bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4489: Effect of cancer-associated fibroblasts on the progression of malignant melanoma cells
Authors: Fujikawa Heishiro , Yashiro Masakazu , Hatano Takaharu , Maeda Shusaku , Motomura Hisashi
Published: August 2018

Introduction

Malignant solid tumor is composed of not only cancer cells but also various types of stromal cells, which include fibroblasts, vascular endothelial cells, and immune cells. Currently, it has been reported that fibroblasts in tumor stroma, namely cancer-associated fibroblasts (CAFs), might affect cancer progression. Some studies suggested that CAFs have the stimulation-activity of the malignant potential of melanoma cells, but some reports mentioned their inhibitory-activity of melanoma cells. In this study, we investigated the effects of CAFs on the proliferation and invasion ability of malignant melanoma cells.

Material and Method

We used 2 malignant melanoma cell lines, MMAc and COLO679. Three CAFs were established from sebaceous carcinoma or skin malignant melanoma. Serum-free conditioned medium (CAF-CM) from CAF was prepared as follows. CAF were seeded into 100 mm plastic dishes with 10 ml of DMEM containing 2% FCS. To obtain CAF-CM, CAF were washed by PBS and then incubated for 3 days with serum-free DMEM. The conditioned medium was collected as CAF-CM. Serum-free DMEM was used as the control. CAFs were used before the 15th passage in culture. The effect of CAF-CM on the proliferation of the melanoma cells was determined by cytometry assay using Coulter Counter. The effect of CAF-CM on the motility of the melanoma cells was examined by Matrigel-invasion assay.

Result

CAF-CM significantly increased the proliferation of melanoma cell, in compared the control. Under the condition of adding 25% of CAF-CM, the number of MMAc cells were increased about 300% after 72 hours of incubation, and 105% after 120 hours of incubation that of COLO679 increased about 56% after 72 hours of incubation, 130% after 120 hours of incubation. CAF-CM significantly increased the invasion of MMAc cells. The invasion of MMAc cells were increased about 200% after 72 hours of incubation following the addiction of 25% CAF-CM. In contrast, CAF-CM did not affect on the invasion of COLO679 cells.

Conclusion

CAFs are associated with the proliferation and invasion of malignant melanoma cells.

Citation Format: Heishiro Fujikawa, Masakazu Yashiro, Takaharu Hatano, Shusaku Maeda, Hisashi Motomura. Effect of cancer-associated fibroblasts on the progression of malignant melanoma 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 4489.
Abstract 4505: Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells
Authors: Fostok Sabreen F. , Bazzoun Dana , Lelievre Sophie A. , El-Sibai Mirvat , Talhouk Rabih S.
Published: August 2018

Studies demonstrated loss of connexin 43 (Cx43) expression in breast cancer cells in-vitro and in-vivo. Our previous studies have shown that silencing Cx43 in HMT-3522 S1 cells, non-neoplastic human mammary epithelial cells (S1 cells), altered apical polarity and mitotic spindle orientation under 3-dimensional (3-D) culture conditions. The purpose of this study is to investigate the effects of Cx43 loss on proliferation and invasion of non-neoplastic breast epithelium. Cx43 shRNA-transfected and control S1 cells were cultured under 2-D and 3-D conditions. Cx43-knockdown S1 cells exhibited enhanced proliferation both in 2-D and 3-D throughout the culture period, as shown by the increased cell counts and acinar size, respectively. In addition, cell cycle entry was enhanced, as indicated by the increased percentages of cells in S and G2/M phases and the reduced percentage of cells in G0/G1 phase. Cx43-silenced S1 cells upregulated the expression of proliferation genes, c-Myc and cyclin D1, in 2-D and 3-D, but maintained β-catenin levels. Furthermore, immunoprecipitation studies showed that Cx43 associated with β-catenin and ZO-2 in 2-D and 3-D cultures of S1 cells, and co-localized with β-catenin in 3-D, suggesting assembly of gap junction complexes in S1 cells. The loss of Cx43 caused mis-localization of β-catenin in 3-D cultures of S1 cells, suggesting involvement of the Wnt/β-catenin pathway downstream of Cx43 signaling. β-Catenin primarily exhibited apico-lateral distribution in S1 cells and re-localized to basolateral membrane domains in Cx43 knockdown cells. Silencing Cx43 enhanced migration of S1 cells by 40%, as shown by time-lapse imaging. In addition, Cx43-silenced S1 cells exhibited loss of the characteristic spheroid morphology and acquired a migratory phenotype in acini. Matrigel-invasion and MMP-9 activity were also enhanced. Cx43-silenced S1 cells were capable of invading the matrigel by two-fold compared to control cells. This was concomitant with enhanced expression and activity levels of Rho GTPases (RhoA, Rac1 and Cdc42), suggesting that Cx43 regulates non-canonical Wnt signaling, as a mechanism to induce migration and invasion. We propose a role for Cx43 loss in triggering proliferation and invasion events in non-neoplastic breast epithelium downstream of disrupted Wnt signaling.

Citation Format: Sabreen F. Fostok, Dana Bazzoun, Sophie A. Lelievre, Mirvat El-Sibai, Rabih S. Talhouk. Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial 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 4505.
Abstract 4513: Notch inhibitors relieve growth suppression by TGF-β and promote growth and metastasis of triple-negative breast cancers
Authors: Saikumar Pothana , Singha Prajjal K. , Pandeswara Srilakshmi , Venkatachalam Manjeri A.
Published: August 2018

The paradoxical behavior of Notch1 and transforming growth factor-beta (TGF-β) is puzzling as they were shown to have both tumor-suppressive and tumor-promoting activities. We addressed the interaction between these two signaling pathways in cancer development and progression in a systematic way. Towards this goal, we utilized primary human mammary epithelial cells (HMEC) and various triple-negative breast cancer (TNBC) cells and examined the molecular crosstalk between TGF-β1 and Notch signaling pathways using biochemical, molecular and cell biology techniques. First, we observed lower expression of Notch1 in most of the TNBC cells compared to normal cells. Most of these TNBC cells also overexpressed TMEPAI (transmembrane prostate androgen induced), which we previously showed switches TGF-β from tumor-suppressive to a tumor-promoting role in breast cancer. Surprisingly, treatment of TNBC with Notch inhibitors increased their growth rate both in the absence and presence of TGF-β, while Notch inhibitors relieved normal cells from TGF-β mediated growth suppression. Both normal and TNBC cells showed increased growth in the presence of Notch inhibitors with decreased TGF-β signaling activity. In fact, normal cells that express very low levels of TMEPAI both at mRNA and protein levels, overexpressed TMEPAI in presence of various Notch inhibitors, suggesting that Notch1 negatively regulates TMEPAI expression. Correspondingly, knockdown of TMEPAI in TNBC cell lines showed increased expression of Notch1 and Jagged 1, suggesting a negative regulation of Notch1 by TMEPAI. In fact, TMEPAI, which binds to E3 ligases, promoted increased turnover of Notch1. In contrast to notch inhibitors, overexpression of active Notch1 in MDA-MB-231 cells inhibited tumor cell growth both in vitro and in vivo. Our results clearly suggest that a crosstalk between TGF-β and Notch1 signaling exists through TMEPAI. Importantly, these studies should be taken into consideration while contemplating the use of Notch inhibitors in triple-negative breast cancer therapy.

Citation Format: Pothana Saikumar, Prajjal K. Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam. Notch inhibitors relieve growth suppression by TGF-β and promote growth and metastasis of triple-negative breast cancers [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 4513.
Abstract 4527: Major pathologic response and biomarker predict survival in lung cancer patients receiving neoadjuvant chemotherapy
Authors: Pataer Apar , Shao Ruping , Correa Arlene M. , Wistuba Ignacio I. , Swisher Stephen G.
Published: August 2018

In a previous study, we determined that major pathologic response (MPR) as indicated by the percentage of residual viable tumor cells predicted overall survival (OS) in patients with non-small cell lung cancer (NSCLC) who received neoadjuvant chemotherapy. In this study, we assessed whether 2 gene and 5 protein biomarkers could predict MPR and OS in 98 NSCLC patients receiving neoadjuvant chemotherapy. We assessed whether gene mutation status or protein expression was associated with MPR or OS. We observed that KRAS mutation tended to be associated with OS (p = 0.06), but EGFR mutation was not associated with OS. We found that patients with high RAD51 expression levels had a poorer prognosis than did those with low RAD51 expression. We also observed that RAD51 expression was associated with MPR. MPR and RAD51 expression were associated with OS in univariate and multivariate analyses (p = 0.04 and p = 0.02, respectively). Combination of MPR with RAD51 is a significant predictor of prognosis in NSCLC patients who received neoadjuvant chemotherapy. No association of MPR or VEGFR2, EZH2, ERCC1, RAD51, or PKR expression with KRAS or EGFR mutation was found. We demonstrated that MPR or RAD51 expression was associated with OS in NSCLC patients receiving neoadjuvant chemotherapy. Prediction of a patient's prognosis could be improved by combined assessment of MPR and RAD51 expression.

Citation Format: Apar Pataer, Ruping Shao, Arlene M. Correa, Ignacio I. Wistuba, Stephen G. Swisher. Major pathologic response and biomarker predict survival in lung cancer patients receiving neoadjuvant chemotherapy [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 4527.
Abstract 4559: KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model
Authors: hao feng , he feng , ning mingxuan , bai zhaoshuai , ning jinying
Published: August 2018

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed on activated T cells. As a secondary costimulatory molecule, activated OX40 can increase effector T-cell survival, proliferation, and memory. Anti-OX40 monoclonal antibodies have shown anti-tumor efficacy. Recent preclinical studies further support the potential synergistic effects of agonist OX40 immunotherapy in combination with cancer vaccination or in sequential co-administration with PD1 antibody to revert PD1 resistance. Several anti-OX40 agonistic monoclonal antibodies are currently tested in early phase cancer clinical trials either in monotherapy or in combination with other immune modulators. KY-B602 is a novel humanized IgG1 anti-OX40 antibody under pre-clinical development. It was generated from hybridoma and humanized by CDR grafting and structure simulation. KY-B602 specifically binds to the extracellular domain of human OX40 with high affinity (KD = 5.1 nM, determined by Biacore). FACS study showed KY-B602 also binds to both human and cynomolgus monkey OX40 over-expressed on 293T cells (EC50 =4.6nM, EC50=6.1nM, respectively). Another, in HT1080 cell reporter assay, KY-B602 significantly increased IL-8 secretion through OX40 pathway. In addition, KY-B602 dose-dependently augmented IL-2 and IFN-gamma secretion from CD4+T cells, that were pre-stimulated with IL-2 and PHA. However, in FACS competition assay, KY-B602 did not affect OX40L binding to OX40, suggesting that KY-B602 binds to a different epitope with that of OX40L. Moreover, KY-B602 inhibits in vivo tumor growth in MC38 syngeneic model. In summary, the current data of KY-B602 support its potency for cancer therapy.

Citation Format: feng hao, feng he, mingxuan ning, zhaoshuai bai, jinying ning. KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model [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 4559.
Abstract 4576: Combination of a label independent CTC enrichment platform with fluorescence in situ hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients
Authors: Ntzifa Aliki , Zavridou Martha , Pollakis Pavlos , Bardi Georgia , Georgoulias Vasilis , Kotsakis Athanasios , Lianidou Evi
Published: August 2018

Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) develop resistance to first (erlotinib/ gefitinib) and second (afatinib) generation EGFR tyrosine kinase inhibitors (EGFR TKIs). Circulating tumor cells (CTCs) may be used as a potential biological material to monitor genetic alterations during the clinical course of the disease. We used the ParsortixTM (ANGLE) system, a label-independent microfluidic-based technology platform, to enrich CTCs from whole blood of NSCLC patients to evaluate the presence of HER-2 and c-MET amplification using fluorescence in situ hybridization (FISH). Materials and methods: Peripheral blood (PB) (15mL) from 42 patients with EGFRmt and resistant to 1st and 2nd generation EGFR TKIs NSCLC was obtained before, during and after treatment with osimertinib. CTCs were isolated using the ParsortixTM system and were further analyzed for HER-2 and c-MET amplification by FISH. Results: PB samples were obtained before (n=42), after one cycle (n=12) and at PD (n=7) during treatment with Osimertinib. One patient had HER-2 amplified CTCs whereas in total, 5 patients had marginally HER-2-positive CTCs; HER-2-positive CTCs were observed in 1/42 baseline sample but in none post-1st cycle and PD samples. Similarly, c-MET amplification was detected in 3/42 patients at baseline whereas in total 4 patients had marginally c-MET-positive CTCs. Treatment with Osimertinib resulted in a decrease of the number of c-MET amplified CTCs in these patients. In all c-MET and HER-2 amplified CTCs there was no HER-2 or c-MET amplification in the primary tumor. c-MET amplification in the primary tumor was detected only in one patient, however this patient had no detectable CTCs. Conclusions: Our preliminary results indicate that in selected patients with EGFRmt NSCLC, detection of HER-2 or c-MET amplified CTCs may be correlated with tumor resistance to EGFR TKIs. The ParsortixTM system can be successfully combined with downstream FISH analysis for the molecular characterization of CTCs in NSCLC patients. More samples are currently being analyzed in order to reveal the implication of HER-2 and c-MET amplification in resistance to therapy with first, second and third generation EGFR TKIs.

Citation Format: Aliki Ntzifa, Martha Zavridou, Pavlos Pollakis, Georgia Bardi, Vasilis Georgoulias, Athanasios Kotsakis, Evi Lianidou. Combination of a label independent CTC enrichment platform with fluorescence in situ hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients [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 4576.
Abstract 458: Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts
Authors: Tan May Leng , Parkinson Eric K. , Yap Lee Fah , Paterson Ian C.
Published: August 2018

Cancer-associated fibroblasts (CAFs) isolated from oral squamous cell carcinoma (OSCC) display an activated phenotype in that they express α-smooth muscle actin (αSMA) and are frequently prematurely senescent. OSCC cell lines can induce senescence in normal fibroblasts in vitro by a mechanism that involves TGF-β and reactive oxygen species. Another phenotype closely associated with senescence is autophagy, but the relationship between autophagy, activation and senescence in CAFs has not been examined in detail. We first examined autophagosome accumulation in normal human oral fibroblasts (NHOFs; n=3) and in a panel of CAFs (n=8) that exhibited different degrees of senescence. Autophagosome accumulation was observed in senescent CAFs, as shown by the increased expression of the LC3B-II protein and by the number of LC3B cytoplasmic puncta, which were quantified using immunofluorescence. The number of LC3B puncta was significantly greater (p<0.01) in senescent CAFs compared to NHOFs, and senescent CAFs contained a higher fraction of autophagic fibroblasts than NHOFs, as indicated by increased number of LC3B puncta per cell. Further, the number of LC3B puncta correlated positively with the degree of senescence, as demonstrated by senescence-associated β-galactosidase (SA β-Gal) activity. To investigate whether autophagy, activation and senescence develop simultaneously when fibroblasts are stimulated to undergo senescence, we examined the temporal induction of these phenotypes in NHOFs in response to TGF-β1. NHOFs were treated with TGF-β1 (4 ng/ml) for up to 10 days to induce autophagy, αSMA expression and SA β-Gal activity. The results showed that autophagic flux preceded fibroblast activation, which in turn was followed by senescence. In summary, our results demonstrate that CAFs from OSCC concomitantly display autophagic, activated and senescent phenotypes and that the induction of autophagy occurs before fibroblast activation and sensecence. Taken together, these data suggest that autophagy, activation and sensecence in fibroblasts may be part of a unified programme leading to the development of CAFs.

Citation Format: May Leng Tan, Eric K. Parkinson, Lee Fah Yap, Ian C. Paterson. Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts [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 458.
Abstract 4582: Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies
Authors: McGrath Lara , Maloney Elizabeth , Stetson Daniel , Lubinski Tristan J. , Kirchner Rory , Dougherty Brian , Barrett J. Carl
Published: August 2018

Introduction Understanding the tumor microenvironment is challenging in situations where a tumor biopsy is unavailable. In these situations, liquid biopsies provide a less invasive alternative that can be collected at multiple timepoints throughout treatment. In particular, analyses of circulating free DNA (cfDNA) from plasma samples can distinguish genomic alterations from shed tumor DNA (ctDNA) within the cfDNA population. In this study, we aim to better understand the origin of cfDNA as well as its potential in understanding the intratumoral immune infiltration by developing and validating a strategy for identifying T cell receptor β (TCR β) rearrangements in the cfDNA from patients with solid malignancies. Methods We devised a target enrichment approach for high-throughput DNA sequencing that employs a hybridization capture strategy to bait germline variable (V) and joining (J) gene sequences in the TCR β loci. The target sequences are proximal to rearrangement sites flanking the CDR3, the principal site of contact for recognition specificity with foreign antigens presented by the major histocompatibility complex (MHC). We controlled for amplification bias by tagging DNA fragments with unique molecular indices (UMI). We validated our approach on dilutions of T cell lines in non-T cell lines, compared T cell levels determined from our method to those derived from flow cytometry, and compared data complexity at DNA input levels from 100 ng to 2 ng. Finally, we analyzed 27 pre-treatment plasma samples from cancer patients with solid malignanices, including quantification of total cfDNA concentrations, analyzing somatic genomic alterations using whole exome sequencing (WES), and employing our method for capture of TCR β rearrangements. Results Results indicate we are able to capture T cell rearrangements from cfDNA in cancer patient plasma. T cell rearrangements were identified in more than 25% of samples (7/27). Interestingly, the total cfDNA levels were not correlated to the number of T cell rearrangements detected, but all samples with detectable somatic alterations in the cfDNA (3/27) were also those with T cells identified. These preliminary results suggest a connection between apoptosis of lymphocytes and solid tumors that shed DNA into the bloodstream, and demonstrate potential utility as liquid biopsy for identifying tumor infiltrating lymphocytes.

Citation Format: Lara McGrath, Elizabeth Maloney, Daniel Stetson, Tristan J. Lubinski, Rory Kirchner, Brian Dougherty, J. Carl Barrett. Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies [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 4582.
Abstract 4596: Early noninvasive prediction of response to targeted therapy in non-small cell lung cancer
Authors: Phallen Jillian A. , Leal Alessandro , Woodward Brian D. , Forde Patrick M. , Naidoo Jarushka , Marrone Kristen , Brahmer Julie , Fiksel Jacob , Palsgrove Doreen N. , Cristiano Stephen , Bruhm Daniel , Weihe Elizabeth , Adleff Vilmos , Keshavarzian Parissa , Anagnostou Valsamo , Scharpf Robert B. , Velculescu Victor E. , Husain Hatim
Published: August 2018

There is an unmet need for improved methods of rapidly identifying responses to targeted therapies. Liquid biopsy approaches have potential as early biomarkers of response based on noninvasive, real-time monitoring of disease burden. We have used the ultrasensitive targeted error correction sequencing (TEC-Seq) approach to analyze 58 cancer driver genes in patients with metastatic non-small cell lung cancer undergoing treatment with targeted tyrosine kinase inhibitors. As a proof-of-principle study, liquid biopsies were obtained from sixteen patients immediately prior to treatment, 6-22 days after treatment, and at serial timepoints until disease progression. Tumor derived alterations and copy number changes were directly detected in plasma and tracked over timepoints analyzed. Based on the dynamics of cell-free circulating tumor DNA (ctDNA) mutations identified, we developed a noninvasive measure of cell-free clonal tumor load (cfTL) to evaluate real-time response to treatment. These analyses revealed that patients with a radiographic response to therapy had a significant drop in cfTL from an average mutant allele fraction of 3.59% to 0.13% within 6-22 days (P < 0.05) as well as in the number of detectable mutations and aneuploidy scores, while radiographic non-responders had limited to no changes. Analyses of residual cfTL 6-22 days after treatment stratified patients into ctDNA responders and ctDNA non-responders. ctDNA responders had improved progression-free survival (12.4 vs 1.7 months, P < 0.001), which was detected on average 38 days earlier and was as predictive as CT imaging. These analyses provide an approach for rapid evaluation of response to targeted therapies and have important clinical implications for the management of cancer patients.

Citation Format: Jillian A. Phallen, Alessandro Leal, Brian D. Woodward, Patrick M. Forde, Jarushka Naidoo, Kristen Marrone, Julie Brahmer, Jacob Fiksel, Doreen N. Palsgrove, Stephen Cristiano, Daniel Bruhm, Elizabeth Weihe, Vilmos Adleff, Parissa Keshavarzian, Valsamo Anagnostou, Robert B. Scharpf, Victor E. Velculescu, Hatim Husain. Early noninvasive prediction of response to targeted therapy in non-small cell 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 4596.
Abstract 4604: Long noncoding RNA Linc00162, as a novel prognostic marker, modulates cancer stem cell properties in triple-negative breast cancer
Authors: Sun Lichao , Lin ChangChing , Burnett Joseph , Yuan Hebao , Sun Duxin
Published: August 2018

Trastuzumab is an effective treatment for HER2+ breast cancer. However, trastuzumab resistance in HER2+ breast cancer may induce the epithelial-to-mesenchymal transition (EMT), transforming HER2+ to triple-negative breast cancer with cancer stem cell properties in some cancer cell populations. Long noncoding RNAs (lncRNAs) have been reported to be upregulated in many types of cancer tissues, and might play significant regulatory roles in cancer progression. However, the mechanisms of lncRNA in trastuzumab resistance still remain largely unknown. Here, we systematically analyzed the levels of LincRNA between BT474 (HER2+trastuzumab-sensitive) and the PTEN-deficient trastuzumab-resistant derivative (BT474-PTEN-LTT) by using RNA-sequencing, and identified that Linc00162 was significantly overexpressed in BT474 PTEN- LTT cells. In vitro functional assay indicated that downregulation of Linc00162 in BT474 PTEN-LTT cells completely inhibited the cell proliferation. To further evaluate the role of Linc00162 in BCSC self-renewal, we found that inhibition of Linc00162 led to a significant reduction in the number and the size of LTT cells sphere by using the mammosphere formation assay. Next, we examined Linc00162 expression levels in breast cancer tissues, and identified that Linc00162 was markedly increased. Clinicopathologic relevance analysis showed that Linc00162 expression was correlated with advanced clinical stage, lymph node metastasis and the depth of invasion. Furthermore, upregulation of Linc00162 was correlated with TRNB phenotype. In Kaplan-Meier survival analysis, high levels of Linc00162 was significantly associated with unfavorable survival in breast cancer patients. These findings suggest that Linc00162 can be used as a clinically relevant novel biomarker in breast cancer and may serve as a target for future drug development.

Citation Format: Lichao Sun, ChangChing Lin, Joseph Burnett, Hebao Yuan, Duxin Sun. Long noncoding RNA Linc00162, as a novel prognostic marker, modulates cancer stem cell properties in triple-negative 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 4604.
Abstract 4607: G1/S transition-related gene mutations associated with survival of hepatocellular carcinoma patients
Authors: Zhang Bingyuan , Song Tianqiang , Yang Pingzhou , Yang Songzhu , Guo Chao , Li Lei , Ren Shuang , Yan Maolin , Hu Weiyu , Guo Honglin , Zhang Yongjian , Li Yanlin , Wang Weifeng , Wang Kai , Yao Ming
Published: August 2018

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies in China and has limited therapeutic options. Genomic alterations in HCC had been widely explored and some prognostic genomic alterations were identified. Different etiologic characteristics were discovered in Chinese HCC patients compared with Western population; however, the genomic profiles of Chinese patients had not been thoroughly elucidated.

Methods: FFPE tumor samples of 94 Chinese HCC patients, including 79 males and 15 females with a median age of 56, were collected for next-generation sequencing (NGS)-based 450 genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, and gene rearrangement and fusions were assessed. Gene Ontology (GO) analysis was conducted with the genes whose variation rates were higher than 5% (N = 22). The TCGA HCC dataset with 198 patients was used to assess the prognostic value of the significantly enriched GO terms.

Results: The top 10 highly mutant genes in the 94 Chinese HCC cohort were TP53 (64.9%), TERT (31.9%), CTNNB1 (23.4%), AXIN1 (16.0%), STK24 (12.8%), RB1 (11.7%), CCND1 (9.6%), ARID1A (9.6%), LRPIB (8.5%) and ARID2 (8.5%). The Gene Ontology analysis revealed these gene alterations were significantly associated with pivotal signal pathways and cell proliferation including phosphatidylinositol phosphorylation, protein kinase B signaling, cell cycle arrest, and G1/S transition of mitotic cell cycle. The G1/S transition-related genes included TP53, TERT, RB1, CCND1 and CDKN2A. Moreover, the TCGA HCC patients with mutations of G1/S phase transition and mitotic cell cycle-related genes had a poor survival (p = 0.029).

Conclusions: Our study showed that Chinese HCC patients had different genomic alteration profile compared with Western population. The most frequent mutant gene in Chinese HCC was TP53 in 65% of the HCC patients, compared to 31% in the TCGA dataset (PMID:28622513). Genomic alterations in G1/S transition of mitotic cell cycle-related genes in Chinese HCC patients might be a negative prognostic factor. Our preliminary result suggested that functional analysis of HCC genomic alteration profile could have prognostic utility.

Citation Format: Bingyuan Zhang, Tianqiang Song, Pingzhou Yang, Songzhu Yang, Chao Guo, Lei Li, Shuang Ren, Maolin Yan, Weiyu Hu, Honglin Guo, Yongjian Zhang, Yanlin Li, Weifeng Wang, Kai Wang, Ming Yao. G1/S transition-related gene mutations associated with survival of hepatocellular carcinoma patients [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 4607.
Abstract 461: An essential role of the iron-sulfur cluster assembly enzyme isu-1 in the aging and stress responses of C. elegans
Authors: Xiao Rui , Sheng Yi
Published: August 2018

Iron is essential for animal cellular homeostasis by acting in the biogenesis of two important redox-reactive prosthetic groups of enzymes: iron sulfur clusters (ISC) and heme. Previous studies have indicated that the misregulated iron metabolism is associated with multiple aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, how iron metabolism modulates the aging process remains largely elusive.In the current study, we aim to reveal the roles and mechanisms of iron metabolism in the aging process of C. elegans, a well-established genetic model organism for aging research. Through our preliminary studies, we have found that the iron-sulfur cluster assembly gene isu-1 plays an important role in the lifespan modulation and stress resistance of C. elegans. Specifically, RNAi knocking-down of isu-1 causes a significantly upregulated mitochondrial unfolded protein response (mitoUPR). Furthermore, the isu-1 RNAi-treated worms are significantly more resistant to heat shock and oxidative stress. Lastly, isu-1 RNAi significantly extends lifespan (~25.7%).Currently, we are examining the transcription factors downstream of isu-1 in regulating aging and stress responses. Surprisingly, the isu-1 RNAi-triggered lifespan extension is independent of insulin and IGF pathway, an evolutionarily conserved master regulator of animal growth and aging. Next, we will characterize the mechanisms underlying the effects of iron-sulfur cluster in animal aging and stress response using various genetic and biochemistry approaches.

Citation Format: Rui Xiao, Yi Sheng. An essential role of the iron-sulfur cluster assembly enzyme isu-1 in the aging and stress responses of C. elegans [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 461.
Abstract 4616: Cancer-associated MUC1 epitope-recognizing antibody as a novel immunohistochemical marker for breast carcinoma
Authors: Shimizu Ai , Hatanaka Kanako , Hatanaka Yutaka , Naruchi Kentaro , Sato Masaharu , Kase Hiroshi , Mitsuhashi Tomoko , Yamashita Hiroko , Matsuno Yoshihiro
Published: August 2018

Background: MUC1 has been used in clinical practice as a serum tumor marker (CA15-3) for monitoring recurrence and response to the treatment of breast cancers. However, it is not so practical to use the conventional anti-MUC1 antibody (C-Ab) for the differential diagnoses among malignant and benign tumors, because of its limited specificity to cancer-associated sugar chain structures. Recently, a novel epitope-defined (ED) antibody, which specifically recognizes MUC1 with cancer-associated carbohydrate antigens including Tn and sialyl-T antigens, was developed by our collaborators. In the present work, we focused on this Tn antigen-recognizing ED antibody (Tn-ED Ab) for MUC1, and examined the potential utility of this novel antibody as an immunohistochemical diagnostic marker of the breast.Design: Tissue microarray (TMA) consisting of invasive lesions and their non-neoplastic counterparts were constructed from 169 lesions of 167 individuals with invasive breast carcinomas (including 2 bilateral cases) resected and diagnosed at Hokkaido University Hospital from 2000 to 2003. We compared MUC1 immunoreactivity of Tn-ED Ab (clone SN-102) to commercially available C-Ab (clone Ma552) using TMA to evaluate its diagnostic performance and to analyze the association with clinicopathologic factors. Results: In invasive cancer parts, the immunoreactive positivity of Tn-ED Ab was observed predominantly in the cytoplasm (146/169) rather than in the cell membrane (5/169). The positive reaction of C-Ab was found in the cytoplasm and the membrane as well (156/169 and 93/169). Tn-ED Ab showed a lower expression rate in non-neoplastic parts and a high specificity in invasive cancer parts. Sensitivity/specificity in invasive parts was 86%/92% for Tn-ED Ab and 92%/47% for C-Ab. Moreover, the invasive cancer group with Tn-ED-positivity was significantly correlated with their molecular subtypes, low Ki-67 indices, and high ages, compared to those with Tn-ED-negativity (p=0.008, p=0.010 and p=0.027, respectively). Conclusion: Compared to C-Ab, the newly-developed Tn-ED Ab for MUC 1 showed a higher specificity for invasive breast cancer with a low reactivity to non-neoplastic components. Future study plans are ongoing to show that this cancer-specific Tn-ED MUC1 Ab could be useful for differentiating malignant from benign intraductal breast lesions as well.

Citation Format: Ai Shimizu, Kanako Hatanaka, Yutaka Hatanaka, Kentaro Naruchi, Masaharu Sato, Hiroshi Kase, Tomoko Mitsuhashi, Hiroko Yamashita, Yoshihiro Matsuno. Cancer-associated MUC1 epitope-recognizing antibody as a novel immunohistochemical marker for breast carcinoma [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 4616.
Abstract 4617: High expression of snail is associated with EZH2 expression and lymph node metastasis in large neuroendocrine lung cancer
Authors: Fang Yueh-Fu , Lee Min-Chia
Published: August 2018

Background Large neuroendocrine lung cancer was less than adenocarcinoma or squamous cell carcinoma of lung. EZH2 expression was high in small cell lung cancer with component of neuroendocrine. There was no study of EZH2 in large neuroendocrine lung cancer. We planned to identify the expression of EZH2 and EMT markers and the clinical outcome in large neuroendocrine lung cancer. Methods and Patient There were 25 resectable large neuroendocrine lung cancer patients in our hospital before 2014. Twenty-two patients' tissue was done for EZH2 and EMT markers. The EZH2-GSK3 and EMT signaling were performed in H1299 and H460 cancer cell lines. EZH2 inhibitor, Akt inhibitor and protease inhibitor was used to validate the cell signaling from EZH2 to EMT markers. Results There were 10 patients had high EZH2 and snail and 10 patients had low EZH2 and snail. The correlation between EZH2 and snail was very high. Snail expression showed high prevalence of lymph node metastasis but no EZH2. The overall survival and progression free survival showed no definite in EZH2 and other insomnia. The recurrence was also not significant difference. The cellular study showed the same result. Conclusion Over expression of snail showed high correlation with EZH2 and was a predictable marker.

Citation Format: Yueh-Fu Fang, Min-Chia Lee. High expression of snail is associated with EZH2 expression and lymph node metastasis in large neuroendocrine 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 4617.
Abstract 463: Glucose-regulated protein 94 modulates the response of osteosarcoma to chemotherapy
Authors: Chien-Yu Huang , Wei Po-Li , Chang Yu-Jia
Published: August 2018

Background: Osteosarcoma (OS) is the most common and most aggressive primary solid malignant bone tumor in children and young adults and has high rates of recurrence and metastasis. The endoplasmic reticulum (ER) stress pathway is important in regulating chemo-responsiveness in cancer. However, the role of glucose-regulated protein 94 (GRP94) in regulating the chemotherapy response of OS has never been explored.

Method: In this study, GRP94-knockdown (GRP94-KD) MG63 OS cells were generated using short hairpin RNAs, and the response to chemotherapy was assessed by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Results: GRP94-KD cells were shown to be more resistant to chemotherapy than scrambled control cells, and the scrambled control cells had higher levels of apoptosis after chemotherapy than GRP94-KD cells.

Conclusion: Therefore, silencing GRP94 may increase resistance to chemotherapy treatment in OS cells by inhibiting the activation of apoptosis. Thus, GRP94 may be a key biomarker for the chemotherapeutic response of OS.

Citation Format: Huang Chien-Yu, Po-Li Wei, Yu-Jia Chang. Glucose-regulated protein 94 modulates the response of osteosarcoma to chemotherapy [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 463.
Abstract 4644: Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development
Authors: Dijkmans Thomas , Basten Sander , Herpers Bram , Yan Kuan , Giesemann Torsten , Schueler Julia , Vader Willemijn , Price Leo
Published: August 2018

Ex vivo cultures of patient-derived tumor enable functional testing of treatment options and optimization of pre-clinical drug development. Patient biopsies or tumor from patient-derived xenografts (PDX) mouse models are a valuable source of human tumor material that can be cultured in 3D for screening of drug efficacy, drug resistance and cellular processes such as proliferation, survival and invasion. A high throughput approach using 384 well plates enables evaluation of multiple drug treatments and dose ranges in parallel. After compound exposure, cultures are fixed and stained with cellular markers. 3D image stack acquisition is followed by ultra-high content multiparametric analysis using the OMiner platform to profile drug responses and quantify tumor spheroid volume, apoptosis and tumor invasion.

Dissected PDX material and tumor biopsy material, were used to establish 3D tumor cultures derived from various indications, including breast, ovarian, cervix, endometrium, stomach, pancreatic, colon, bladder and lung cancer. These were exposed to standard-of-care chemotherapeutics (e.g. 5-FU, taxanes, platinum compounds, anthracyclines, alkylating agents), small molecules (e.g. erlotinib, lapatinib, trametinib), targeted therapies (PARPi; niraparib, olaparib, rucaparib), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. The tumor culture response was measured, generating dose-dependent profiles for relevant features.

Patient-derived 3D tumor cultures were tested with standard-of-care and novel therapeutic agents and high content analysis was used to evaluate drug sensitivity. This method enables both the in vitro selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Correlation of drug sensitivity in 3D cultures from fresh patient tumor biopsies, on the other hand, can be used for development of predictive diagnostics and also provides a unique source of patient material for drug discovery and development.

Citation Format: Thomas Dijkmans, Sander Basten, Bram Herpers, Kuan Yan, Torsten Giesemann, Julia Schueler, Willemijn Vader, Leo Price. Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development [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 4644.
Abstract 4647: Identifying drug targets in sarcoma using machine learning and cell phenotype-based compound screening
Authors: Lachacz Eric J. , Wu Zhi Fen , Bixby John L. , Lemmon Vance P. , Merajver Sofia D. , Al-Ali Hassan , Soellner Matthew B.
Published: August 2018

High-throughput phenotypic screens that incorporate compound biochemical activity annotations are positioned for novel target discovery in cancer. We used machine learning approaches to correlate kinome-wide profiling data of a collection of kinase inhibitors with phenotypic cell proliferation data for the same compounds. We assembled a library of profiled kinase inhibitors with diverse chemotypes and kinome-wide selectivities and determined their anti-proliferative activities in a panel of sarcoma cell lines. Using a previously published machine learning algorithm, we related the compound inhibition profiles across 237 kinases to their abilities to inhibit cell proliferation. This identified Protein Kinase D (PRKD) as a putative novel target kinase selectively in synovial sarcoma cell lines, such that its inhibition leads to a decrease in proliferation in these cells. Next, we reasoned that our approach could be leveraged to identify targets that, when co-inhibited, induce a synergistic phenotype. This would enable rational design of drug combinations for further testing as opposed to the labor-intensive, random, pairwise testing commonly performed. To identify targets synergistic with PRKD, we performed a screen of a synovial sarcoma cell line in the presence of a selective PRKD inhibitor. Several kinases became prioritized as new targets in this “synergy screen.” Combining selective inhibitors for each synergistic target, as defined by Chou-Talalay, (here, CDK and AKT) with PRKD inhibitors synergistically reduced synovial sarcoma cell proliferation. Conversely, combining PRKD inhibitors with a selective inhibitor of a kinase that was deprioritized in the synergy screen (p38 MAPK) resulted in non-synergistic or even antagonistic effects. Overall, our approach provides a promising framework to identify new targets of cancer subtypes and a novel methodology to identify new combinational strategies for treatment.

Citation Format: Eric J. Lachacz, Zhi Fen Wu, John L. Bixby, Vance P. Lemmon, Sofia D. Merajver, Hassan Al-Ali, Matthew B. Soellner. Identifying drug targets in sarcoma using machine learning and cell phenotype-based compound screening [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 4647.
Abstract 4653: Intracellular Delivery of SOCS3 Suppresses Pancreatic Cancer Progression
Authors: Lee Kuysook , Shin Seulmee , Hwang Yunseo , Choi Youngsil , Jo Daewoong
Published: August 2018

Background: JAK/STAT signaling, regulated by SOCS family proteins, plays a role in the progression of the inflammation, growth and metastasis of pancreatic adenocarcinoma. SOCS3 suppresses JAK kinase activity and degrades the activated cytokine receptor complex, and its loss contributes to pancreatic cancer development. We have developed advanced macromolecule transduction domains (aMTDs) to deliver recombinant proteins into to cells and tissues. In principle, aMTD enabled SOCS3 could provide an effective treatment for pancreatic tumors that depends on JAK/STAT signaling for growth or survival.

Objective: The present study utilized aMTD sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against pancreatic cancer.

Methods: Improved cell-permeable SOCS3 proteins, with and without a 12-amino acid aMTD sequence, were expressed at high yields in E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers.

Results: The iCP-SOCS3 exhibited high solubility, cell-/tissue-permeability and biological activity in pancreatic cancer cell lines (BxPC3, HPAC and PANC-1) with decreased endogenous SOCS3 levels, hyperactived JAK/STAT signaling and hypermethylated SOCS3 promoter region. The iCP-SOCS3 suppressed cancer-associated phenotypes (i.e., up to 90% inhibition of cell viability, 52% induction of apoptosis, 14.8% inhibition of cell cycle progression, 54% inhibition of migration and 61.5% inhibition of invasion) in pancreatic cancer cells dependent on elevated JAK/STAT signaling, whereas, these effects were not observed in non-cancer cells (H6c7, pancreatic duct epithelial cell) or with proteins lacking the aMTD sequence. In contrast, gemcitabine, a conventional pancreatic cancer therapeutic, showed undesirable cytotoxic effects including decreased cell viability and induced apoptosis in non-cancer cells. Furthermore, iCP-SOCS3 (30 mg/kg) induced tumor regression (-168% at day 18) in PANC-1 CDX model. In BxPC-3 orthotopic CDX model, iCP-SOCS3 suppressed tumor growth by 98% which was significantly higher compared to the suppressed tumor growth induced by gemcitabine (77%). Correlating to the tumor suppression, iCP-SOCS3 significantly decreased the secretion of TNF-α and the expression of p-STAT1, p-STAT3 and VEGF in tumor tissues.

Conclusion: These results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide a novel protein therapy against pancreatic cancer.

Citation Format: Kuysook Lee, Seulmee Shin, Yunseo Hwang, Youngsil Choi, Daewoong Jo. Intracellular Delivery of SOCS3 Suppresses Pancreatic Cancer 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 4653.