bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2311: A peptide derived from a photosynthetic cupredoxin protein, auracyanin, induces a p53-independent apoptosis in cancer cells
Authors: Naffouje Samer A. , Yamada Tohru , Gupta Tapas K. Das
Published: July 2017

Cupredoxins are a family of low-molecular weight, water soluble, copper-containing redox proteins involved mainly in the electron transfer chain in prokaryotes. Azurin is a member of the cupredoxin family secreted by Pseudomonas aeruginosa, of which a 28-amino acid sequence (p28) was identified to acquire the capability of preferential entry into cancer cells of various histogenesis. Upon entry, it exerts a cytostatic effect in these cancer cells via a p53-mediated pathway. Our aim was to study whether the other members of the cupredoxin family possess a similar function to that of azurin in the eukaryotic cancer cells. Based on structural and sequence analyses, helical regions of auracyanin A & B were shown to resemble p28 closely. Thus, we studied the biologic function of two 28-amino-acid peptides derived from auracyanin A (aurA; pI 4.5, MW 2.6 kD), and auracyanin B (aurB; pI 3.4, MW 2.7 kD). Cancer growth and MTT assays demonstrated that aurB, but not aurA, induced cytotoxicity in various cancer cell lines in a dose-dependent (range 0.1uM-100uM) and time-dependent (~72 hours) pattern, including p53-null (prostate cancer PC-3) and dn-p53 (breast cancer MDD-2 and ovarian cancer SKOV-3) in which p28 did not demonstrate a significant growth inhibition. Annexin-V assays showed a dose-dependent apoptosis in prostate cancer cell lines of various p53 statuses (wt-p53 LNCaP, mut-p53 DU-145, p53-null PC-3) following treatment with aurB at 5-50uM for 48 hours, whereas p28 did not demonstrate an apoptotic activity in p53-null cell lines, suggesting an aurB-induced apoptosis via a p53-indepdnent pathway. In contrast, aurA did not appear to induce apoptosis in any of the above cell lines. Induction of apoptosis was mediated through depolarization of the mitochondrial membrane and caspase cascade activation in cancer cells. Moreover, fluorescence microscopy demonstrated that aurB, unlike p28, does not acquire a similar pattern of preferential entry into cancer cells. This proposes that aurB acquires the potential as an anticancer cell penetrating peptide, and differs from p28 in its mode of entry and p53-mediated action. Our studies pertaining to azurin and auracyanin, suggest that cupredoxins, as a family of prokaryotic metalloproteins, could represent a fountainhead of numerous derived peptides that hold promise as novel and efficient antineoplastic agents against various cancer cell lines.

Citation Format: Samer A. Naffouje, Tohru Yamada, Tapas K. Das Gupta. A peptide derived from a photosynthetic cupredoxin protein, auracyanin, induces a p53-independent apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2311. doi:10.1158/1538-7445.AM2017-2311
Abstract 2315: Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs
Authors: Kleemann Michael , Bereuther Jeremias , Fischer Simon , Marquart Kim , Hänle Simon , Unger Kristian , Jendrossek Verena , Riedel Christian U. , Handrick René , Otte Kerstin
Published: July 2017

Ovarian cancer is a common human cancer with a poor outcome and a high risk of death. Due to resistances to current standard therapies (carboplatin and paclitaxel for 3 to 6 cycles) additional therapeutic approaches are required and novel specific biomarkers for ovarian cancer patients are necessary. MicroRNAs (miRNA) are conserved, small noncoding RNAs regulating gene expression by binding to their target mRNA which have been shown to regulate biological processes including apoptosis. Apoptosis is a conserved, irreversible process that allows cells to undergo a highly regulated form of cell death. However, the final role of miRNAs in apoptotic signaling has not yet been fully determined. Modulating the expression of key molecular components of the cell death machinery is an attractive strategy for cancer therapy overcoming chemo drug resistance. To extend the pool of potential miRNA as anticancer agents and biomarkers, we transferred a high content miRNA screening for pro-apoptotic miRNAs to human cancer cell lines. Based on the high recovery rate of apoptosis inducing miRNAs in SKOV3 cells analyses were extended to multiple ovarian carcinoma cell lines with different genetic background. After transfection of miR-1912-5p the apoptosis rate increased strongly in cisplatin-resistant cells. In combination treatment with carboplatin the apoptotic effect increased after transfection of miR-1912-5p in the cisplatin-resistant A2780 cells. Furthermore, miR-1912-5p, miR-147b and miR-3073a showed significant apoptosis induction in the various ovarian cancer cell lines alone and in combined therapy with carboplatin. Western Blot analysis revealed an enhanced expression of the pro-apoptotic proteins Bak1 and Bax and a decrease inBcl2 and Bcl-xL. We characterized the miRNAs in regard to their endogenous expression and detected an enhanced expression after apoptosis induction by chemotherapeutic drugs. Finally, analysis of The Cancer Genome Atlas data showed an influence on the median survival of ovarian cancer patients (> 69 years). Our data indicate a potential benefit of tissue specific pro-apoptotic miRNAs in regard to cisplatin-resistant cells. Furthermore the study identified novel pro-apoptotic miRNAs with their potential use as novel therapeutic entities or prognostic biomarkers for ovarian cancer.

Citation Format: Michael Kleemann, Jeremias Bereuther, Simon Fischer, Kim Marquart, Simon Hänle, Kristian Unger, Verena Jendrossek, Christian U. Riedel, René Handrick, Kerstin Otte. Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2315. doi:10.1158/1538-7445.AM2017-2315
Abstract 2316: Elevated H-Ras suppresses death receptor-mediated apoptosis in cancer cells
Authors: Kim Su-Ryun
Published: July 2017

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. Despite its selectivity in killing cancer cells over most normal cells, recombinant human TRAIL or its receptor agonists (monoclonal antibodies against DR4 or DR5) encountered resistance in many tumor cells while the underlying mechanisms remain partially understood. Here, we show that wild-type H-Ras GTPase is systemically upregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. The elevated H-Ras expression correlated with a deficiency of DR4 and DR5 on plasma membrane in TRAIL-resistant cell lines. Notably, knockdown of H-Ras in TRAIL-resistant cells successfully restored the surface expression of DR4 and DR5, thereby sensitizing the cells to TRAIL-induced apoptosis. Consistently, ectopic expression of H-Ras in TRAIL-sensitive cells reduced surface DR4 and DR5 which was associated with a loss of TRAIL sensitivity. By contrast, the status of K-Ras or its mutations was not casually linked to TRAIL receptor expression or TRAIL sensitivity across the panel of cancer cell lines tested. These data suggest that H-Ras may play a distinct role to negatively regulate TRAIL receptors and apoptosis. The upregulated H-Ras could be a predictor of tumor resistance to DR-targeted agents and a potential therapeutic target for combinational therapy to achieve better treatment outcomes.

Citation Format: Su-Ryun Kim. Elevated H-Ras suppresses death receptor-mediated apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2017-2316
Abstract 2320: Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells
Authors: Ku Jin Mo , Hong Se Hyang , Kim Myeong-Sun , Kim Hyo In , Kang Soo-Yeon , Lee Kangwook , Choi Yu-Jeong , Cheon Chunhoo , HUANG CHING WEN , Ko Youme , Sasaki Yui , Kang Sohyeon , Kim Ji Hye , Seo Hye Sook , Kim Tai Young , Shin Yong Cheol , Ko Seong-Gyu
Published: July 2017

Objective: We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells.

Methods: Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by annexin VFITC/PI assay.

Results: More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus.

Conclusion: Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat Adriamycin-resistant patients.

Note: This abstract was not presented at the meeting.

Citation Format: Jin Mo Ku, Se Hyang Hong, Myeong-Sun Kim, Hyo In Kim, Soo-Yeon Kang, Kangwook Lee, Yu-Jeong Choi, Chunhoo Cheon, CHING WEN HUANG, Youme Ko, Yui Sasaki, Sohyeon Kang, Ji Hye Kim, Hye Sook Seo, Tai Young Kim, Yong Cheol Shin, Seong-Gyu Ko. Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2320. doi:10.1158/1538-7445.AM2017-2320
Abstract 2322: Antitumor activity of spicatoside A, a steroidal saponin, via induction of switch from autophagy to apoptotic cell death
Authors: Kim Won Kyung , Pyee Yuna , Park Hyen Joo , Hong Ji-Young , Lee Sang Kook
Published: July 2017

The antitumor activity of spicatoside A (SA), a steroidal saponin isolated from the tuber of Liriope platyphylla (Liliaceae), and its underlying mechanisms were investigated in HCT116 human colorectal cancer cells. SA induced autophagy and apoptotic cell death and inhibited tumor growth in a nude mouse xenograft model implanted with HCT116 cells. Treatment with SA for 24 h enhanced the formation of acidic vesicular organelles in the cytoplasm, indicating the induction of the onset of autophagy. This event was associated with the regulation of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3)-II, p62, beclin 1, lysosomal-associated membrane protein 1, and cathepsin D by inhibiting the PI3K/Akt/mTOR signaling pathway, regulating mitogen-activated protein kinase signaling, and increasing p53 levels. However, a prolonged exposure to SA resulted in apoptosis characterized by the accumulation of a sub-G1 cell population and an annexin V/propidium iodide-positive cell population. Apoptosis induced by SA was associated with the regulation of apoptotic proteins including Bcl-2, Bax, and Bid, the release of cytochrome c into the cytosol, and the accumulation of cleaved poly ADP-ribose polymerase (PARP). Further study revealed that cleavage of beclin 1 by caspase-8 plays a critical role in the SA-mediated switch from autophagy to apoptosis. Taken together, these findings highlight the significance of SA in the modulation of crosstalk between autophagy and apoptosis, as well as the potential use of SA as a novel candidate in the treatment of human colorectal cancer cells. Acknowledgement: This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No. 2009-0083533)

Citation Format: Won Kyung Kim, Yuna Pyee, Hyen Joo Park, Ji-Young Hong, Sang Kook Lee. Antitumor activity of spicatoside A, a steroidal saponin, via induction of switch from autophagy to apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2017-2322
Abstract 2326: A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis
Authors: Dowling Catriona M. , Hemann Micheal , Bradner James , Letai Anthony , Chonghaile Triona Ni
Published: July 2017

Triple negative breast cancer (TNBC) lacks expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2). Importantly, there have been fewer advances in the treatment of TNBC with the mainstay of treatment being cytotoxic chemotherapy. All too often though tumor responds poorly to chemotherapy, or relapse and resistance follow an initial response. We previously found in a range of hematological and solid tumors that chemoresistant cancers are far from the mitochondrial apoptotic threshold. To identify novel therapeutics for the treatment of chemoresistant breast cancer, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill independent of the mitochondrial pathway of apoptosis. To perform this screen we used TNBC line MDA-MB-231 in which the pro-death proteins BAX/BAK were knocked down. BAX/BAK are essential for mitochondrial apoptosis. To enrich for compounds that were not non-specifically toxic, we simultaneously counter-screened against the non-transformed breast epithelial cell line MCF10a. We screened a total of 30,000 compounds in duplicate across the two cell lines. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From the cherry pick screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound. This signature enables classification of the compound into known or novel class of compounds. The lead compound signature was most like that of histone deacetylase inhibitors (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound selectively inhibited HDAC6. Importantly, we validated that the lead compound caused an increase in acetylated tubulin in cells, a target of HDAC6. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis.

Citation Format: Catriona M. Dowling, Micheal Hemann, James Bradner, Anthony Letai, Triona Ni Chonghaile. A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2017-2326
Abstract 2333: Modulation of Bax and mTOR for cancer therapeutics
Authors: Li Rui , Ding Chunyong , Zhang Jun , Xie Maohua , Park Dongkyoo , Ding Ye , Chen Guo , Zhang Guojing , Gilbert-Ross Melissa , Zhou Wei , Marcus Adam , Sun Shi-Yong , Chen Zhuo , Sica Gabriel , Ramalingam Suresh , Magis Andrew , Fu Haian , Khuri Fadlo , Curran Walter , Owonikoko Taofeek , Shin Dong , Zhou Jia , Deng Xingming
Published: July 2017

Pharmacologic manipulation of the serine (S)184 phosphorylation site of Bax protein to functionally regulate its proapoptotic activity is an attractive anticancer strategy. We recently identified three small molecule Bax agonists. SMBA1 was selected as the lead compound based on its chemical structure and its drug-like properties, from which a more effective analog, CYD-2-11, was generated. CYD-2-11 targets the structural pocket around S184 in the C-terminal tail of Bax, directly activating its proapoptotic activity via 6A7 conformational change and formation of Bax homo-oligomers in mitochondrial membranes. CYD-2-11 suppresses tumor growth in SCLC, NSCLC and patient-derived lung cancer xenografts as well as the genetically engineered mutant KRAS-driven lung cancer model with no significant normal tissue toxicity. Inhibition of mTOR by RAD001 enhances S184 Bax phosphorylation in lung cancer cell lines and tumor tissues from lung cancer patients treated with RAD001, which inactivates the propaoptotic function of Bax, contributing to rapalog-resistance. Combined CYD-2-11 and RAD001 treatment not only displays strong synergistic activity against lung cancer but also overcomes rapalog-resistance in vitro and in vivo. Therefore, a mechanism-driven combination of Bax agonist and mTOR inhibitor represents a highly attractive therapeutic strategy to improve lung cancer patient outcomes.

Citation Format: Rui Li, Chunyong Ding, Jun Zhang, Maohua Xie, Dongkyoo Park, Ye Ding, Guo Chen, Guojing Zhang, Melissa Gilbert-Ross, Wei Zhou, Adam Marcus, Shi-Yong Sun, Zhuo Chen, Gabriel Sica, Suresh Ramalingam, Andrew Magis, Haian Fu, Fadlo Khuri, Walter Curran, Taofeek Owonikoko, Dong Shin, Jia Zhou, Xingming Deng. Modulation of Bax and mTOR for cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2333. doi:10.1158/1538-7445.AM2017-2333
Abstract 2336: Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer
Authors: Onyeagucha Benjamin Chidi , Subbarayalu Panneerdoss , Rajamanickam Subapriya , Abdelfattah Nourhan , Timilsina Santosh , Guzman Rosa M. , Zeballos Carla , Eedunuri Vijay , Bansal Sanjay , Bansal Hima , Mohammad Tabrez A. , Chen Yidong , Rao Manjeet K.
Published: July 2017

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3’UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn’t have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation.

Citation Format: Benjamin Chidi Onyeagucha, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Nourhan Abdelfattah, Santosh Timilsina, Rosa M. Guzman, Carla Zeballos, Vijay Eedunuri, Sanjay Bansal, Hima Bansal, Tabrez A. Mohammad, Yidong Chen, Manjeet K. Rao. Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2017-2336
Abstract 2341: In situ labelling identifies a novel landscape of nuclear CDK2 substrates
Authors: Clurman Bruce E. , Chi Yong
Published: July 2017

Cellular signal transduction pathways rely heavily on protein kinase-mediated phosphorylation events. Comprehensive identification of the physiologic targets of protein kinases is key to understanding their functions and elucidating cellular signalling pathways. Various direct and indirect approaches, combined with advanced mass spectrometry technologies, have made it possible to identify many candidate targets of a given kinase, but have also yielded many false-positive identifications, largely due to technical limitations. Identifying direct and physiological kinase targets thus remains a difficult task. We developed a mass spectrometry-based in situ approach that utilizes substrate thiophosphorylation and ATP analog-sensitive CDK2 (AS-CDK2) to identify over one hundred potential cyclin-dependent kinase 2 (CDK2) substrate in intact nuclei. More than one-third of these candidates are known CDK substrates, indicating significant enrichment for physiologic CDK2 substrates, and we also we identified and validated new CDK2 substrates. Importantly, many of these new CDK2 substrates are chromatin-associated proteins with roles in histone modification, DNA replication, and DNA repair, which likely reflects our use of AS-CDK2 within its normal nuclear context, and regulated by endogenous cyclins. These methods should be broadly applicable to the study of other kinases with complex substrate networks.

Citation Format: Bruce E. Clurman, Yong Chi. In situ labelling identifies a novel landscape of nuclear CDK2 substrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2017-2341
Abstract 235: Probiotics ameliorate Porphyromonas gingivalis-promoted pancreatic cancer progression in oncogenic Kras transgenic mice
Authors: Jan Ming-Shiou , Chen Wan-Ting , Chen Yu-Jen , Lin Chia-Wei , Chang Wen-Wei , Tsai Chung-Hung , Peng Jia-Shiou , Hsu Li-Jin
Published: July 2017

Periodontal disease has been shown to play important roles in the pathogenesis of many chronic diseases. Recent epidemiological studies and microbiome analysis have suggested that periodontal disease may be associated with the progression of pancreatic cancer. Our recent study has demonstrated that oral-smear of a periodontitis pathogen Porphyromonas gingivalis (P. gingivalis) significantly induced pancreatic cancer deterioration in the KrasG12D/-Pdx-1-Cre transgenic mouse model. Higher expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-Cadherin, αSMA, SNAIL-1, Vimentin and ZEB1, were observed in P. gingivalis-treated KrasG12D/-Pdx-1-Cre mice than the untreated controls. In an attempt to develop a prophylactic treatment for pancreatic cancer, the mice were fed with a combination of two probiotics, Lactobacillus reuteri and Lactobacillus paracasei, for four consecutive weeks after oral-smear of P. gingivalis. These probiotic strains have been demonstrated to have the efficacy of either anti-inflammation or enhancement of cell-mediated immunity, respectively. Our results demonstrated that oral administration of the probiotics significantly suppressed oncogenic Kras-induced pancreatic cancer development and reversed P. gingivalis-stimulated cancer deterioration in mice. Significantly reduced expression of the aforementioned EMT markers in pancreatic cancer tissues was observed in KrasG12D/-Pdx-1-Cre transgenic mice treated with the probiotics. Our results suggest that the microbiota profile is very important for pancreatic cancer development. The manipulation of microbiota for enhancing antitumor immunity may be an emerging strategy for cancer prevention.

Citation Format: Ming-Shiou Jan, Wan-Ting Chen, Yu-Jen Chen, Chia-Wei Lin, Wen-Wei Chang, Chung-Hung Tsai, Jia-Shiou Peng, Li-Jin Hsu. Probiotics ameliorate Porphyromonas gingivalis-promoted pancreatic cancer progression in oncogenic Kras transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 235. doi:10.1158/1538-7445.AM2017-235
Abstract 2350: In vivo E2F reporting on efficacious dosing schedules of MEK plus CDK4/6 inhibition in melanoma
Authors: Teh Jessica , Nikbakht Neda , Purwin Timothy , Chervoneva Inna , Patel Prem , Davies Michael , Aplin Andrew
Published: July 2017

Pharmacological targeting of cyclin dependent kinases 4 and 6 (CDK4/6) could represent a viable therapeutic option in combination with BRAF and/or MEK inhibitors in different genetic subsets of melanoma. Indeed, continuous and concurrent dosing of MEK inhibitor (MEKi) plus a CDK4/6 inhibitor (CDK4/6i) leads to melanoma regressions in in vivo models and delays the onset of MEKi resistance. Current scheduling in breast cancer patients for the CDK4/6i, palbociclib, is intermittent due to adverse effects resulting in neutropenia. It is unclear what the most efficacious schedules are of palbociclib plus a MEKi. Utilizing an E2F reporter system, we sought to analyze the efficacy of different CDK4/6i plus MEKi schedules in a quantitative and temporal manner. Intermittent dosing (3 weeks on/1 week off) of both CDK4/6i and MEKi combination therapy resulted in tolerant tumors and rapid reactivation of E2F activity during drug holiday. Continuous MEKi with intermittent CDK4/6i led to more complete responses as compared to continuous CDK4/6i with intermittent MEKi. Weight loss of mice was also evident in the continuous CDK4/6i plus intermittent MEKi arm suggesting adverse events related to continuous CDK inhibition. Importantly, functional proteomic analysis revealed distinct mechanisms of acquired resistance/drug tolerance that arose from the three scheduling arms. Here, we report that upregulation of S6 phosphorylation is associated with acquired resistance to MEK plus CDK4/6 inhibition and can be overcome with an mTOR inhibitor. Taken together, in vivo reporting allows for quantitative measurement of pathway activity associated with inhibitor resistance and can be utilized to optimize combination schedules to improve the therapeutic index in patients.

Citation Format: Jessica Teh, Neda Nikbakht, Timothy Purwin, Inna Chervoneva, Prem Patel, Michael Davies, Andrew Aplin. In vivo E2F reporting on efficacious dosing schedules of MEK plus CDK4/6 inhibition in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2350. doi:10.1158/1538-7445.AM2017-2350
Abstract 2351: The novel potential of palbociclib (CDK4/6 inhibitor) in the treatment of triple-negative breast cancer
Authors: Asano Yuka , Kashiwagi Shinichiro , Goto Wataru , Takahashi Katsuyuki , Takashima Tsutomu , Noda Satoru , Onoda Naoyoshi , Tomita Shuhei , Hirakawa Kosei , Ohira Masaichi
Published: July 2017

Background: The effectiveness of palbociclib (CDK4/6 inhibitor) for estrogen receptor positive breast cancer has been demonstrated by large-scale clinical studies, with the drug garnering attention as a key drug for breast cancer subtypes with endocrine sensitivity in the future. According to PALOMA-3 trial, palbociclib has been demonstrated to contribute to the extension of progression-free survival in patients with advanced hormone receptor-positive and HER2-negative metastatic breast cancer after endocrine therapy. On the other hand, in the case of triple-negative breast cancer (TNBC), luminal AR (LAR) related to androgen signaling is believed to have endocrine activity. Previous clinical data revealed that palbociclib shows high sensitivity in luminal breast cancer cell lines with endocrine activity, with effectiveness also expected in LAR. In this study, we created TNBC cell lines that forcibly express AR and examined the effectiveness of palbociclib for TNBC.

Materials and Methods: MCF-7 and T-47D were used as luminal breast cancer cell lines, while MDA-MB-231 and BT-549 were used as TNBC cell lines. In addition, we created TNBC cell lines that forcibly express AR, called AR-MDA-MB-231, by the transfection of pEGFP-C1-AR Plasmid Vector using Lipofectamine® 3000 Reagent. We confirmed the expression of AR by qRT-PCR as well as Western blotting and examined the impact of palbociclib on proliferation as well as apoptosis of breast cancer cell lines.

Results: AR was found to have been expressed only in luminal breast cancer cell lines but not TNBC cell lines. It was confirmed that AR was expressed in AR-MDA-MB-231 which are stable cell lines with the properties of LAR. In a CCK assay, palbociclib showed high sensitivity in AR-MDA-MB-231 as in luminal breast cancer cell lines. Furthermore, in an apoptosis assay using FACS and cell cycle assay, apoptosis was induced in AR-MDA-MB-231 and cell cycle arrest at the G1S check point was confirmed.

Conclusion: palbociclib (CDK4/6 inhibitor) showed effectiveness for TNBC cell lines that compulsively express AR, suggesting it may be one treatment option for TNBC in the future.

Note: This abstract was not presented at the meeting.

Citation Format: Yuka Asano, Shinichiro Kashiwagi, Wataru Goto, Katsuyuki Takahashi, Tsutomu Takashima, Satoru Noda, Naoyoshi Onoda, Shuhei Tomita, Kosei Hirakawa, Masaichi Ohira. The novel potential of palbociclib (CDK4/6 inhibitor) in the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2017-2351
Abstract 2359: Inhibition of the ATR kinase enhances therapeutic efficacy of cisplatin in ATM low uterine carcinosarcoma cells
Authors: Penick Emily R. , Mhawech-Fauceglia Paulette , Bateman Nicholas , Conrads Kelly , Litzi Tracy , Tian Chunqiao , Hamilton Chad A. , Darcy Kathleen , Maxwell George , Conrads Thomas
Published: July 2017

Objective: Uterine carcinosarcoma (UCS) is an aggressive malignancy, making up less than 5% of uterine cancers. Pharmacologic inhibition of ATR kinase synergistically enhances the ability of cisplatin to kill carcinoma cells of many solid tumor types and is further enhanced in ATM low carcinoma cells. It is unknown whether ATR inhibition (ATRi) offers a therapeutic opportunity in UCS. The goal of this study was to evaluate ATM expression in UCS tumors and determine the response of an ATM low UCS model to ATRi + cisplatin combination.

Methods: ATM expression was evaluated in full tissue sections from 74 UCS tumors by immunohistochemistry (IHC) with an ATM specific monoclonal antibody (clone Y170, Abcam), and was categorized as negative vs any positive staining in the carcinomatous (C) vs sarcomatous (S) components. In vitro models of UCS: SK-UT-1, SK-UT-1B, KLE, and RL95-2 cell lines were treated with an ATRi (AZD6738), cisplatin, and ATRi + cisplatin. ATM low models were generated in RL95-2 cells using short hairpin RNA (shRNA) lentivirus targeting ATM and a non-target control. Cells were subjected to cisplatin and ATRi dose response analyses.

Results: Negative IHC expression of ATM protein was observed in 24% of the C vs 31% of the S components of the 74 UCS cases (Table 1). ATRi increased the sensitivity of uterine carcinosarcoma cell line models to cisplatin. Western blot confirmed a 57% knockdown in ATM in RL95-2 cells infected with shRNA for ATM and treatment with ATRi +cisplatin therapy showed ~20% increase in cell death in ATM low vs control cells lines, p=0.02.

Conclusions: IHC analyses of UCS tumors showed an average of ~27% of tumors have loss of ATM in C and S components. Preliminary evidence shows that ATRi increases the sensitivity of UCS cell models to cisplatin therapy, which is further increased in ATM low uterine carcinoma cells. These findings suggest a novel therapeutic opportunity for ATRi + cisplatin therapy in UCS patients with low ATM expressing tumors.

Immunohistochemistry-based quantification of ATM expression in uterine carcinosarcoma tumorsATMUterine CarcinosarcomaIntensityCarcinomatous ComponentSarcomatous component0-131 (41.9%)33 (44.6%)018 (24.3%)23 (31.1%)113 (17.6%)10 (13.5%)2-343 (58.1%)41 (55.4%)211 (14.9%)10 (13.5%)332 (43.2%)31 (41.9%)TOTAL7474

Citation Format: Emily R. Penick, Paulette Mhawech-Fauceglia, Nicholas Bateman, Kelly Conrads, Tracy Litzi, Chunqiao Tian, Chad A. Hamilton, Kathleen Darcy, George Maxwell, Thomas Conrads. Inhibition of the ATR kinase enhances therapeutic efficacy of cisplatin in ATM low uterine carcinosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2017-2359
Abstract 2360: Regulation of the MAPK phosphatase MKP-1 by the E3 ligase GP78 in cancer cells
Authors: Kho Dhonghyo , Wu Gen Sheng
Published: July 2017

Mitogen-activated protein kinase (MAPK) phosphatase 1 (DUSP1/MKP-1) is a member of the threonine-tyrosine dual-specificity phosphatase family that is endogenous inhibitor for extracellular signal-regulated protein kinases (ERKs), p38 MAPKs, and JNKs signaling. MKP-1 plays a key role in determining the magnitude and duration of MAPK activation in response to extracellular stimuli. In human cancers, abnormal expression of DUSP1 is associated with tumor development, chemoresistance and prognosis. However, the mechanism by which MKP-1 is regulated is not fully understood. Here, we show that GP78, an E3 ligase, plays an important role in regulation of MKP-1 protein levels. Specifically, we found that MKP-1 interacts with GP78 both in vitro and in vivo. Their interaction occurs between the N-terminal, Rhodanese domain of MKP-1 and C-terminal E3 ligase domain of GP78. We also found that K230, 280, and 289 residues on MKP-1 are responsible for its ubiquitination. Importantly, this interaction causes MKP-1 ubiquitination and subsequent degradation. Furthermore, we showed by overexpression and knockdown studies that GP78 downregulates the level of MKP-1 protein in cancer cells. Therefore, we identify a novel mechanism by which GP78 controls MKP-1 protein levels and suggest that this regulatory mechanism may play an important in cancer.

Citation Format: Dhonghyo Kho, Gen Sheng Wu. Regulation of the MAPK phosphatase MKP-1 by the E3 ligase GP78 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2360. doi:10.1158/1538-7445.AM2017-2360
Abstract 2362: Mechanistic insights into phosphoinositide 3-kinase α
Authors: Maheshwari Sweta
Published: July 2017

Phosphoinositide 3-kinases (PI3Ks) are at the center of mTOR pathway that plays key roles in regulating cell survival, proliferation, protein synthesis and vesicle trafficking. Dysregulation of the mTOR pathway has been implicated in various diseases such as cancer, diabetes, arthritis, inflammation and respiratory illnesses. PI3Ks have dual kinase specificity with a lipid kinase activity that phosphorylates the 3’-hydroxyl of phosphoinositides, and a protein kinase activity including auto-phosphorylation. In this study, we explored the roles of residues of the catalytic domain and regulatory subunit of human PI3Kα in the lipid and protein phosphorylation. We used site-directed mutagenesis and kinetic assays to precisely map the molecular basis of substrate recognition and catalysis in PI3Kα. Kinetic analysis of K776, located in the P-loop of PI3Kα, reveals that a lysine at this position is essential for the recognition of the lipid and ATP substrates. This same residue plays an important role in the auto-phosphorylation of the enzyme. Replacement of histidine residues–H936 and H917 in the activation and catalytic loops respectively with alanine residues results in a dramatic change in the kinetics of PI3Kα. While H936A inactivates the lipid kinase activity without affecting auto-phosphorylation, H917A abolishes both the lipid and protein kinase activities of PI3Kα. On the basis of kinetic and structural analysis, we proposed possible mechanistic roles of these critical residues in catalysis.

Citation Format: Sweta Maheshwari. Mechanistic insights into phosphoinositide 3-kinase α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2017-2362
Abstract 2364: Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling
Authors: Moharram Sausan A. , Rönnstrand Lars , Kazi Julhash U.
Published: July 2017

Type III receptor tyrosine kinases (RTKs) including FLT3 and KIT play a major role in cell differentiation, proliferation, and survival of hematopoietic stem cells. FLT3-ITD and KIT-D816V mutations are the most common oncogenic mutations in FLT3 and KIT found in hematological cancers. These mutations lead to constitutive activation of proliferative and survival signals. Tyrosine kinase inhibitors (TKIs) in combination with chemotherapy display promising results in a clinical setting, but patients develop resistant disease after short-term treatment,. Hence, proteins that regulate the activity of RTKs can be alternative targets for patients carrying these mutations. Activation of FLT3 and KIT results in phosphorylation on several tyrosine residues that recruit SH2 domain-containing signaling proteins. In this study we identified Src-like adaptor protein 2 (SLAP2) as a potent FLT3 and KIT interacting protein. The interaction requires an intact SH2 domain of SLAP2 as well as phosphorylation of the receptor. Overexpression of SLAP2 in murine proB Ba/F3 cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation. SLAP2 displayed a similar inhibitory potential in cells expressing KIT-D816V. SLAP2 partially blocked phosphorylation of several FLT3 and KIT downstream signaling proteins such as AKT, ERK and p38. Moreover, SLAP2 expression inhibited FLT3-ITD-mediated STAT5 phosphorylation and KIT-D816V-mediated STAT3 phosphorylation. SLAP2 expression significantly accelerated ubiquitination-mediated degradation of FLT3 and KIT. Collectively, these data suggest that SLAP2 negatively regulates FLT3 and KIT signaling and therefore, modulation of SLAP2 expression levels may become a potential target for anticancer therapy.

Citation Format: Sausan A. Moharram, Lars Rönnstrand, Julhash U. Kazi. Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2017-2364
Abstract 2371: Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop
Authors: Kazi Julhash U. , Rönnstrand Lars
Published: July 2017

Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling.

Citation Format: Julhash U. Kazi, Lars Rönnstrand. Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2017-2371
Abstract 2379: Nuclear lemur tyrosine kinase-2 regulates RNA polymerase II dependent transcription in prostate cancer
Authors: Shah Kalpit , Foley Justin , Nickerson Michael L. , Dean Michael , Bradbury Neil
Published: July 2017

The Androgen Receptor (AR), a DNA-binding transcription factor plays a key role in the development and maintenance of prostate epithelia by modulating expression of growth-promoting genes. AR has been proposed to regulate gene expression by enhancing the efficiency of RNA polymerase II (RNAPII) dependent transcription elongation. Dysregulation of this AR-dependent transcriptional activity has been implicated not only in prostate cancer but also androgen-independent prostate cancer. Recently, we showed that protein expression of Lemur Tyrosine Kianse-2 (LMTK2), a serine-threonine kinase is downregulated in prostate cancer. Importantly, our study ascribed a novel role for LMTK2, as a negative regulator of AR-dependent transcriptional activity in prostate epithelial cells. However, the mechanism through which LMTK2 is able to regulate AR remains to be determined. Here, we show that LMTK2 is not only present in cytoplasmic fraction but also, within the nucleus of mammalian cells where it co-localizes with AR, newly transcribed mRNA and with RNA polymerase II (RNAPII). Interestingly, our data reveals that LMTK2 colocalizes with elongating RNAPII, phosphorylated on serine 2 of the carboxyl-terminal domain. This presents an interesting possibility that LMTK2 might be able to regulate transcription of AR-dependent genes by interacting with the elongating RNAPII and transcription & epigenetic factors. As such, our study potentially identifies LMTK2 as an important player regulating the AR-RNAPII mediated transcription machinery in mammalian cells.

Citation Format: Kalpit Shah, Justin Foley, Michael L. Nickerson, Michael Dean, Neil Bradbury. Nuclear lemur tyrosine kinase-2 regulates RNA polymerase II dependent transcription in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2017-2379
Abstract 2380: Insertion mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the D835Y mutation in acute myeloid leukemia
Authors: Marhäll Alissa , Fischer Thomas , Heidel Florian , Kazi Julhash U. , Rönnstrand Lars
Published: July 2017

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of deaths. Mutation in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent factor in this heterogeneous disease. The major mutations in FLT3 can be categories as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs are subdivided in to two groups depending on their location: the juxtamembrane domain (ITD-JM) and the tyrosine kinase domain (ITD-TDK). Although, ITD-JM has been characterized well the ITD-TKD has not yet been studied well due to its recent discovery. For this reason, we compared ITD mutations in TKD and JM, as well as the most frequently occurring point mutation located in the TKD, D835Y. The purpose of this study was to understand whether it is the mutation’s nature or location that plays the driving role in leukemogenesis. We used a cytokine-dependent mouse pro-B cell line, BaF3, to overexpress different FLT3 mutants. We first examined the colony formation capacity in semisolid cytokine- and serum-free medium. The assay resulted in indistinguishable number and size of colonies for both ITD-JM and ITD-TKD, while D835Y-TKD transfected cells failed to form colonies suggesting that the ITD-TKD mutations have stronger transforming potential than other TKD mutations. In addition to colony formation assays, cell proliferation and survival was significantly higher in ITD-TKD expressing cells compared to cells expressing D835Y-TKD. Finally, we showed that phosphorylation of STAT5 and AKT is increased in ITD-TKD, while other FLT3 downstream signaling remained unaffected. All together, our data suggest that ITD-TKD displays higher oncogenic potential than other TKD mutants.

Citation Format: Alissa Marhäll, Thomas Fischer, Florian Heidel, Julhash U. Kazi, Lars Rönnstrand. Insertion mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the D835Y mutation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2017-2380