bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3125: Therapeutic implications of mTORC1 and mTORC2 inhibitors in genetically heterogeneous glioblastoma
Authors: Amin Anubhav G. , Wang Arthur , Braun Alex , Tobias Michael , Murali Raj , Jhanwar-Uniyal Meena
Published: July 2017

Glioblastoma (GB) is the most common malignant primary brain tumor, which is characterized by marked intra-tumor genetic heterogeneity in receptor tyrosine kinases (RTK) genes. Aberrations in the RTK/PI3K/Akt pathways in GB leads to an abnormal signaling pathway of mechanistic target of Rapamycin (mTOR). mTOR exists in two distinct complexes, namely, mTORC1 and mTORC2, which are involved in regulation of cell survival, growth, and motility. In this investigation, we test the hypothesis that the genetic heterogeneity of GBM cells and its stem cells with respect to the expression of EGFR and/or PDGFRα may define the treatment efficacy of RTK inhibitors with or without PI3K/ mTOR inhibitors. We demonstrated that GB tumors over-expressed PDGFRα and EGFR at range of 80% and 30%, respectively, with varying degree of overlap. Combined treatment with the inhibitors of PDGFRα (AC 710) or EGFR (gefitinib), with or without inhibitors of mTORC1 (rapamycin) or mTORC1/2 (Torin1 and Torin 2), led to suppression of cell proliferation, cell cycle, cell migration and drug resistance in GB cells to varying degrees that correlated with EGFR or PDGFRα expression. Downstream signaling pathway substrates of mTORC 1 and 2, namely pS6k and pAkt, respectively, were suppressed by combined treatments of RTK and mTORC1/2 inhibitors. Furthermore, GB stem cell self-renewal and growth was also suppressed with combined treatments. The combination of AC710 plus mTORC1/2 inhibitor, Torin2, was the most effective. These findings underscore the usefulness of determining PDGFRα and EGFR status when considering viable PI3K/mTOR inhibitors for targeted treatment of GB.

Citation Format: Anubhav G. Amin, Arthur Wang, Alex Braun, Michael Tobias, Raj Murali, Meena Jhanwar-Uniyal. Therapeutic implications of mTORC1 and mTORC2 inhibitors in genetically heterogeneous glioblastoma [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 3125. doi:10.1158/1538-7445.AM2017-3125
Abstract 313: Tbata induces G2/M cell cycle arrest and sensitizes osteosarcoma cells to etoposide in a p53-independent manner
Authors: Miljkovic Milos D. , Flomerfelt Francis A. , Gress Ronald E.
Published: July 2017

Thymus, brain, and testes-associated (Tbata) is a negative control cell-cycle gene highly expressed in murine thymic epithelial cells (TECs). Tbata protein binds to Uba3, inhibiting formation of Nedd8 E1 and subsequent target degradation via neddylation of several cell cycle control proteins needed for G2/M transition, which may be a major mechanism of TEC growth arrest during thymic involution. Etoposide is a cytotoxic drug which targets the enzyme toposimerase II — increased 2-3 fold during the G2 phase. To further characterize effect of Tbata on the cell cycle, we modified p53 wild-type U2OS cells derived from human osteosarcoma to express a Tbata/mCherry fusion protein when mifepristone is added to culture media. Tbata-expressing cells identified on flow cytometry did exhibit growth arrest, with quantitative assessment of DNA content in U2OS cells by flow cytometry establishing that 32% of Tbata/mCherry-expressing cells were in the G2 phase when exposed to mifepristone for 24 hours, compared to 11% of mCherry-expressing cells. Gene expression studies were consistent with these results. Cells expressing Tbata/mCherry were also more sensitive to etoposide at 0.5, 1, and 5 times the IC50 dose. To test whether p53 function was required, we further modified the cells to overexpress a dominant negative p53 mutant along with Tbata/mCherry. Similar G2 arrest and increased sensitivity to etoposide were observed, indicating that the effects of Tbata did not require normal p53 function. Potential Tbata analogues or mimetics may therefore be used as an adjunct to G2-targeted chemotherapy.

Citation Format: Milos D. Miljkovic, Francis A. Flomerfelt, Ronald E. Gress. Tbata induces G2/M cell cycle arrest and sensitizes osteosarcoma cells to etoposide in a p53-independent manner [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 313. doi:10.1158/1538-7445.AM2017-313
Abstract 3135: Benzaldehyde suppresses the binding activity of overexpressed 14-3-3ζ to multiple signaling proteins in cancer cells
Authors: Saitoh Jun , Saya Hideyuki
Published: July 2017

Benzaldehyde is the simplest aromatic aldehyde constituent of almonds and many fruits. Anticancer effect of Benzaldehyde was first reported in 1980, and multi-institutional clinical trials were performed in those days in Japan. However, trial was over without determination of effectiveness, only its’ safety was confirmed. The underlying mechanism why Benzaldehyde specifically suppresses growth of some particular cancer cells but not that of normal cells has not been elucidated. Therefore, we attempted to clarify the mechanism of anticancer effect of Benzaldehyde. We have previously reported that, in pancreatic cancer cell BxPC3 and in non-small cell lung cancer cell A549, Benzaldehyde inhibits PI3K/AKT/mTOR, STAT3, NFκB and ERK pathways, those are major signaling pathways activated in cancer cells. Effects of Benzaldehyde on multiple signaling pathways are found to be derived from regulation of 14-3-3 family proteins which interact with phosphorylation sites of various proteins of multiple signals. In BxPC3 cells, Benzaldehyde treatment reduced the phosphorylation levels of 14-3-3-binding sites. Moreover, we ectopically expressed seven isoforms of 14-3-3 in HEK293T cells and found that Benzaldehyde treatment significantly suppressed association of 14-3-3ζ with client proteins such as mTOR, Rictor, cRaf, STAT3 and FOXOs. The interaction of other isoforms of 14-3-3 with their client proteins was also partially reduced. But, the expression levels of those seven 14-3-3 isoforms were not significantly changed. Here we further analysis the Benzaldehyde-mediated 14-3-3ζ regulation, in HEK293 cells overexpressing 14-3-3ζ. Benzaldehyde significantly suppressed mTOR pathway while it was not observed in parental HEK293 cells. Furthermore, we performed pull down assay by using 14-3-3ζ GST tagged recombinant proteins and lysates of HEK293T and BxPC3 and found that Benzaldehyde treatment suppressed the interaction of 14-3-3ζ with cRaf, TSC2 and other 14-3-3ζ client proteins. From these data, we confirmed that Benzaldehyde is an inhibitor of the overexpressed 14-3-3ζ. Recent reports have shown that 14-3-3ζ is overexpressed in many cancers and acts as a signaling hub controlling the network of oncogenic pathways, suggesting that overexpressed 14-3-3ζ associates with carcinogenesis, metastasis and resistance for chemotherapy and radiation. Hence, Benzaldehyde is considered to be a new molecular target agent of 14-3-3ζ which promote and maintain cancer signals.

Citation Format: Jun Saitoh, Hideyuki Saya. Benzaldehyde suppresses the binding activity of overexpressed 14-3-3ζ to multiple signaling proteins 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 3135. doi:10.1158/1538-7445.AM2017-3135
Abstract 3137: Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung cancer
Authors: Bae Kieun , Lee Minkyong , Yoon Daseul , Kim Yun-Hee , Yoon Kyong-Ah
Published: July 2017

BCAR4 was identified as tamoxifen resistance related gene in breast cancer due to its function to activate estrogen-independent cell growth. Instead of estrogen receptor, ERBB2 and ERBB3 genes were activated by BCAR4 in tamoxifen resistant cells. Recently, the function of BCAR4 as a long non-coding RNA (lncRNA) was reported to regulate a non-canonical Hedgehog/GLI2 pathway in breast cancer. However, the association of BCAR4 and lung cancer has not been identified. Here, we describe the oncogenic effect of BCAR4 in human lung cancer. Expression pattern of BCAR4 was examined in lung cancer cell lines, primary tumor tissues, and adjacent normal tissues. BCAR4 was highly expressed in 71% of lung cancer cell lines and especially in 43% of cancer tissues of lung adenocarcinoma patients who did not harbor the activational mutations of EGFR nor KRAS. Exogenous expression of BCAR4 protein promoted cell growth of non-tumorigenic bronchial epithelial cell line, BEAS-2B, and lung cancer cell line, NCI-H1299. We also confirmed the oncogenic feature of BCAR4 by colony forming assay using stably expressing cells. These effects were notably reduced after knockdown of BCAR4 by small interfering RNA. BCAR4 encoded a functional protein and its overexpression resulted in the enhanced migration than controls cells. This result proposed a similar role of BCAR4 in lung cancer considering its previously reported role in breast cancer metastasis. We also detected a fusion gene of BCAR4 in a lung cancer tissue by analyzing RNA-sequencing data. BCAR4 protein as well as its fusion form showed the improved tumorigenic feature in the mouse xenograft model with lung cancer cells. We compared the gene expression pattern in BCAR4 overexpressed cells to identify the possible mechanism of the oncogenic effect of BCAR4. Cyclin D1, Cyclin E, and MMP1 mRNA and protein levels were significantly increased after BCAR4 overexpression. However, downstream genes of GLI2 signaling were not altered by protein expression of BCAR4 arguing an alternative role for lncRNA BCAR4. Our results suggest a novel oncogenic role of BCAR4 protein in lung cancer.

Citation Format: Kieun Bae, Minkyong Lee, Daseul Yoon, Yun-Hee Kim, Kyong-Ah Yoon. Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung 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 3137. doi:10.1158/1538-7445.AM2017-3137
Abstract 3140: MUC1 expression as a prognosis marker and a new therapeutic target in patient with duodenal adenocarcinoma
Authors: Shiba Satomi , Miki Atsushi , Ohzawa Hideyuki , Teratani Takumi , Sakuma Yasunaru , Kitayama Joji , Lefor Alan Kawarai , Sata Naohiro
Published: July 2017

Background: Mucin1 (MUC1) is a glycoprotein as a member of the mucin family. The expression has been associated with poor prognosis in patients with several types of cancer. Duodenal cancer is rare, therefore details are not known including carcinogenesis and clinical treatment. We verify the association of MUC1 expression and prognosis in patient with duodenal adenocarcinoma. We also assess the function of MUC1 in vitro. Materials and Methods: Between 1989 and 2014, eighteen patients of duodenal adenocarcinoma with surgery at Jichi Medical University Hospital were included in this study. MUC1 expression was evaluated immunohistochemically with anti-MUC1 antibody to examine the association of MUC1 expression and prognosis clinicopathologically. A human-derived duodenal cancer cell line, HuTu-80, is used for the experiments of invasiveness and proliferation in vitro. The invasive capacity was evaluated by transfection of siRNA into HuTu80 with matrigel invasion assay. Viability assay with the anti-MUC1 peptide GO201 was assessed to determine whether MUC1 associate with cell growth of duodenal adenocarcinoma. Results: Seven cases were MUC1 positive and 11 were MUC1 negative immunohistochemically. TMN staging was not associated with MUC1 expression. Kaplan-Meir analysis showed a poor prognosis with MUC1 positive patients in cumulative overall survivals (MUC1 positive : MUC1 negative, 18M : 52M; HR:20.6, 95%CI 3.247-130.7,p=0.002). Lymph node metastasis of MUC1 positive patients (71.4%) was significantly higher than MUC1 negative patients (9%) (p = 0.01). In vitro experiment, the expression of MUC1 was confirmed by RT-PCR and flowcytometry. Knockdown experiment with transduction of siRNAs to HuTu80 decreased the expression of the MUC1 gene. Transduction of siRNAs significantly reduced invasive capacity (47.02±21.65%, 95%CI 3.046-102.9, p= 0.04). Growth of HuTu80 cells was inhibited by GO201 (5 μM : 95%CI 0.109-0.3565, 10μM :95%CI 0.024-0.389 p<0.0001). Conclusions: MUC1 expression is a predictive marker for poor prognosis in patients with duodenal adenocarcinoma. Functional Interference of MUC1 core protein reduces cell growth and invasiveness. Taken together, MUC1 is associated with malignant potential and a novel target for the treatment.

Citation Format: Satomi Shiba, Atsushi Miki, Hideyuki Ohzawa, Takumi Teratani, Yasunaru Sakuma, Joji Kitayama, Alan Kawarai Lefor, Naohiro Sata. MUC1 expression as a prognosis marker and a new therapeutic target in patient with duodenal adenocarcinoma [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 3140. doi:10.1158/1538-7445.AM2017-3140
Abstract 3146: Targeting ANXA3 in combination with sorafenib for the treatment of hepatocellular carcinoma
Authors: Tong Man , Luk Steve , Che Noelia , Ding Jin , Lee Terence KW , Ma Stephanie
Published: July 2017

Sorafenib is the only FDA-approved tyrosine kinase inhibitor for targeted therapy in advanced HCC. Nevertheless, its efficacy is limited with only a modest improvement in patient outcome, likely due to acquired resistance. In-depth understanding of the molecular mechanism of sorafenib resistance is warranted for the development of novel treatment strategies. Recent studies by us and others have characterized liver tumor-initiating cells (T-ICs) to be a possible source of resistant and recurrent tumors and a plausible target for HCC treatment. Our group has previously identified CD133 to be a functional marker of liver T-ICs and found annexin a3 (ANXA3) to regulate cancer and stem cell-like properties in this subset of cells. Interestingly, our recent observations also found CD133+ liver T-ICs to be more resistant to sorafenib. Sorafenib resistant clones, established in HepG2 and Huh7 cells by continuous exposure to increasing concentrations of sorafenib, displayed enhanced abilities to migrate, invade, self-renew, and initiate tumor formation in immunodeficient mice, as well as higher expression of stemness associated genes. These two sorafenib resistant cell lines and two other sorafenib resistant HCC patient-derived xenografts established in a similar manner were also found to be enriched for CD133 and ANXA3 expression. Sorafenib resistant clones with ANXA3 stably suppressed were re-sensitized to sorafenib treatment and had diminished ability to migrate, invade, self-renew and initiate tumor growth in vivo, further substantiating the role of ANXA3 in mediating sorafenib resistance in HCC. Mechanistically, an activated PKC/ERK/FRA2 signaling axis was found to be responsible for driving this phenomenon. Clinically, ANXA3 expression was also found to have prognostic value as a higher ANXA3 expression in HCC patients who have received sorafenib treatment was correlated with poor overall survival. The combinatorial use of a homemade ANXA3 neutralizing antibody and sorafenib on HCC patient derived xenografts is now being investigated as a potential new treatment regimen for combating sorafenib resistance in HCC.

Citation Format: Man Tong, Steve Luk, Noelia Che, Jin Ding, Terence KW Lee, Stephanie Ma. Targeting ANXA3 in combination with sorafenib for the treatment of hepatocellular 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 3146. doi:10.1158/1538-7445.AM2017-3146
Abstract 3156: Multiple acquired resistance mechanisms against third generation EGFR-TKI osimeritinib in non-smal cell lung cancer cells
Authors: Namba Kei , Shien Kazuhiko , Yoshioka Takahiro , Torigoe Hidejiro , Sato Hiroki , Yamamoto Hiromasa , Soh Junichi , Tsukuda Kazunori , Toyooka Shinichi
Published: July 2017

Background Osimertinib (AZD9291; Tagrisso) is a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) known to be effective against the EGFR-T790M variant, which is accounts for half of the acquired resistance mechanisms to the first generation EGFR-TKIs. However, resistance to osimertinib is likely to progress and the study of potential osimertinib-resistance mechanisms in advanced is necessary. In this study, we investigated the molecular and cellular profiles of the acquired resistance cells to osimertinib in EGFR-mutant non-small cell lung cancers. Materials and Methods Five EGFR-mutant cell lines were exposed to osimertinib by stepwise escalation or high-concentration exposure methods, and resistant sublines to osimertinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Results EGFR-C797S mutation which was reported to be a major mechanism of resistance to osimertinib in clinical samples was not detected in established resistance cells by using direct sequencing. Several osimertinib-resistance cell lines displayed MET amplification, and some of these cells were sensitive to the combination of osimertinib plus MET inhibitor crizotinib. However, one cell line that displayed MET amplification was not sensitive to this combination therapy. On the other hand, several osimertinib-resistance cell lines displayed epithelial-to-mesenchymal transition (EMT) features. The HCC827-derived subline established by the high-concentration exposure method exhibited not only EMT features but also cancer stem cell (CSC)-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) and ATP binding cassette subfamily B member 1 (ABCB1) overexpression. Conclusion We established osimertinib-resistant cells and found that MET amplification, EMT, and CSC -like features were observed. These findings may provide clues to overcome acquired resistance to EGFR-TKIs. In some cell lines, however, the mechanisms of acquired resistance to osimertinib were not revealed yet.

Citation Format: Kei Namba, Kazuhiko Shien, Takahiro Yoshioka, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Kazunori Tsukuda, Shinichi Toyooka. Multiple acquired resistance mechanisms against third generation EGFR-TKI osimeritinib in non-smal cell lung 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 3156. doi:10.1158/1538-7445.AM2017-3156
Abstract 3170: MCAM modulates small cell lung cancer chemoresistance via PI3k/Akt/Sox2 signaling pathway
Authors: Tripathi Satyendra C. , Fahrmann Johannes F. , Celiktas Muge , Aguilar Mitzi , Marini Kieren D. , Jolly Mohit K. , Katayama Hiroyuki , Wang Hong , Murage Eunice N. , Dennison Jennifer B. , Watkins D. Neil , Levine Herbert , Ostrin Edwin J. , Taguchi Ayumu , Hanash Samir M.
Published: July 2017

Despite favorable responses to initial therapy SCLC relapse occurs within a year exhibiting a multidrug resistant phenotype. Due to limited accessibility of patient tissues for research purpose, SCLC patient derived xenografts (PDXs) have provided the best opportunity to address this limitation. We sought to identify novel mechanisms involved in SCLC chemoresistance. Through in-depth proteomic profiling, we identified MCAM as a markedly upregulated surface receptor in chemoresistant SCLC cell lines that exhibited a mesenchymal phenotype and in chemoresistant PDXs compared to matched treatment-naïve tumors. MCAM is a cell membrane protein whose expression has been implicated in multiple human cancers. MCAM expression is also detected in lung adenocarcinoma; however, its expression and role in SCLC is still not been explored. MCAM knockdown in chemoresistant cells reduced cell proliferation and decreased the IC50 inhibitory concentration of chemotherapeutic drugs. MCAM was found to modulate sensitivity of SCLC cells to chemotherapeutic drugs through up-regulation of MRP1/ABCC1 expression and of the PI3K/AKT pathway in a SOX2 dependent manner. Metabolomic profiling revealed that MCAM can modulate glutamic acid and lactate production in chemoresistant cells with a distinct metabolic phenotype sustaining low oxidative phosphorylation. MCAM may serve as a novel therapeutic target to overcome chemoresistance in SCLC.

Citation Format: Satyendra C. Tripathi, Johannes F. Fahrmann, Muge Celiktas, Mitzi Aguilar, Kieren D. Marini, Mohit K. Jolly, Hiroyuki Katayama, Hong Wang, Eunice N. Murage, Jennifer B. Dennison, D. Neil Watkins, Herbert Levine, Edwin J. Ostrin, Ayumu Taguchi, Samir M. Hanash. MCAM modulates small cell lung cancer chemoresistance via PI3k/Akt/Sox2 signaling pathway [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 3170. doi:10.1158/1538-7445.AM2017-3170
Abstract 3178: The p53 tumor suppressor protein paradoxically drives chemo-resistance in human medulloblastoma cells through suppressing the mTOR AKT pathway
Authors: Naeem Aisha , Choudhry Muhammad U. , Avantaggiati Maria L. , Rodriguez Olga C. , Albanese Chris
Published: July 2017

Medulloblastoma (MB) accounts for approximately 25% of childhood brain tumors with 70% of the cases occurring in children under 10. Prognosis for children less than three years old is considerably worse and, due to its destructive effects on the developing nervous system, irradiation is largely avoided in this age group. The p53 pathway is considered a key determinant of anti-tumor responses in many tumors; however, its role in the regulation of cell survival, chemo-sensitivity and chemo-resistance in MB is much less well defined. It has been shown that p53 pathway defects, mutations and nuclear levels increase significantly from MB diagnosis to relapse and correlate with an adverse prognosis. We recently reported on the highly novel and unexpected finding that both the genetic (sh-RNA) and chemical silencing of p53 led to a significant increase in cell death by the drug VMY in MB cell lines with elevated basal p53 (e.g. in D556 cells which express wild type p53 and in DAOY cells that contain mutant p53) as measured by colony forming assays, DNA degradation assays and annexin-V staining, suggesting a surprising commonality in the p53 signaling in both cell lines despite the differences in their p53 status. Equally surprising was the observation that the silencing of p53 in D556 cells enhanced cell death by the clinically used drugs doxorubicin and vincristine. Conversely, we now find that suppressing p53 with sh-RNA in D283 cells, which express much lower levels of wild type p53 compared to D556, resulted in the more classical chemoresistance profile, suggesting a form of p53 ‘addiction’ in D556 and DAOY cells. Mechanistically, we observed that suppressing p53 with sh-RNA in D556 cells treated with VMY or doxorubicin significantly increased the levels of phosphorylated Chk1, S6K, gH2AX, and MDM2. Importantly, phospho-mTOR levels were also significantly increased in the D556/p53 sh-RNA cells and the suppression of mTOR enhanced chemoresistance. These somewhat paradoxical findings suggest that activated Akt/mTOR may induce MB cell death and that suppression of mTOR by p53 enhances chemoresistance. Our data provide new mechanistic insights into the role of p53 in primitive neuroectodermal tumors and may provide new approaches for enhancing the clinical outcome of patients with MB.

Citation Format: Aisha Naeem, Muhammad U. Choudhry, Maria L. Avantaggiati, Olga C. Rodriguez, Chris Albanese. The p53 tumor suppressor protein paradoxically drives chemo-resistance in human medulloblastoma cells through suppressing the mTOR AKT pathway [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 3178. doi:10.1158/1538-7445.AM2017-3178
Abstract 3184: Autophagy regulation by vacuolar-ATPases: A contributing mechanism of chemo-resistance in ovarian cancer
Authors: Kulshrestha Arpita , Katara Gajendra K. , Ibrahim Safaa A. , Gilman-Sachs Alice , Beaman Kenneth D.
Published: July 2017

In ovarian cancer (OVCA), the main obstacle in the clinical usefulness of platinum based drugs is the eventual emergence of chemo-resistant tumors. Exposure of cancer cells to platinum drugs such as cisplatin elicits a stress response that leads to the induction of coping mechanisms favoring cancer cell survival. Autophagy induction is the primary protective mechanism in cancer cells that also contributes to cisplatin resistance. A tightly regulated pH is imperative for autophagy and the role of major pH regulators is therefore vital to this process. Vacuolar ATPases (V-ATPases) are the multi-subunit proton pumps that regulate intra/extra-cellular pH in cancer. Its `a2` isoform (V-ATPase-a2) is located at the plasma membrane and early/late endosomal compartments. We previously found that this isoform is overexpressed on cisplatin resistant cells (cis-R) and its inhibition sensitizes cis-R cells to platinum drugs. In continuation with our efforts, the present study was performed to understand what alterations in the autophagy process occur upon V-ATPAse-a2 inhibition and how these contribute to cisplatin sensitization. Expression profiling of ovarian cancer cells lines (A2780-cis R and TOV-112D- cis R) revealed that upon a2 inhibition, the basal levels of autophagosome membrane protein, LC3B were enhanced compared to control cis-R cells as determined by flow cytometry and western blot analysis. Upon cisplatin treatment, the LC3B levels were further enhanced in a2 inhibited cis-R cells compared to control cells. Further, we observed a significantly increased expression of the autophagy protein, beclin-1 in these cells as compared to control cells. In addition, the inhibition of V-ATPase-a2v increased the protein levels of SQSTM1/p62, a known substrate for the autophagy process. The enhanced LC3B and P62 levels suggest an accumulation of the autophagosomes due to an altered autophagy flux upon V-ATPase-a2v inhibition. The study provides a rationale for the utility of V-ATPase-a2 inhibitors in combination with standard drugs as a novel strategy to improve the treatment efficacy of the chemoresistant ovarian cancer.

Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Safaa A. Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. Autophagy regulation by vacuolar-ATPases: A contributing mechanism of chemo-resistance in ovarian 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 3184. doi:10.1158/1538-7445.AM2017-3184
Abstract 3188: ER-stress induced translational regulation by RGS2 leads to tumor-promoting microenvironment and chemoresistance
Authors: Min Hye-Young , Park Shin-Hyung , Lee Ho Jin , Cho Jaebeom , Noh Myungkyung , Hyun Seung Yeob , Lee Ho-Young
Published: July 2017

Lung cancer is the leading cause of the cancer-related death worldwide. Despite extensive efforts for the cure of lung cancer, the effectiveness of currently available therapeutic regimens has been marginal and limited, and recurrence is still an inevitable consequence of anticancer therapies. Therefore, mechanistic understanding on the progression and relapse of lung cancer and finding out relevant targets are essential for development of efficacious therapeutic strategies. We have investigated mechanisms of chemoresistance by using subsets of lung cancer cell lines carrying acquired resistance to chemotherapies. These chemoresistant sublines of NSCLC cells exhibited a prominent downregulation of in vitro tumor activities, including proliferation, metabolism, and protein synthesis, but displayed increased tumor growth in vivo. Additional studies revealed that the chemoresistant sublines experienced increased secretion of various soluble factors that led to increases in proliferation and stemness of drug-naïve cancer cells and recruitment of macrophages, establishing tumor microenvironment (TME) prone to tumor recurrence. We further demonstrate a pivotal role of regulator of G protein signaling 2 (RGS2) in tumor-promoting cell-cell communications, ultimately leading to chemoresistance. RGS2 disrupted chemotherapy-induced apoptosis and secretion of soluble factors involved in cellular communications in TME by regulating unfolded protein responses, a typical cellular event associated with downregulation of protein synthesis. Mechanistically, RGS2 induced proteasome-mediated protein degradation of a component of eukaryotic translation initiation factor complexes through direct interaction, resulting in blockade of the CAP-dependent protein translation. RGS2 expression was elevated in tumors derived from patients with lung cancer compared with normal counterparts and significantly associated with poor clinical outcomes. These findings suggest that RGS2 is a novel biomarker responsible for chemoresistance by establishing tumor-promoting TME and can be a potential therapeutic target for the treatment of lung cancer with chemoresistance.

Citation Format: Hye-Young Min, Shin-Hyung Park, Ho Jin Lee, Jaebeom Cho, Myungkyung Noh, Seung Yeob Hyun, Ho-Young Lee. ER-stress induced translational regulation by RGS2 leads to tumor-promoting microenvironment and chemoresistance [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 3188. doi:10.1158/1538-7445.AM2017-3188
Abstract 319: Cholesterol inhibition reduces Hh mediated chondrosarcoma
Authors: Wei Qingxia , Ramu Eyal , AL-Jazrawe Mushriq , Poon Raymond , Wunder Jay , Alman Benjamin
Published: July 2017

Constitutive activation of Hh signaling is a common occurrence in chondrosarcoma(CSA). Gene profiling analysis showed that Gli transcription regulates genes that govern cholesterol homeostasis, which alters cholesterol accumulation in chondrocytes; a higher level of Gli-mediated transcription results in accumulation of intracellular cholesterol. Here we determined if targeting cholesterol-processing genes downstream of Hh signalling could be used as a novel treatment approach. With institutional review board approval, human CSA samples were obtained fresh from surgery. For in vitro studies, CSA explants of 2mmx2mm x2 mm cubic in size establihed as organ cultures. For in vivo studies, one million CSA cells were subcutanously injected into NSG mice. Cells from five CSAs were treated both in vitro and in vivo with a hedgehog inhibitor, Cur61414, a cholesterol inhibitor, Lovastatin, or both. In vitro, CSA explants were treated for 48 hrs at concentration of 20 μM of each drug. In vivo, mice were treated with 4.5mg/kg/day of Cur61414, Lovastatin, or both by intraperitoneal injection for 4 wks. At the end of treatment, the explants or xenografts were harvested and processed for further analysis. RT-PCR was used to meausre the expression of Hh and cholesterol target genes. Tumor size was meausred from the xenografts. Blockade of Hh signaling significantly decreased Gli1 gene expression by 30%, increased 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression for more than 300% indicating decreased intracellular cholesterol. Treatment with the cholesterol inhibitor Lovastatin increased expression of HMGCR for more than 500%. The combination of Hh and cholesterol blockage resulted in increased expression of HMGCR for more than 3400%. Analysis of chondrosarcoma xenografts in vivo showed a significnat decrease in tumor size with Lovastatin (32% decline), 3 folds reducation of Brdu(+) cells, and 2.4 fold increase of Caspase-3 + cells, treatment with Cur 61414 reduced the tumor growth by 5% with no significant reduction of Brdu(+) cells but 2.6 fold increase of Caspase-3 (+) cells . The combination treatment of lovastatin and Cur61414 on xenografts resulted in a significnat decrease in tumor size (32% decline), 3 fold reduction of Brdu (+) cells, but no significant changes of Caspase-3 (+) cells . These data suggest that cholesterol functions downstream of Hh signaling pathway in CSA. The more effective reduction in tumor growth with cholesterol inhibition compared to Hh blockade suggests cholesterol blockade is an effective therapeutic approach.

Citation Format: Qingxia Wei, Eyal Ramu, Mushriq AL-Jazrawe, Raymond Poon, Jay Wunder, Benjamin Alman. Cholesterol inhibition reduces Hh mediated chondrosarcoma [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 319. doi:10.1158/1538-7445.AM2017-319
Abstract 3201: Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma
Authors: Barbhuiya Mustafa A. , Mirando Adam C. , Simons Brian W. , Lemtiri-Chlieh Ghali , Green Jordan J. , Popel Aleksander S. , Pandey Niranjan B. , Tran Phuoc T.
Published: July 2017

Hepatocellular carcinoma (HCC) related mortality ranks second worldwide and stands one step behind lung cancer. Due to the asymptomatic nature of the cancer, early diagnosis is a major problem. Currently only liver transplantation is curative for early stage hepatocellular cancer. There are other multidisciplinary treatment options like radiation and chemotherapy available for advanced patients. However, chemotherapy resistance is a common problem in the treatment of liver cancer. Newer therapeutics are needed for better management of advanced HCC. Angiogenesis and lymphangiogenesis are processes that are vital for tumorigenesis and metastasis. HCC is known to be a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. We explored the therapeutic potential of the anti-angiogenic and anti-lymphangiogenic, biomimetic peptide SP2043 developed by our group in HCC. Hepatocellular carcinoma cell lines HuH-7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration following treatment. Furthermore, SP2043 was found to impair microvascular endothelial cell (MEC) tube formation induced by HepG2 tumor conditioned media and to significantly inhibit HepG2 tumor xenograft growth compared to untreated controls. The peptide drug was also found to improve the survival of autochthonous Myc induced HCC in a transgenic mouse model from two weeks to four weeks. We discovered the peptide to be a multi-modal anti-angiogenic drug that also inhibits IGF1R and MET signaling pathways in HCC cell lines. We also found that SP2043 treatment reduced the microvascular density in both subcutaneous and autochthonous liver tumors with reduced tumor cell proliferation and increased apoptosis. This study shows the therapeutic potential of SP2043 in the treatment of hepatocellular carcinoma.

Note: This abstract was not presented at the meeting.

Citation Format: Mustafa A. Barbhuiya, Adam C. Mirando, Brian W. Simons, Ghali Lemtiri-Chlieh, Jordan J. Green, Aleksander S. Popel, Niranjan B. Pandey, Phuoc T. Tran. Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular 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 3201. doi:10.1158/1538-7445.AM2017-3201
Abstract 3207: Targetting proteasome function by inhibition of the proteasome deubiquitinase USP14
Authors: Linder Stig T. , D'Arcy Padraig , Zhang Xiaonan
Published: July 2017

The 20S proteasome core particle has evolved as an important target for anti-cancer drug development. We previously identified the small molecule b-AP15 as a novel class of proteasome inhibitors that function by abrogating the deubiquitinating (DUB) activity of the 19S regulatory particle. An optimised lead of b-AP15 (VLX1570) has been approved by the FDA for clinical studies and is currently in clinical trials for relapsed multiple myeloma. VLX1570 has also shown promising activity in Ewings sarcoma and paediatric leukemias. b-AP15 and VLX1570 bind to the proteasome DUB ubiquitin-specific protease-14 (USP14) in vitro and in exposed cells (CETSA). Binding and inhibition of enzymatic activity is believed to be due to targeting Cys114 in the active site. The cell death response to b-AP15/VLX1570 is distinct from that of 20S proteasome inhibitors. Thus, the sensitivity to these compounds is unaffected by overexpression of BCL2-family proteins and unaffected by p53 status. We here show that b-AP15 induces direct effects on mitochondria, possible explaining this phenomenon. We also provide additional understanding of drug-target interactions using a series of compounds identified by screening. Our findings demonstrate promising antiproliferative activities of USP14 inhibitors in vitro and in vivo.

Citation Format: Stig T. Linder, Padraig D'Arcy, Xiaonan Zhang. Targetting proteasome function by inhibition of the proteasome deubiquitinase USP14 [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 3207. doi:10.1158/1538-7445.AM2017-3207
Abstract 3208: AZD1775 modulates kinetochore component Wee1-CDC2 and NF-κB signaling as potential therapeutic targets in head and neck squamous cell carcinoma
Authors: Hu Zhengbo , Saleh Anthony , Chen Jianghong , Sophie Carlson , An Yi , Paul Clavijo , Wang Ru , Cornelius Shaleeka , Chen Zhong , Waes Carter Van
Published: July 2017

Recent findings in The Cancer Genome Atlas (TCGA) analysis of head and neck squamous cell carcinomas (HNSCC) revealed frequent alterations affecting the cell cycle and NF-κB pathway, which are critically involved in cancer cell proliferation and survival. We hypothesized that common mechanism(s) could coordinate cell cycle and NF-κB cell survival signaling in HNSCC. Recently, we established an NF-κB β−lactamase reporter UM-SCC-1 cell line and performed genome-wide RNAi screening to explore this hypothesis. We identified genes involved in the cell cycle and related to components of the kinetochore that co-modulate NF-κB activity. Among these targets, WEE1 kinase is currently under investigation as a therapeutic target of inhibitor AZD1775 in HNSCC. We found that WEE1-CDC2 axis protein levels were higher in a panel of HNSCC lines than in normal human oral keratinocytes (HOK). To further investigate the potential cross-talk between WEE1 cell cycle regulation and NF-κB activation, we showed that treatment of HNSCC cells with AZD1775 decreased NF-κB transcriptional activation and decreased TNFα-induced phosphorylation of IKKs and RELA subunit. Furthermore, AZD1775 inhibited NF-κB regulated proteins modulating the cell cycle and survival, including p21, CyclinD1, BCL2 and BCL-XL. These changes also correlate with alteration in cell cycle and survival detected by flow cytometry, as tumor cells treated by AZD1775 exhibited increased G2/M phase and sub-G0 fragmented DNA, and decease in G1/S phases. WEE1 inhibitor AZD1775 inhibited tumor cell proliferation and colony formation in a dose-dependent manner, and exhibited combinatory effects with cisplatin chemotherapy in tumor xenografts. Thus, we identified the novel cross-talk between WEE1-CDC2 and NF-κB signaling, potentially implicating decreased NF-κB prosurvival signaling in the anti-tumor activity of Wee1 inhibitors. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, and 74; NIDCD core facility: advanced imaging, ZIC DC000081).

Citation Format: Zhengbo Hu, Anthony Saleh, Jianghong Chen, Carlson Sophie, Yi An, Clavijo Paul, Ru Wang, Shaleeka Cornelius, Zhong Chen, Carter Van Waes. AZD1775 modulates kinetochore component Wee1-CDC2 and NF-κB signaling as potential therapeutic targets 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 3208. doi:10.1158/1538-7445.AM2017-3208
Abstract 3218: Molecular function of PARP inhibition in glioblastoma
Authors: Aldrighetti Christopher , Huebert Barbara , Quenet Delphine
Published: July 2017

Glioblastoma (GBM) is the most common primary malignant brain tumor, whose major biomarkers are the DNA methylation status of the promoter for O6-methylguanine-DNA-methyltransferase (MGMT) and the loss/mutation of the tumor suppressor phosphatase and tensin homolog (PTEN). Despite treatment combining surgery, radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ), patient survival remains poor and recurrence is virtually inevitable, due to TMZ resistance. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear protein involved in multiple facets of DNA repair and transcriptional regulation. This enzyme catalyses the transfer and polymerization of ADP ribose units from NAD+ to form branched polymers of ADP-ribose, called poly(ADP-ribose) (PAR), covalently attached to heterologous acceptor proteins or PARP-1 itself. There is a growing interest in PARP inhibitors (PARPi) for their clinical potential in cancer treatment. Because of the importance of PARP-1 activity in the maintenance of genomic integrity and DNA repair, treatment with PARPi as adjuvant to TMZ chemotherapy can be beneficial in GBM. PARPi are currently in clinical trials for patients with GBM (NCT02152982, NCT01390571, NCT01514201), and one of them is a phase II/III study of the PARPi veliparib for patients with newly-diagnosed GBM (NCT02152982). However, the molecular role of PARP-1 and its activity in GBM remains elusive. Using cellular and molecular approach, we are assessing the effectiveness of the PARPi veliparib compared to standard treatment with TMZ in PTEN-mutated GBM and TMZ-resistant GBM cell lines. By understanding the biology of PARP inhibition in GBM, we will establish a specific molecular signature that predicts the efficacy of PARPi treatment in patients diagnosed with GBM.

Citation Format: Christopher Aldrighetti, Barbara Huebert, Delphine Quenet. Molecular function of PARP inhibition in glioblastoma [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 3218. doi:10.1158/1538-7445.AM2017-3218
Abstract 3231: Identifying high quality, potent and selective pyrimidinylthienopyrrolone inhibitors of ERK1/2 kinase: LY3214996
Authors: Zhao Gaiying , McMillen William T. , Cai Shufen , Zhao Baohui , Whitesell Matthew , Wu Wenjuan , Huss Karen , Anderson Bryan , Yuan Xiu-Juan , Jaken Susan , Kindler Lisa , Flack Robert S. , McCann Denis , Mathes Brian , Dropsey Andrew J. , Walgren Jennie , Yuen Eunice , Manro Jason , Gong Xueqian , Cortez Guillermo , McLean Johnathan , Rodriguez Michael J. , Tiu Ramon V. , Bhagwat Shripad V. , Joseph Sajan
Published: July 2017

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. ERK inhibitors have the potential to address resistance caused by ERK reactivation. Herein, a potent, selective small molecule ERK1/2 inhibitor is described. LY3214996 possesses an optimal balance of potency (hERK1 IC50 5 nM, hERK2 IC50 5nM, pRSK IC50 0.43 µM), solubility (FaSSIF solubility at pH 6.5 0.133 µM), PK properties (dog, AUCoral 23800 nM*hr, CL 12.1 mL/min/kg, bioavailability 75.4%), IVTI (TED50 =16 mg/kg pRSK1) and demonstrated significant in vivo efficacy in several human cancer xenograft models. LY3214996 is currently undergoing early clinical evaluation.

Citation Format: Gaiying Zhao, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Lisa Kindler, Robert S. Flack, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jennie Walgren, Eunice Yuen, Jason Manro, Xueqian Gong, Guillermo Cortez, Johnathan McLean, Michael J. Rodriguez, Ramon V. Tiu, Shripad V. Bhagwat, Sajan Joseph. Identifying high quality, potent and selective pyrimidinylthienopyrrolone inhibitors of ERK1/2 kinase: LY3214996 [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 3231. doi:10.1158/1538-7445.AM2017-3231
Abstract 3236: Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery
Authors: Jordan Allan M. , Newton Rebecca , Waszkowycz Bohdan , Bayliss Richard , Begum Habiba , Burschowsky Daniel , Echalier Aude , Hitchin Samantha , Hutton Colin , Johns Shaun , Jones Stuart , Lin Li-Ying , Richards Mark , Seewooruthun Chitra , Stowell Alex , Waddell Ian , Watson Mandy , Ogilvie Donald
Published: July 2017

Activating gene fusions in the RET receptor tyrosine kinase have been found to drive 1-2% of lung adenocarcinomas and therefore offer an attractive target for targeted therapy. Whilst non-selective tyrosine kinase inhibitors with RET activity are efficacious in this setting, their use is generally limited by dose limiting toxicity associated with their more potent activity versus other targets, specifically KDR (VEGFR2) in the case of cabozantinib and vandetanib. Given this limitation, there is considerable interest in developing more selective inhibitors of RET kinase. Tyrosine kinase inhibitors are prone to early clinical failure due to mutations in the kinase ATPase binding domain, which render the kinase catalytically active but no longer sensitive to drug treatment. Such mutations often occur in the so-called “gatekeeper” region and in this specific case, resistance is predicted to arise from a Val-Met or Val-Leu mutation at residue 804. Through a combination of computational methods, structural biology and drug design, we have identified and further optimized a series of inhibitors of the V804M mutant RET kinase which show sub-micromolar cellular activity in cells driven by V804M RET. Moreover, these agents show excellent selectivity against the wtRET kinase and KDR. As such, these agents may offer valuable start-points for second-generation RET inhibitors for use in patents who relapse after treatment with first generation selective RET inhibitors.

Citation Format: Allan M. Jordan, Rebecca Newton, Bohdan Waszkowycz, Richard Bayliss, Habiba Begum, Daniel Burschowsky, Aude Echalier, Samantha Hitchin, Colin Hutton, Shaun Johns, Stuart Jones, Li-Ying Lin, Mark Richards, Chitra Seewooruthun, Alex Stowell, Ian Waddell, Mandy Watson, Donald Ogilvie. Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery [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 3236. doi:10.1158/1538-7445.AM2017-3236