bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3452: N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway
Authors: Hung Chun-Hua , Yeh Hsuan-Heng , Wang Hao-Chen , Lin Chien-Chung , Tsai Tsung-Lin , Huang Wei-Lun , Chang Chuan-Fa , Su Wu-Chou
Published: May 2015

Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and modulating anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been deduced in early studies; however, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remain unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we had screened the expression of glycosyltransferases using qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells compared to normal bronchial cell. We then reduced core fucosylation on lung cancer cell-secreted IL-6 by silencing FUT8 with shRNA transduction. Subsequently, cells were treated with conditional media containing fully-glycosylated or core fucose-depleted IL-6 to uncover the potential influences on cellular signaling from glycosylation on IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. The nuclear retention of STAT3 was concordant with the prolonged STAT3 activation in cells treated with fully-gycosylated IL-6. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on the regulation of JAK/STAT pathway.

Citation Format: Chun-Hua Hung, Hsuan-Heng Yeh, Hao-Chen Wang, Chien-Chung Lin, Tsung-Lin Tsai, Wei-Lun Huang, Chuan-Fa Chang, Wu-Chou Su. N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2014-3452
Abstract 3490: Measurement of Gifinitib (ZD1839) effect on electrophysiological properties of head and neck cancer cells using Dielectrophoresis (DEP)
Authors: Mahabadi Sina , Hughes Michael Pycraft , Labeed Fatima Hanna
Published: May 2015

Introduction:

Dielectrophoresis (DEP) is a physical phenomenon which can be used to determine the dielectric properties of the cells through applying a non-uniform electric field. Electrophysiological properties such as the conductivity and permittivity of the membrane and cytoplasm can be determined by this method, which has already been applied in cancer research.

Gifinitib (Iressa) or ZD1839 is one of the most commonly used drugs for treatment of cancer such as breast and head and neck. This drug damages the local tumours through blocking the activity of EGFR (epidermal growth factor receptor) which is one of the most common factors in proliferation of the cancer cells. In this study, the effect of Gifinitib (Iressa) treatment was investigated through analysis of the electrophysiological properties of the HN5 (Head and Neck Carcinoma cells) using a DEPtech 3DEP dielectrophoresis analysis device.

Results:

The cell membrane capacitance for HN5 cells post twenty two hours of drug treatment became substantially lower. Significant changes were also observed in the cytoplasm over the 22-hour period.

Conclusion:

The results indicated a substantial reduction in the membrane parameters of HN5 cells, and these results can be attributed to the interaction between the drug and the EGFR in these cells. DEP appeared to be a reliable method for assessing the action of EGFR inhibitors for drug discovery.

Citation Format: Sina Mahabadi, Michael Pycraft Hughes, Fatima Hanna Labeed. Measurement of Gifinitib (ZD1839) effect on electrophysiological properties of head and neck cancer cells using Dielectrophoresis (DEP). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2014-3490
Abstract 3540: Overexpression of incrna h19 enhances development and metastasis of gastric cancer
Authors: Li Jiangfang , Li Hao , su Liping , Zhu Zheng-gang , Liu Bingya
Published: May 2015

Background: Long non-coding RNAs (lncRNAs) have been shown to play key roles in the progression and metastasis in some carcinomas in recent studies. We found that the expression of long non-coding RNA H19 (H19) was much higher in gastric cancer tissues than that in paired noncanerous tissues. But its mechanism in gastric cancer remains unknown. The object of this study is to explore the role of H19 and it derived miR-675 in gastric cancer and to discover the downstream target genes. Methods: We detected the expression of H19 in 74 gastric tissues together with paired noncancerous tissues and followed-up median 27 months. Then, we investigated the role of H19 and miR-675 by knock-down their expression in MKN45 cell line and ectopic expression in SGC7901 cell line in vitro and in vivo. Finally, we built the co-expression network of H19 and demonstrate the target genes by RIP and DLR. Results: The overall survival (OS) in the patients with low H19 expression (ratio<6) is much longer than that with high H19 expression (ratio≥6). Overexpression of H19 can enhance the characteristics of gastric cancer cells such as proliferation, migration, invasion Tumor formation and metastasis. We built the co-expression network of H19 and demonstrated ISM1 was the binding protein of H19. The expression of ISM1 was positive correlated with that of H19. CALN1 was the target gene of miR-675. The expression of CALN1 was negative correlated with that of miR-675. Conclusion: Overexpression of H19 correlates with bad prognosis in gastric cancer. H19 acts as an oncogene in gastric cancer to enhance the proliferation, migration, invasion, and metastasis. MiR-675 also can enhance gastric cancer progression and metastasis. In additional, H19 RNA is actively bind to protein ISM1 however miR-675 targets the protein CALN1. These suggested that H19 effect on GC both through encoding miR-675 and up-regulation ISM1.

Citation Format: Jiangfang Li, Hao Li, Liping su, Zheng-gang Zhu, Bingya Liu. Overexpression of incrna h19 enhances development and metastasis of gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2014-3540
Abstract 3546: Transcriptional activation of Thioredoxin interacting protein by small activating RNA exerts cell growth inhibition in A549 human lung cancer cells
Authors: Park Ki Hwan , Kang Moo-Rim , Han Sang-Bae , Yun Jieun , Lee Chang Woo , Oh Soo Jin , Kim Youngsoo , Kang Jong Soon
Published: May 2015

TXNIP (also known as VDUP1 or TBP-2) has been identified as a tumor suppressor gene (TSG) and downregulated in various solid tumors and hematological malignancies. In recent years, a therapeutic application of small activating RNA for cancer treatment has been suggested. Therefore, we designed small activating RNA targeting TXNIP promoter (hsaTXNIP-834) and investigated the effect of hsaTXNIP-834 on cell growth inhibition in A549 human lung carcinoma. Transfection of hsaTXNIP-834 induced transcriptional activation of TXNIP gene and protein expression in a dose dependent manner in A549 cells. Transfection of hsaTXNIP-834 also stimulated demethylation of CpG island region of TXNIP gene promoter. In addition, hsaTXNIP-834 leaded to cell growth inhibition in a dose dependent manner. Further study demonstrated that hsaTXNIP-834 induces G0/G1 and G2/M phase cell cycle arrest. Moreover, hsaTXNIP-834 induced p53 expression and reduced phosphorylation of RB protein. However, co-transfection of TXNIP or Ago2 siRNA reversed cell growth inhibition by hsaTXNIP-834. Collectively, our results suggest that hsaTXNIP-834 inhibits the growth of A549 lung carcinoma cells by inducing G0/G1 and G2/M phase cell cycle arrest and this is mediated, at least in part, by transcriptional activation of TXNIP by hsaTXNIP-834.

Citation Format: Ki Hwan Park, Moo-Rim Kang, Sang-Bae Han, Jieun Yun, Chang Woo Lee, Soo Jin Oh, Youngsoo Kim, Jong Soon Kang. Transcriptional activation of Thioredoxin interacting protein by small activating RNA exerts cell growth inhibition in A549 human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3546. doi:10.1158/1538-7445.AM2014-3546
Abstract 4436: Neddylation of ribosomal protein S27-like and RPS27 regulates survival of breast cancer cells
Authors: Zhao Yongchao , Xiong Xiufang , Sun Yi
Published: May 2015

Neddylation plays a distinct role in stabilization of a subset of ribosomal proteins. How ribosomal proteins are neddylated and whether their neddylation plays any biological roles in cell growth and survival remains elusive. Here we report that ribosomal protein S27-like (RPS27L) and its family member RPS27 is subjected to neddylation by MDM2 E3 ubiquitin ligase, and deneddylation by NEDP1. Disrupting neddylation with MLN4924, a newly discovered small molecule inhibitor of neddylation pathway, destabilized RPS27L and RPS27. Moreover, silencing of RPS27L and RPS27 sensitized breast cancer cells to MLN4924-induced apoptosis. Our results suggest that neddylation stabilizes RPS27L and RPS27, and confers the survival of breast cancer cells.

Citation Format: Yongchao Zhao, Xiufang Xiong, Yi Sun. Neddylation of ribosomal protein S27-like and RPS27 regulates survival of breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4436. doi:10.1158/1538-7445.AM2014-4436
Abstract 4437: Fbxw8 targets hematopoietic progenitor kinase 1 for proteasomal degradation
Authors: Wang Hua , Chen Yue , Lin Ping , Li Lei , Zhou Guisheng , Liu Guangchao , Logsdon Craig , Jin Jianping , Abbruzzese James L. , Tan Tse-Hua , Wang Huamin
Published: May 2015

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasomemediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7 ubiquitin ligase targeted HPK1 for degradation via the 26S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1.

In addition, we showed that Thr355 of HPK1 is a key PP4 dephosphorylation site, through which Fbxw8 and PP4 regulates HPK1 stability. Our study demonstrated that CUL7 ubiquitin ligase targets HPK1 for degradation which requires HPK1 kinase activity and autophosphorylation. Fbxw8-mediated HPK1 degradation revealed a direct link and novel role of CUL7 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.

Citation Format: Hua Wang, Yue Chen, Ping Lin, Lei Li, Guisheng Zhou, Guangchao Liu, Craig Logsdon, Jianping Jin, James L. Abbruzzese, Tse-Hua Tan, Huamin Wang. Fbxw8 targets hematopoietic progenitor kinase 1 for proteasomal degradation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4437. doi:10.1158/1538-7445.AM2014-4437
Abstract 444: BRM/SMARCA2 is a critical synthetic lethal target in BRG1-deficient cancers
Authors: Jaskelioff Mariela , Hoffman Gregory , Rahal Rami , Xiang Kay , Haas Kristy , Saenz-Vash Veronica , Zhai Huili , Keen Nicholas , Stegmeier Frank , Jagani Zainab
Published: May 2015

The mammalian SWI/SNF complexes (mSWI/SNF) regulate chromatin structure through ATP-dependent nucleosome remodeling and thereby control key cellular processes. Recent cancer genome studies have revealed a significant frequency of mutations in several components of the mSWI/SNF complexes including the catalytic subunit BRG1. Emerging evidence is supportive of tumor suppressor roles for mSWI/SNF subunits, yet the inactivating nature of these mutations presents a challenge for devising targeted therapeutic strategies against cancers harboring such mutations. In this study, we interrogated epigenetic cancer dependencies by screening a deep-coverage design (DeCoDe) shRNA library targeting the epigenome across a broad range of 50+ cancer cell lines. Strikingly, this unbiased screen revealed that silencing of the SWI/SNF ATPase subunit BRM/SMARCA2, selectively inhibits the proliferation of BRG1-deficient cancer cells. BRM knockdown selectively induced cell cycle arrest in BRG1-mutant cancer cells and significantly impaired the growth of BRG1-mutant lung tumor xenografts. BRM is the paralog of BRG1, suggesting a model in which mSWI/SNF mutations lead to a hypomorphic complex that promotes tumorigenesis but cannot tolerate complete inactivation. Thus, targeting mSWI/SNF subunits that exhibit redundant activities to the residual mutated complexes may present a general strategy for SWI/SNF mutated cancers.

Citation Format: Mariela Jaskelioff, Gregory Hoffman, Rami Rahal, Kay Xiang, Kristy Haas, Veronica Saenz-Vash, Huili Zhai, Nicholas Keen, Frank Stegmeier, Zainab Jagani. BRM/SMARCA2 is a critical synthetic lethal target in BRG1-deficient cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 444. doi:10.1158/1538-7445.AM2014-444
Abstract 4448: Overexpression of ECT2 promotes proliferation and metastasis of UPSC
Authors: Mach Claire M. , Magliaro Thomas J. , Anderson Matthew L.
Published: May 2015

Introduction: UPSC is a rare but aggressive malignancy that accounts for no more than 5 to 10% of uterine cancers, but more than 40% of associated uterine cancer deaths. The molecular events responsible for the poor clinical outcomes observed with UPSC are largely unknown. Epithelial cell transforming sequence 2 oncogene (ECT2) is a guanine nucleotide exchange factor (Rho-GEF) that catalyzes the exchange of GDP for GTP by Rho family GTPases. High levels of ECT2 expression have been reported in brain, lung and breast cancers, where they were shown promote metastasis. However, the role of ECT2 in UPSC has not been previously explored.

Methods: After obtaining IRB permission, ECT2 mRNA and protein expression were measured in flash frozen specimens of normal proliferative endometrium (n=8) and UPSC (n =8) by quantitative real-time PCR (qPCR) and Western blot. Established cultures of UPSC cell lines (UPSC-ARK1, UPSC-ARK2) were transfected with either an siRNA targeting ECT2 or a non-targeting control (Dharmacon) using Lipofectamine 2000 (Invitrogen). Reduced expression of ECT2 following transfections with ECT2 siRNAs was confirmed by qPCR and Western blot. Standard MTS and Caspase 3/7 assays (Promega) were utilized to measure proliferation and apoptosis. Colony formation and Boyden chamber assays were used to measure metastatic capacity, migration and invasion in vitro.

Results: Our data indicate that ECT2 is overexpressed >4-fold in nearly all UPSC specimens tested (n=8, p<0.001). We also found that ECT2 is robustly expressed in both the UPSC-ARK1 and UPSC-ARK2 cell lines. Utilizing these 2 cell lines, we found that knockdown of ECT2 significantly decreased proliferation (UPSC-ARK1: 0.62-fold versus control; n=3, p<0.05; UPSC-ARK2: 0.34-fold versus control, p<0.05) and increased rates of apoptosis (UPSC-ARK1: 1.3-fold; n=3, p<0.05; UPSC-ARK2: 2.2-fold, n=3, p<0.05) in both cell lines tested. Decreased ECT2 expression targeted by transfection with siRNAs led to decreased rates of colony formation in UPSC-ARK1 and UPSC-ARK2 cell lines. Knockdown of ECT2 also significantly reduced rates of migration and invasion observed in vitro with both cell lines.

Conclusions: Overexpression of ECT2 plays a critical role in promoting the growth and metastasis of UPSC. Targeting ECT2 expression may provide an effective strategy for improving outcomes for UPSC and other human cancers where hyperactivation of metastasis-promoting Rho GTPases are observed.

Citation Format: Claire M. Mach, Thomas J. Magliaro, Matthew L. Anderson. Overexpression of ECT2 promotes proliferation and metastasis of UPSC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4448. doi:10.1158/1538-7445.AM2014-4448
Abstract 4449: Tissue- and isoform-specific Ras expression during development
Authors: Newlaczyl Anna U. , Coulson Judy M. , Prior Ian A.
Published: May 2015

Ras GTPases are proto-oncogenes that transduce intracellular signals important for diverse cellular processes, such as proliferation and differentiation. The four main Ras isoforms, H-Ras, KRas-4A, KRas-4B and N-Ras, are well conserved across the species and are often mutated in human cancers. However, distinct isoforms preferentially contribute to different cancer types, suggesting non-redundant function.

In order to study Ras isoform prevalence within different tissues and lineages, the transcript copy numbers were examined throughout development using a series of mouse embryo, postnatal and adult tissues. Quantitative RT-PCR was used to measure Ras isoforms in 16 different adult tissues and in 8 of these tissues at 10 developmental time points (n=3). The data showed that Ras abundance is both tissue-specific, with 10-60 copies of total Ras per 1pg RNA in adult tissues, and isoform-specific. KRas-4B and N-Ras were the major isoforms in all tissues. In general Ras isoform expression levels were stable through development except for KRas-4A that showed dramatic upregulation over time in stomach, intestine and kidneys. In heart, KRas-4A transcripts were detected only at and just before birth. Ongoing quantitative proteomics experiments will test whether transcript expression patterns persist at the protein level.

We are building the first complete map of developmental Ras isoform expression. Importantly, establishing baseline expression in normal tissues will give clues to questions about isoform-specific Ras coupling to different cancers.

Citation Format: Anna U. Newlaczyl, Judy M. Coulson, Ian A. Prior. Tissue- and isoform-specific Ras expression during development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2014-4449
Abstract 4455: Targeting a novel kras-integrin-linked kinase regulatory circuitry in pancreatic cancer
Authors: Chu Po-Chen , Yang Ming-Chen , Kulp Samuel K. , Chen Ching-Shih
Published: May 2015

Activating KRAS mutations are the most frequent genetic abnormality in over 90% of pancreatic cancers. Evidence indicates that these mutations not only play a crucial role in initiating pancreas carcinogenesis, but also are required for pancreatic tumor maintenance. From a clinical perspective, oncogenic KRAS represents a therapeutically relevant target in pancreatic cancer, of which the proof-of-concept was demonstrated by the effectiveness of siRNA-mediated silencing of KRAS to suppress pancreatic tumor growth in vivo. Nonetheless, attempts to develop small-molecule agents to inhibit oncogenic KRAS activity have proven futile to date. Consequently, recent strategies have focused on targeting downstream effectors of KRAS signaling, including Raf, MEK, and PI3K kinase, each of which, however, shows limited clinical benefit as single agent. In this study, we have identified a novel KRAS-E2F1-ILK-hnRNP A1 regulatory circuitry that governs the expression of oncogenic KRAS. Integrin-linked kinase (ILK) is a serine/threonine kinase that mediates a diversity of cellular functions including cell survival, cell-matrix interactions, angiogenesis and also plays a role in epithelial to mesenchymal transition (EMT) in cancer cells. Dysregulation of ILK expression has been observed in several tumors including breast, ovary, melanoma, lung, prostate and pancreas and reported to be correlated with tumor progression, metastasis and chemoresistance to gemcitabine in pancreatic adenocarcinoma cells, but the mechanisms by which ILK is required for the tumorogenesis in pancreatic cancer are yet to be understood. In this circuitry, KRAS induces ILK expression via an E2F1-dependent mechanism, which, in turn, stimulates KRAS expression through hnRNP A1 upregulation. Mechanistically, hnRNP A1 binds and relaxes the G-quadruplex structure at the KRAS promoter, thereby enhancing the transcription initiation of the KRAS gene. As a result, disruption of this circuitry via siRNA-mediated knockdown of any of these intermediary effectors (E2F1, ILK, or hnRNP A1), or pharmacological inhibition of ILK by T315, a novel ILK inhibitor developed in our laboratory, led to suppression of KRAS expression and reversal of the mesenchymal phenotype of pancreatic cancer cells. The therapeutic relevance of ILK in regulating this regulatory circuitry is also evident in the suppressive effect of ILK knockdown on epidermal growth factor (EGF)-induced KRAS expression. Together, these findings provide a rationale for targeting ILK as a novel strategy to suppress oncogenic KRAS signaling.

Citation Format: Po-Chen Chu, Ming-Chen Yang, Samuel K. Kulp, Ching-Shih Chen. Targeting a novel kras-integrin-linked kinase regulatory circuitry in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4455. doi:10.1158/1538-7445.AM2014-4455
Abstract 4465: Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems
Authors: Okeley Nicole M. , VanEpps Heather A. , Zhang Xinqun , Setter Jocelyn R. , Burke Patrick J. , Hamilton Joseph Z. , Lyon Robert P.
Published: May 2015

The previously described β-glucuronide linker system (Jeffrey S.C. et al, Bioconj. Chem. 17, 831-840, 2006) was designed to release the toxic payload from an antibody-drug conjugate via the activity of β-glucuronidase, a known lysosomal enzyme. This linker system was shown to be suitable for the generation of potent ADCs utilizing the microtubule disrupting agent monomethylauristatin E (MMAE) as the delivered payload, and is thus an alternative to the protease-cleavable linker valine-citrulline-p-amino benzyl alcohol (vc-PAB). We have recently observed that ADCs utilizing the β-glucuronide linker system to deliver an MMAE payload exhibit potent cytotoxic activity in some cell lines that are resistant to conjugates utilizing the vc-PAB linker to deliver the same drug. To better understand this phenomenon, we have used mass spectrometry to compare the concentration and distribution of MMAE released from both glucuronide and vc-PAB linked ADCs in cell culture. Our data demonstrate that cellular accumulation of released MMAE is considerably greater when delivered by a glucuronide ADC relative to a vc-PAB ADC. We have also employed live-cell fluorescence microscopy to monitor the process of enzymatic cleavage of glucuronide and vc-PAB conjugates. The results of these studies suggest that the two linker systems deliver drug via distinct cellular mechanisms, possibly due to unique intracellular locations of drug release. We propose a model for how these unique linker systems can lead to differences in cellular accumulation of free drug and ultimately increased cytotoxicity.

Citation Format: Nicole M. Okeley, Heather A. VanEpps, Xinqun Zhang, Jocelyn R. Setter, Patrick J. Burke, Joseph Z. Hamilton, Robert P. Lyon. Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2014-4465
Abstract 4470: Elucidating the role of drug-linker hydrophobicity in the disposition of antibody-drug conjugates
Authors: Doronina Svetlana O. , Setter Jocelyn R. , Bovee Tim D. , Anderson Martha E. , Jonas Mechtild , Daniho Steven , Kostner Heather , Senter Peter D. , Lyon Robert P.
Published: May 2015

The in vitro potency of antibody-drug conjugates (ADCs) is generally proportional to the level of drug loading, with higher drug per antibody levels producing more potent ADCs. However, this intuitive relationship often fails to translate in vivo, presumably because plasma clearance of the ADC also increases with drug loading, resulting in substantially reduced exposure to highly loaded ADCs (Hamblett, KJ et al, Clin. Cancer Res. 2004, 10: 7063-7070). We postulated that this accelerated clearance effect is not purely a function of the number of drugs per antibody, but also of the intrinsic hydrophobicity of each drug-linker. Collectively, drug-linker hydrophobicity and number of drugs per antibody determine the overall hydrophobicity of the ADC. To test this hypothesis, we designed and prepared a series of drug-linkers within the auristatin class that varied in hydrophobicity, and used them to produce a corresponding series of homogeneous 8-loaded ADCs. The pharmacokinetic profiles of these ADCs were determined and found to correlate well with their relative retention times by hydrophobic interaction chromatography. We further postulated that hydrophobicity-driven clearance is likely to be mediated by cells of the mononuclear phagocytic system (MPS). To identify the specific cell populations responsible for accelerated ADC clearance, we performed an immunohistochemistry-based biodistribution study which has provided clear evidence for the cellular sites of hydrophobic ADC clearance which are not operative on native antibodies or hydrophilic ADCs. Using the information gained from these studies, we have successfully prepared a hydrophilic auristatin drug-linker which enables the preparation of ADCs uniformly loaded with 8 drugs per antibody at defined cysteine sites. These ADCs evade clearance by the MPS, maintain native antibody pharmacokinetics, and translate their high in vitro potency into the in vivo setting.

Citation Format: Svetlana O. Doronina, Jocelyn R. Setter, Tim D. Bovee, Martha E. Anderson, Mechtild Jonas, Steven Daniho, Heather Kostner, Peter D. Senter, Robert P. Lyon. Elucidating the role of drug-linker hydrophobicity in the disposition of antibody-drug conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2014-4470
Abstract 4474: Protein-photosensitizer nanoparticles for the treatment of cancer
Authors: Benitez Marimar , Molina Anna M. , Griebenow Kai
Published: May 2015

Photodynamic therapy is a treatment that uses photosensitizer drugs to produce reactive oxidative species that kill cells when exposed to light of a specific wavelength. Our research project focuses on the development of protein nanoparticles as delivery systems for photosensitizers in cancer treatments, with the objective to overcome the lack of target specificity resulting in inappropriate drug retention by non-target tissues. The main goal is to produce nanoparticles that selectively accumulate in the target tissue due to their size, are effectively internalized into the cells via receptor-mediated endocytosis and disintegrate in the reducing environment of the cell, resulting in the activation of the drug. The synthesis method has been optimized to obtain nanoparticles of the desired size that disintegrate rapidly in the intracellular space. The photosensitizer is covalently bound to prevent non-specific release, and folate has been immobilized on the nanoparticle surface. The reversible activation of the drug was confirmed by fluorescence experiments and the therapeutic efficiency of the system was evaluated in HeLa cells. The system dark toxicity and phototoxicity was evaluated in vitro, as well as the internalization, the intracellular localization, and the mechanism of cell death.

Note: This abstract was not presented at the meeting.

Citation Format: Marimar Benitez, Anna M. Molina, Kai Griebenow. Protein-photosensitizer nanoparticles for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2014-4474
Abstract 4476: Targeted delivery of curcumin to improve therapeutic outcome in breast cancer
Authors: Singh Rajesh , Lillard James W.
Published: May 2015

Conventional chemotherapeutics offered to treat breast cancer (BrCa) are not selectively targeted. Due to this fact, they are toxic to the normal cells. It is imperative to identify new anti-cancer agents and/or methods to selectively target BrCa cells without affecting normal cells. In this regard, Curcumin, a plant polyphenol, has shown very promising results. Despite the proven anti-tumor effects of curcumin, clinical use of this compound is restricted due to its poor bioavailability. To surmount these problems, we developed nanoparticles, loaded with curcumin, using a planetary-balled milling (PBM) technique with particles tagged with folate for selective targeting to BrCa cells, which express high affinity folate receptors. Multispectral imaging, using an Amnis ImageStream system, was used to measure the response of curcumin-loaded PBM nanoparticles, and free curcumin on various BrCa cell lines. Curcumin-loaded folic acid-coated PBM nanoparticles selectively targeted and were taken-up by BrCa cells and induced apoptosis. Furthermore, our results indicate that targeted delivery of curcumin using PBM nanoparticles was 60 % more effective in killing BrCa cells at concentrations lower than the IC50 of free curcumin.

Citation Format: Rajesh Singh, James W. Lillard. Targeted delivery of curcumin to improve therapeutic outcome in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2014-4476
Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models
Authors: Adler Alexander P. , Daly Christopher , Parveen Asma A. , Nevins Thomas , Shan Jing , Fairhurst Jeanette , Huang Tammy , Martin Joel , Papadopoulos Nicholas , Yancopoulos George D. , Thurston Gavin , Thurston Gavin , Eichten Alexandra
Published: May 2015

Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy.

Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492
Abstract 4507: SL-501, a next-generation targeted therapy directed to IL-3R, inhibits the growth of tyrosine kinase inhibitor-resistant CML cells
Authors: Brooks Christopher , Hoberman Kenneth , Bergstein Ivan , Rowinsky Eric
Published: May 2015

Although imatinib and other tyrosine kinase inhibitors (TKIs) achieve high response rates in patients with chronic myeloid leukemia (CML), patients can fail these therapies or develop specific mutations that render TKIs ineffective. Moreover, TKIs have limited effectiveness against CML cancer stem cells (CSCs). It was previously shown that SL-401, a novel targeted therapy directed to the interleukin 3 receptor (IL-3R), inhibits the growth of CML tumor bulk and CSCs, both of which over express IL-3R. SL-501 is a next-generation targeted therapy also directed to IL-3R. SL-501 has approximately 4-fold increased binding affinity for IL-3R and a more than 30-fold increase in potency against acute myeloid leukemia (AML) cells compared to SL-401. In this study, we assessed the activity of SL-501 against a panel of TKI-sensitive and TKI-resistant CML cell lines. KBM5, KBM5-STI, K562, and BV173 cells were treated with SL-501 (range: 5 - 21,000 pg) for 24 or 48 hours and assessed for viability and apoptosis. We found that SL-501 reduced the absolute cell number of all cell lines tested in a dose-dependent manner with IC50s of 20 pM, 21 pM, 31.5 nM, and 3.9 nM, respectively; these concentrations are readily achieved at tolerable doses in the clinic. Moreover, SL-501 significantly reduced the number of imatinib-resistant KBM5-STI viable cells by more than 5-fold after 48 hours. Using caspase and lactate dehydrogenase (LDH) release assays, we also found that SL-501 alone induced apoptosis of all cell lines tested by more than 40% after 24 hours. The combination of SL-501 with TKIs (imatinib, dasatinib, or nilotinib) had an enhanced effect on apoptosis in KBM5, K562, and BV173 cells 24 hours post-treatment. Taken together, these findings indicate that SL-501, a novel next-generation IL-3-targeted therapeutic, may be effective at treating TKI-resistant CML. These promising results together with the IL-3R expression pattern on CML blasts and CSCs warrant further development of SL-501 in CML and, in particular, TKI-resistant CML.

Citation Format: Christopher Brooks, Kenneth Hoberman, Ivan Bergstein, Eric Rowinsky. SL-501, a next-generation targeted therapy directed to IL-3R, inhibits the growth of tyrosine kinase inhibitor-resistant CML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2014-4507
Abstract 4510: RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors
Authors: Niederfellner Gerhard , Bauss Frieder , Imhof-Jung Sabine , Hesse Friederike , Kronenberg Sven , Staak Roland , Lechmann Martin , Krippendorff Ben , Richter Wolfgang , Mateus Rita , Kollmorgen Gwendlyn , Brinkmann Ulli , Onda Masanori , Pastan Ira , Bosslet Klaus
Published: May 2015

Attaching toxic payloads to antibodies has recently been established as a breakthrough in cancer therapy. Most of the currently developed antibody drug conjugate programs represent targeted chemotherapy with microtubule polymerization inhibiting drugs and hence share resistance mechanisms and side effects with classical chemotherapeutics like Taxol. Inhibition of protein synthesis by Pseudomonas Exotoxin (PE) is a more powerful mode of action that interferes with all hallmarks of cancer cells, not just with proliferation. However, its clinical use has been limited by immunogenicity as shown for SS1P, a mesothelin targeted immunotoxin. We have developed a novel de-immunized PE fusion protein (RG7787) for treatment of mesothelin-positive tumors.

In order to de-immunize PE, point mutations have been introduced into domain III and the entire domain II has been deleted reducing the size of the effector moiety to approximately 24 kD. PE24 fusions with a disulfide-stabilized Fv fragment have previously been shown to be much better tolerated in rodents, but this could, at least in part, also be attributed to a much reduced serum half-life and exposure. In order to also de-immunize the targeting moiety of RG7787 and restore PK parameters similar to those of SS1P, we substituted the mouse dsFv moiety by a humanized Fab fragment. We show that RG7787 has indeed similar PK properties to SS1P in mouse and cyno.

Cell viability assays show that RG7787 has similar cytotoxic potency as SS1P on different cell lines. In-vivo equipotency to SS1P in different xenograft models has been achieved at ∼3 fold higher doses indicating that tumor penetration of SS1P might be slightly better. However, RG7787 is up to tenfold better tolerated in rodents and cynomolgus monkeys, indicating that the therapeutic window is improved. RG7787 achieves potent tumor growth inhibition and even tumor regressions in several xenograft models. We also observed clearly synergistic efficacy with Taxol treatment in different tumor models making this a promising combination for clinical trials.

Citation Format: Gerhard Niederfellner, Frieder Bauss, Sabine Imhof-Jung, Friederike Hesse, Sven Kronenberg, Roland Staak, Martin Lechmann, Ben Krippendorff, Wolfgang Richter, Rita Mateus, Gwendlyn Kollmorgen, Ulli Brinkmann, Masanori Onda, Ira Pastan, Klaus Bosslet. RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2014-4510
Abstract 4523: Chaetoglobosin K, a dual Akt and JNK inhibitor, modulates Akt phosphorylation in an mTORC2 independent manner
Authors: Ali Amna , Matesic Diane F.
Published: May 2015

Chaetoglobosin K (ChK) is a natural indolylcytochalasin that has been reported to inhibit the proliferation of human lung and ovarian carcinoma cells in vitro. We have previously shown that ChK concomitantly down-regulates Akt and JNK activation, and have recently determined that the inhibition of Akt by ChK is not via direct inhibition of the upstream enzyme PI3 kinase. The reduction in Akt activation after ChK treatment is also not related to changes in phosphorylation of the upstream modulators PDK1 or PTEN. The mammalian target of rapamycin (mTOR) is a kinase that regulates cell growth and protein synthesis and can be both upstream (mTORC2) and downstream (mTORC1) of Akt. The purpose of this study was to determine whether ChK: 1) decreases the activation of upstream mTORC2, as this may lead to a decrease in Akt phosphorylation; 2) alters the phosphorylation of downstream mTORC1 mediators 4E-BP1 and p70 S6 kinase. In these studies, ras-transformed rat liver epithelial cells were treated with either a non-cytotoxic dose of ChK or vehicle. Western blot analysis was performed using phosphorylation site-specific antibodies to monitor changes in 4E-BP1, p70 S6 kinase, and mTOR protein activation levels. The results of this study indicate that ChK treatment did not decrease mTORC2 activation levels and suggest that ChK decreases Akt phosphorylation through an alternate mechanism. ChK treatment decreased the phosphorylation of 4E-BP1 and p70 S6 kinase, confirming that ChK modulates downstream Akt/mTOR pathway effectors. Further investigations are warranted to determine the exact mechanism by which ChK down-regulates Akt, as well as JNK, activation in tumorigenic cells.

This project was funded by NIH grant 1R15CA135415

Citation Format: Amna Ali, Diane F. Matesic. Chaetoglobosin K, a dual Akt and JNK inhibitor, modulates Akt phosphorylation in an mTORC2 independent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2014-4523
Abstract 4525: mTOR complex inhibition as a novel therapeutic strategy in high-grade papillary serous ovarian cancer
Authors: Musa Fernanda , Alard Amandine , David-West Gizelka , Giuroiu Iulia , Blank Stephanie , Pothuri Bhavana , Curtin John P. , Schneider Robert
Published: May 2015

Objectives:

To compare mTOR complex 1 inhibition (RAD001) vs. mTOR complex 1/2 inhibition (PP242) as single agents and with Carboplatin (CPP) in a preclinical model of serous ovarian cancer (OVCA).

Methods:

In vitro: OVCAR3 and SKOV3 cell lines were exposed to the following treatment conditions: RAD001 or PP242 as single agents, vehicle, CPP alone, and RAD001 or PP242 followed by CPP. Colony forming assays (CFAs) were performed and quantified. mRNA levels of AKT, downstream targets of mTOR and the DNA repair response (ATR, ATM, BRCA1/2) were quantified by qRTPCR. Immunoblots characterized the protein expression of key components of mTOR and DNA repair pathways. Non-parametric analyses were used to compare results across groups on SPSS. In vivo: OVCAR3 cells expressing F-Luciferase were injected IP into SCID-BG mice. 15 wks post-injection, mice were exposed to the treatment conditions described above for 4 weeks. Tumor growth and response to treatment were assessed using bioluminescence imaging (IVIS). Results were analyzed on Living Image and Prism6.

Results:

In vitro: OVCAR3 and SKOV3 cells are highly sensitive to mTOR inhibition. CFAs showed significantly decreased colony counts and diameter in groups exposed to either PP242 or RAD001 vs. control, an effect that was potentiated by CPP (Fig 1). qRTPCR revealed a significant decrease in 4EBP1 mRNA with mTOR inhibition (p<0.0001), but no change in other biomarkers. Although treatment with both mTOR inhibitors resulted in decreased expression of p-S6 by immunoblots, treatment with the dual inhibitor (PP242) caused a decrease in p-AKT, p-4EBP1, total and p-CHK1, and total and p-BRCA1. The levels of these proteins were not changed by addition of CPP despite the significant effect observed in the functional assay (CFA). In vivo: Treatment with CPP + PP242 was associated with a longer median survival than other treatment groups (Table 1). A decrease in tumor burden was seen on IVIS and tumor flux (photons/sec) at the end of treatment was significantly lower in mice treated with CPP+PP242 compared to other groups (p<0.0001).

Conclusions:

Our preclinical model supports the concurrent use of dual mTOR inhibitors and platinum chemotherapy in the treatment of OVCA. mTOR complex 1/2 inhibition impairs the DNA repair response and correlates with improved survival in a murine model.

Citation Format: Fernanda Musa, Amandine Alard, Gizelka David-West, Iulia Giuroiu, Stephanie Blank, Bhavana Pothuri, John P. Curtin, Robert Schneider. mTOR complex inhibition as a novel therapeutic strategy in high-grade papillary serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4525. doi:10.1158/1538-7445.AM2014-4525