bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3928: Sertraline in melanoma treatment: TCTP as a therapeutic target
Authors: Boia-Ferreira Marianna , Basílio Alana , Baldissera Antonielle B. , Matsubara Fernando H. , Appel Marcia H. , Costa Cleber R. da , Chaim Olga M. , Gremski Luiza H. , Veiga Silvio S. , Senff-Ribeiro Andrea
Published: August 2018

Background: Sertraline (an SSRI antidepressant) binds directly to TCTP protein and decreases its intracellular levels. TCTP (Translationally Controlled Tumor Protein) is an antiapoptotic protein highly conserved through phylogeny. TCTP overexpression was detected in several tumor types. Silencing TCTP was shown to induce tumor reversion, a process overriding at the molecular level the malignant process. There is a reciprocal repression between TCTP and P53.

Methods: We evaluated the role of TCTP in melanoma using sertraline and siRNA. Cell viability, migration and clonogenicity were assessed in human and murine melanoma cell in vitro. Sertraline was evaluated in vivo in a murine melanoma model and compared to dacarbazine, a major a chemotherapeutic agent used in melanoma treatment. Long-term effect of sertraline was evaluated keeping cells on regular culture for 5 days after a 72h-treatment and then assessing proliferation, clonogenicity and in vivo growth.

Results: (i) In human melanoma cell lines, sertraline treatment and decrease of TCTP levels (by siRNA) are related to the inhibition of clonogenicity and migration; (ii) TCTP levels are related to melanoma malignancy and invasiveness (in B16F10/F1 murine model); (iii) knockdown of TCTP triggers decrease of proliferation and migration on murine melanoma cells; (iv) sertraline decreases TCTP mRNA levels in B16F10 cells and results in inhibition of clonogenicity; (v) sertraline presents remarkable in vivo antitumoral effects in B16F10/C57BL6 model, greater than dacarbazine, the main chemotherapeutic agent used for melanoma; (vi) sertraline treatment is capable of a long-term effect on melanoma cells, suggesting that the drug can trigger tumor reversion, a reprogram of tumor cells to a less malignant phenotype. Our data could pave the way for new approaches in the treatment of melanoma, a pathology whose prognostic is poor in late stages, and the efficiency of the current available chemotherapeutic treatment is very low.

Conclusion: Altogether, these results indicate that sertraline could be effective against melanoma and TCTP can be a target for melanoma treatment, opening up new therapies for melanoma treatment.

Citation Format: Marianna Boia-Ferreira, Alana Basílio, Antonielle B. Baldissera, Fernando H. Matsubara, Marcia H. Appel, Cleber R. da Costa, Olga M. Chaim, Luiza H. Gremski, Silvio S. Veiga, Andrea Senff-Ribeiro. Sertraline in melanoma treatment: TCTP as a therapeutic target [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 3928.
Abstract 3949: p53-restoring small molecule CB001 suppresses tumor growth and metastasis
Authors: Zhang Shengliang , Zhou Lanlan , Wagner Jessica , Dicker David T. , El-Deiry Wafik S.
Published: August 2018

The uncontrolled growth and metastasis of tumor cells leads to morbidity and mortality in patients with cancer. The tumor suppressor p53, as a guardian of genome integrity, plays an important role in suppressing tumor growth and metastasis. However, loss of p53 pathway signaling frequently occurs in advanced tumors. Dysfunction of the p53 pathway is due to either p53 gene mutation or inactivation of p53. Mutations not only result in loss of wild-type p53 function, but also endow mutant p53 with a gain-of function, which further confers tumor aggressiveness and metastasis. Restoring p53 pathway signaling is an attractive strategy for suppressing tumor growth and metastasis in cancer therapy. Small-molecule CB001 restores p53 pathway signaling in p53-deficient colorectal cancer cells. We examined p53 pathway signaling based on the p53-responsive bioluminescence and the protein level of p53 targets. CB001 restores p53 pathway signaling in a p53-dependent and -independent manner in cancer cells. CB001 restores p53 pathway signaling via p73 in mutant p53-expressing cancer cells, and activates the p53 pathway in wild-type p53-expressing cancer cells. Correlated with restoration of p53 pathway signaling, CB001 induces cell death in cancer cells, but not in normal cells. Neither phosphorylation of H2AX in cancer cells nor DNA intercalation was observed upon CB001 treatment at the tested doses, suggesting little or no genotoxicity of CB001 in human colorectal cancer cells. Furthermore, both cell scratch assay and Boyden chamber assay reveal that CB-001 inhibits cancer cell migration. We further demonstrate that CB001 induces cell death and inhibits cell migration via p73 in p53-mutant colorectal cancer cells. These results taken together

suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth and metastasis through restoring p53 pathway in cancer cells.

Citation Format: Shengliang Zhang, Lanlan Zhou, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. p53-restoring small molecule CB001 suppresses tumor growth and metastasis [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 3949.
Abstract 3953: The role of USF1 in the regulation of lipogenesis and breast cancer tumor progression
Authors: Lewis Jessica , Mahmud Iqbal , Liao Daiqing
Published: August 2018

Breast cancer accounts for nearly one-quarter of all cancer diagnoses and is the principal cause of cancer-related mortality in women worldwide. Triple negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with ER+, PR+ and HER2+ breast cancer subtypes. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. USF1 (upstream stimulatory factor 1), a gene linked to drive lipogenesis and cellular proliferation, is over-expressed in human malignancies, yet its contribution to cancer remains unclear. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), we found that USF1 expression is significantly higher in TNBC tumor samples and cell lines. Also, USF1 gene expression positively correlates with key lipogenic enzymes. Significantly, we found that high expression of USF1 in breast cancer correlates with decreased patient survival. We found that USF1 shRNA expression resulted in cell death in breast cancer cell lines. We therefore hypothesize that USF1 drives de novo lipogenesis to promote breast tumorigenesis. Further studies are underway to determine the mechanisms by which USF1 promotes de novo lipogenesis, tumorigenesis and metastatic progression. Our studies will shed lights on the roles of USF1 in breast cancer tumor biology and undercover potential novel anticancer drug targets. (Supported by McKnight Foundation, Bankhead-Coley Cancer Research Program and James and Esther King Biomedical Research Program, Florida Department of Health.)

Citation Format: Jessica Lewis, Iqbal Mahmud, Daiqing Liao. The role of USF1 in the regulation of lipogenesis and breast cancer tumor progression [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 3953.
Abstract 3955: PIK3R1 loss activates AKT and STAT3 signaling in ovarian cancer
Authors: LI Xinran , Mak Victor CY , Zhou Yuan , Lu Yiling , Cheung Annie NY , Mills Gordon B , Cheung Lydia WT
Published: August 2018

The phosphoinositide 3-kinase (PI3K) regulatory subunit p85α is one of the critical gatekeepers of PI3K pathway activation through stabilizing and inhibiting the catalytic subunit p110. p85α is encoded by PIK3R1, which is frequently mutated or deleted in multiple cancer types including ovarian cancer, providing justification of exploring the approaches to target the aberrations. In this study, we observed a spectrum of oncogenic phenotypes in ovarian cancer cells upon knockdown of PIK3R1 in vitro and in vivo, indicating the functional significance of PIK3R1 loss in ovarian cancer. Strikingly, PIK3R1 loss not only led to AKT activation (canonical signaling of PI3K), but also unexpectedly JAK2/STAT3 signaling activation (non-canonical signaling of PI3K). PIK3R1 loss induced translocation of STAT3 into the nucleus, where gene transcription was promoted and thereby the malignant properties. Using in vitro 3D culture spheroid assay, we found that PIK3R1 loss sensitized ovarian cancer cells to AKT inhibitors and JAK2/STAT3 inhibitors. More importantly, the combination of AKT and STAT3 inhibitors resulted in synergistic loss of cell viability. The demonstrated signaling alterations and the associated therapeutic susceptibility downstream of PIK3R1 loss may open new avenue for the treatment of PIK3R1 loss-bearing cancer. [This study was supported by Research Grants Council of the Hong Kong SAR, China, Project No. 27103616]

Citation Format: Xinran LI, Victor CY Mak, Yuan Zhou, Yiling Lu, Annie NY Cheung, Gordon B Mills, Lydia WT Cheung. PIK3R1 loss activates AKT and STAT3 signaling in ovarian 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 3955.
Abstract 397: Systematic functional annotation of somatic mutations in cancer
Authors: Liang Han , Ng Patrick Kwok-Shing , Li Jun , Jeong Kang Jin , Lu Yiling , Yi Song , Sahni Nidhi , Mills Gordon
Published: August 2018

Recent sequencing studies have identified thousands of unique somatic mutations across patient tumors. However, the functional impact of the vast majority of these mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant open reading frame generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of downstream signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. We show that our platform has higher sensitivity for characterizing weak drivers than pooled screening. Our data are accessible through the user-friendly, open-access data portal we created. Our study will facilitate the discovery of novel biomarkers, improvement of existing prediction algorithms, and development of new therapeutic approaches.

Citation Format: Han Liang, Patrick Kwok-Shing Ng, Jun Li, Kang Jin Jeong, Yiling Lu, Song Yi, Nidhi Sahni, Gordon Mills. Systematic functional annotation of somatic mutations in 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 397.
Abstract 398: Functional coding and noncoding drivers of EMT-mediated acquired drug resistance
Authors: Bainer Russell O. , Wilson Catherine , Callow Marinella , Feng Siyu , Costa Michael , Watanabe Colin , Mayba Oleg , Lin Eva , Martin Scott , Yauch Bob , Bourgon Richard , Klijn Christiaan
Published: August 2018

In response to diverse stimuli, tumor cells can undergo a process resembling the epithelial-mesenchymal transition (EMT) observed during development. During this transition, epithelial-like tumor cells acquire mesenchymal-like physiologic features, become more motile and invasive, and acquire insensitivity to many therapeutic agents. While the importance of this phenomenon to cancer treatment and patient outcomes is well known, considerable heterogeneity exists across experimental models of EMT, and diverse mechanisms have been proposed to explain its various associated malignant phenotypes. Consequently, the extent to which these processes are shared uniformly across instances of EMT, as well as their relative importance in treatment contexts, remains unclear. We have used RNAseq, ATACseq, and parallelized high-throughput cell viability screening to identify differences in gene expression levels, chromatin accessibility, and drug sensitivity that correlate with EMT in a series of cell line models. By contrasting these changes across different genetic backgrounds and mechanisms of EMT induction, we are able to identify sets of elements in the transcribed and noncoding genome that are characteristic of different modes of EMT induction. Then, by comparing these candidate sets with observed differences in acquired drug sensitivity in the respective models, we were able to define a set of genetic elements whose biologic activity is likely to influence the efficacy of these compounds. Finally, we used a series of targeted CRISPR-based knockout screens to assess the relative importance of these elements to the viability of isogenic epithelial-like and mesenchymal-like cells, alone and in the presence of multiple therapeutic drugs. Using this approach, we were able to validate known mechanisms of acquired drug resistance and identify new candidate effectors. Notably, we identify multiple candidate noncoding elements containing CTCF binding sites that appear to meaningfully influence cell viability in a drug-specific manner, potentially implicating genome conformational changes in acquired therapeutic resistance.

Citation Format: Russell O. Bainer, Catherine Wilson, Marinella Callow, Siyu Feng, Michael Costa, Colin Watanabe, Oleg Mayba, Eva Lin, Scott Martin, Bob Yauch, Richard Bourgon, Christiaan Klijn. Functional coding and noncoding drivers of EMT-mediated acquired drug resistance [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 398.
Abstract 4001: Gremlin-1 augments the estrogen-related receptor α signaling: Implications for progression of breast cancer in synergistic manner
Authors: Park Sin-Aye , Choi Bae-Jung , Kim Wonki , Surh Young-Joon
Published: August 2018

Gremlin-1 (GREM1), one of the bone morphogenic protein (BMP) antagonists, is known to be involved in organogenesis, tissue differentiation, and pathogenesis of several renal disorders including fibrosis, inflammation, and diabetes. Recently, it has also been reported that aberrant epithelial GREM1 expression initiates colonic tumorigenesis. However, the role of GREM1 in breast cancer remain largely elusive. We observed that GREM1 expression was highly increased in human breast cancer cells and tissues obtained from breast cancer patients. Furthermore, its overexpression is associated with poor prognosis of breast cancer patients, especially those with estrogen receptor (ER)-negative tumors. Suppression of GREM1 expression significantly inhibited the proliferation of SKBR3 human breast cancer cells in culture and xenograft mammary tumor growth while its overexpression enhanced their viability and invasiveness. Estrogen-related receptor α (ERRα) is an orphan nuclear hormone receptor and has recently attracted attention as a novel therapeutic target in the management of triple-negative breast cancers. We observed that ERRα binds directly to GREM1 promoter and modulates GREM1 expression. The mRNA level of GREM1 was reduced by an inverse agonist of ERRα, XCT790 and also by siRNA silencing of ERRα. Conversely, the overexpression of ERRα with its co-factor peroxisome proliferator-activated receptor gamma co-activator 1-α increased the mRNA level of GREM1. Surprisingly, the transcriptional activity of ERRα was found to be regulated by GREM1. The promoter activity of ERRα and the mRNA levels of ERRα target genes were highly increased in GREM1-overexpressing breast cancer cells. Treatment of MCF-7 cells with recombinant GREM1 protein also enhanced the ERRα promoter activity and transcription of its target genes. Taken together, our study identifies for the first time that the GREM1 can act as a downstream signaling molecule of the transcription factor ERRα and suggests a positive feedback loop between ERRα and GREM1, highlighting GREM1 as a potential diagnostic and therapeutic target for the treatment of ER-negative breast cancer.

Citation Format: Sin-Aye Park, Bae-Jung Choi, Wonki Kim, Young-Joon Surh. Gremlin-1 augments the estrogen-related receptor α signaling: Implications for progression of breast cancer in synergistic manner [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 4001.
Abstract 4018: Mutations of HER2 at L755 residue results in HER2 nuclear accumulation and enhances breast cancer stem cell activity
Authors: Wang Wen-Ling , Nie Lei , Huynh Kieu-Thanh , Chen Jhen-Yu , Yao Jing-Han , Hung Mien-Chie , Huang Wei-Chien
Published: August 2018

Background: HER2 plays a critical role in tumor progression and cancer stemness in breast cancer cells. Somatic mutations in HER2 gene have been observed in low HER2-expressing cancer cells and are correlated with insensitization to tyrosine kinase inhibitors. One of the most frequently altered residue is L755, which is located at kinase domain and near a putative nuclear export signal sequence, increasing kinase activity and is associated with lapatinib resistance. Moreover, the correlation between HER2 nuclear localization and drug-resistance and poor clinical outcome has been demonstrated in cancer patients. However, the roles of HER2 mutations in its nuclear distribution and its ability to enhance cancer stemness remain unknown.

Methods: We hypothesized that activation of HER2 mutations on amino acid L755 might affect HER2 cellular localization. To investigate the cellular localization of HER2 and the role of L755 mutations in expansion of cancer stem cells, T47D cell line clones expressing wile-type (WT) and mutant HER2 (L755P and L755S) were established. The cellular fractionation, immunofluorescence staining, and western blot analysis were employed to verify the cellular distribution of HER2 L755 mutations. Biochemical strategies were further performed to examine the interaction of HER2 L755 mutant and cellular transporters nuclear importers or exporters.

Results: Our results showed that ectopically expressed HER2 L755P and L755S mutants exhibited a higher nuclear retention via interrupting nuclear exporting complex formation. HER2 L755P and HER2 L755S showed higher abilities to associate with CRM1 and RanBP3 but reduced the binding capacity with Ran. The increases in tyrosine phosphorylation of CRM1 and RanBP3 were found by HER2 mutants and cause the defect in the formation of nuclear exporting complex. Additionally, the HER2 L755P and HER2 L755S-expressing cell lines showed higher levels of cancer stem cell markers, CD44 and ALDH1 and increased mammosphere formation.

Conclusion: HER2 L755P and L755S mutations enhance the HER2 nuclear localization and breast cancer stemness. This study provided new insight into the role of HER2 mutation in cancer stemness, and was helpful to develop novel prognostic markers for the therapeutic efficacy in breast cancer patients.

Citation Format: Wen-Ling Wang, Lei Nie, Kieu-Thanh Huynh, Jhen-Yu Chen, Jing-Han Yao, Mien-Chie Hung, Wei-Chien Huang. Mutations of HER2 at L755 residue results in HER2 nuclear accumulation and enhances breast cancer stem cell activity [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 4018.
Abstract 403: Analysis of the functional consequences of single nucleotide polymorphisms in CYP3A4 gene to prostate cancer in men of African ancestry
Authors: Rotimi Solomon O. , Ogo Chidiebere Ndukwe , Ogunlana Olubanke Olujoke , Chinedu Shalom Nwodo , Akinremi Titi , Fatiregun Omolara , Buraimoh Funlayo , Alabi Adewunmi , Tijani Kehinde , Salako Ayo , Omonisi Emmanuel , Agaba Ruth , Odedina Folakemi
Published: August 2018

Prostate cancer (CaP) is the leading cause of cancer death among Black men and its pathogenesis is linked to testosterone metabolism. CYP3A4 deactivates testosterone and the polymorphisms of its gene are known to alter testosterone metabolism. In this study, computational techniques were used to investigate the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) in the CYP3A4 gene in African Americans exome sequence. A case-control study was also carried out on 100 prostate cancer patients and 100 controls, among Yoruba men in Nigeria, to investigate the allelic variants of CYP3A4*1B (rs2740574 A>G) using polymerase chain reaction restriction fragment length polymorphism. A total of 22 nsSNPs were found in CYP3A4 gene of African American; out of which, 13 mutations (Q472R, M445T, K421R, R403C, I396T, L373F, R372I, I335T, T309I, V191A, D174H, R162W and S134C) were predicted to have deleterious effects on the CYP3A4 protein. The protein structural analysis of these amino acid variants revealed that M445T mutation could result in an increased free energy with a significant (p<0.05) loss of allosteric site at M445, altered metal binding, and loss of catalytic site at R446. T309I mutation which also resulted in increase free energy was found to be associated with a loss of catalytic site at E308. All the Nigerian samples were homozygous for the variant CYP3A4*1B allele. Hence, the presence of GG homologous variant in the Yoruba population of Nigeria, coupled with the computationally predicted deleterious mutations, could contribute to the high prevalence and aggression of CaP in black men. These genetic variants identified through in silico analysis can provide a better understanding of the wide range of disease susceptibility associated with this gene in Black men and aid the development of new molecular diagnostic markers for CaP particularly in men of West African ancestry.

Citation Format: Solomon O. Rotimi, Chidiebere Ndukwe Ogo, Olubanke Olujoke Ogunlana, Shalom Nwodo Chinedu, Titi Akinremi, Omolara Fatiregun, Funlayo Buraimoh, Adewunmi Alabi, Kehinde Tijani, Ayo Salako, Emmanuel Omonisi, Ruth Agaba, Folakemi Odedina. Analysis of the functional consequences of single nucleotide polymorphisms in CYP3A4 gene to prostate cancer in men of African ancestry [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 403.
Abstract 4032: Pharmacogenomic analysis of acquired resistance can reveal effective drug combination in glioblastoma
Authors: Li Xiaolong , Martinez-Ledesma Juan , Zhang Chen , Gao Feng , Yung W.K. Alfred , Koul Dimpy
Published: August 2018

Targeted cancer therapies have produced substantial clinical responses, but glioblastoma multiforme (GBM) has poor response due to developing resistance. The goal is thus to design strategies to overcome resistance to targeted drugs. To address this, we have embarked on a new type of screening for resistant targets, by developing acquired resistant glioma models and using pharmacogenomic platform to identify the resistant targets. We have established several patient-derived in vivo glioma stem cells (GSC) models using a PI3K specific inhibitor BKM-120 by a selective pressure after a chronic drug treatment. The resistant cells were subjected to RNA-seq analysis as well as pharmacologic analysis utilizing high-throughput kinase drug library screening. Analysis of integrated RNA-seq and kinase drug library screening of resistant cells identified multiple effective drug combination. Activation of Aurora kinase A (Aurora A)/Polo-like kinase 1 (Plk1)/cyclin-dependent kinase (CDK) signaling pathway was predominant in resistant GSCs. Concomitant targeting of PI3K together with Aurora A, Plk1 or CDK1 had a synergistic growth inhibitory effect. In addition, the combined inhibition of Plk1 and PI3K resulted in tumor growth suppression and significant delay of acquired resistance in vivo. These observations suggest that cell cycle pathway serves as a major survival signal upon PI3K inhibition and provides a rationale for the combined use of PI3K and cell cycle inhibition to prevent acquired resistance in human GBM.

Citation Format: Xiaolong Li, Juan Martinez-Ledesma, Chen Zhang, Feng Gao, W.K. Alfred Yung, Dimpy Koul. Pharmacogenomic analysis of acquired resistance can reveal effective drug combination in glioblastoma [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 4032.
Abstract 4038: A functional genomics approach to the identification of genes involved in resistance to Oxaliplatin in colorectal cancer
Authors: Invrea Federica , Cavicchioli Francesca , Petti Consalvo , Russo Rosalia , Isella Claudio , Medico Enzo
Published: August 2018

Currently, the first line therapy for metastatic colorectal cancer (CRC) is the FOLFOX regimen, which includes Oxaliplatin in combination with Fluorouracil and Leucovorin. Responses to FOLFOX range from overt regressions (35%) to full resistance (25%), and eventually all patients become resistant. Indeed, the molecular mechanisms of Oxaliplatin resistance remain mostly to be understood. To identify genes involved in sensitivity/resistance to Oxaliplatin, we designed an integrated approach including forward genetic screens and computational analyses. The pipeline includes: (i) stable transduction of CRC cells with pooled shRNA libraries; (ii) Oxaliplatin treatment for six weeks to select a drug-resistant subpopulation; (iii) identification of shRNA constructs enriched in the Oxaliplatin-resistant subpopulation by next-generation sequencing analysis. We focused the shRNA libraries targeting phosphatases and ubiquitin conjugation pathway genes. The first are involved in several biological processes, reversing the biochemical activity of kinases and typically acting as feedback loop regulators. The second direct proteins to degradation by the proteasome regulating homeostasis, cell cycle, and DNA repair pathways. HCT116 CRC cells, markedly sensitive to Oxaliplatin, were transduced in duplicate with both shRNA libraries. After Oxaliplatin treatment for six weeks, resistant populations emerged from the library-transduced cells, but not from control-transduced cells. To reveal shRNA constructs responsible of such phenotype, we extracted genomic DNA from transduced cells and, upon amplification of shRNA constructs by targeted PCR and next-generation sequencing, we compared the repertoire of shRNA sequences in HCT116 cells selected with Oxaliplatin with that of cells grown in standard medium. Screening hits were then prioritized for functional characterization, resulting in a phosphatase (CDC25C) and an ubiquitin ligase candidate (DCAF17). In conclusion, we successfully setup a functional genomic screening for acquired resistance to Oxaliplatin in CRC. Specific shRNA constructs from both the phosphatase and ubiquitin ligase libraries were enriched after Oxaliplatin treatment, highlighting candidate genes whose loss of function could potentially drive resistances to Oxaliplatin.

Citation Format: Federica Invrea, Francesca Cavicchioli, Consalvo Petti, Rosalia Russo, Claudio Isella, Enzo Medico. A functional genomics approach to the identification of genes involved in resistance to Oxaliplatin in colorectal 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 4038.
Abstract 4083: Intrahepatic cholangiocarcinoma and gallbladder carcinoma mouse model based on transplantation of syngeneic tumor-initiating cells
Authors: Kasuga Akiyoshi , Semba Takashi , Takaishi Hiromasa , Kanai Takanori , Saya Hideyuki , Arima Yoshimi
Published: August 2018

Biliary tract cancers (BTCs) are relatively rare in the Western world, but the rates of BTCs have risen worldwide in the past few decades. BTCs, which arise from epithelial cells in bile ducts, include intrahepatic and extrahepatic cholangiocarcinomas, gallbladder carcinomas, and ampullary carcinomas. The differences between these subtypes are not fully understood because of the limited number of genetically engineered mouse models for BTCs. Genetic mutations in the KRAS gene and the epigenetic changes in Ink4a/Arf genes have been frequently detected in BTCs, and it has been considered that those alterations are associated with the development and maintenance of human BTCs. To clarify the biological features of intrahepatic cholangiocarcinomas and gallbladder carcinomas, we developed syngeneic mouse models based on a combination of organoid 3D cultures and the transplantation of oncogene-expressed syngeneic bile duct epithelial cells. We isolated the epithelial cell adhesion molecule (EpCAM)-positive intrahepatic bile duct epithelial cells and the gallbladder epithelial cells from the Ink4a/Arf knockout C57BL/6 mice, and the cells were maintained as organoids under 3D culture conditions. Those cells were then infected with retroviruses expressing KRASG12V. Lethal metastatic adenocarcinomas having differentiated components formed in our mice when we transplanted the KRASG12V-expressing cells into the livers of wild-type C57BL/6 mice. The tumors resembled human desmoplastic intrahepatic cholangiocarcinomas and gallbladder carcinomas. Our mouse models can be useful for investigating pathogenesis and therapeutic strategies for human BTCs.

Citation Format: Akiyoshi Kasuga, Takashi Semba, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Yoshimi Arima. Intrahepatic cholangiocarcinoma and gallbladder carcinoma mouse model based on transplantation of syngeneic tumor-initiating 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 4083.
Abstract 4087: Investigating the novel role of Hippo signaling in mouse pancreas
Authors: Gao Ming , Liu Jun , Sharkey Francis E. , Crawford Howard C. , Johnson Randy L. , Chen Yidong , Wang Pei
Published: August 2018

Background: Large tumor suppressor kinase 1 and 2 are the core kinases of the Hippo signaling pathway, and play critical roles in regulating cell growth and organ size during animal development. Inactivation of the Hippo signaling pathway has been demonstrated to initiate tumor development in certain organs. However, the function of Lats1&2 in the pancreas is still elusive. Methods and results: To investigate the function of Lats1&2 in the pancreas, we generated mice with adult pancreatic acinar cell-specific deletion of Lats1&2 genes. Interestingly, instead of enlarging of the pancreas or pancreatic tumorigenesis, the deletion of Lats1&2 genes in pancreatic acinar cells resulted in severe inflammation of the pancreas. To further examine the mechanisms, we took advantage of a Rosa26 reporter to trace individual Lats1&2 null cells in vivo. We found that the loss of Lats1&2 did not affect cell growth directly but activated pancreatic stellate cells. This was followed by immune cell infiltration and acinar-to-ductal metaplasia in Lats1&2 null pancreas. Our RNA seq data revealed several cytokines and chemokine genes, such as ctgf, spp1, cxcl12, cxcl16, were gradually unregulated with time in Lats1&2 null pancreas before immune cell infiltration. Finally, we revealed the phenotype of Lats1&2 DKO mouse was in a YAP1/TAZ-dependent manner. Conclusion and significance: In our present study, our data suggested that deletion of the Lats1&2 genes in acinar cells caused pancreatitis through activation of pancreatic stellate cells directly. Our study discovered a new mechanism of the inflammatory response initiated by pancreatic epithelial cells and regulated by the Hippo signaling pathway, which is likely to be useful for the identification of new strategies for controlling pancreatic inflammation and prevention of pancreatic cancer.

Citation Format: Ming Gao, Jun Liu, Francis E. Sharkey, Howard C. Crawford, Randy L. Johnson, Yidong Chen, Pei Wang. Investigating the novel role of Hippo signaling in mouse pancreas [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 4087.
Abstract 4088: Metabolic disease drivers of liver cancer, a new disease paradigm
Authors: Briggs Michael R. , Deshpande Dipti , Siegmund Nikole
Published: August 2018

Much of the global burden of HCC until recently has been caused by alcohol abuse and hepatitis viruses (HepB & HepC). With a vaccine for HepB being widely used in Asian countries and multidrug cocktails to effectively cure much HepC, these drivers of HCC will be reduced in the future. Ironically, HCC continues to be one of the few cancers growing in incidence. It is postulated that the global obesity epidemic is fueling a new etiology of HCC, through fatty liver disease, nonalcoholic steatohepatitis (NASH) and cirrhosis. Liver and pancreatic cancers are projected to continue to increase in incidence for the foreseeable future, and distressingly, for the first time, children are being diagnosed with NASH. We have undertaken the development of a mouse model of NASH leading to HCC and performed a longitudinal analysis of liver samples through the phenotypes of obesity and hyperglycemia, inflammation and fibrosis leading to hyperproliferative disease and HCC to begin to map out the pathways and biomarkers of this metabolically driven liver cancer for comparison to human clinical data and biomarkers of disease progression to HCC.

Citation Format: Michael R. Briggs, Dipti Deshpande, Nikole Siegmund. Metabolic disease drivers of liver cancer, a new disease paradigm [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 4088.
Abstract 4090: Generation of a new mouse to model pancreatic cancer-induced cachexia
Authors: Talbert Erin E. , Ladner Katherine J. , Cuitino Maria Cecilia , Leone Gustavo W. , Guttridge Denis C.
Published: August 2018

Of all cancers, pancreatic cancer causes the third most fatalities, and incidence rates of this devastating disease continue to rise. Further, pancreatic cancer presents with the highest incidence of cachexia, a syndrome of weight loss due to depletion of skeletal muscle and adipose tissue. Clinically, cachexia is associated with increased mortality, poor quality of life, poor treatment responses, and increased treatment complications. Although clearly the best way to prevent cachexia is to address the cancer, pancreatic cancer has proven challenging to treat, with a dismal 9% 5-year survival rate. We believe effective anti-cachexia therapy will improve the care of pancreatic cancer patients, but the development of these therapies will require animal models that accurately recapitulate the etiology of pancreatic cancer-induced cachexia. To improve on the current, non-pancreas tumor xenograft models of cachexia, we generated a genetically engineered mouse model (GEMM) of pancreatic cancer, where induction of Kras and loss of the tumor suppressor Pten occurs in the pancreas of postnatal mice, which we refer to as the KPP model. KPP mice progressively lose skeletal muscle and adipose mass as a result of their cancer. We find that KPP mice exhibit histologic features of pancreatic ductal adenocarcinoma and reach endpoint criteria at an average of 107 days of age. Beginning at approximately 75 days of age, KPP mice undergo progressive loss of existing skeletal muscle mass, resulting in decreased muscle function. We also find that muscle loss from KPP mice exhibits a gene expression signature that closely aligns with the gene expression signature in muscle from cachectic pancreatic cancer patients. In summary, we expect KPP mice to serve as a useful model to elucidate the underlying mechanisms of muscle loss in pancreatic cancer-induced cachexia and to evaluate potential anti-cachexia therapies.

Citation Format: Erin E. Talbert, Katherine J. Ladner, Maria Cecilia Cuitino, Gustavo W. Leone, Denis C. Guttridge. Generation of a new mouse to model pancreatic cancer-induced cachexia [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 4090.
Abstract 4099: Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis
Authors: Regenbrecht Christian RA , Gambara Guido , Pachmayr Eva , Silvestri Alessandra , Dahlmann Mathias , Brzezicha Bernadette , Buettner Britta , Rau Beate , Keilholz Ulrich , Stein Urike , Walther Wolfgang
Published: August 2018

For patients with colorectal cancer (CRC) peritoneal metastases (PM) represent a terminal tumor stage with limited therapeutic options. To increase therapeutic efficacy and overall survival, availability of appropriate patient-derived 3D cell culture (PD3D) and patient-derived xenografts (PDX) models of PM could help improving the predictability of drug response for a specific tumor, but also foster the identification of novel biomarkers and therapeutic targets for CRC-PM patients. In this context, we generated the first scaffold-based PD3D and matching PDX models of CRC-PM as platform to test for chemotherapy response and to identify novel biomarkers.

For model establishment, surgical specimens were processed either for PD3D cell culture or transplanted subcutaneously (s.c.) onto immunocompromized NOD scid gamma (NSG) mice. For 3D cell culture models, tissue was dissected, digested and filtered before embedding into a scaffold matrix. For PDX models engrafted tumors were transferred to NMRI nu/nu mice for further passaging. They were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of both sibling models was evaluated on a panel of conventional chemotherapeutic and of targeted drugs.

The same panel of drugs was used in 15 matched PD3D/PDX models and revealed individual response patterns both in PD3D and PDX. Most interestingly, different drug response pattern was observed in models derived from tumor tissue of the omentum vs. tissue from the peritoneum of the same patient.

Our results demonstrate, that matched models maintain basic characteristics such as the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical platform for translational studies of potential clinical use.

Citation Format: Christian RA Regenbrecht, Guido Gambara, Eva Pachmayr, Alessandra Silvestri, Mathias Dahlmann, Bernadette Brzezicha, Britta Buettner, Beate Rau, Ulrich Keilholz, Urike Stein, Wolfgang Walther. Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis [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 4099.
Abstract 4109: Multi-modality imaging to interrogate lipidome changes during melanoma progression in zebrafish
Authors: Henderson Fiona , Johnston Hannah , Jones Emrys , Foster Duncan , Nagaraju Raghavendar T. , Green Michael , Fairclough Michael , Ramirez Irene Barinaga-Rementeria , He Shuning , Snaar-Jagalska B.Ewa , Hollywood Katherine A. , Dunn Warwick , Spaink Herman P. , Lorigan Paul , Claude Emmanuelle , Williams Kaye , Hurlstone Adam , McMahon Adam
Published: August 2018

Lipids play diverse roles in the body, including cell structure, cell signalling, and energy production; all of which are altered in cancer. Lipid metabolism is therefore a potential therapeutic target, and here has been investigated using a transgenic zebrafish model. This study combines transcriptome and lipidomic analyses for the first time, with in-vivo positron emission tomography (PET) and ex-vivo desorption electrospray ionisation-mass spectrometry (DESI-MS), to investigate lipid metabolism changes in melanoma. Most lipids are made of fatty acids, and the methods by which tumours acquire fatty acids can be altered compared to normal tissue. Here, the PET tracer [18F]-FTHA (a fatty acid analogue) has been used to demonstrate free fatty acid uptake in melanoma tumours in-vivo. Additionally, the excellent spatial resolution of mass spectrometry imaging has been utilised by imaging the FTHA ex-vivo by DESI-MS, allowing more precise spatial correlation with the histology, and revealing the heterogeneous nature of this fatty acid uptake. These imaging data complement the findings of the transcriptome analyses which show upregulation of genes associated with fatty acid uptake. Integrating transcriptome and lipidome data analyses revealed dysregulation of glycerophospholipid pathways, and was supported by DESI-MS data, which revealed heterogeneous changes in lipid metabolism.

Citation Format: Fiona Henderson, Hannah Johnston, Emrys Jones, Duncan Foster, Raghavendar T. Nagaraju, Michael Green, Michael Fairclough, Irene Barinaga-Rementeria Ramirez, Shuning He, B.Ewa Snaar-Jagalska, Katherine A. Hollywood, Warwick Dunn, Herman P. Spaink, Paul Lorigan, Emmanuelle Claude, Kaye Williams, Adam Hurlstone, Adam McMahon. Multi-modality imaging to interrogate lipidome changes during melanoma progression in zebrafish [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 4109.
Abstract 4137: Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma
Authors: Flynn Andrea T. , Liu Kangning , Vu Annette , Hogarty Michael D.
Published: August 2018

The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma.

Citation Format: Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma [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 4137.
Abstract 4146: N6Methyladenosine RNA demethylase ALKBH5 as a novel therapeutic target for osteosarcoma
Authors: Yadav Pooja , Subbarayalu Panneerdoss , Abdelfattah Nourhan , Eedunuri Vijay K. , Chen Yidong , Rao Manjeet K.
Published: August 2018

Osteosarcoma (OS) is the most prevalent primary bone malignancy that affects children and young adults. Despite several years of research, survival outcome of OS patients has not improved in last three decades. OS is treated with multi-modal chemotherapy, which is highly toxic and does not work well for metastatic and chemo-resistant tumors. Currently there is no FDA approved drug that can serve as an alternative to chemotherapy, warranting an urgent need to find more efficacious and targeted therapeutics for OS. Here, we report that RNA demethylase AlkB Homolog 5 (ALKBH5) may serve as a novel therapeutic adjuvant for treating OS. N6 Methyladenosine (m6 A) is the most common internal mRNA modification, which is modulated by the multi-component RNA methyltransferase complex, RNA demethylase (ALKBH5) and m6A readers . We show that ALKBH5 is amplified in sarcomas and its expression is highly elevated in osteosarcoma patients. We demonstrate that silencing of ALKBH5 inhibits the OS growth and migration without affecting the viability of normal human fetal osteoblast cells. Our results reveal that ALKBH5 depletion impairs the cell cycle progression and induces apoptosis in OS cells. Interestingly, we demonstrate that reduction in ALKBH5 levels suppresses the DNA damage repair capacity of osteosarcoma cells rendering them sensitive to DNA damaging agent like Doxorubicin. Using DR-GFP reporter-based homologous recombination (HR) assay, we show that ALKBH5 depletion leads to reduced HR-mediated DNA repair capacity of osteosarcoma cells. Supporting this, we observed significantly reduced expression of several genes that are known to play critical roles in cell cycle progression and DNA damage repair. In summary, this study shows that ALKBH5 is a critical regulator of OS growth and chemosensitivity. Approaches aimed at silencing ALKBH5 can be potentially used to inhibit osteosarcoma growth and progression as well as sensitize osteosarcoma cells to DNA damaging agents.

Citation Format: Pooja Yadav, Panneerdoss Subbarayalu, Nourhan Abdelfattah, Vijay K. Eedunuri, Yidong Chen, Manjeet K. Rao. N6Methyladenosine RNA demethylase ALKBH5 as a novel therapeutic target for 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 4146.