bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4303: Vopp1 physically interacts with Wwox and inhibits its apoptotic function in breast cancer
Authors: Lallemand François , Bonin Florian , Lidereau Rosette , Driouch Keltouma
Published: July 2017

WWOX is a tumor suppressor gene spanning the fragile site FRA16D and targeted by loss of heterozygosity or homozygous deletion in several tumor types. Wwox protein consists of two N-terminal WW domains that mediate protein-protein interactions and a C-terminal short-chain Dehydrogenase/Reductase domain (SDR). The WW1 module acts as a versatile platform linking WWOX with numerous multiprotein complexes. Thus, Wwox suppressor activity relies on its binding capacities to various partners. Wwox has been shown to interact with known proto-oncogenes such as p73, deltaNp63, AP2γ, c-jun, Erbb4, c-Met, Runx2 and Dvl-2. At the cellular level, Wwox has been shown to be a pro-apoptotic protein that induces cell death by acting in both the nucleus and cytoplasm. More recently, emerging evidence also suggest an important role of WWOX in DNA damage response. By using a yeast two-hybrid system and co-immunoprecipitation assays, we characterized Vopp1 (Vesicular Over-expressed in cancer Pro-survival Protein 1) as a new molecular partner of the tumor suppressor Wwox. VOPP1 gene localizes at the frequently amplified 7p11.2 locus and is often co-amplified with EGFR. It is overexpressed in multiple malignancies such as glioblastoma, gastric, head and neck and lung cancers. In this study, we demonstrate that VOPP1 physically interacts with Wwox in breast cancer cells. Upon binding, Wwox is recruited to the Vopp1-containing lysosomal compartment. This recruitment inhibits Wwox-mediated apoptosis at least in part by preventing Wwox-p73 interaction. In addition, Vopp1 acts as an oncogene as shown by its capacity to potentiate cellular transformation. Moreover, in breast cancer clinical samples, VOPP1 overexpression was detected predominantly in tumors retaining Wwox expression. Vopp1 overexpression is associated to a reduced survival of breast cancer patients, especially patients with luminal B tumor types. Remarkably, the VOPP1/WWOX expression ratio showed a significantly worst prognosis outperforming either gene alone. These findings highlight the importance of Wwox compartimentation in addition to Wwox loss in human cancer pathogenesis and define Vopp1 as a novel negative regulator of Wwox whose overexpression induces breast carcinogenesis by affecting the tumor suppressive activity of Wwox.

Citation Format: François Lallemand, Florian Bonin, Rosette Lidereau, Keltouma Driouch. Vopp1 physically interacts with Wwox and inhibits its apoptotic function 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 4303. doi:10.1158/1538-7445.AM2017-4303
Abstract 4304: Feedback-phosphorylation of MKK4 by MAPKs promotes apoptosis
Authors: Moriizumi Hisashi
Published: July 2017

Feedback-phosphorylation of MKK4 by MAPKs promotes apoptosis Moriizumi Hisashi, Takanori Nakamura and Mutsuhiro Takekawa Div. of Cell Signaling and Mol. Med., IMS, Tokyo Univ. The mitogen-activated protein (MAP) kinase pathway is an important system for cellular responses to extracellular stimuli. Each of the MAPK pathways is composed of MAPKKK, MAPKK and MAPK. In mammalian cells, at least three subfamilies of MAPKs, namely ERK, p38 and JNK, are present. The ERK pathway is activated in response to mitogenic stimuli and plays a key role in proliferation and differentiation. In contrast, the p38 and JNK pathways [collectively called stress-activated protein kinase (SAPK) pathways] regulate cellular stress responses such as apoptosis and cell cycle arrest. MKK4 is unique among the members of the mammalian SAPKK family (i.e., MKK3/4/6/7) in its ability to phosphorylate and activate both p38 and JNK. Interestingly, inactivating mutations of the MKK4 gene are frequently found in many types of cancer. Although MKK4 has been suggested as a tumor suppressor, the molecular mechanism by which MKK4 inhibits tumorigenesis remains unclear. Here we demonstrate that MKK4 can be phosphorylated at specific serine and/or threonine residues by downstream MAPKs(named the “feedback-phosphorylation”)and thereby regulates stress-induced apoptotic cell death. By generating antibodies specific to these phosphorylation sites, we found that under stress conditions both Thr and Ser residues of MKK4 were phosphorylated by JNK, while only the Ser residue was phosphorylated by ERK in response to mitogenic stimuli. In order to clarify physiological significance of the feedback-phosphorylation, we then generated a phosphorylation-deficient mutant of MKK4 (MKK4-AA) by substituting both Thr and Ser residues with Ala. Although the feedback-phosphorylation did not affect the enzymatic activity, stability and subcellular localization of MKK4, we found that cells expressing MKK4-AA mutant suppressed apoptosis induced by various stresses stimuli such as UV irradiation and anticancer drug treatment. These results suggest that feedback-phosphorylation of MKK4 regulates stress-induced apoptosis. Moreover, we are currently investigating the mechanism of how MKK4 affects apoptotic cell death in response to feedback-phosphorylation.

Citation Format: Hisashi Moriizumi. Feedback-phosphorylation of MKK4 by MAPKs promotes 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 4304. doi:10.1158/1538-7445.AM2017-4304
Abstract 4308: Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma cells
Authors: Jo Eunbi , Jang Hyun Jin , Jang Ik-Soon
Published: July 2017

Forkhead transcription factor (Foxo3a) is a critical effector of JNK-mediated tumor suppression. However, it is not clear whether the caveolin-1 (CAV1)-mediated JNK/Foxo3a pathway is involved in cancer cell apoptosis. We found that cordycepin upregulates CAV1 expression, which was accompanied by JNK phosphorylation (p-JNK), and, subsequently Foxo3a translocation into the nucleus, resulting in the upregulation of Bax protein expression. Furthermore, we found that CAV1 overexpression upregulated p-JNK whereas CAV1 siRNA downregulated p-JNK. Additionally, SP600125, a specific JNK inhibitor, significantly increased Foxo3a phosphorylation, which attenuated Foxo3a translocation into the nucleus, indicating that CAV1 mediates JNK regulation of Foxo3a. Foxo3a siRNA downregulated Bax protein and attenuated A549 apoptosis, indicating that the CAV1-mediated JNK/Foxo3a pathway induces the apoptosis of A549 lung cancer cells. Cordycepin significantly decreased tumor volume in nude mice. Taken together, these results indicate that cordycepin promotes CAV1 upregulation to enhance JNK/Foxo3a signaling pathway activation inducing apoptosis in lung cancer cells and support its potential as a therapeutic agent for lung cancer.

Citation Format: Eunbi Jo, Hyun Jin Jang, Ik-Soon Jang. Cordycepin induces apoptosis by caveolin-1-mediated JNK regulation of Foxo3a in human lung adenocarcinoma 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 4308. doi:10.1158/1538-7445.AM2017-4308
Abstract 4313: Protein synthesis modulates paraptotic death induced by inhibition of cyclophilins in glioblastoma (GBM) cells
Authors: Wang Lin , Gundelach Justin , Bram Richard
Published: July 2017

In this study, we investigated the mechanisms underlying cell death induced by the small molecule cyclophilin inhibitor NIM811. NIM811 effectively killed GBM cells through a non-apoptotic pathway, as there was no nuclear shrinkage or caspase activation. Instead, death was preceded by dramatic cytosolic vacuolization, stemming from enlargement of the endoplasmic reticulum (ER). RNA-seq revealed upregulation of genes pertinent to the unfolded protein response (UPR) and autophagy. Western blotting for UPR and autophagy mediators also supported a transient UPR signaling and compromised autophagy during prolonged NIM811 treatment. By utilizing a bicistronic reporter plasmid, we independently quantitated Cap-dependent and Cap-independent translation. We further assessed the long-term effects mediated by different short-term paraptosis inhibitors via colony formation assay. We find that NIM811 initiates paraptosis in GBM cells, due to an abnormal upregulation of protein translation that caused accumulation of misfolded or unfolded proteins in ER. Transient activation of pro-survival autophagy and UPR signals were shutdown during prolonged treatment with NIM811, thus allowing cell death to occur. Cycloheximide was previously reported by others to suppress paraptosis. In our studies, we find instead that it temporarily delayed vacuole formation, but actually enhanced paraptotic cell death in the long term. The mTOR inhibitors rapamycin or torin-2 rescued cells from NIM811 induced paraptosis by sustaining autophagy and the UPR, and specifically restrained cap-dependent translation. We propose that ER vacuolization could be a protective response generated by cells to isolate defective proteins within the ER in order to prevent misfolded aggregates to interfere with cellular homeostasis. For that reason, simply inhibiting vacuole formation without clearing protein accumulation in the ER was insufficient to prevent cell death. These findings aid our understanding of cellular mechanisms that contribute to non-apopotic death. Moreover, this work underlines the importance of evaluating cellular responses in cell death experiments over multiple time frames to judge the true impact.

Citation Format: Lin Wang, Justin Gundelach, Richard Bram. Protein synthesis modulates paraptotic death induced by inhibition of cyclophilins in glioblastoma (GBM) 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 4313. doi:10.1158/1538-7445.AM2017-4313
Abstract 4314: Exclusive delivery of mazF in cancer cells by Listeria monocytogenes
Authors: Saffarian Maryam , Tzeng Jeremy
Published: July 2017

Programmed cell death (PCD), an active process that leads to cell suicide, is a critical mechanism in every organism. MazF, a bacterial ribonuclease protein, can trigger PCD in bacterial cells and also induce Bak-dependent apoptosis in mammalian cells. This bacterial ribonuclease cleaves mRNAs at ACA sequences leading to inhibition of protein synthesis in cells. Hence, we hypothesized that the overexpression of MazF proteins in cancer cells could result in the induction of apoptosis. In the present study, the ACA-less mazF gene was inserted downstream of the Internal Ribosome Entry Site (IRES) and under the control of T7 promoter. The plasmid was electroporated into Listeria monocytogenes harboring pCSA1 that encodes T7 RNA polymerase under the control of the listeria actA promoter. Subsequently, the bacteria were functionalized with Trastuzumab (Herceptin®) to deliver the mazF mRNA into HER2/neu breast cancer cells. The results showed that MazF protein has an ability to induce apoptosis in HER2/neu breast cancer cells. This finding is the first report of the application and delivery of MazF as an anti-cancer agent for the induction of apoptosis in HER2/neu breast cancer cell line. This research suggests cancer therapy that targets only cancer cells but not healthy cells and offers more advantages, e.g., inexpensiveness, target tissue specificity, easy and safe delivery, in comparison to other gene therapy vectors or direct protein administration.

Citation Format: Maryam Saffarian, Jeremy Tzeng. Exclusive delivery of mazF in cancer cells by Listeria monocytogenes [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 4314. doi:10.1158/1538-7445.AM2017-4314
Abstract 4315: PTBP1 modulation of MCL1 mRNA regulates sensitivity to antitubulin chemotherapeutics
Authors: Placzek William J. , Cui Jia
Published: July 2017

Background: Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic Bcl-2 family protein, is a key regulator of intrinsic apoptosis. Normal cells require strict control over MCL1 expression and aberrant MCL1 expression has been shown linked to the emergence of various diseases and chemoresistance. Previous studies have detailed how MCL1 expression is regulated by multiple mechanisms both transcriptionally and translationally. However, characterization of the post-transcriptional regulators of MCL1 mRNA is limited. Polypyrimidine tract binding protein 1 (PTBP1) is a known regulator of post-transcriptional gene expression that can control mRNA splicing, translation, stability, and localization. In the described study we characterize the impact that PTBP1 has on MCL1 expression, its molecular mechanisms, and its roles on cellular apoptosis.

Methods: Cross-linking Immunoprecipitation (CLIP)-seq data analysis and RNA immunoprecipitation (RIP) were performed to analyze protein interactions with RNA and map the RNA binding sites on the genes of interests. RNA half-life was measured by treating cells with Actinomycin D and extracting total RNA at different time point. Cell viability was measured by the MTS assay and apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining followed by flow cytometry analysis.

Results: Our studies demonstrate that PTBP1 binds to MCL1 mRNA and that knockdown of PTBP1 up-regulates MCL1 expression in cancer. Mechanistically, PTBP1 silencing stabilizes MCL1 mRNA and increases MCL1 mRNA accumulation in cytoplasm. We further identify that this post-transcriptional regulation is mediated by microRNAs and Argonaute2 (Ago2). Moreover, we show that PTBP1 down-regulation is anti-apoptotic to antitubulin chemotherapeutics in a MCL1-dependent manner.

Conclusions: Our findings suggest that PTBP1 is a novel post-transcriptional regulator of MCL1 mRNA by which it controls apoptotic response to antitubulin chemotherapeutics.

Note: This abstract was not presented at the meeting.

Citation Format: William J. Placzek, Jia Cui. PTBP1 modulation of MCL1 mRNA regulates sensitivity to antitubulin chemotherapeutics [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 4315. doi:10.1158/1538-7445.AM2017-4315
Abstract 4319: Analysis of Bcl-2 family members and protein-protein interactions using novel multiplex immunoassays
Authors: Maheshwari Reeti , Schluter Melissa , Pepin Danielle , Hwang Joseph
Published: July 2017

Bcl-2 family members play a very important role in intrinsic apoptotic pathways. The family consists of more than 17 members that can be functionally separated into pro-apoptotic and anti-apoptotic subfamilies. Interactions between these family members determine cell fate, and dysregulation can lead to tumor growth and tumor cell survival. There are several investigational drugs for cancer treatment targeting Bcl-2 family members in clinical trials, driving the need for robust assays to measure Bcl-2 family members and their interactions. Though existing assays measure Bcl-2 family members, there is a lack of reliable assays to evaluate total proteins and protein-protein interactions simultaneously. To address this issue, we have developed two Luminex®-based multiplex immunoassay panels that allow for the simultaneous detection of multiple components of the Bcl-2 family in a single well, including Total Bcl-2, Bcl-xL, Mcl-1, BAD, BIM, and BAX as well as protein interactions like NOXA/Mcl-1, Mcl-1/BIM, and Bcl-xL/BAD. Using these panels, we analyzed changes in the expression and interactions of Bcl-2 family members in response to known apoptotic drugs, including Camptothecin (topoisomerase inhibitor), Anisomycin (protein translation inhibitor), and AT101 (BH3 mimetic drug). Camptothecin and Anisomycin each elicited a significant decrease in the levels of Mcl-1. Additionally, the Mcl-1/BIM interaction was disrupted by both Camptothecin and Anisomycin, perhaps a consequence of the reduced Mcl-1 levels. However, expression of the other anti-apoptotic proteins was unaffected by any of the three drugs. Interestingly, all three treatments had different effects on the Mcl-1/NOXA interaction. Mcl-1/NOXA signal was increased by AT101, decreased by Anisomycin, and showed a dose dependent effect with Camptothecin. In contrast to their disparate effects on the Mcl-1/NOXA interaction, all three drugs acted in a similar manner in blocking the Bcl-xL/BAD interaction without affecting the total levels of either protein. Overall, these results illustrate the dynamic responses of the Bcl-2 family to apoptosis-inducing drugs. The novel multiplex immunoassays described here provide a powerful tool for studying the underlying mechanisms regulating the Bcl-2 family of proteins.

Citation Format: Reeti Maheshwari, Melissa Schluter, Danielle Pepin, Joseph Hwang. Analysis of Bcl-2 family members and protein-protein interactions using novel multiplex immunoassays [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 4319. doi:10.1158/1538-7445.AM2017-4319
Abstract 432: xCT inhibition disrupts redox homeostasis in CD44v-expressing tumor cells showing glutaminolytic metabolism in head and neck squamous cell carcinoma
Authors: Okazaki Shogo , Tsuchihashi Kenji , Sampetrean Oltea , Saya Hideyuki , Nagano Osamu
Published: July 2017

Metabolic changes favoring aerobic glycolysis and glutaminolysis may influence the sensitivity of tumor cells to anticancer drugs. We previously showed that sulfasalazine, an inhibitor of xCT-dependent cystine transport, selectively induces oxidative damage in undifferentiated tumor cells that express CD44 variant (CD44v) at a high level, without affecting CD44v-negative differentiated tumor cells, in head and neck squamous cell carcinoma (HNSCC). However, whether metabolic changes contribute to the xCT dependency of CD44v-expressing HNSCC cells has remained unknown. Here we show that CD44v-expressing HNSCC cells manifest a highly glutaminolytic phenotype and constitutively generate reactive oxygen species through glutamine metabolism pathway, and that xCT plays a key role in maintaining redox homeostasis in these cells. Inhibition of xCT suppressed the consumption of glutamine without affecting that of glucose in these cells, indicating that CD44v-expressing HNSCC cells also rely on xCT for maintenance of glutaminolysis. Furthermore, administration of sulfasalazine, but not that of the anticancer drug cisplatin, selectively depleted CD44v-expressing undifferentiated tumor cells positive for the glutamine transporter ASCT2 in human HNSCC tumors formed in nude mice. Targeting of xCT is thus a potential therapeutic approach for ASCT2-expressing HNSCC tumors in which cellular metabolism is shifted toward enhanced glutaminolysis.

Citation Format: Shogo Okazaki, Kenji Tsuchihashi, Oltea Sampetrean, Hideyuki Saya, Osamu Nagano. xCT inhibition disrupts redox homeostasis in CD44v-expressing tumor cells showing glutaminolytic metabolism in head and neck squamous cell carcinoma [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 432. doi:10.1158/1538-7445.AM2017-432
Abstract 4320: The Survivin-targeting miR-542-3p overcomes erbB3 signaling-mediated chemo-resistance and enhances the antitumor activity of paclitaxel against erbB2-positive breast cancer
Authors: Lyu Hui , Wang Shuiliang , Huang Jingcao , Wang Bolun , Liu Bolin
Published: July 2017

Elevated expression of erbB3 interacts with erbB2 in breast cancer cells to confer chemo-resistance via induction of Survivin. However, the underlying mechanism of erbB3 signaling-induced upregulation of Survivin remains elusive. Ectopic expression or downregulation of erbB3 in erbB2-positive breast cancer cells did not alter Survivin mRNA levels and had no significant effect on Survivin protein stability. We thus hypothesize that the erbB3 signaling modulates certain miRNAs that target Survivin to influence its protein translation. Here we showed that overexpression of erbB3 decreased and specific knockdown of erbB3 enhanced expression of two Survivin-targeting miRNAs, miR-203 and miR-542-3p in breast cancer cells. While the specific inhibitor of either miR-203 or miR-542-3p clearly attenuated an anti-erbB3 antibody (Ab)-mediated reduction of Survivin, a more profound effect on Survivin expression was observed with inhibition of miR-542-3p than miR-203 inhibition. Consistently, miR-542-3p mimic was much more effective than miR-203 mimic not only to downregulate Survivin, but also to enhance paclitaxel-induced apoptosis in the otherwise paclitaxel-resistant breast cancer cells. Moreover, using a murine tumor xenografts model established from a human breast cancer cell line expressing both erbB2 and erbB3, we discovered that the combinations of miR-542-3p mimic and paclitaxel, as compared to either agent alone, significantly inhibited tumor growth in vivo. Collectively, these data demonstrate that the erbB3 signaling upregulates Survivin via inhibition of miR-203 and miR-542-3p in erbB2-positive breast cancer cells. Since miR-542-3p has three binding sites on the 3’UTR of erbB3 mRNA, the miR-542-3p mimic serves as an excellent replacement therapeutic agent to downregulate Survivin, and thereby significantly enhances the antitumor activity of paclitaxel against erbB2-positive breast cancer.

Citation Format: Hui Lyu, Shuiliang Wang, Jingcao Huang, Bolun Wang, Bolin Liu. The Survivin-targeting miR-542-3p overcomes erbB3 signaling-mediated chemo-resistance and enhances the antitumor activity of paclitaxel against erbB2-positive 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 4320. doi:10.1158/1538-7445.AM2017-4320
Abstract 4333: The role of cancer-associated fibroblast-induced chemokine ligand 11 in tumor microenvironment contributes to the progression of oral cancer
Authors: Nieh Shin
Published: July 2017

Oral squamous cell carcinoma (OSCC) is one of the leading causes of cancer-related death in Taiwan and worldwide. The prognosis of OSCC is usually poor because of its propensity of extensive invasion, local recurrence and frequent regional lymph node metastasis, even at initial diagnosis. Recent studies showed carcinoma-associated fibroblasts (CAFs), a major type of tumor-surrounding stromal cell, generate certain mediators through which CAFs interact with tumors and contribute to cancer progression in numerous cancers. The orchestration between CAFs and cancer cells is complex and its underlying mechanism needs to be explored. In the present study, we used organotypic culture to investigate CAFs that promote aggressive behavior of cancer cells. Using microarray analysis, we detected abundant expression of chemokine (C-C motif) ligand 11(CCL11), also named Eotaxin, secreted by CAFs and further be identified as a critical mediator in CAF-induced invasiveness. We planned to validate that CCL11 played a major role in the crosstalk between fibroblasts and OSCC cells via the paracrine manner. CCL11was found upregulated in CAFs than in normal fibroblasts via Western blot analysis. Cells lines of OSCC, Fadu and TW204, treated with recombinant CCL11 increased capabilities of sphere formation in ten days. Administration of CCL11 promoted migration and invasion abilities through induction of the epithelial-to-mesenchymal transdifferentiation with corresponding morphological alterations of cancer cells. Counteracting CCL11 activity diminished the aggressive phenotype of cancer cells induced by CAFs. We further studied the relationship between the expression of CCL11 in both CAFs and OSCC cells and clinical outcome in the patients with OSCC. As a result, a high CCL11 expression level was associated with poor prognosis in terms of nodal metastasis and survival. These results indicate that CAFs promote cancer invasiveness via a paracrine effect on microenvironmental CCL11 signaling and suggest that CCL11 is a potential prognostic biomarker that may be considered in therapeutic strategies for the treatment of patients with OSCC.

Citation Format: Shin Nieh. The role of cancer-associated fibroblast-induced chemokine ligand 11 in tumor microenvironment contributes to the progression of oral 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 4333. doi:10.1158/1538-7445.AM2017-4333
Abstract 4341: Mesenchymal stem cells promote epithelial-mesenchymal transition of colon cancer cells via direct cell-to-cell contact
Authors: Takigawa Hidehiko , Kitadai Yasuhiko , Kuwai Toshio , Yuge Ryo , Tanaka Shinji , Chayama Kazuaki
Published: July 2017

We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) migrated to tumor stroma and promoted tumor growth and metastasis. We evaluated the proliferation and migration ability in directly and indirectly co-culture assay to clarify the mechanism of interaction between cancer cells and MSCs. Proliferation and migration ability of cancer cells were increased by direct co-culture with MSCs but not by indirect co-culture of MSCs. Thus, we thought that direct contact between cancer cells and MSCs is important to their interaction. We performed microarray analysis of gene expression in KM12SM colon cancer cells directly co-cultured with MSCs. Expression of epithelial-mesenchymal transition (EMT) related genes such as fibronectin (FN), SPARC and Galectin 1 was increased by direct co-culture with MSCs. We also confirmed the upregulation of these genes with real time PCR, and these genes were not increased in cancer cells indirectly co-cultured with MSCs. Among these EMT related genes upregulated by direct co-culture with MSCs, we examined immune localization of FN, well-known EMT marker. In co-culture assay in chamber slides, expression of FN in cancer cluster was seen only at the edge where cancer cells directly contacted with MSCs. FN expression by cancer cells was increased at the tumor periphery and invasive edge in orthotopic nude mice tumors and human colon cancer tissues, respectively. These results suggest that mesenchymal stem cells induce epithelial-mesenchymal transition of colon cancer cells via direct cell-to-cell contact and may play an important role of colon cancer metastasis.

Citation Format: Hidehiko Takigawa, Yasuhiko Kitadai, Toshio Kuwai, Ryo Yuge, Shinji Tanaka, Kazuaki Chayama. Mesenchymal stem cells promote epithelial-mesenchymal transition of colon cancer cells via direct cell-to-cell contact [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 4341. doi:10.1158/1538-7445.AM2017-4341
Abstract 435: Regulation of macropinocytosis-dependent cell survival in pancreatic cancer cells
Authors: Kim Sung Eun , Wang Man-Tzu , McCormick Frank
Published: July 2017

Cancer cells utilize multiple scavenging mechanisms to support growth in nutrient-poor, hypoxic environments. Signaling pathways to accommodate these mechanisms may not be required for oncogenesis per se but are crucial for cancer cell survival. These processes, known as non-oncogene addiction, can be conferred by specific oncogenes that reprogram metabolism or by the tumor microenvironment. Understanding how these pathways are regulated can provide novel opportunities for therapeutic intervention.

Studies showed that KRAS-mutant cancer cells, including pancreatic ductal adenocarcinoma (PDAC), upregulate macropinocytosis, a caveolin- and clathrin-independent endocytic process, to import extracellular protein to support growth upon nutrient depletion. Similarly, high levels of macropinocytic uptake are observed in PDAC murine models and also in human PDAC specimens. This process serves as a scavenging mechanism for cancer cells to survive and proliferate in nutrient-deprived tumor microenvironments. Thus, uncovering the key players of this pathway and the mechanisms underlying their regulation will be important to find potential therapeutic targets.

Here, we used the Informer set of small molecules to screen regulators of survival in culture conditions supplemented with either glutamine or extracellular protein. The Informer set drug library consists of small molecules including FDA-approved drugs and clinical candidates that have high selectivity for their targets and regulate many cellular pathways. We find that survival of cells grown in conditions supplemented with glutamine or exogenous protein is differentially regulated by distinct drug subsets. This suggests that different pathways of nutrient uptake, processing, and utilization are used in cells depending on the nutrient source. Further examination to determine the mechanism of pathway inhibition and applicability may unravel potential uses for therapeutics.

Citation Format: Sung Eun Kim, Man-Tzu Wang, Frank McCormick. Regulation of macropinocytosis-dependent cell survival in pancreatic 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 435. doi:10.1158/1538-7445.AM2017-435
Abstract 4358: The immunomodulatory anticancer agent RRx-001 induces a vaccine-like interferon response through epigenetic induction of viral mimicry
Authors: Zhao Hongjuan , Ning Shoucheng , Nolley Rosalie , Scicinski Jan , Oronsky Bryan , Knox Susan J. , Peehl Donna M.
Published: July 2017

RRx-001, sourced from the aerospace industry and currently in phase II clinical trials, is a novel anti-cancer agent that mediates immunomodulatory effects, either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a single chemotherapeutic agent to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. In this study, we identified another mechanism, viral mimicry, which refers to the ‘unsilencing’ of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anti-cancer activity of RRx-001. Specifically, RRx-001 inhibited the growth of human colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases Dnmt1 and Dnmt3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5 μM RRx-001 for 24 hours significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4 weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retroviruses was induced by RRx-001 through demethylation of the promoter of endogenous retrovirus genes as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA. In conclusion, transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this “pseudo-pathogenic” stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination.

Citation Format: Hongjuan Zhao, Shoucheng Ning, Rosalie Nolley, Jan Scicinski, Bryan Oronsky, Susan J. Knox, Donna M. Peehl. The immunomodulatory anticancer agent RRx-001 induces a vaccine-like interferon response through epigenetic induction of viral mimicry [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 4358. doi:10.1158/1538-7445.AM2017-4358
Abstract 4359: Systematic approach to methylation biomarker development in chronic lymphocytic leukemia, breast and lung cancers
Authors: Wojdacz Tomasz K. , Daugaard Iben , Kjeldsen Tina , Hager Henrik , Kristensen Thomasz , Kristensen Louise , Møller Michael B. , Hansen Lise Lotte
Published: July 2017

There is no doubt that testing for disease specific methylation changes can guide disease risk assessment, facilitate detection of the disease, support prognostication and personalization of treatment as well as guide post treatment patient care (1). Nevertheless, the use of methylation biomarkers in standard patient care is still marginal. Difficulties in implementation of methylation biomarker testing in standard in-vitro diagnostics are mainly attributed to challenges in establishment of the systematic approach allowing for efficient discovery and subsequent validation of clinical utility of potential disease specific methylation biomarkers. We have combined state-of-the-art genome wide methylation screening technologies (including: newest Illumina MethylationEPIC 850k BeadChip) enabling methylation biomarker discovery with the cost and time efficient locus specific technologies to streamline the development, validation and implementation of methylation biomarkers for clinical disease management. With the use of technologies that in an affordable fashion enable screening for genome wide methylation changes in a substantial number of clinical samples, we were able to discover clinically relevant disease specific methylation signatures e.g. in CLL. The high technical complexity of the genome wide screen technologies prevents them for being straightforward applicable in diagnostic settings. Thus we used techniques such as Methylation-Sensitive High Resolution Melting (MS-HRM) (2) to develop assays fulfilling the requirements for diagnostic applications. Subsequently, we evaluated the clinical relevance of the most promising biomarker candidates in chronic lymphocytic leukaemia, breast and lung cancers, using these assays (3, 4). Overall, our workflow allows fast and efficient validation of not only already existing biomarker candidates but also discovery of new disease specific methylation changes that show promise for clinical implementation.

References:

(1) Wojdacz TK. Expert Rev Mol Diagn. 2012 Jan;12(1):39-47 (2) for BLUEPRINT consortium, Nature Biotechnology, Vol. 34, Nr. 7, s. 726-737. (3) Daugaard I et al., Sci Rep. 2016 Oct 26;6:35807. (4) Wojdacz TK et al.. Breast Cancer Res. 2014 Feb 3;16(1):R17

Note: This abstract was not presented at the meeting.

Citation Format: Tomasz K. Wojdacz, Iben Daugaard, Tina Kjeldsen, Henrik Hager, Thomasz Kristensen, Louise Kristensen, Michael B. Møller, Lise Lotte Hansen. Systematic approach to methylation biomarker development in chronic lymphocytic leukemia, breast and lung cancers [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 4359. doi:10.1158/1538-7445.AM2017-4359
Abstract 4360: Epigenetic alteration at HBV integrants is associated with methylation at flanking human genome
Authors: Watanabe Yoshiyuki , Oikawa Ritsuko , Yamamoto Hiroyuki , Itoh Fumio
Published: July 2017

Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis.

Citation Format: Yoshiyuki Watanabe, Ritsuko Oikawa, Hiroyuki Yamamoto, Fumio Itoh. Epigenetic alteration at HBV integrants is associated with methylation at flanking human genome [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 4360. doi:10.1158/1538-7445.AM2017-4360
Abstract 4378: Gastric cancer organoid culture shows preserved genomic stability in long-term passage
Authors: Yue Sarah Siu Kuen , Yan Helen Hoi Ning , Siu Hoi Cheong , Ho Siu Lun , Tsui Wai Yin , Chan Dessy , Chan Annie Shuk Yee , Lee Bernard Chi Hang , Chan Anthony Kin Wang , Leung Suet Yi
Published: July 2017

With recent advances in 3D organoid culture techniques, normal epithelial and tumor cells can be directly cultured from clinical specimens in vitro and expanded long-term with a very high success rate. Thus, this constitutes a good platform for various mechanistic functional studies and clinical applications. A previous karyotyping and gene expression profiling study has indicated that organoid culture of normal epithelial cells can be propagated for long periods of time without genomic alterations. However, data on the genomic stability of tumor organoids in long-term culture are not yet available. We have successfully established organoid cultures derived from either primary tumor or lymph node metastasis from 4 gastric cancer patients. For each sample, we performed long-term culture for at least 6 months. DNA was extracted from blood, as well as the early and late passages of these organoids, and submitted for whole-exome sequencing (WES), achieving a mean coverage approaching 50X. Somatic mutations detected in early versus late passage gastric cancer organoids were compared. Furthermore, we examined the organoids for copy number variations based on the coverage and loss of heterozygosity (LOH) information derived from exome sequencing.

We detected between 80 to 228 somatic mutations in the 4 early passage organoids, in which over 84-99% of them were retained during long-term culture. We detected 16, 20, 31 and 98 new mutations in the 4 long-term cultures, respectively. The one case with a substantially large number of new mutations (n=98) appeared to have emerged from a subclone of TP53 wild-type cells in a TP53 mutant tumor. Notably, the C>T mutation was the most dominant mutation spectrum in this case, constituting 48%. Despite the presence of frequent long segment LOH and chromosomal aberrations in early passage organoids, indicating the presence of chromosomal instability, these patterns of aberrations were stably maintained in long-term passage.

Overall, with the current organoid culture protocol, tumor genomes are mostly stably maintained with the accumulation of only a small number of new mutations. Our results indicate that this is a reliable and stable in vitro cell culture model for various cancer-related and clinical studies.

Citation Format: Sarah Siu Kuen Yue, Helen Hoi Ning Yan, Hoi Cheong Siu, Siu Lun Ho, Wai Yin Tsui, Dessy Chan, Annie Shuk Yee Chan, Bernard Chi Hang Lee, Anthony Kin Wang Chan, Suet Yi Leung. Gastric cancer organoid culture shows preserved genomic stability in long-term passage [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 4378. doi:10.1158/1538-7445.AM2017-4378
Abstract 4398: Identification of bile duct cancer specific fusion genes in patient tissues
Authors: Kim Kahee , Jung Dawoon Esther , Seo Mi-kyoung , Kim Sang Woo , Song Si Young
Published: July 2017

Gene fusion occurs when a part of one gene fuses with or attaches to a part of another gene by genome rearrangement and the result in gene fusion may possess oncogenic properties; fused gene may be translated into a unique protein that may promotes cancer properties. Bile duct cancer are one of the most lethal cancer types with low 5-year survival rates, but lack of proper diagnostic or prognostic markers. Our aim was to investigate bile duct cancer specific fusion genes found in patient’s specimens. We extracted total RNA from five bile duct cancer tissues and normal tissues from the same patients. We performed RNA sequencing and the result data was analyzed using ChimeraScan, Jaffa or Fusionchacher softwares to detect gene fusion. To identify the cancer somatic fusion gene, the fusion occurred in normal tissues and previously reported fusion genes found in normal tissues were excluded and the fusion occurred in coding region was included. As a result, we found 52 bile duct cancer tissue specific- fusion genes. We analyzed the expression of selected fusion genes in 30 bile duct cancer patients and cancer cell lines by qRT-PCR. As a result we observed two novel bile duct cancer specific fusion genes. We cloned one of the fusion genes and transfected into the bile duct cancer cell lines SNU-1196 to assess, and increase in cell motility was observed in vitro. These findings indicate the presence of novel fusion genes as well as its possible application for the early diagnosis or prognosis of bile duct cancer.

Citation Format: Kahee Kim, Dawoon Esther Jung, Mi-kyoung Seo, Sang Woo Kim, Si Young Song. Identification of bile duct cancer specific fusion genes in patient tissues [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 4398. doi:10.1158/1538-7445.AM2017-4398
Abstract 4405: A new energy restriction mimetic decreases survival and FOXM1 expression in pancreatic cancer cells
Authors: Scolaro Laura , Lanza-Jacoby Susan
Published: July 2017

Pancreatic cancer is a deadly disease with only 6% of the patients living longer than 5 years. Even if the disease is diagnosed when it is localized, the 5-year survival rate is approximately 19%. A characteristic of cancer cell metabolism is a shift from oxidative phosphorylation to aerobic glycolysis known as the Warburg effect. It is well known that calorie restriction (CR) is a powerful intervention that suppresses tumorigenesis in animal models by targeting glycolysis and oncogenic signaling pathways. Recent studies have shown that the metabolic responses characteristic of CR can be obtained with the use of energy restriction mimetics (ERM). The purpose of this study is to evaluate a new ERM, OSU-CG5 in altering growth of pancreatic cancer cells and nutrient-sensitive signaling pathways.

Tumors including those of the pancreas have uncontrolled proliferation necessitating increased fuel demands by increasing glucose availability and altering metabolic signaling pathways. Since ERM have been reported to target glucose transport, we evaluated whether OSU-CG5 alters glucose transport (Glut) proteins and glucose uptake.

Glut1, but not Glut2, 3, 4, and 6, were overexpressed in Panc-1 cells with a 6-fold higher expression compared to HPNE cells. Glut proteins were not altered in MiaPaca cells. OSU-CG5 did not alter Glut1 protein expression in either cell line. Silencing Glut1 also had no effect on the cytoxicity of OSU-CG5. OSU-CG5 did not alter glucose uptake when measured in different concentrations of glucose in the media. Cell survival, as measured with Pico green, was decreased after OSU-CG5 treatment with an IC 50 of 3uM when cultured in physiological concentration of 5 and 10mM of glucose.

FOXM1 is unregulated in pancreatic cancer and a promoter of the Warburg effect. We found that OSU-CG5 significantly decreased expression of FOXM1 in Panc1 and MiaPaca cells. Our findings indicate that OSU-CG5 does not inhibit the growth of Panc1 and MiaPaca pancreatic cancer cells by decreasing glucose uptake but may target the FOXM1 signaling pathways.

Citation Format: Laura Scolaro, Susan Lanza-Jacoby. A new energy restriction mimetic decreases survival and FOXM1 expression in pancreatic 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 4405. doi:10.1158/1538-7445.AM2017-4405