bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-222: Culturing cancer cells from liquid biopsies for precision cancer therapy
Authors: Liu Xuefeng
Published: August 2018

Liquid biopsies include circulation tumor cells (CTC), ctDNA/RNA, exosomes from blood or tumor relevant information from other liquid samples (urine, pleural effusion, ascites, saliva, etc.). Culturing cancer cells from liquid biopsies is a simple, non-invasive technique that has the potential to advance the diagnosis, treatment and monitoring of cancer patients. In this study, we described several approaches that allow to enrich cancer cells from urine (for bladder cancer), blood (for prostate cancer), and pleural effusion (for non-small cell lung carcinoma). These cancer cells from liquid biopsies were cultured and further propagated in suspension and/or adherent cultures using a specialized cell culture medium. We also confirmed genetic alterations in both original tumors and cancer cells from liquid biopsies, for example, mutant EGFR for lung cancer, mutations of hTERT promoter for bladder cancer. We also reported that these cancer cells from liquid biopsies were used for identify therapies for the patients. For example, a woman patient with metastatic NSCLC harboring mutant EGFR was primarily resistant to TKI treatment, we were able to identify a combination of chemotherapy for the patient. Cancer cells from urine samples were used for predicting recurrence of the bladder cancer or monitoring treatment. We also used these cultured cancer from original tumors and liquid biopsies for study of tumor heterogeneity.

Citation Format: Xuefeng Liu. Culturing cancer cells from liquid biopsies for precision cancer therapy [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 LB-222.
Abstract LB-246: TIGAR amplification promotes glutamine addition in the progression of esophageal squamous cell carcinoma
Authors: Chu Jiahui , Shao Mingming , Chang Jiang , Cui Qionghua , Peng Linna , Luo Yingying , Tan Wen , Wu Chen , Lin Dongxin
Published: August 2018

BACKGROUND: Our previous whole-genome sequencing in esophageal squamous-cell carcinoma (ESCC) has identified 23 focal regions with recurrent gain of copy number that contain 1591 genes. Further integrative analysis of copy number alterations with mRNA expression levels showed 141 copy-number amplified genes that had higher mRNA levels, suggesting an important role of these genes in ESCC. This study was to investigate the functional roles of these genes and their potential as biomarkers and therapeutic targets for ESCC.

METHODS: An siRNA library was used to knock down all these 141 genes in ESCC cells and change of the malignant phenotype of these cancer cells was examined by using a high-throughput imaging method and in vitro proliferation and colony formation assays. The acting mechanism of the interest genes on malignant phenotypes of cells was explored using biochemical and molecular biological assays. We also examined the effect of the amplified genes on sensitivity of cancer cells to anticancer agents in vitro and in vivo in mice.

RESULTS: We found that, among the 141 examined genes, TIGAR was the top one influencing ESCC cell proliferation in vitro and in vivo in xenografts. We confirmed that TIGAR can inhibit glycolysis and divert the carbon flux into the PPP to produce ribose-5-phosphate (R5P) for nuclear synthesis in cells. Overexpression of TIGAR may activate AMPKα that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain energy balance in cells with a reduced glycolysis ability, which was responsible for energy production under normal conditions. We found that ESCC cells overexpressing TIGAR had an addiction to glutamine metabolism and were sensitive to glutaminase inhibitors. Glutamine restriction or glutaminase inhibitors had a synergistic effect with chemotherapy drugs, such as DDP and 5-Fu, on killing of ESCC cells, suggesting that glutaminase inhibitors may be used as adjuvant agents for treatment of ESCC with TIGAR amplification.

CONCLUSION: These results suggest that amplification or overexpression of TIGAR may prevent ESCC cells from killing by anticancer drugs and thus may represent a therapeutic target for ESCC.

Citation Format: Jiahui Chu, Mingming Shao, Jiang Chang, Qionghua Cui, Linna Peng, Yingying Luo, Wen Tan, Chen Wu, Dongxin Lin. TIGAR amplification promotes glutamine addition in the progression of esophageal squamous cell 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 LB-246.
Abstract LB-250: Oncogenic activation of mitochondrial phosphocreatine energy shuttle creates a druggable metabolic liability in cancer cells
Authors: Hitosugi Taro , Kurmi Kiran
Published: August 2018

Many important metabolic processes such as energy homeostasis, regulation of reactive oxygen species (ROS) levels, production of signaling metabolites, and synthesis of precursors for macromolecules occur in mitochondria of cancer cells. However, targeting mitochondrial metabolism in cancer has been challenging due to toxicity in normal cells, which also depend on mitochondrial metabolism. Therefore, identification of tumor specific mitochondrial metabolic alterations would be crucial to develop drugs that can selectively target cancer cells while sparing normal tissue.

Here, by using targeted metabolite profiling and mitochondria-specific phospho-proteomics approach, we identified Mitochondrial Creatine Kinase (MtCK) 1 as a metabolic target in HER2+ breast cancer cells. We show that the oncogenic HER2-ABL axis stabilizes MtCK1 through tyrosine 153 (Y153) phosphorylation to activate the PCr energy shuttle pathway that transfers ATP from mitochondria to cytosol. Inhibition of the PCr energy shuttle pathway either by genetically depleting MtCK1 or by pharmacologically with a creatine analogue cyclocreatine, decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cancer cells and reduces tumor growth in vivo without obvious toxicity. These findings suggest that, once ATP is produced in the mitochondria, it is equally important for breast cancer cells to transfer this energy from mitochondria to the cytosol for maintenance of various biochemical processes and tumor growth. In summary, our study reveals MtCK1 stabilization by Y153 phosphorylation as a key molecular mechanism underlying the intersection between oncogenic HER2 signaling and mitochondrial energy metabolism, which creates a druggable metabolic vulnerability in cancer. Since cyclocreatine has been used as a dietary sport supplement, cyclocreatine might be able to inhibit trastuzumab-resistant tumors with much less toxicity in patients as compared to conventional anticancer drugs.

Citation Format: Taro Hitosugi, Kiran Kurmi. Oncogenic activation of mitochondrial phosphocreatine energy shuttle creates a druggable metabolic liability in cancer 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 LB-250.
Abstract LB-299: Cynomolgus monkey plasma cell gene signature to quantify the in vivo activity of a half-life extended anti-BCMA BiTE® for the treatment of multiple myeloma
Authors: Goyos Ana , Li Chi-Ming , Deegen Petra , Bogner Pamela , Thomas Oliver , Wahl Joachim , Goldstein Rebecca , Friedrich Maththias , Coxon Angela , Balazs Mercedesz , Arvedson Tara
Published: August 2018

Background: Multiple myeloma (MM) is a hematologic malignancy where abnormal plasma cells (PC) accumulate in the bone marrow (BM). Patients often develop relapsed/refractory disease leading to significant morbidity and mortality. Elimination of residual disease is required for curative treatments. Patient outcome can be improved by using T-cell engaging agents, such as bispecific T-cell engager (BiTE®) antibody constructs, to redirect T cells to tumor cells. B-cell maturation antigen (BCMA), a TNF receptor superfamily member, supports PC survival and is widely expressed on MM and normal PC.

Methods: A half-life extended (HLE) anti-BCMA BiTE® (BCMA HLE-BiTE®) antibody construct was generated and characterized in vitro, and in vivo in a mouse xenograft model and in non-human primates (NHP). To quantify on-target activity of the BCMA HLE-BiTE® in NHP it was necessary to follow changes in PC numbers. Because PC are rare (0.1 - 3%) in the BM and blood, detection using traditional assays is not possible. To address this limitation, a PC gene signature was defined and used to develop a quantitative ddPCR assay (sensitivity = 0.01%) to assess the effect of the BCMA HLE-BiTE® in a 12-day repeat-dose NHP study.

Results: A BCMA HLE-BiTE® was selected that demonstrated cytotoxic activity using human effector cells (EC50: 0.13 ± 0.07 pM in MM.1R cells) and comparable activity using NHP effector cells. In a sub-cutaneous mouse xenograft model in which BCMA-positive NCI-H929 cells and human PBMC were co-administered, the BCMA HLE-BiTE® completely inhibited tumor formation, in contrast to vehicle controls with/without PBMC. The serum half-life of the BCMA HLE-BiTE® in NHP was 112 hours following a single 15 µg/kg IV dose. The PK/PD relationship in NHP was next evaluated in a 12-day repeat-dose study. A dose-dependent PK behavior as well as depletion of PC in blood and BM was observed, reaching 93% depletion in the blood and 85% in the BM in the high-dose group on day 12.

Conclusions: These results demonstrate that the BCMA HLE-BiTE® is potent and effective at inducing cytotoxicity of target cells in vitro and in vivo, and may be suitable for once-weekly dosing in MM patients. This molecule is being evaluated in humans: trial NCT03287908.

Citation Format: Ana Goyos, Chi-Ming Li, Petra Deegen, Pamela Bogner, Oliver Thomas, Joachim Wahl, Rebecca Goldstein, Maththias Friedrich, Angela Coxon, Mercedesz Balazs, Tara Arvedson. Cynomolgus monkey plasma cell gene signature to quantify the in vivo activity of a half-life extended anti-BCMA BiTE® for the treatment of multiple myeloma [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 LB-299.
Abstract LB-312: GRK5 activity mediates in vivo and in vitro prostate cancer progression and chemoresistance
Authors: Black Joseph B. , Purayil Hamsa Thayele , Mahmud Iqbal , Liao Daiqing , Daaka Yehia
Published: August 2018

Despite decades of research targeting the activity of the androgen receptor (AR), patients diagnosed with locally advanced and metastatic prostate cancer face an incurable disease. The Daaka lab investigates constituents of G-protein-coupled receptor signaling cascades for their ability to provide mitogenic signaling, which promotes PCa progression. We previously identified G protein-coupled receptor 5 (GRK5) for its ability to regulate PCa progression, independent of AR activity. GRK5 partially partitions to the nucleus, wherein it has been shown to regulate the transcriptome in non-PCa models. To globally elucidate the mechanistic impact of GRK5 on PCa progression, we are investigating the impact of GRK5 on the PCa transcriptome. We hypothesize that GRK5's regulation of the PCa transcriptome promotes tumor progression. To assay the effect of GRK5 on the PCa transcriptome, RNA sequencing was performed in two cell lines: PC3 Control (PC3 shGFP) and PC3 GRK5 Knockdown (PC3 shGRK5).  Ontologic analysis identified the epithelial–mesenchymal transition (EMT) as being affected when GRK5 is depleted. The epithelial–mesenchymal transition (EMT) is highly associated with promoting PCa progression and chemoresistance, thus we selected this pathway for further investigation. Confirmatory western blot analysis and quantitative PCR (qPCR) validated that depleting GRK5 suppresses the expression of the mesenchymal markers Vimentin and N-Cadherin. Similarly, overexpression of GRK5 increases the expression of these mesenchymal markers. Stable overexpression of GRK5 promotes cells to develop a spindle-like morphology, indicative of a mesenchymal state. GRK5 mediates this mesenchymal transition through increasing the expression of the EMT transcription factor, Twist1. Further analysis identifies that GRK5 activity promotes in vitro invasion. Cell lines overexpressing GRK5 demonstrate an increase in resistance to docetaxel, the mainstay chemotherapy for advanced PCa. Overexpression of GRK5 promotes lymphatic intravasation. Overexpression of mutated forms of GRK5 that are relegated to the nucleus are able recapitulated all aforesaid changes, arguing that the nuclear activity of GRK5 mediates this effect. Collectively, this data presents a novel mechanism promoting PCa progression, independent of AR activity.

Citation Format: Joseph B. Black, Hamsa Thayele Purayil, Iqbal Mahmud, Daiqing Liao, Yehia Daaka. GRK5 activity mediates in vivo and in vitro prostate cancer progression and chemoresistance [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 LB-312.
Abstract LB-327: Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake
Authors: Fletcher Alice , Thompson Rebecca , Read Martin , Turnell Andrew , Smith Vicki , McCabe Chris J.
Published: August 2018

Ablative radioiodide therapy is an effective treatment for thyroid cancer, but around a quarter of patients do not accumulate therapeutically-sufficient doses of radioiodide due to decreased expression and/or plasma membrane (PM) localization of the sodium iodide symporter (NIS). Putative use of radioiodide therapy in breast cancer is hampered by low levels of NIS membrane localisation. Currently, the regulation of NIS trafficking is ill-defined. Mass spectrometry was performed on proteins co-immunoprecipitating with lentivirally expressed NIS in whole cell and plasma membrane extracts. NIS activity was significantly altered by ADP-ribosylation factor 4 (ARF4) and valosin containing protein (VCP) in TPC1 thyroid and MDA-MB-231 breast cancer cells lentivirally-expressing NIS. ARF4 downregulation markedly decreased radioiodide uptake, and VCP downregulation increased radioiodide uptake in thyroid and breast cells. Transient overexpression of these genes significantly reversed siRNA effects on NIS function. Co-immunoprecipitation assays confirmed NIS interacts with ARF4 and VCP in vitro, and proximity ligation assays revealed the subcellular sites of interaction. Critically, human primary thyroid culture experiments confirmed that ARF4 and VCP regulate endogenous NIS function in vivo. In addressing the mechanisms of NIS trafficking we identified a C-terminal VXPX motif of NIS which is required for Arf4 regulation, whilst TIRF microscopy revealed coincident Arf4 and NIS localization to shared PM vesicles. Pharmacological inhibitor studies identified that the VCP inhibitors Eeyarastatin-1 and NMS-873 overcome VCP inhibition of NIS function. VCP, which is central to ER-associated Degradation (ERAD), selectively targeted a dimeric form of NIS, which we now identify through co-immunoprecipitation, 3D modelling and Förster resonance energy transfer (FRET) assays. TCGA data analysis of 58 matched papillary thyroid cancers revealed that Arf4 was significantly repressed and VCP highly upregulated in thyroid cancer, providing a putative explanation for repressed NIS function. Our data suggest a possible model for NIS trafficking: NIS dimerises in the ER, a process monitored by VCP and the ERAD pathway, with correctly processed and folded NIS being trafficked to the PM via C-terminal interaction with Arf4. We thus now identify two new potential therapeutic targets for enhancing radioiodide uptake in patients with radioiodide-refractory thyroid cancer.

Citation Format: Alice Fletcher, Rebecca Thompson, Martin Read, Andrew Turnell, Vicki Smith, Chris J. McCabe. Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake [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 LB-327.
Abstract LB-360: JNKs-mediated phosphorylation of human stromal xbp1s regulates multiple myeloma growth & osteolysis
Authors: Chen Risheng , Xu Guoshuang , Beaino Wissam , Lui Kai , Zeng Xuemei , Yates Nathan , Chong Rong , Verdelis Konstas , Roodman G. David , Toscani Denise , Giuliani Nicola , Lin Yan , Anderson Carolyn J. , Ouyang Hongjiao
Published: August 2018

Spliced X-box binding protein-1 (XBP1s) is a major effector of the unfolded protein response (UPR) signaling. We previously reported that human XBP1s in bone marrow stromal cells plays an essential role in regulating stromal support of multiple myeloma bone disease (MMBD). Here we report that human XBP1s (hXBP1s) is a physiological & direct phosphorylation substrate of JNKs. This phosphorylation greatly increases hXBP1s protein degradation by the E3 ubiquitin ligase β-TrCP. Mutation of the phosphorylation site of JNKs to alanine largely prevents JNK phosphorylation of XBP1s & JNK-induced protein degradation of hXBP1s. Further, we found that bone marrow stromal cells (BMSCs) harboring this XBP1s mutant are resistant to JNK phosphorylation and provided significantly greater support of MM growth and MM-induced bone destruction both in vitro & in vivo, compared with counterparts expressing WT hXBP1s. Finally, we observed that JNK-mediated phosphorylation of hXBP1s is associated with favorable therapeutic responses of MM patients to combination therapy of bortezomib, &/or dexamethasone &/or thalidomide, MM drugs known to stimulate JNK. These results suggest p-hXBP1sJNK is a potential predictive biomarker for MM patients' response to the drug treatment. Taken together, our study provides the first evidence that hXBP1s is a novel physiological & direct target of JNK phosphorylation and demonstrates the pathophysiological significance of this event in the hXBP1s-mediated bone microenvironment support of MM growth and MM-induced bone destruction.

Citation Format: Risheng Chen, Guoshuang Xu, Wissam Beaino, Kai Lui, Xuemei Zeng, Nathan Yates, Rong Chong, Konstas Verdelis, G. David Roodman, Denise Toscani, Nicola Giuliani, Yan Lin, Carolyn J. Anderson, Hongjiao Ouyang. JNKs-mediated phosphorylation of human stromal xbp1s regulates multiple myeloma growth & osteolysis [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 LB-360.
Abstract LB-361: Tumor-derived microvesicles promote tumor immune escape through PD-1/PD-L1 interaction
Authors: Kim Dong Ha , Choi Yun Jung , Kim Seon Ye , Lee Jung-Eun , Sung Ki Jung , Lee Jae Cheol , Rho Jin Kyung
Published: August 2018

PD-1/PD-L1 pathway blockade is a promising new cancer therapy. Although PD-1/PD-L1 therapy has yielded clinical benefits in several types of cancer, further studies are required to clarify predictive biomarkers for drug efficacy and to understand the fundamental mechanism of PD-1/PD-L1 interaction between host and tumor cells. Here, we show that microvesicles (MVs) derived from lung cancer cells express PD-L1 and play a role in immune escape by reducing T cell activity and promoting tumor growth. The abundance of PD-L1 on MVs represented the quantity of PD-L1 expression on cell surfaces. MVs containing PD-L1 inhibited IFN-γ secretion by Jurkat T cells. The IFN-γ secretion was restored by PD-L1 knockout or masking on the MVs. Both the forced expression of PD-L1 on cells without PD-L1 and treatment with MVs containing PD-L1 enhanced tumor growth in vivo. PD-L1 was present on MVs isolated from the plasma of patients with non-small cell lung cancer, and its abundance in the MVs was correlated with the PD-L1 positivity on tumor tissues. MVs could impair immune functions by reducing cytokine production and inducing apoptosis in CD8+ T cells. Our findings indicate that tumor-derived MVs expressing PD-L1 may be an important mediator of tumor immune escape.

Citation Format: Dong Ha Kim, Yun Jung Choi, Seon Ye Kim, Jung-Eun Lee, Ki Jung Sung, Jae Cheol Lee, Jin Kyung Rho. Tumor-derived microvesicles promote tumor immune escape through PD-1/PD-L1 interaction [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 LB-361.
Abstract LB-365: Microscopic imaging for understanding gold nanorods-protein interactions in inhibiting cancer cell migration
Authors: Wu Yue , Ali Moustafa R. , Fang Ning , El-Sayed Mostafa A.
Published: August 2018

Metastasis is the primary cause of cancer-related deaths. Current clinical treatments for anti-metastasis, however, are not effective. Gold nanorods (AuNRs), which are safe within biological systems, were recently discovered to inhibit cancer cell migration and prevent metastasis. However, the mechanism of how AuNRs inhibit cancer cell migration remains largely unexplored. The migration of cancer cells from one site to another requires dramatic remodeling of cytoskeleton proteins. Our recent studies have shown a great impact of AuNRs treatment on proteins that regulate the cytoskeleton. However, the interaction of AuNRs and the cytoskeletal structures are not well studied due to the lack of advanced microscopic technologies at single nanoparticle-single protein level. Herein, we used cutting-edge imaging techniques to enable the study of the direct correlation between cytoskeleton filaments (microtubule) and AuNRs . Differential interference contrast (DIC) (for imaging AuNRs) and the stochastic optical reconstruction microscopy (STORM) (for imaging proteins) were combined to resolve the interaction of how AuNRs interact with the cytoskeleton filaments, disrupt the cytoskeleton structures, including microtubule and therefore inhibit cell migration. In conclusion, we are able to visualize the interaction of AuNRs with cytoskeleton filaments at nanometer resolution with their accurate intracellular location, which is beneficial for the future studies of nanoparticle-cell interactions.

Citation Format: Yue Wu, Moustafa R. Ali, Ning Fang, Mostafa A. El-Sayed. Microscopic imaging for understanding gold nanorods-protein interactions in inhibiting cancer cell migration [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 LB-365.
Abstract LB-376: Clinico-genomic analysis of temozolomide-induced hypermutation in both molecular subtypes of IDH-mutant glioma
Authors: Grimmer Matthew R.
Published: August 2018

Hypermutated gliomas arising from treatment with alkylating agents, such as temozolomide (TMZ), are genetically distinct, recurrent tumors about which very little is known. Here, we have identified common and subtype-specific patterns in the tumor evolution of IDH-mutant, low-grade astrocytoma (1p/19q-intact) and oligodendroglioma (1p/19q-codeleted) from 57 glioma patients. Hypermutation involved unanimous defects in mismatch repair in both subtypes, with unique mismatch repair alterations differentiating hypermutation events in serial recurrences. Hypermutation was associated with malignant progression in astrocytoma, and can differentiate high-grade regions from low-grade regions within the same tumor. Hypermutated oligodendroglioma tumors associated with newly-arising p53 mutations and mirrored the early branching evolution of astrocytoma tumors. Results from this longitudinal clinical-genomic study suggest prominent roles for MMR and p53 in the establishment and maintenance of hypermutation. The evolution of TMZ-related tumor antigens across serial tumor recurrences will be presented.

Citation Format: Matthew R. Grimmer. Clinico-genomic analysis of temozolomide-induced hypermutation in both molecular subtypes of IDH-mutant glioma [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 LB-376.
Abstract LB-380: TMB calling in absence of matched normal: Learning germline behavior
Authors: Tian Yiqing , Jones Wendell , Mola Natalie , Weigman Victor J.
Published: August 2018

In most standard cases, mutational burden is derived from samples where mutational calling is performed in tumor and matched normal specimens. While this is ideal to get a true sense of tumor-derived mutations, it creates limitations due to cost of extra sequencing or in many trials, unavailable for genomic profiling. Without this normal material, there are increased difficulties of eliciting the accurate somatic mutation calls away from natural variation. This is even further complicated by the fact that different tumor types have different spectra of mutations so a method built on an individual indication have risks of overfitting. We present here the results of our investigations of machine learning to build reliable germline variant estimates appropriate for use in calculating burden. In this draft, we examine two tumor mutation classification methods based on our latest TMB tool: random forest and neural network across multiple indications in TCGA. We compare calls made by these methods against burden scores that we have generated from our own paired tumor/normal method (sister submission) to determine accuracy. Using sampled tumor results from each indication, different models were built across both random forest and neural networks, showing accuracy in some indications, up to 98%. Looking to work to create and indication-independent classifier, validation results show we can reach an average of 89% accuracy where training is built across all indications. In all cases, cross validation was performed to prove the consistency of the classification method and test the algorithm on our latest tumor only data set against what was generated from paired results.

Citation Format: Yiqing Tian, Wendell Jones, Natalie Mola, Victor J. Weigman. TMB calling in absence of matched normal: Learning germline behavior [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 LB-380.
Abstract LB-390: A transfer RNA derived small RNA affects translation in rapidly dividing cells and a target for hepatocellular carcinoma
Authors: Kim Hak Kyun , Fuchs Gabriele , Wang Shengchun , Wei Wei , Zhang Yue , Park Hyesuk , Roy-Chaudhuri Biswajoy , Li Pan , Xu Jianpeng , Chu Kirk , Zhang Feijie , Chua Mei-sze , So Samuel , Zhang Cliff , Sarnow Peter , Kay Mark A.
Published: August 2018

There are tens of thousands of different transfer RNA-derived small RNAs (tsRNAs) of 18-40 nucleotides in length in mammalian cells. In recent years there is accumulating evidence suggesting that these RNAs can play different yet important roles in gene regulations. Nevertheless, in most cases, the biological roles of these RNAs have yet to be defined. Using a variety of locked nucleic acid/antisense oligonucleotide (LNA/ASO)-mediated strategies, we found that inhibition of one specific 3'tsRNA, induces apoptosis in rapidly dividing cells. Inhibition of the tsRNA but not the mature tRNA reduced the translation of ribosomal protein S28 (RPS28) and S15 (RPS15) mRNAs and led to a block in pre-18S ribosomal RNA processing, and ultimately a decrease in the number of 40S ribosomal subunits. Using a modified ChIRP method, we found that the tsRNA binds to ribosomal protein S28 mRNA and by genetic complementation analyses established the binding sites contained within the mRNAs. Furthermore, we established that tsRNA binds to duplexed target sites in RPS28 and RPS15 mRNAs and enhances their translation by perhaps helping to unfold their secondary structures. The systemic delivery of a specific anti-tsRNA oligonucleotides into mice (the tsRNA sequence is the same in mouse and humans) did not cause liver injury in normal mice but induced apoptosis and a significant growth retardation of patient-derived orthotopic hepatocellular carcinomas surgically implanted into mouse livers. Our result establishes a newly defined post-transcriptional mechanism of gene regulation and provides a novel target for cancer therapeutics.

Citation Format: Hak Kyun Kim, Gabriele Fuchs, Shengchun Wang, Wei Wei, Yue Zhang, Hyesuk Park, Biswajoy Roy-Chaudhuri, Pan Li, Jianpeng Xu, Kirk Chu, Feijie Zhang, Mei-sze Chua, Samuel So, Cliff Zhang, Peter Sarnow, Mark A. Kay. A transfer RNA derived small RNA affects translation in rapidly dividing cells and a target for hepatocellular 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 LB-390.
Abstract LB-393: LINC01137 functions as a ceRNA promoting SMAD3 signaling and ESCC metastasis
Authors: Shao Mingming , Chu Jiahui , Cui Qionghua , Peng Linna , Luo Yingying , Chang Jiang , Tan Wen , Wu Chen , Lin Dongxin
Published: August 2018

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the leading cause of cancer-related death in China and some other parts of the world. Studies have shown that certain long non-coding RNAs (lncRNAs) are involved in cancer development and progression. In this study, we sought to identify lncRNAs that are associated with metastasis of ESCC and to explore their functions and therapeutic implications.

METHODS: We performed RNA sequencing and analyzed the association of lncRNAs with ESCC survial time. The significantly associated lncRNAs that were overexpressed in ESCC were selected for further study. Knockdown or overexpression of interest lncRNAs were performed to investigate their effects on ESCC cell invasion and metastasis in vitro and in vivo in mice xengraft models. Biochemical assays such as RNAi, RNA immunoprecipitation, RNA pulldown and chromatin immunoprecipitation assays were used to examine the interactions among lncRNAs, mRNAs and microRNAs and between proteins and DNA. Patient survival time differentiated by RNA expression levels was estimated by Kaplan-Meier plotting and hazard ratios for death were calculated by Cox models.

RESULTS: RNA sequencing identified 9 overexpressed lncRNAs were associated with ESCC survival. Among these lncRNAs, LINC01137 had the strongest effects on invasion and metastasis of ESCC cell lines in vitro and in vivo in mice. We found that ESCCwith high expression of LINC01137 had significantly more lymph node metastases compared with ESCC with low expression of LINC01137. Functional analyses demonstrated that LINC01137 may compete with HOXA1 mRNA for miR-608 binding. Higher expression level of LINC01137 in cells may prevent miR-608 from inhibiting HOXA1 translation. We found that HOXA1 is a transcription activator of SMAD2/3 and knockdown of LINC01137 substantilly reduced the SMAD3 signaling. Treatment of ESCC cells overexpressing LINC01137 with Halofuginone, a plant derivative that can inhibit SMAD3 phosphorylation, significantly reduced the abilities of migration and invasion.

CONCLUSION: These results suggest that LINC01137 might function as a ceRNA competing with miR-608 for suppressing HOXA1 expression, which promotes SMAD3 signaling to enhance metastasis of ESCC cells.

Citation Format: Mingming Shao, Jiahui Chu, Qionghua Cui, Linna Peng, Yingying Luo, Jiang Chang, Wen Tan, Chen Wu, Dongxin Lin. LINC01137 functions as a ceRNA promoting SMAD3 signaling and ESCC 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 LB-393.
Abstract SY19-02: Novel mechanisms of acquired resistance to selective CDK4/6 inhibition
Authors: Shapiro Geoffrey I.
Published: August 2018

CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptor (ER)-positive breast cancer, so that understanding mechanisms of resistance is a pressing issue. We have identified identified increased CDK6 expression as a key determinant of acquired resistance after exposure to palbociclib in ER-positive breast cancer cells. Increased CDK6 in resistant cells is dependent on TGF-β pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediated transfer of the resistance phenotype between neighboring cell populations, causing previously sensitive cells to acquire CDK4/6 inhibitor resistance. We confirmed these data in pretreatment and postprogression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating that this resistance mechanism is clinically relevant. Additionally, this CDK4/6 inhibitor resistance phenotype can be reversed in vitro and in vivo by a prolonged drug holiday, which restored drug sensitivity. CDK4 is also emerging as a target in KRAS-mutant non-small cell lung cancer (NSCLC). We have demonstrated that KRAS-mutant NSCLC cell lines are initially sensitive to palbociclib, but readily acquire resistance associated with increased expression of CDK6, D-cyclins, and cyclin E. Resistant cells also demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating palbociclib resistance. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A genetically engineered mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2 and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 that confers palbociclib resistance, and indicate that CDK4/6 inhibition acts to promote MAP kinase dependence.

Citation Format: Geoffrey I. Shapiro. Novel mechanisms of acquired resistance to selective CDK4/6 inhibition [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 SY19-02.
Abstract SY38-01: Inflammation-induced neutrophil extracellular traps (NETs) awaken dormant cancer in the lung microenvironment
Authors: Egeblad Mikala
Published: August 2018

To elucidate mechanisms of cancer metastasis and recurrence from minimal residual disease, we use intravital imaging allowing for long-term visualization of cancer cells and surrounding host cells in living mice. Using intravital imaging, we found that neutrophils, the most abundant type of leukocytes in blood, form neutrophil extracellular traps (NETs) near disseminated cancer cells in the lung of mice during experimental inflammation. NETs are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes in response to, e.g., infections. However, we found that metastatic breast cancer cells themselves can induce neutrophils to form metastasis-supporting NETs, in the absence of infection. We also found NETs in clinical samples of metastatic, triple-negative human breast cancer. Using mouse models, we determined that NETs promote metastasis by facilitating proteolytic extracellular matrix remodeling. Pharmacologic inhibition of NET formation or digestion of NETs with DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs is an important host-mediated mechanism of promoting metastasis and a potential therapeutic target.

Citation Format: Mikala Egeblad. Inflammation-induced neutrophil extracellular traps (NETs) awaken dormant cancer in the lung microenvironment [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 SY38-01.
Abstract A07: DNA methylation predicts response of triple-negative breast cancer to all-trans retinoic acid treatment
Authors: Coyle Krysta M. , Dean Cheryl A. , Vidovic Dejan , Weaver Ian , Giacomantonio Carman , Helyer Lucy , Marcato Paola
Published: August 2018

We have identified a novel, epigenetic-based strategy to identify breast cancer patients who will benefit from an existing anticancer agent, all-trans retinoic acid (atRA). Triple-negative breast cancer (TNBC) is among the most aggressive breast cancers and lacks targeted therapies. TNBCs are a heterogeneous group of breast cancers with a wide range of gene expression profiles and drug sensitivities. Profiling these differences will lead to the development of more effective treatment options for TNBC. We hypothesize that atRA can be an effective therapy for a subset of TNBC patients. To test our hypothesis, we profiled the response of 12 TNBC cell lines to atRA using in vivo tumor growth assays. We found that atRA treatment had a range of effects on the tumor growth of TNBC cell lines: significantly decreasing tumor growth in HCC70, SUM149, and HCC1937, while increasing the growth of MDA-MB-231, MDA-MB-436, and Du4475 tumors. Gene expression and methylation analysis of these 12 cell lines revealed subtype-specific expression of atRA-inducible genes due to silencing by DNA methylation, e.g., of the atRA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in cell lines where atRA promotes tumor growth, but is hypomethylated and expressed in cells that can be effectively treated with atRA. Use of the distinct gene expression and methylation profiles allowed us to accurately predict the response of 4 patient-derived xenografts (PDXs) to RA treatment. Continued classification of TNBCs by gene expression and DNA methylation can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs.

Citation Format: Krysta M. Coyle, Cheryl A. Dean, Dejan Vidovic, Ian Weaver, Carman Giacomantonio, Lucy Helyer, Paola Marcato. DNA methylation predicts response of triple-negative breast cancer to all-trans retinoic acid treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A07.
Abstract A15: DNA damage response-targeted molecules for breast cancer therapy
Authors: Valenea Alfredo , Grindrod Scott , Jung Mira
Published: August 2018

Breast cancer genes 1 and 2 (BRACA1 and BRACA2) are known to be linked to breast cancer risk. BRCA1 is an ATM substrate and plays critical roles in the DNA damage response (DDR) and maintenance of genomic stability. Furthermore, recent studies have shown that the aberrant expression of DNA damage repair transducer, ataxia-telangiectasia mutated (ATM), is associated with breast cancers. To develop target-specific drugs, we rationally designed a dual-function small molecule, SP-1-303, by conferring ATM activation and HDAC inhibition activity. The compound was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor at an IC50 of 120 nM. Western analysis revealed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and ATM activation (p-ATM/S1987). Testing two ER-positive BC cell lines (MCF7 and T-47D), two triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effect of SP-1-303 on cell growth was examined by cytotoxicity assays. SP-1-303 demonstrated cytotoxicity (IC50) of ER-positive BC cells in a range of 0.2-0.3 μM, while the values for TNBC cells are in a range of 1-3 μM and 12 μM for normal breast epithelial cells, respectively. Furthermore, proteomic analysis of MCF7 cells following SP-1-303 treatment identified differential effects on expression of various DDR molecules and chromatin regulators, including ATM, BRCA1, TP53BP1, NBN, histones, and chromatin remodelers. Taken together, SP-1-303 shows specificity for ER-positive breast cancers, potentially by modulating the DDR signaling pathway.

Citation Format: Alfredo Valenea, Scott Grindrod, Mira Jung. DNA damage response-targeted molecules for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A15.
Abstract A24: SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression
Authors: Khokhar Zunair Shakeel , Kim Alexander J. , Pires Eusebio , D’Souza Ryan
Published: August 2018

The matrix metalloproteinase- Sperm Acrosomal SLLP1 Binding (SAS1B) is an ideal molecular and cellular candidate for both female contraception and for cancer therapies. SAS1B translation is restricted among adult tissues to the ovary and oocytes, and SAS1B first appears in oocytes of follicles at the primary-secondary transition and also in tumors of varied types and stages. Research conducted using SAS1B antibodies suggested that the protein is localized at the cell membrane of cancer cells and can get internalized in a classical endocytosis assay and can be killed using an immunotoxin linked onto antibodies. The current study aims at deciphering whether the ASTL gene or the SAS1B protein is important for tumor progression, invasion and metastasis through cell biology and molecular biology tools and ensuring tumor cell killing properties via an ADC mediated killing using biochemistry and molecular biology tools. PCR, immunohistochemistry, Immunofluorescence, and Western Blots revealed that a non cancerous breast cell line MCF10A5E is SAS1B null and can be used to study cancer like properties once stably transfected with the SAS1B construct. Previous work in the lab indicated that an SAS1B-ADC strategy is a viable immunotherapy that could benefit patients who have failed to respond to chemotherapy, if indeed SAS1B plays a role in cancer development and progression. The outcomes of a SAS1B-ADC in a breast cancer model will be presented.

Citation Format: Zunair Shakeel Khokhar, Alexander J. Kim, Eusebio Pires, Ryan D’Souza. SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A24.
Abstract A26: The metastatic potential of breast cancer cells was suppressed by NLRP3 inflammasome inhibition in macrophages
Authors: Lee Jin Y. , Lee Hye E. , Lee Joo Y.
Published: August 2018

Tumor microenvironment favors tumor cells to promote tumor growth, migration, invasion, and angiogenesis. IL-1beta, one of the inflammatory cytokines associated with tumor microenvironment, plays an important role in the development and progress of tumor. The activation of inflammasome is critical to secrete mature IL-1beta through stepwise reactions to activate capspase-1. Therefore, we investigated whether the metastatic potential of breast cancer cells was regulated by NLRP3 inflammasome activation of macrophages in tumor microenvironment. The activation of NLRP3 inflammasome was induced by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of 4T1 breast cancer cells was determined by migration and invasion assays with transwell system. Conditioned media from ATP-stimulated wild-type macrophages increased the migration and invasion of breast cancer cells (4T1) while conditioned media from NLRP3 knockout macrophages stimulated with ATP failed to induce the migration and invasion of breast cancer cells. Celastrol, an inhibitor of NLRP3 inflammasome, reduced the potency of macrophages to stimulate the migration and invasion of 4T1 breast cancer cells. The suppression of NLRP3 inflammasome by celastrol was mediated by the blockade of potassium efflux. The results demonstrate that the activation of NLRP3 inflammasome in macrophages leads to the enhancement of metastatic potential of breast cancer cells. The results would provide a novel anticancer strategy to modulate tumor microenvironment by suppressing NLRP3 inflammasome and consequently reducing IL-1beta production. (This study was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (Bio & Medical Technology Development Program, NRF-2017M3A9F5028608; Basic Research Laboratory Program, NRF-2017R1A4A1015036.)

Citation Format: Jin Y. Lee, Hye E. Lee, Joo Y. Lee. The metastatic potential of breast cancer cells was suppressed by NLRP3 inflammasome inhibition in macrophages [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A26.
Abstract A46: KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer
Authors: Jia Yunlu , Linbo Wang
Published: August 2018

Krüppel-like factor 4 (KLF4) has critical roles in breast cancer development and progression and several solid tumors. Tamoxifen (TAM) resistance represents a daunting challenge to the successful treatment of breast cancer. KLF4 expression, function, and regulation in the efficacy of TAM therapy in breast cancer have yet to be demonstrated. Here, we investigated the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 was correlated with increased TAM sensitivity in breast cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERa and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness of MCF-7/TAM and T47D cells to TAM. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion, and migration ability. Besides, KLF4 expression was downregulated in breast cancer tumor tissues and high expression of KLF4 linked to favorable outcome. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and P38 were relatively activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors can significantly suppress cell viability. Knockdown of KLF4 activated ERK and P38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and P38 signaling and resulted in enhanced sensitivity to TAM. Therefore, our findings suggest that KLF4 contributes to TAM efficiency in breast cancer via phosphorylation modification of ERK and P38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting the KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for TAM-resistant patients.

Note: This abstract was not presented at the conference.

Citation Format: Yunlu Jia, Wang Linbo. KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A46.