.
Abstract 147: Targeting USP22 suppresses tumorigenicity and enhances sensitivity to cisplatin in cancer-initiating cells from lung adenocarcinoma through downregulating ALDH1A3
Authors: Zhang Keqiang , Yun Xinwei , Wang Jinhui , Bonner Melisa , Yang Lu , Wu Jun , Raz Dan J.
Published: August 2018

We previously found that loss of monoubiquitination of histone H2B (H2Bub1) was associated with poor differentiation and enhanced malignancy of lung adenocarcinoma. Herein, we investigated the expression and roles of USP22, an H2Bub1 deubiquitinase, in cancer-initiating cells (CICs) with stem cell-like characteristics from primary lung adenocarcinoma. CICs from patient-derived cancer tissues were isolated and characterized, and the association of USP22 with CICs was investigated using flow cytometry, aldehyde dehydrogenase (ALDH) activity, Western blot, and fluorescence microscope assays. The impact of USP22 on stem cell-like characteristics and cisplatin resistance in CICs was examined by in vitro tumorsphere formation, in vivo transplantation, and apoptosis assays. We identified that USP22 is predominantly expressed in CICs, a subpopulation of cells with high expression of CD133 stem cell biomarker. The expression of USP22 in CICs is dramatically reduced upon serum-induced differentiation. Moreover, knockdown of USP22 significantly suppressed in vitro tumorsphere formation and in vivo xenograft growth in NOD-SCID mice. Notably, USP22 is dramatically elevated in tumorsphere cells that survived cisplatin treatment, while knockdown of USP22 significantly sensitizes tumorsphere cells to cisplatin. Interestingly, ALDH1A3, a dominant isoform of ALDH implicated in enhancing cisplatin resistance in lung adenocarcinoma, is significantly downregulated upon knockdown of USP22 in tumorsphere cells. Furthermore, knockdown of ALDH1A3 significantly sensitizes tumorsphere cells to cisplatin. Therefore, our data indicate that USP22 plays critical roles in tumorigenicity and cisplatin resistance in lung adenocarcinoma, and targeting USP22 may represent a potential therapeutic approach to eliminate CICs in lung adenocarcinoma through downregulation of ALDH1A3 expression.

Citation Format: Keqiang Zhang, Xinwei Yun, Jinhui Wang, Melisa Bonner, Lu Yang, Jun Wu, Dan J. Raz. Targeting USP22 suppresses tumorigenicity and enhances sensitivity to cisplatin in cancer-initiating cells from lung adenocarcinoma through downregulating ALDH1A3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 147.
Abstract 1490: Molecular function of the read-through transcript PRR5-ARHGAP8
Authors: Sethi Jasmeen K. , Wong Oscar G. , Wong Esther S. , Cheung Annie N.
Published: August 2018

Background: Ovarian carcinoma is one of the most fatal malignancies in females. From the analysis of RNA-seq we have previously conducted to study the transcriptome of ovarian carcinoma upon knockdown of transcription factor hepatic nuclear factor 1β (HNF-1β), PRR5-ARHGAP8 was identified as the only read-through transcript not targeted for the nonsense-mediated decay pathway. Read-through transcription refers to the splicing of the intergenic region from two adjacent genes resulting in a chimeric transcript comprising of one or more exons from both genes. Although several of such transcripts are found in the transcriptome, their significance remains an enigma. Our aim was to ascertain the differential expression of the read-through transcript PRR5-ARHGAP8 in ovarian cancers and to determine its molecular functions.

Materials & Methods: The deregulation of PRR5-ARHGAP8 was validated by RT-qPCR in several ovarian carcinoma cell lines. The expression of this fusion transcript and the individual genes were studied in immortalised human ovarian surface epithelial (HOSE) cell lines, ovarian carcinoma cell lines, and matched tumour and non-tumour pairs from frozen patient samples. Overexpression of this transcript was achieved by transient transfection in HEK293T cells to study its effect on proliferation using MTT assays. RNA and protein lysates were also obtained at several time points to evaluate the pathways affected by RT-qPCR and western blot, respectively.

Results: There was an approximate 3- to 9-fold upregulation of PRR5-ARHGAP8 transcript with a decrease in the expression of PRR5 and ARHGAP8 transcripts in OVMANA, OVISE and KK ovarian carcinoma cell lines upon the knockdown of HNF-1β. Moreover, higher transcript levels of PRR5, ARHGAP8 and PRR5-ARHGAP8 were observed in ovarian carcinoma cell lines compared to HOSE cell lines and in ovarian cancer samples compared to the non-tumour counterparts. Overexpression of PRR5-ARHGAP8 in HEK293T cells resulted in the decrease of phosphorylated Akt protein expression and a reduction of cell viability. RT-qPCR analysis showed a decrease in E-cadherin and an increase in vimentin expression upon PRR5-ARHGAP8 overexpression.

Discussion: The present data suggests PRR5-ARHGAP8 is highly abundant in ovarian carcinomas and may mediate its effects on proliferation via the MAPK pathway and on migration by regulating the epithelial to mesenchymal transition. Overall, this study provides novel insights into the biological relevance of read-through transcription in carcinogenesis.

Citation Format: Jasmeen K. Sethi, Oscar G. Wong, Esther S. Wong, Annie N. Cheung. Molecular function of the read-through transcript PRR5-ARHGAP8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1490.
Abstract 1492: High-efficiency, strand-specific, low-bias library preparation method for transcriptome profiling of low-input RNA
Authors: Nowosielska-Vecchietti Anetta
Published: August 2018

RNA Sequencing (RNA-seq) is a powerful technique for transcriptome analysis. It is widely used for gene expression analysis, detection of mutations, fusion transcripts, alternative splicing, and the study of post transcriptional modifications. RNA-seq is emerging as a powerful tool for molecular diagnostics as well, providing insights into the biological pathways and molecular mechanisms associated with disease progression including cancer. Recent improvements in next generation sequencing technologies (NGS) and sample barcoding strategies have resulted in significant cost reduction. As RNA-seq is increasingly adopted for molecular diagnostics, the quality and reproducibility of library preparation methods become more important. In addition, demand for library preparation methods that produce successful NGS libraries from very low input RNA or precious clinical samples is increasing.

To achieve these goals, we have developed an RNA-seq library preparation method which can be used across a wide range of input RNA (5-1000 ng total RNA). GC content analysis, gene body coverage and gene expression correlation show that these important parameters remain consistent across varying inputs, even though input amounts vary by two orders of magnitude. As a result, our method has increased sensitivity and specificity for low-abundance transcripts, and reduced PCR duplicates and sequence bias, delivering high quality strand-specific data. Our method is compatible with poly A-tail enriched and ribosomal RNA depleted samples as well as RNA extracted from FFPE (formaldehyde fixed paraffin embedded) tissue samples.

Citation Format: Anetta Nowosielska-Vecchietti. High-efficiency, strand-specific, low-bias library preparation method for transcriptome profiling of low-input RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1492.
Abstract 1494: SOX18: A novel master regulator of high-grade serous ovarian tumorigenesis
Authors: Liu Annie Y. , Vavra Kevin C. , Corona Rosario I. , Abassi Forough , Fonseca Marcos , Segato Felipe , Freedman Matthew L. , Gayther Simon A. , Noushmehr Houtan , Lawrenson Kate
Published: August 2018

High-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer. Recent data indicate that fallopian tube secretory epithelial cells (FTSECs) represent the cell of origin for HGSOC. While many studies have characterized molecular features associated with HGSOC biology, the master transcription factors (TFs) that drive disease development are not known. To identify master TFs for HGSOC, we first performed an integrative analysis of gene expression profiles from 73 FTSEC samples and 394 HGSOCs from The Cancer Genome Atlas (TCGA) project. We then integrated these data with chromatin immunoprecipitation sequencing (ChIP-seq) analyses performed on primary HGSOC samples to select active chromatin regions marked by H3k27ac and to identify super-enhancer (SE) regions. These analyses identified overexpressed TFs that coincide with tumor-specific SEs, a hallmark of master TFs. We identified multiple putative master TFs including SOX18, which was highly overexpressed in HGSOCs relative to normal FTSECs (log2 fold change in expression = 3.5, p = 2.5x10-19). SOX18 is a TF that regulates development of blood and lymphatic vessels, but its association with HGSOC development has not been reported before. We examined the expression of SOX18 in ~8,000 tumors representing 17 tumor types from TCGA. SOX18 expression was significantly elevated in HGSOC relative to any other tumor types, suggesting it is highly specific to this cancer. We found that SOX18 is highly expressed in the HGSOC cell lines UWB1.289, Kuramochi, and EFO21, and we then performed lentiviral-mediated shRNA knockdown of SOX18 in these cell lines to establish the effects of SOX18 depletion on neoplastic phenotypes. In ongoing experiments, we are performing gene expression profiling and H3k27ac ChIP-seq analysis after SOX18 knockdown to identify SOX18 target genes and characterize the landscape of SOX18 binding sites in HGSOC. These studies will likely identify novel molecular biomarkers that may represent much-needed therapeutic targets for HGSOC.

Citation Format: Annie Y. Liu, Kevin C. Vavra, Rosario I. Corona, Forough Abassi, Marcos Fonseca, Felipe Segato, Matthew L. Freedman, Simon A. Gayther, Houtan Noushmehr, Kate Lawrenson. SOX18: A novel master regulator of high-grade serous ovarian tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1494.