bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4360: Altering the regulation of KRAS GTPase cycle via Src and SHP2 creates a potential therapeutic vulnerability for pancreatic cancer
Authors: Gebregiworgis Teklab , Marshall Christopher B. , Kano Yoshihito , Radulovich Nikolina , Tsao Ming-Sound , Ohh Michael , Ikura Mitsuhiko
Published: August 2018

More than 30% of all human cancers have Ras mutations and more than 95% of pancreatic cancers harbor KRAS mutations. However, therapeutically targeting cancers driven by oncogenic Ras mutations is still an ongoing investigation. KRas is a small GTPase found in either a GDP-bound inactive form or a GTP-loaded activated form. The activated form of KRas localizes on the membrane where it binds and activates downstream effector proteins such as Raf kinases. GDP-bound Ras can be activated by nucleotide exchange of GTP for GDP, a reaction that is catalyzed by guanine nucleotide exchange factors (GEFs). Ras is inactivated by hydrolysis of GTP, which is assisted by GTPase activating proteins (GAPs). It was recently reported that Ras can be tyrosyl phosphorylated by Src and dephosphorylated by SHP2 (PNAS 2014;111(36):E3785-94; Nat Commun 2015;6:8859). Here we have characterized the structural and functional alterations of Src-phosphorylated KRas to mechanistically explain the impact of tyrosyl phosphorylation on the GTPase cycle. Our NMR and MS analyses show that Src phosphorylates KRas at Tyr32 and Tyr64, which perturbs the chemical shifts of several residues in each of the two "switch" regions that mediate interactions with effectors and regulators. Using real-time NMR and biolayer interferometry (Octet) assays, we demonstrated the negative impact of KRas phosphorylation on the GTPase cycle as well as BRAF binding. Conversely, the SH2 domain containing inositol 5-phosphatase 2 (SHP2) dephosphorylates KRAS and reverses Src-induced phosphorylation. In vivo, either the pharmacologic inhibition or genetic ablation of SHP2 promotes cell death in several KRAS mutant cancers. Our findings reveal that altering the KRas GTPase cycle regulation via the balance of Src and SHIP2 activities may create a therapeutic vulnerability for pancreatic cancer.

Citation Format: Teklab Gebregiworgis, Christopher B. Marshall, Yoshihito Kano, Nikolina Radulovich, Ming-Sound Tsao, Michael Ohh, Mitsuhiko Ikura. Altering the regulation of KRAS GTPase cycle via Src and SHP2 creates a potential therapeutic vulnerability for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4360.
Abstract 4365: Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma
Authors: Lin Wan-Hsin , Feathers Ryan , Lewis-Tuffin Laura , Anastasiadis Panos
Published: August 2018

The RhoA GTPase is thought to play a crucial role in cancer development. Its activation is tightly controlled by numerous guanine nucleotide-exchange factors (GEFs). We previously showed that Syx, a RhoA-GEF, promotes junctional integrity in endothelial cells and governs cell polarity and directed migration in glioblastoma (GBM) cells. GBM is a grade IV glioma with a median survival of approximately 14 months. Importantly, the gene locus of Syx (Plekhg5) is located within chromosome 1p36, a region deleted in oligodendroglioma (a type II/III glioma) and associated with better prognosis, suggesting a role of Syx in GBM aggressiveness. Here we show that Syx is a potential target for the treatment of GBM. Knockdown of Syx resulted in altered expression of cell cycle regulators, including cyclins A, B, and E, cyclin-dependent kinase inhibitors (CDIs), namely p21 and p27, and mitosis modulators CDC20 and survivin. These defects resulted in G2/M arrest and dysfunction of cell division, leading to improper chromosome segregation and DNA double-strand breaks. However, DNA damage response could not be activated effectively for DNA repair, leading to cellular apoptosis. Consistent with these findings, we used an orthotopic patient-derived xenograft GBM model to show that Syx silencing results in decreased tumor growth accompanied by increased animal survival. Agents that cause DNA damage and/or disruption of DNA repair pathways are often used for cancer therapy. We thus tested the possibility of targeting Syx for GBM treatment, and showed that Syx knockdown in combination with temozolomide, a first-line drug for GBM, synergistically inhibit cell growth. In summary, this is the first study showing the role of Syx in GBM cell growth and that targeting this signaling pathway can be beneficial for GBM therapy.

Citation Format: Wan-Hsin Lin, Ryan Feathers, Laura Lewis-Tuffin, Panos Anastasiadis. Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4365.
Abstract 4368: PAK4-NAMPT dual inhibition as a feasible strategy for treatment of resistant pancreatic neuroendocrine tumors
Authors: Mpilla Gabriel , Muqbil Irfana , Aboukameel Amro , Philip Philip A. , Senapedis William , Baloglu Erkan , Landesman Yosef , Kauffman Michael , Shacham Sharon , Mohammad Ramzi M. , Azmi Asfar S.
Published: August 2018

Our studies in pancreatic neuroendocrine tumor (pNET) cell lines demonstrate hyper-activation of the Rho GTPase effector p21 activated kinase 4 (PAK4) and the nicotinamide adenine dinucleotide (NAD) salvage pathway rate limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). The PAK4 protein is known to regulate a myriad of signaling proteins in the mTOR pathway including mTORC1, mTORC2, PI3K, and IGF-1. Similarly, NAMPT is recognized to regulate mTOR through energy sensor protein, AMPK. In this study, pNET cell lines QGP-1 and Bon-1 were subjected to either PAK4 RNA interference (RNAi), PAK4-NAMPT dual inhibitors (the clinical compound KPT-9274 or an analog KPT-7523) or the PAK4 specific inhibitor, PF-3738309 and NAMPT specific inhibitor FK866 in the presence or absence of mTOR inhibitors (everolimus or INK128). Gene expression profiling and phospho-proteomic analyses were performed to capture molecular changes post single or combination treatments. The anti-tumor activity of KPT-9274-everolimus was evaluated in a subcutaneous xenograft mouse model derived from QGP-1 and Bon-1 cells. PAK4 RNAi suppressed proliferation and restored everolimus sensitivity in pNET cell lines. The dual inhibitors were effective in reducing proliferation and inducing apoptosis. KPT-9274 or KPT-7523 could synergistically enhance the anti-tumor activity of everolimus or INK128 in pNET cell lines [CI <1]. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers such as mTORC1, mTORC2, PI3K, ERK, FAK, RICTOR, β-catenin and IGF-1. In addition, KPT-9274 and analogs suppressed the steady state level of NAD and ATP. Importantly, KPT-9274 given i.v. or orally at the maximum tolerated dose (140 mg/kg, once/day for 5 days/week for 4 weeks) dramatically inhibited the growth of QGP-1 and Bon-1 tumors. This is the first study demonstrating the role of PAK4 and NAMPT in pNETs. KPT-9274 is currently in a Phase I trial of patients with advance solid malignancies or NHL (NCT02702492). Our pre-clinical work establishes a solid rationale for a Phase II clinical study of KPT-9274 and an mTOR inhibitor combination for the treatment of difficult to treat pNETs.

Citation Format: Gabriel Mpilla, Irfana Muqbil, Amro Aboukameel, Philip A. Philip, William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Asfar S. Azmi. PAK4-NAMPT dual inhibition as a feasible strategy for treatment of resistant pancreatic neuroendocrine tumors [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 4368.
Abstract 437: Leveraging new methods in single-cell copy number analysis and clonotype detection to uncover and characterize hidden subclones within standard cell lines
Authors: Villarreal Enrique I. Velazquez , Kumar Vijay , Yin Yifeng , Carpten John D. , Craig David W.
Published: August 2018

Single-cell sequencing is an important and powerful tool for improving our understanding of cancer heterogeneity. While previous reports have largely focused on single-cell RNA-seq, we examined the utility of novel approaches for single-cell copy-number analysis and clonotype detection using 10x Chromium™ Technology capable of processing thousands of single cells. In contrast to bulk sequencing, this technology is able to call cell clonotypes down to 1% of 1,000 cell inputs, while providing detection of CNVs down to 100 Kb events on clones consisting of 10 or more cells. As a pilot study, we processed 300 single cells from COLO829 through the system, generating a barcoded short-read library, which was sequenced to a raw depth of approximately 600 million reads. Previous efforts had characterized COLO829 by bulk whole-genome sequencing of tumor/normal cell-line lineages grown and sequenced across four laboratories to help establish tumor/normal reference standards. We used barcode-aware bioinformatic analysis to recover reads derived from single cells, analyzing them in 20 Kb cassettes of reads providing both regional copy number and genome-wide ploidy estimates. This high resolution of CNV detection is enabled by aggregation of reads across cells within the same clone. Somewhat surprisingly, we find the presence of multiple clones within a single cell line growth evident by large-scale copy number changes, in some cases spanning entire chromosome arms. We show an ability to better resolve absolute copy number, whereas previous analysis approaches on bulk sequencing have lagged due to a reliance on indirect inferences. Consideration of prior publications on this cell line are consistent with the undescribed existence of clonal heterogeneity in these, and when considered with these data provide greater insight into copy number-driven events. Overall, our results suggest that single-cell copy number provides unique and important insight into larger-scale events, providing unique and distinct insight into cancer heterogeneity.

Citation Format: Enrique I. Velazquez Villarreal, Vijay Kumar, Yifeng Yin, John D. Carpten, David W. Craig. Leveraging new methods in single-cell copy number analysis and clonotype detection to uncover and characterize hidden subclones within standard cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 437.
Abstract 4370: Regulation of AGO2-KRAS interaction through epidermal growth factor receptor
Authors: Siebenaler Ronald F. , Shankar Sunita , Tien Jean C. , Dommeti Vijaya L. , Mody Malay , Kumar-Sinha Chandan , Chinnaiyan Arul M.
Published: August 2018

The RAS gene family is among the most commonly mutated genes within cancer. While much research has elucidated its major functions and downstream pathways, little progress has been made in successfully targeting RAS mutations. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the Switch II domain of KRAS. We have found that stable knockdown of AGO2 in KRAS-dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. In addition, we observed a decrease in microRNA unwinding in the presence of mutant KRAS, suggesting this interaction inhibits the endogenous RNAi function of AGO2. Despite this clear connection between KRAS and AGO2 in KRAS mediated oncogenesis, the precise function of this interaction remains unclear in both normal and cancer biology.

In order to identify endogenous regulators of AGO2-RAS, we investigated the ability of EGFR signaling to modulate the AGO2-RAS interaction. We established two mouse embryonic fibroblast cell lines (NIH-3T3 and MEF) with complete knockout of AGO2. When compared to normal control cells, we found that knockout of AGO2 resulted in an increase in WT RAS-GTP activation levels, phosphorylation of Y1068-EGFR, and MAPK/ERK and PI3K/AKT signaling. Rescue of AGO2 knockout resulted in a return to normal levels of active RAS-GTP, pEGFR, and downstream signaling.

Recent studies have described EGFR phosphorylation of AGO2 under hypoxic cell conditions, resulting in the inhibition of AGO2 association with RISC members. In order to better characterize the relationship between EGFR-AGO2-RAS, we found that overnight serum starvation followed by stimulation with EGF led to a decrease in AGO2-RAS co-IP in WT KRAS cells. Blocking AGO2 phosphorylation with a Y393F mutant of AGO2 prevented AGO2-RAS dissociation following EGFR stimulation. While WT KRAS cell lines displayed regulation of AGO2-RAS via EGFR, the phosphorylation of AGO2Y393 was unable to disrupt the interaction of AGO2 with mutant KRAS following stimulation with EGF. Together these results suggest a unique EGFR-AGO2-RAS signaling axis, and its dysregulation by mutant KRAS could increase oncogenic growth through promotion of AGO2-RAS interaction in cancer.

Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Malay Mody, Chandan Kumar-Sinha, Arul M. Chinnaiyan. Regulation of AGO2-KRAS interaction through epidermal growth factor receptor [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 4370.
Abstract 4372: SET/PP2A/SHP-1/Lyn oncogenic signaling contributes to tumor aggressiveness in diffuse large B cell lymphoma
Authors: Chen Ji-Lin , Tsai Wen-Chun , Lan Tien-Yun , Huang Chun-Teng , Chu Pei-Yi , Lee Chia-Han , Lau Ka-Yi , Wang Wan-Lun , Chen Kuen-Feng , Shiau Chung-Wai , Liu Chun-Yu
Published: August 2018

Purpose:

Diffuse large B-cell lymphoma (DLBCL) is aggressive non-Hodgkin lymphoma. It is a heterogeneous disease and is classified as germinal center B-cell like (GCB) and activated B-cell like (ABC) subtypes according to gene-expression profiling. The oncogenic pathways in DLBCL are potential targets for new therapies. In present study, the role and regulation of SET/PP2A/SHP-1/Lyn axis in DLBCL were investigated.

Experimental design: U2932, OCL-Ly3, OCI-Ly7, SU-DHL-6 and DB DLBCL cell lines were used for this study. TD-19, a novel SET/PP2A protein-protein interaction inhibitor, was used to address the molecular events of SET signaling. Cell viability was measured by MTT assay. The apoptotic cells were examined by PI/Annexin V staining and Western blotting. SHP-1, Lyn and pLyn were analyzed by immunohistochemistry on lymphoma tissue microarrays from DLBCL patients.

Results:

We first examined the effects of two different SET inhibitors, TD-19 that is a SET/PP2A protein protein interaction inhibitor, and FTY720 which is a sphingosine analogue that targets SET, on DLBCL cells. TD-19 and FTY720 significantly decreased cell viability and induced apoptosis. SET inhibition activated PP2A and SHP-1 by reducing pPP2AY307 and pSHP-1S591 and inactivated Lyn by decreasing pLynY396. Overexpression of SET rescued these molecular events, and promoted cell growth and migration. Interestingly, we observed the level of SET was declined by TD-19 and TFY720 treatment. We hypothesized Lyn might up-regulate SET expression. Overexpression of Lyn increased SET level, and was accompanied with phosphorylation of PP2A (pPP2AY307) and SHP-1 (pSHP-1S591). Lyn elevated cell growth and migration and suppressed PP2A and SHP-1 activities. Moreover, exogenous SET restored the FTY720- and TD-19-suppressed pSHP-1 and pLyn. Immunohistochemically, high SHP-1 level was linked to low pLyn level of patient with DLBCL and vice versa.

Conclusion: This study established the existence of positive feedback of SET/PP2A/SHP-1/Lyn axis in DLBCL cells, and targeting SET could disrupt this regulation.

Citation Format: Ji-Lin Chen, Wen-Chun Tsai, Tien-Yun Lan, Chun-Teng Huang, Pei-Yi Chu, Chia-Han Lee, Ka-Yi Lau, Wan-Lun Wang, Kuen-Feng Chen, Chung-Wai Shiau, Chun-Yu Liu. SET/PP2A/SHP-1/Lyn oncogenic signaling contributes to tumor aggressiveness in diffuse large B cell lymphoma [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 4372.
Abstract 4378: Novel CARP-1- H2AX interaction regulates apoptosis in breast cancer cells
Authors: Sekhar Sreeja C. , Cheriyan Vino T. , Rishi Arun K.
Published: August 2018

CCAR1/CARP-1 is a peri-nuclear phosphoprotein that regulates apoptosis signaling by breast cancer therapeutic Doxorubicin and a novel class of CARP-1 Functional Mimetic (CFM) compounds. Prior reports have established that Doxorubicin exposure causes DNA damage in cells. Phosphorylation of histone 2AX (often referred as γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Although, currently it is unclear whether CFMs also induce DNA damage, we found that Doxorubicin or CFM-4.16 treatments caused elevated CARP-1 and γH2AX in HBC cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in HBC cells undergoing apoptosis. Knock-down of CARP-1 abolished increase of CARP-1 and γH2AX, their co-localization, and apoptosis in CFM-4.16 or Doxorubicin-treated HBC cells. We found that CARP-1 directly binds with H2AX, and the H2AX-interacting epitope is located within CARP-1 amino acids 600 to 652. We generated HBC cells that have stable expression of myc-tagged, wild-type CARP-1 (1-1150) or CARP-1 (1-1150; Δ600-652) mutant. Further analyses revealed that CARP-1 (1-1150), but not CARP-1 (1-1150; Δ600-652) mutant, interacted with H2AX. Moreover, HBC cells expressing CARP-1 (1-1150; Δ600-652) mutant were resistant to apoptosis by Doxorubicin or CFM-4.16, and had diminished levels of γH2AX, when compared with their counterparts expressing wild-type CARP-1. We have earlier reported involvement of the Stress-Activated Protein Kinases (SAPKs) such as p38 and JNKs in apoptosis by Doxorubicin or CFMs. Since the PI3K family of serine-threonine kinases ATM/ATR, as well as JNK SAPKs, phosphorylate serine-139 of H2AX following DNA damage, our current studies are investigating whether H2AX binding with CARP-1 serves to facilitate phosphorylation of CARP-1 and H2AX proteins for transduction of apoptosis signaling.

Citation Format: Sreeja C. Sekhar, Vino T. Cheriyan, Arun K. Rishi. Novel CARP-1- H2AX interaction regulates apoptosis in breast 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 4378.
Abstract 4381: Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of cFLIPL expression
Authors: Jang Ik-Soon , Jang Hyun-Jin , Jo Eunbi
Published: August 2018

Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIPL negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIPL inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIPL) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIPL, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of cFLIPL prevented TNF-α-induced JNK inactivation, whereas c-FLIPL overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIPL expression, thus inducing TK-10 cell apoptosis.

Citation Format: Ik-Soon Jang, Hyun-Jin Jang, Eunbi Jo. Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of cFLIPL expression [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 4381.
Abstract 4383: CD44s is a crucial ATG7 downstream regulator for formation of sphere, a key stem-like property, invasion and lung metastasis of human bladder cancer/BC cells
Authors: Hua Xiaohui , Che Xun , Xu Jiheng , Huang Chuanshu , Huang Grace , Dai Wei
Published: August 2018

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its specific shRNA dramatically reduced Sphere formation and invasion in vitro, as well as lung metastasis in vivo in invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 and CD133, while ectopic introduction of CD44s, but not CD133, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44's protein, resulting in the stabilization of CD44s protein. Moreover, inhibition of ATG7 stabilized AUF1 protein and in turn reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, thereby reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

Citation Format: Xiaohui Hua, Xun Che, Jiheng Xu, Chuanshu Huang, Grace Huang, Wei Dai. CD44s is a crucial ATG7 downstream regulator for formation of sphere, a key stem-like property, invasion and lung metastasis of human bladder cancer/BC 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 4383.
Abstract 4384: The differential induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells in response to RBBP6 silencing is associated with p53 expression
Authors: Moela Pontsho , Motadi Lesetja Raymond
Published: August 2018

Breast cancer remains a female-related health problem on a global scale, accounting for over a million newly estimated cases that are still on the rise. More than 50% cases of breast tumorigenesis are due to mutations or inactivation of p53, a tumor suppressor gene involved in the regulation of cell cycle and apoptosis. In breast cancer, the frequency of TP53 mutations varies greatly between the heterogeneous subtypes, with basal-like breast cancers having the highest frequency whereas the luminal subtypes have been shown to generally express wt. p53. Active p53 is responsible for sensing any cellular stress signal with a potential to damage DNA, thus deciding the cell's fate depending on whether the damage is reversible or not. Therefore cellular p53 levels remain in check by the MDM2 regulatory protein, which promotes the degradation of unwanted p53 via a negative feedback loop mechanism using its E3 ligase activity. However, this interaction between MDM2 and wild-type p53 is cancer promoting in the same way that E6 oncoprotein is capable of leading to the degradation of wild-type p53 in cervical cancer. Negative regulators of p53, therefore, threaten cancer repression and play a role in drug resistance among cancer patients. Of particular interest in this study is RBBP6, another negative regulator of p53 that uses the E3 ligase activity as well in order to promote p53 degradation. Previous studies have shown that silencing RBBP6 in breast cancer restores p53 followed by apoptosis induction. However, it is unclear whether the observed apoptosis is linked to the recovered p53. To test this hypothesis, we aim to analyze the differential susceptibilities of MCF-7 and MDA-MB-231 cells expressing wt. p53 and mt. p53, respectively, to RBBP6 silencing. Real-time cell analysis revealed a clear distinction between the growth of the two cell lines, with MCF-7 showing a significantly reduced proliferation in comparison to MDA-MB-231. Apoptosis induction was also high in MCF-7 AND lower is MDA-MB-231, as was further confirmed by the differential expression of pro-apoptotic genes between the two cell lines. These findings suggest that difference in p53 statuses of the two cell lines play a role in apoptosis induced by RBBP6 silencing. These findings are necessary for reaching a better understanding of molecular mechanisms that impact patient response and resistance to candidate therapeutics and may be helpful in developing more effective treatment strategies for different subtypes of breast cancer.

Citation Format: Pontsho Moela, Lesetja Raymond Motadi. The differential induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells in response to RBBP6 silencing is associated with p53 expression [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 4384.
Abstract 4388: Anti-cancer effects of oleuropein olive leaf extract in K562 leukemia cells
Authors: Fagan Sophia , Fulco Natalie , Gilbertson Justin , Dyke Aaron R. Van , Phelan Shelley A.
Published: August 2018

Olive leaves come from a Mediterranean plant that many believe to have positive effects on human health, including anti-cancer properties. Oleuropein is one of the two main phenolic compounds derived from olive leaves, and it has been shown to increase apoptosis in many types of cancer cells. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, suggesting that the compound could be effective in anti-cancer treatments. The purpose of this study was to examine the effects of oleuropein extract on cell growth and viability in K562 human leukemia cells, and begin to investigate possible molecular mechanisms of action. We purified our own extract from olive leaves for these studies. Oleuropein was microwave extracted from olive leaf powder using methanol and water as solvent. The oleuropein was successfully identified using HPLC to characterize elution time in comparison to an authentic standard. HPLC was then used in purification and fractions were lyophilized. Isolated oleuropein was greater than 94% in comparison to the authentic standard. We next demonstrated that K562 cells treated with 50-150 ug/ml of oleuropein exhibited significant reduction in cell growth and cell viability after four days when compared to 70% ethanol-treated controls. This effect was markedly greater after 6 days . Current studies are being conducted to determine the most effective concentration and duration of treatment for oleuropein-induced cell death. We are also examining cellular cyytotoxicity and cell cycle progression in these experiments using LDH assay and cell cycle staging, and further studies are being conducted to examine inflammatory and antioxidant pathways for their involvement in the anti-cancer effect of the compound in these cells. Given the observation that many of the cytotoxic effects of oleuropein are cancer-cell specific, this research provides a promising new avenue for the use of natural products as anti-cancer agents.

Citation Format: Sophia Fagan, Natalie Fulco, Justin Gilbertson, Aaron R. Van Dyke, Shelley A. Phelan. Anti-cancer effects of oleuropein olive leaf extract in K562 leukemia 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 4388.
Abstract 4397: Tak-1 inhibition re-sensitizes cancer cells to TNFα
Authors: Scarneo Scott , Sell Madeline , Totzke Juliane , Carlson David , Hughes Phillip , Haystead Tim
Published: August 2018

Identifying new pharmaceutical targets in cancer has been a long lasting goal of academia. Complex genomic changes coupled with a cancer cell's ability to adapt make tumors particularly difficult to target. To this end, we have identified the TAK1 kinase in the TNFα pathway as a potential mediator of cell survival or apoptosis. Here we evaluate the apoptotic effects of a novel TAK1 inhibitor, Takinib. The selectivity of Takinib was established with an IC50 of 9nm for TAK1, followed by an IC50 for IRAK4 and IRAK1 at 120nm and 390nm respectively. Treatment of MDA-MB-231 cells with Takinib on its own is not sufficient to induce consequential cell death. However, combination therapy with TNFα induces caspase 3/7 activity and subsequent cell death in a dose-dependent manner. Genetic knock out of TAK1 expression in MDA-MB-231 cells with the CRISPR/Cas9 system significantly sensitized previous resistant cells to TNFα. Furthermore, we have shown that activation of human THP-1 cells with LPS is sufficient to produce TNFα levels necessary to facilitate Takinib mediated cell death, indicating that potential immunotherapy applications may be possible. Together these findings support the idea that TAK1 may be an effective target in the TNFα pathway, shifting the cellular mechanisms from pro survival to pro apoptosis.

Citation Format: Scott Scarneo, Madeline Sell, Juliane Totzke, David Carlson, Phillip Hughes, Tim Haystead. Tak-1 inhibition re-sensitizes cancer cells to TNFα [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 4397.
Abstract 44: Characterization and targeting of a temporal micro-metastatic signature in human brain metastases
Authors: Singh Mohini , Venugopal Chitra , Tokar Tomas , McFarlane Nicole , Subhapanditha Minomi , Bakhshinyan David , Qazi Maleeha , Vora Parvez , Savage Neil , Murty Naresh K. , Jurisica Igor , Singh Sheila K.
Published: August 2018

Metastases to the brain (BM) are the most common neoplasms to affect the adult central nervous system, arising in 40% of cancer patients and at a rate 10 times greater than primary brain tumors. Despite the prevalence and poor survival rates, therapeutic strategies for BM remain limited, and a substantial cause is the lack of proper preclinical models available to interrogate the intricacies of BM development. Previous work in our lab utilized BM samples from patient-derived lung-to-brain metastases to successfully establish clinically relevant in vivo models of BM. Here we further characterized the cells responsible for BM, termed brain metastasis initiation cells (BMIC). Patient-derived BMICs were injected via intracranial (BT), intracardiac (IC) and intrathoracic (IT) routes into NOD/SCID mice and re-isolated at different stages of metastatic progression. We isolated cells from primary lung tumors (LT), cells injected via intra-thoracic route that crossed the blood brain barrier to seed the brain forming micro-metastases (BMIT), and cells injected via intracardiac route that seeded large macro-metastases (BMIC). Through RNA sequencing we determined cells from BMIT (micro-metastasis stage) to retain a vastly different genetic profile compared to BMICs isolated at other stages of metastasis. Several of these genes belonged to pathways implicated in autonomic central nervous system neoplasms and neural development. Through connectivity mapping of BMIT profiles we discovered drugs that could inhibit the micro-metatsasis signature, and further in vitro validation revealed apomorphine to reduce BMIC sphere formation and proliferation. In vivo treatment with apomorphine blocked both micro- and macro-metastatic stages of our BMIC model. Of 5 BMIT genes identified to be specifically targeted by apomorphine, KIF16B, TESK2 and SEPW1 were shown to have significant value when applied as an independent prognostic signature in a cohort of lung adenocarcinoma patients. Future work will further validate the efficacy of apomorphine in targeting BMICs in primary lung cancer patient samples. With this work we present a possible new avenue for therapeutic targeting toward the prevention of BM development, where it is anticipated to transform a uniformly fatal disease into one that is eminently more treatable.

Citation Format: Mohini Singh, Chitra Venugopal, Tomas Tokar, Nicole McFarlane, Minomi Subhapanditha, David Bakhshinyan, Maleeha Qazi, Parvez Vora, Neil Savage, Naresh K. Murty, Igor Jurisica, Sheila K. Singh. Characterization and targeting of a temporal micro-metastatic signature in human brain metastases [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 44.
Abstract 4400: MicroRNA loaded circular dumbbell RNAs, a novel antiangiogenic approach
Authors: Gnanamony Manu , lakka Sajani S. , Libes Jaime , Lin Julian , Joseph Pushpa A. , Gondi Christopher S.
Published: August 2018

Angiogenesis is a tightly regulated biological process where new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer; therefore, angiogenesis has been actively explored as a drug target for cancer therapy. Many patients treated with endogenous angiogenesis inhibitors show increased survival; however, eventual resistance to therapies is associated with disease progression and death. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. We therefore propose a novel and ‘out of the box' approach that utilizes dumbbell-RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. Dumbbell RNAs are a stable and safe alternative to viral, non-viral, and naked plasmid-based gene-transfer systems. They integrate the advantages of durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA. In this study, we have successfully constructed a plasmid expressing miR-34a dbRNA using the permuted intron-exon (PIE) method. We have also modified and optimized a cost effective protocol to purifiy dbRNA from bacterial culture with high purity. To test the efficacy of miR-34a dbRNA, we used the pancreatic cancer cell line MIA-PaCa2. When transfected into MIA-PaCa2 cell line we observed increased expression of miR-34a. Further, miR-34a overexpression caused a 3- fold and 2.5-fold decrease in VEGFA and VEGFB RNA expression respectively. Functional validation on the effect of miR-34a on angiogenesis was performed on Human umbilical vein endothelial cells (HUVEC) using the tube formation assay, where miR-34a dbRNA showed significant decrease in tube formation when compared to cells treated with scrambled dbRNA. We will further validate these results in vivo using Zebrafish angiogenesis model. In conclusion, our study demonstrate for the first time a novel approach to block angiogenesis using miR-34a loaded dbRNA. Our data also show that this approach may be used in targeting multiple molecules/pathways, and when used in combination with existing therapies can improve the efficacy of current treatment modalities.

Citation Format: Manu Gnanamony, Sajani S. lakka, Jaime Libes, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. MicroRNA loaded circular dumbbell RNAs, a novel antiangiogenic approach [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 4400.
Abstract 4404: Identification of small molecules that inhibit miR-21 activity in breast cancer
Authors: Guiraldelli Michel F. , Schwartz Alexander O. , Markus Michelle A. , Jiang Jun , Boyd Justin D. , Radetich Branko , Monahan William E. , Nirmala Nanguneri , Pontin Johan A. , Cohen Dalia
Published: August 2018

In cancer, microRNA (miR) dysregulation can lead to tumor development, invasion and metastasis. miRs are small single-stranded non-coding RNAs that bind to target mRNAs and stop protein expression. miRs are transcribed as a long transcript that fold to form secondary hairpin structures called pri-miRs that are subsequently processed by the microprocessor complex (Drosha and DGCR8) resulting in the formation of the precursor miRs (pre-miRs). Pre-miRs are exported to the cytoplasm via Exportin 5 and RanGTP. In the cytoplasm, pre-miRs are cleaved by Dicer resulting in double stranded molecule. This miR duplex is assembled into the RNA-induced silencing complex (RISC) and targets specific mRNAs. In breast cancer, miR-21 expression levels are increased, and are correlated with tumor grade. The use of small molecules targeting the miR process to modulate levels of mature miR is a novel approach for developing therapeutics. Inactivation of any of the processing steps of miR would result in an inactive mature miR or lower significantly its level. Using a parallel screening approach employing cell biology, NMR and chemistry, we identified several small molecules that bind and inhibit the activity of miR-21. Further characterization of one of our hits in MCF-7 cells showed a dose dependent inhibition of miR-21 activity. A decrease in proliferation with a concomitant increase in apoptosis signal was observed in cells treated with the compound. Analysis of miR-21 processing species by qPCR in MCF-7 cells showed a decrease on pri, pre-and mature miR-21 relative to cells treated with vehicle only. Furthermore, the levels of PDCD4, a direct downstream effector of miR-21, increased upon treatment with the compound. NMR experiments showed that this compound binds to a specific sequence and that the flanking C-G bases are necessary for its binding. These results support the feasibility of targeting miRs by small molecule as a novel therapeutic modality.

Citation Format: Michel F. Guiraldelli, Alexander O. Schwartz, Michelle A. Markus, Jun Jiang, Justin D. Boyd, Branko Radetich, William E. Monahan, Nanguneri Nirmala, Johan A. Pontin, Dalia Cohen. Identification of small molecules that inhibit miR-21 activity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4404.
Abstract 4409: Developing brain tumor-specific gold-liposomal nanocarriers for a noninvasive delivery of microRNA inhibitors
Authors: Grafals Nilmary , Quiñoes-Diaz Blanca I. , Valle Janixa Del , Barletta-Bonano Gabriel L. , Vivas-Mejía Pablo E.
Published: August 2018

MicroRNAs are non-coding RNA molecules that regulate more than 60% of the protein coding genes and their dysregulation has proven to be involved in the initiation, progression and maintenance of malignant brain tumors such as glioblastoma multiforme (GBM). Targeting microRNAs in GBM has shown therapeutic value, although their translation to the clinic have been halted due to their rapid renal clearance, short circulatory life and inability to reach the brain tumor after systemic administration. Over the last years, nanodelivery systems such as liposomes (Lip) and gold nanoparticles (AuNPs) have gained acceptance for the treatment of cancer. This project is focused on developing anti-miRNA gold carriers further encapsulated inside nanoliposomes that are labeled with blood brain barrier specific peptides (Apolipoprotein E and Rabies Virus Glycoprotein). The synthesis of these dual gold-liposomal particles has three important steps: (a) Peptide Pegylation, (b) anti-microRNA conjugation to gold nanoparticles and, (c) nanoparticle assembly. To achieve each of them the corresponding peptide was conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)]-Malemide (DSPE-PEG-Mal) by a thioester bond and incorporated into DOPC and cholesterol lipid mixture. Afterwards, anti-microRNAs were functionalized to 15 nm gold nanoparticles (AuNPs) to obtain RNAi-AuNPs. Finally, the nanoparticles of interest where produce: RNAi-AuNP-Lip, RNAi-AuNP-Lip-APOE and RNAi-AuNP-Lip-RVG. These nanoparticles where characterized by dynamic light scattering, zeta potential and encapsulation efficiency. Additionally, we determine their toxicity in U-87 GBM cell lines. Our results obtained show sizes that range from 30 nm, 60 nm and 95 nm for gold-RNAi, gold-liposomes and gold-liposome-peptides respectively. These particles are slightly negative and have encapsulation efficiencies that are greater than 50%. Cell viability analysis also show that gold nanoparticles carrying microRNA inhibitor negative controls are not toxic to GBM cell lines. Additional studies for cell internalization, reactive oxygen species generation and RNA delivery and release are ongoing now. Developing systemic viable microRNA based therapeutics its a big challenge, but they are crucial to assure the patient a non-invasive therapy that could improve their quality of life and their overall survival.

Citation Format: Nilmary Grafals, Blanca I. Quiñoes-Diaz, Janixa Del Valle, Gabriel L. Barletta-Bonano, Pablo E. Vivas-Mejía. Developing brain tumor-specific gold-liposomal nanocarriers for a noninvasive delivery of microRNA inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4409.
Abstract 4414: Radiation-induced miR-5094 expression inhibits cell proliferation by targeting STAT5b
Authors: Ding Nan , Hua Junrui , He Jinpeng , Wei Wenjun , Pei Hailong , Zhou Guangming , Wang Jufang , Wani Altaf A. , Zhu Qianzheng
Published: August 2018

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that alter protein expression and regulate multiple intracellular processes including cellular stress response. We previously uncovered several novel miRNAs, whose expressions are induced by X-rays irradiation in HeLa cells, using a miRNA array. Here, we examined the function of miR-5094 during cellular radiation response. With software predictions and confirmatory experiments, we identified the signal transduction and activator of transcription 5b (STAT5b), a known regulator of proliferation and survival, as a target of miR-5094. The study also showed that both miR-5094 induction by X-rays irradiation and miR-5094 mimics transfection decreased STAT5b expression, down-regulated STAT5b downstream genes, and consequently, reduced the cell survival and cell viability. Knockdown of miR-5094 by miRNA inhibitor after radiation partially reversed these suppressive effects. As STAT5b plays an important role in lymphocyte proliferation, we further confirmed the proliferation inhibitory effects of miR-5094/STAT5b pathway on Jurkat cells and human peripheral blood CD4+ T cells during cell radiation response. In conclusion, our study provided the first evidence that radiation induces upregulation of miR-5094, which in turn inhibits cell proliferation via targeting STAT5b.

Citation Format: Nan Ding, Junrui Hua, Jinpeng He, Wenjun Wei, Hailong Pei, Guangming Zhou, Jufang Wang, Altaf A. Wani, Qianzheng Zhu. Radiation-induced miR-5094 expression inhibits cell proliferation by targeting STAT5b [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 4414.
Abstract 4415: Characterization of tumor-derived exosomes in BRCA-associated breast tumors
Authors: Shin Vivian Y. , Siu Man-Ting , Cheuk Isabella , Kwong Ava
Published: August 2018

Background: BRCA-mutated tumors are usually with higher grade than sporadic tumors and the biology is poorly understood. Exosomes are small membrane-derived vesicles that function to mediate cell-cell communication via the transfer of tumor-promoting microRNAs (miRNAs), RNAs and proteins. Interestingly, different spectrum of miRNAs is released by cancer cells to promote tumor growth and metastasis in breast cancer. However, the selectivity of miRNA released from tumor-derived exosomes and its relevance in cancer treatment has not been widely studied. Hence, this study is sought to characterize the role of exosomal-miRNA (exo-miR) for the development of BRCA-associated tumors. Methods: Plasma samples from patients with BRCA-positive and non-BRCA carriers were recruited and selected from the Hong Kong Hereditary Breast Cancer Family Registry. By using miRCURY LNA array, the exosomal-miRNA expressions in plasma of patient with BRCA-positive (n=4) and non-carriers (n=4), as well as healthy controls (n=4) were profiled. Selected miRNAs were further validated in pre- and post-operative plasma and paired primary tissues (n=40) using real-time RT-PCR. Functional study of miRNA was also carried out in the cell lines. Results: LNA array data showed that differential expressed BRCA-associated exo-miRNAs were identified. Upregulated miRs (miR-106a, miR-20a, miR-23a, miR-451 and miR-486) were validated in BRCA-positive, BRCA-negative and healthy controls by real-time RT-PCR. In addition, the expression levels of miR-106a, miR-451 and miR-486 were significantly lower in post-operative plasma of BRCA-carriers than non-carriers. Importantly, the expression of miR-451 was reduced after tumor resection in BRCA mutation carriers. In parallel, high expression of exo-miR-106a, miR-20a, miR-23a, miR-451 and miR-486 were also seen in breast cancer cell lines (MB-231 and MB-468) relative to normal breast cells (MCF-10A). We found that cells transfected with miR-451 inhibitor significantly reduce cell proliferation. Conclusions: These preliminary results reveal differential exo-miRNA profiles between BRCA carriers and non-carriers with breast cancer, and identify potential targets of dysregulated miRNAs which is crucial in disease progression of BRCA-associated breast cancers.

Citation Format: Vivian Y. Shin, Man-Ting Siu, Isabella Cheuk, Ava Kwong. Characterization of tumor-derived exosomes in BRCA-associated breast tumors [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 4415.