bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3351: NF-κB signaling between stromal macrophages and breast cancer cells
Authors: Bradford Jennifer W.
Published: August 2015

It has become increasingly clear that the tumor microenvironment directly interacts with tumor cells to promote tumor growth and progression. Tumor associated macrophages (TAMs) have emerged as key components of the tumor microenvironment, and their presence in breast cancer is associated with poor prognosis. There is evidence that nuclear factor -kappaB (NF-κB) is an important signaling pathway between cancer cells and the stromal compartment. Disruption of NF-κB communication between TAMs and breast tumors may weaken the pro-tumorigenic influences of TAMs, and therefore make the tumor more susceptible to standard treatments.

Our work reveals that when breast cancer cells and bone marrow derived macrophages (BMDMs) are co-cultured in transwell plates, activated canonical NF-κB and STAT3 levels dramatically increase in both cell types. This signaling interaction was confirmed by co-culturing human blood derived macrophages with breast cancer cells. BMDMs co-cultured with breast cancer cells acquire a mixed M1/M2 activation phenotype, and also have skewed inflammatory cytokine production. In basal-like mouse tumors, active NF-κB levels are much higher in the CD11b+ (TAM) fraction compared to the CD11b- fraction. Even more so than BMDMs, TAMs isolated from mouse basal-like tumors have high NF-κB activity, and can promote elevated NF-κB and STAT3 activation and cytokine production in breast cancer cells when co-cultured in transwell plates. BMDMs genetically lacking p65 have altered inflammatory cytokine production when compared to wild type BMDMs, which remains skewed when the cells are co-cultured with breast cancer cells. Because the NF-κB pathway can be targeted therapeutically, understanding how NF-κB activation influences breast tumor-TAM communication is important for developing new treatment options for aggressive cancers.

Citation Format: Jennifer W. Bradford. NF-κB signaling between stromal macrophages and breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3351. doi:10.1158/1538-7445.AM2015-3351
Abstract 3364: Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma
Authors: Lou Yanyan , Diao Lixia , Roger Parra Cuentas Edwin , Denning Warren L. , Chen Limo , Fan Youhong , Rodriguez Jaime , Byers Lauren , Wang Jing , Papadimitrakopoulou Vassiliki , Carmen Behrens , Wistuba Ignacio I. , Hwu Patrick , Heymach John V. , Gibbons Don L.
Published: August 2015

Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with immunomodulatory agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune-based approaches, such as immune checkpoint blockade, is critical to successful translation of these agents. By using integrated gene array analysis in three large independent NSCLC patient datasets along with immunohistochemistry study (IHC), reverse phase protein array (RPPA) and in vivo animal study, we demonstrated that epithelial-mesenchymal transition (EMT) is highly associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. Our data revealed immune activation and simultaneous development of multiple immune suppressive mechanisms, including elevation of immune checkpoints such as PD-L1, PD-L2, PD-1, TIM-3, BTLA and CTLA-4, as well as an increase in tumor infiltrating CD4+Foxp3+ regulatory T cells, IL-6 and indoleamine 2,3-dioxygenase (IDO) in lung adenocarcinomas with a mesenchymal phenotype. Our data suggested that EMT might represent a potential biomarker to select the patients who will benefit from immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly other cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Yanyan Lou, Lixia Diao, Parra Cuentas Edwin Roger, Warren L. Denning, Limo Chen, Youhong Fan, Jaime Rodriguez, Lauren Byers, Jing Wang, Vassiliki Papadimitrakopoulou, Behrens Carmen, Ignacio I. Wistuba, Patrick Hwu, John V. Heymach, Don L. Gibbons. Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2015-3364
Abstract 3371: Periostin secreted by breast cancer cells inhibits macrophage phagocytosis and adhesion to fibronectin
Authors: Coleman Michelle M. , Helms Rachel S. , Dreau Didier
Published: August 2015

Interactions between cancer cells and immune cells are critical to the development of breast cancer metastases. We have previously shown that breast cancer cells secrete periostin, an extracellular matrix protein implicated in tumor progression. Here we evaluated 1) the effect of macrophage secretions on 4T1 breast cancer cell production of periostin and 2) the effect of periostin on key macrophage functions. In vitro, periostin secretion by highly metastastic 4T1 mammary cancer cells was investigated by ELISA. Following a 48-hour incubation with conditioned media obtained from J774A-1 monocyte cells, 4T1 cells secreted significantly higher periostin concentrations (p<0.05). Next, the effect of prolonged incubation with periostin on proliferation, adhesion and phagocytosis of macrophages was investigated using mouse J774A-1 and RAW264.7 monocyte cells and mouse primary bone marrow macrophages. In the conditions tested, periostin did not affect macrophage proliferation. Incubation with periostin significantly decreased macrophage adhesion to fibronectin-coated vessels (p<0.05). Incubation with periostin also significantly inhibited macrophage phagocytosis of polymer beads (p<0.05). Together, these observations indicate that the secretion of periostin by 4T1 cells is, in part, stimulated through paracrine communication with macrophages, and that periostin inhibits both macrophage adhesion and phagocytosis.

Citation Format: Michelle M. Coleman, Rachel S. Helms, Didier Dreau. Periostin secreted by breast cancer cells inhibits macrophage phagocytosis and adhesion to fibronectin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3371. doi:10.1158/1538-7445.AM2015-3371
Abstract 3374: Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP6 induction
Authors: Lee Geun Taek , Kwon Seok Joo , Kim Isaac Yi
Published: August 2015

Prostate cancer (CaP) is the second leading cause of cancer death in American men. Traditional androgen ablation is a first-line therapy in relapsed or metastatic prostate cancer cases, but it is eventually rendered ineffective by the development of castration-resistant prostate cancer (CRPC). Once CRPC develops, treament options include taxol-based chemotherapy and second-line hormonal agents, but these have limited effectiveness. In the present study, we have investigated the role of Bone Morphogenetic Protein-6 (BMP6) in the development of castration resistant prostate cancer (CRPC) in the context of bone metastases. We initially investigated the clinical course of 158 men with advanced CaP who were treated with primary androgen deprivation therapy. To elucidate the underlying mechanism of CRPC in the context of bone metastases, we examined the impact of bone stromal cells on CaP in the absence of androgens using a co-culture model.

In these patients, we found that the median time to PSA progression was significantly shorter when bone metastases were present (14 months [95% CI, 10.2-17.8 months] vs. 57 months [95% CI, 19.4-94.6 months]). These results suggest that bone-tumor interactions may accelerate castration resistance. Mechanistically, gene expression analysis using quantitative polymerase chain reaction (Q-PCR) arrays showed a dramatic induction of BMP6 by CaP cell lines in the presence of bone stromal cells. Further studies revealed that WNT5A derived from bone stromal cells induced the expression of BMP6 by CaP cells; BMP6 in turn stimulated cellular proliferation of CaP cells in an androgen-deprived media via a physical interaction between Smad5 and β-catenin. Intracellularly, WNT5A increased BMP6 expression via PKC/NF-κB pathway in CaP cell lines. Taken together, these observations suggest that bone-CaP interaction leads to castration resistance via WNT5A/BMP6 loop. We have herein demonstrated that the bone-CaP interaction promotes resistance to androgen deprivation. At the cellular and molecular level, this castration-resistance is mediated by bone stroma-derived WNT5A that leads to the induction of BMP6; BMP6, in turn, permits CaP cells to proliferate in the absence of androgens by interacting with β-catenin. Targeting WNT5A/BMP6/β-catenin may be a viable therapeutic option in men with CaP skeletal metastases and CRPC.

Citation Format: Geun Taek Lee, Seok Joo Kwon, Isaac Yi Kim. Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP6 induction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2015-3374
Abstract 3399: Using protein chips to develop a highly specific HER2 antibody for HER2 amplification testing
Authors: Zhou Lixin , Yuan Kehu , Ren Fangfang , Qi Lili , Li Zhongwu , Wu Guiyin , Huang Xiaozheng , Shen Yi , Zhao Min , Fu Wei , Liu Huibo , Chu Boyang , Wang Guangli , Shu Youmin , Ma Donghui , He Wei-Wu , Chen Jian
Published: August 2015

Human epidermal growth factor receptor 2 (HER2) is an orphan receptor tyrosine kinase member of the EGFR families and is found to be a key tumor driver gene. In breast cancer and gastric cancer, HER2 amplification can be effectively treated by its neutralizing antibody, trastuzumab. In clinic, the HER2 immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted to another family member HER4. IHC staining also indicated that it has strong non-specific cytoplasmic and nucleus staining in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have successfully identified and confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and unclear staining of 4B5 on normal gastric epithelium. Finally, we have developed a novel HER2 mouse monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. UMAB36 could provide an alternative high specific IHC reagent for HER2 amplification testing in gastric cancer population.

Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Qi, Zhongwu Li, Guiyin Wu, Xiaozheng Huang, Yi Shen, Min Zhao, Wei Fu, Huibo Liu, Boyang Chu, Guangli Wang, Youmin Shu, Donghui Ma, Wei-Wu He, Jian Chen. Using protein chips to develop a highly specific HER2 antibody for HER2 amplification testing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2015-3399
Abstract 3406: Enhancer of zeste homolog 2 (EZH2) expression and proliferation index (Ki-67) in neuroendocrine tumors of the gastrointestinal tract and pancreas
Authors: Qin Jia , Agrawal Raag , Echevarria Miguel G. , Martello LAura A. , Haseeb M.A , Gupta Raavi
Published: August 2015

EZH2 is a polycomb group protein that is involved in the progression of multiple human cancers including prostatic, endometrial, and colonic adenocarcinomas. Its expression has been shown to increase from typical carcinoid to atypical carcinoid and further to small cell/large cell neuroendocrine carcinomas of the lung. Here we examine the EZH2 expression and the proliferation index (PI) by Ki-67 expression in neuroendocrine tumors of the gastrointestinal tract (GI) and pancreas.

We retrieved 72 cases of GI and pancreatic neuroendocrine tumors of grades G1 (n = 56), G2 (n = 6) and G3 (n = 10) which were graded according to the WHO classification. Expression of EZH2 and Ki-67 was analyzed by immunohistochemistry using monoclonal antibodies (EZH2 at 1:200; Ki-67 at 1:70). Expression of EZH2 was determined to be low (10%), and was compared with the respective PI.

All G1 tumors had low EZH2 expression and a low PI (20%). In G2 tumors, EZH2 expression was intermediate (1-10%) and PI was between 0-8%. One of the G2 tumors had a low PI of 0, however, the EZH2 expression was intermediate (3%). There was increased EZH2 expression relative to increasing tumor grade from G1 to G3 (p <0.05).

EZH2 expression increases incrementally from G1 neuroendocrine tumors to G2 and further to G3 tumors, suggesting an association or a role in progression of neuroendocrine tumors. There is strong correlation between the expression of EZH2 and PI in G1, G2 and G3 tumors. EZH2 appears to be a better predictor of tumor grade in G2 tumors as compared to PI. Thus, EZH2 could be used as an additional diagnostic marker in examining atypical GI neuroendocrine tumors.

Citation Format: Jia Qin, Raag Agrawal, Miguel G. Echevarria, LAura A. Martello, M.A Haseeb, Raavi Gupta. Enhancer of zeste homolog 2 (EZH2) expression and proliferation index (Ki-67) in neuroendocrine tumors of the gastrointestinal tract and pancreas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2015-3406
Abstract 3408: Nedd4-1 is an exceptional prognostic biomarker for gastric cardia adenocarcinoma and functionally associated with metastasis
Authors: Sun Aiqin , Yu Guanzhen , Dou Xiaoyan , Yan Xiaowei , Yang Wannian , Lin Qiong
Published: August 2015

Background: Gastric cardia adenocarcinoma (GCA) is the most aggressive subtype of gastric carcinoma. New molecular markers and therapeutic targets are needed for diagnosis, prognosis and treatment of GCA. This study is to establish the E3 ubiquitin ligase Nedd4-1 as a prognostic biomarker to predict the survival and guide the treatment of GCA patients.

Methods: Expression of Nedd4-1 in 214 GCA tumor samples was detected by immunohistochemistry staining (IHC) using tissue microarray assay (TMA). Association of Nedd4-1 with cumulative survival of the TNM stages I-III patients and clinicopathological characteristics was statistically analyzed. The role of Nedd4-1 in gastric cancer cell migration and invasion were determined by transwell and wound healing assays.

Results: Nedd4-1 is overexpressed in 83% of the GCA tumors. The 5-year survival rate in Nedd4-1 negative GCA patients is as high as 96%. Log-rank analysis indicated that overexpression of Nedd4-1 is inversely correlated with cumulative survival (χ2 = 21.885, p<0.001). Multivariate logistic regression analysis showed that overexpression of Nedd4-1 is associated with an extremely low GCA survival rate with a hazard ratio (HR) = 0.068 (p = 0.008) in TNM stages I-III patients. Statistical analysis of association of Nedd4-1 overexpression with clinicopathological characteristics revealed that overexpression of Nedd4-1 is tightly associated with TNM stage (p<0.001). Knockdown of Nedd4-1 in gastric cancer cell lines AGS and N87 dramatically inhibited the gastric cancer cell migration and invasion.

Conclusions: Our results indicate that Nedd4-1 is an exceptional prognostic biomarker for GCA and suggest that Nedd4-1 may play an essential role in GCA metastasis.

Citation Format: Aiqin Sun, Guanzhen Yu, Xiaoyan Dou, Xiaowei Yan, Wannian Yang, Qiong Lin. Nedd4-1 is an exceptional prognostic biomarker for gastric cardia adenocarcinoma and functionally associated with metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3408. doi:10.1158/1538-7445.AM2015-3408
Abstract 3410: Onodera's prognostic nutritional Index predicts survival in patients with unresectable advanced or recurrent colorectal cancer
Authors: Ikeya Tetsuro
Published: August 2015

Background: Nutrition and immunity significantly affect the progression of cancer in cancer patients. Therefore, the evaluation of the nutritional and immune status would be useful as a prognostic factor and to determine the optimal treatment strategy for patients with unresectable metastatic colorectal cancer who are receiving chemotherapy. The aim of this retrospective study was to evaluate the prognostic significance of the nutritional and immune status in patients with unresectable metastatic colorectal cancer treated with chemotherapy. Methods: We retrospectively reviewed 80 patients with colorectal cancer. A total of 22 patients had metachronous unresectable cancer and 58 patients had synchronous unresectable cancer. All patients underwent combination chemotherapy with oxaliplatin or irinotecan plus 5-fluorouracil/leucovorin as first-line chemotherapy for unresectable metastatic colorectal cancer. We then examined the correlations between the Onodera's Prognostic Nutritional Index (OPNI) and the patients’ clinicopathological features. The OPNI was calculated as follows: 10 × serum albumin (g/dl) + 0.005 × total lymphocyte count (per mm3). According to the receiver operating characteristic (ROC) curve analysis, the cut-off value for OPNI was 44.5. Results: Patients with a pretreatment OPNI of ≧44.5 demonstrated a longer OS than those with a pretreatment OPNI of <44.5. Moreover, we categorized these patients into four groups according to the combination of the pre- and post-treatment OPNI. The patients in the group with both OPNIs ≥ 44.5 exhibited a better prognosis compared to the other group (p = 0.001). Conclusion: The OPNI is considered to be a useful marker for predicting the long-term outcome in patients who receive chemotherapy for unresectable metastatic colorectal cancer.

Citation Format: Tetsuro Ikeya. Onodera's prognostic nutritional Index predicts survival in patients with unresectable advanced or recurrent colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2015-3410
Abstract 3425: Overexpression of TRIM44 contributes to malignant outcome in upper GI tract cancer
Authors: Kawaguchi Tsutomu
Published: August 2015

Recent studies have shown that some members of the tripartite motif-containing protein (TRIM) family, which function as important regulators for carcinogenesis. In this study, we tested whether TRIM44 (11p13) acts as a cancer-promoting gene through overexpression in gastric cancer (GC) and esophageal squamous cell carcinoma (ESCC). We analyzed 7 GC and 14 ESCC cell lines for in vitro analysis. We also analyzed 113 GC and 68 ESCC primary tumors, which were curatively resected in our hospital between 1998 and 2008. As the results, expression of the TRIM44 protein was detected in GC and ESCC cell lines (2/7 cell lines; 29% and 8/14; 57%, respectively) and primary tumor samples of GC and ESCC (24/113 cases; 24% and 39/68; 57%, respectively). Knockdown of TRIM44 expression using several specific siRNAs inhibited cell proliferation and migration and invasion in TRIM44-overexpressing GC cell, and inhibited cell migration and invasion in TRIM44-overexpressing ESCC. Using immunohistochemical analysis, overexpression of the TRIM44 was significantly correlated with an advanced type of macroscopic appearance, lymphatic invasion, and higher recurrence rate in GC, and with the status of the lymph node metastasis in ESCC. TRIM44-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors in GC and ESCC tumors. Moreover, TRIM44 positivity was independently associated with worse outcome in multivariate analysis in GC tumors. These findings suggest that TRIM44 plays a crucial role in tumor progress through its overexpression, and highlight its usefulness as a novel predictor and potential therapeutic target in GC and ESCC.

Citation Format: Tsutomu Kawaguchi. Overexpression of TRIM44 contributes to malignant outcome in upper GI tract cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2015-3425
Abstract 3426: Occult and clinical pancreatic cancers in elderly patients
Authors: Matsuda Yoko , Suzuki Akemi , Hamashima Yuri , Hamayasu Hideki , Yoshimura Hisashi , Ishiwata Toshiyuki , Arai Tomio
Published: August 2015

Objectives: This study aimed to investigate the clinicopathological features of pancreatic cancer at different stages using autopsy results. Methods: We retrospectively evaluated 8399 consecutive cases of autopsy performed between 1972 and 2013 at our geriatric hospital. Results: Macroscopic pancreatic lesions were detected in 6.13% of cases. Primary and secondary pancreatic tumors were observed in 2.88% and 2.10% of cases, respectively. Most primary tumors were invasive ductal adenocarcinomas (193 cases, 2.31%, mean patient age: 78.09 years) with a peak incidence at 50-59 years. Occult invasive ductal adenocarcinoma was discovered incidentally in 15 cases (Table), with distant metastasis present in 26.67% of those. Microscopically, occult and advanced tumors exhibited similar characteristics such as hyalinized fibrous stroma; necrosis; and invasion into vessels, peripancreatic fat tissues, and extrapancreatic nerve plexus. Mucin 1 and 2 immunohistochemical expression levels were also similar. Occult cancer incidence increased with age. Patients aged ≥85 years had shorter survival, a small tumor size, and a low incidence of lymph node metastasis. Approximately 8% of pancreatic invasive ductal adenocarcinomas progressed asymptomatically and were discovered incidentally at autopsy. Conclusions: Pancreatic cancers in elderly patients tend to progress asymptomatically, but once symptoms develop, they are more often fatal than those in younger patients.

Comparison of clinical and occult invasive ductal adenocarcinoma cancer cases (193 cases)Clinical cancer casesOccult cancer casesP valueAge, years (mean ± S.D.)77.67 ± 8.6583.13 ± 8.130.02*Size, cm (mean ± S.D.)5.88 ± 2.683.03 ± 1.91<0.01*MUC1 score2.00 ± 0.871.90 ± 0.830.75MUC2 score0.05 ± 0.180.28 ± 0.530.09Number of cases17815SexMale7780.45Female1017Main locationPancreatic head1076<0.01*Pancreatic body502Pancreatic tail177No data40PathologyWell43100.01*Moderately773Poorly391Adenosquamous carcinoma110Anaplastic carcinoma81Lymph node metastasisNegative239<0.01*Positive1556Stage (UICC)I07<0.01*II234III10IV1544

Citation Format: Yoko Matsuda, Akemi Suzuki, Yuri Hamashima, Hideki Hamayasu, Hisashi Yoshimura, Toshiyuki Ishiwata, Tomio Arai. Occult and clinical pancreatic cancers in elderly patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2015-3426
Abstract 3441: Proteomic profiling identifies PTK2/FAK as a targetable marker of radioresistance in head and neck cancer
Authors: Skinner Heath D. , Giri Uma , Yordy John S. , Story Michael D. , Wang Jing , Byers Lauren A. , Williams Michelle D. , El-Naggar Adel K. , Woo Sang H. , Yang Liang P. , Fan You , Pickering Curtis R. , Myers Jeffrey N. , Heymach John V.
Published: August 2015

One of the primary treatments for head and neck squamous cell carcinoma (HNSCC) is radiotherapy. Despite decades of study, few if any targetable biomarkers for resistance to this modality. To systematically evaluate resistance to radiotherapy in HNSCC, we evaluated 50 HNSCC cell lines using reverse phase protein array (RPPA). Utilizing this method we identified 11 protein markers with altered expression in radioresistant HNSCC cell lines (FDR 1%), including PTK2/FAK. Both PTK2/Fak gene expression and copy number were highly correlated to PTK2/Fak protein expression and both were associated with radioresistance in these cell lines. Additionally, in clinical samples from the TCGA, PTK2/Fak copy number was highly associated with gene expression, with high expression levels in HNSCC tumors. To validate the association between PTK2/Fak and clinical radioresistance in HNSCC, we examined PTK2/FAK copy number in two separate cohorts of HNSCC patients treated uniformly with radiotherapy (n = 39 and n = 44). In both cohorts, PTK2/Fak amplification was associated with treatment failure following radiotherapy (p = 0.04 and p = 0.03 respectively and p = 0.004 for the combined population). Additionally, in a separate cohort of HNSCC patients treated with radiotherapy, high levels of PTK2/Fak gene expression were associated with failure following radiotherapy (p = 0.02). Additionally, in vitro pharmacologic inhibition of PTK2/FAK function led to radiosensitization in multiple HNSCC cell lines, primarily via induction of G2/M arrest, with minimal apoptosis observed. Taken together, these data identify a validated, targetable biomarker of radioresistance in HNSCC.

Citation Format: Heath D. Skinner, Uma Giri, John S. Yordy, Michael D. Story, Jing Wang, Lauren A. Byers, Michelle D. Williams, Adel K. El-Naggar, Sang H. Woo, Liang P. Yang, You Fan, Curtis R. Pickering, Jeffrey N. Myers,, John V. Heymach. Proteomic profiling identifies PTK2/FAK as a targetable marker of radioresistance in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3441. doi:10.1158/1538-7445.AM2015-3441
Abstract 3442: Ganetespib sensitizes colorectal cancer in vitro and in vivo to a clinically relevant regimen of radiation and chemotherapy
Authors: Nagaraju Ganji Purnachandra , Patel Kirtesh , Wen Jing , Diaz Roberto , Bassel El-Rayes F.
Published: August 2015

Purpose: We describe the efficacy of the second generation HSP90 inhibitor ganetespib in combination with radiation and chemotherapy against colorectal adenocarcinoma in vitro and in vivo.

Methods and Materials: Ganetespib's effects on colorectal adenocarcinoma proliferation and ability to sensitize to radiation and/or chemotherapy were analyzed in the colorectal cell lines HCT-116 and HT-29. Ganetespib's ability to modulate apoptosis when combined with radiation and chemotherapy was characterized, and its effects on the AKT, ERK and PI3K pathways were investigated. Tumor growth delay of colorectal adenocarcinoma with ganetespib alone and in combination with radiation and chemotherapy was analyzed in xenografts. Lastly, immunohistochemical analyses of HSP90 and p-AKT levels where performed in paired normal and tumor colorectal tissue samples from 10 patients.

Results: At the clinically achievable dose of 50 nM, ganetespib inhibited colorectal cancer cells proliferation (p<0.014), potentiated 5-FU chemotherapy and 2Gy of ionizing radiation (p<0.0001), increased pro-apoptotic signaling proteins, and decreased levels of p-AKT, PI3K, and p-ERK in vitro. Combinations of ganetespib, 5-FU, and radiation therapy exhibited synergistic efficacy in human xenograft models in vivo (p<0.0001). Finally, HSP90 and p-AKT were both co-overexpressed in human colorectal cancer tissue samples relative to normal colon tissue. Conclusion: Altogether, these data show the clinically relevant combination of ganetespib with chemotherapy and radiation is effective in colorectal adenocarcinoma in vitro and in vivo. We are currently evaluating ganetespib's safety with chemotherapy and radiation therapy in a phase I study of patients with localized colorectal adenocarcinoma.

Citation Format: Ganji Purnachandra Nagaraju, Kirtesh Patel, Jing Wen, Roberto Diaz, El-Rayes F. Bassel. Ganetespib sensitizes colorectal cancer in vitro and in vivo to a clinically relevant regimen of radiation and chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3442. doi:10.1158/1538-7445.AM2015-3442
Abstract 3448: Overexpression of the steroid inactivating UGT2B28 enzyme is associated with high circulating androgens, tumor aggressiveness and adverse prostate cancer outcome
Authors: Belledant Anaïs , Hovington Hélène , Brisson Hervé , Têtu Bernard , Fradet Yves , Lacombe Louis , Caron Patrick , Guillemette Chantal , Lévesque Eric
Published: August 2015

Androgen inactivation occurs mainly through the glucuronidation conjugative reaction mediated by uridine diphospho-glucuronosyltransferase enzymes, namely UGT2B28. This metabolic pathway controls systemic and local androgen bioavailability along with other biotransformation and transport pathways. In this study, we examined the relationship between UGT2B28 protein expression, circulating steroid levels and prostate cancer phenotype. To study distribution of the UGT2B28 protein in prostatic tissues, we used a specific antipeptide antibody displaying no cross-reactivity against highly identical UGT2B family members. Findings revealed that UGT2B28 cytoplasmic staining of secretory cells was distinctive of early stage tumorigenesis (i.e. prostatic intraepithelial neoplasia) and cancer cells with a variable nuclear staining, whereas normal prostate presented only nuclear enzyme expression. Using tissue microarray analysis, we analyzed 239 localized prostate cancer cases that underwent radical prostatectomy. Compared to patients with prostate tumors displaying absent or weak UGT2B28 staining, those with tumors presenting strong UGT2B28 nuclear expression had significantly lower PSA levels at diagnosis (20%; p = 0.043). UGT2B28 expression in the cytoplasm was also correlated with Gleason scores (p≤0.03) and nodal invasion status (p = 0.024). After adjustments for known prognostic markers including PSA, Gleason score, margin and nodal status, tumor size, smoking and germline UGT2B28 deletion status, overexpression of the UGT2B28 protein was an adverse and independent prognostic factor associated with disease recurrence and/or death (HR = 2.82; 95%CI, 1.09-7.36; p = 0.033). Remarkably, prostatic overexpression of UGT2B28 concurs with 30% higher circulating levels of testosterone (p = 0.004) and dihydrotestosterone (p = 0.002), measured by mass spectrometry. Our observations demonstrate that an increased expression of the UGT2B28 enzyme is associated with circulating steroid hormone levels, disease aggressiveness and risk of cancer progression.

Citation Format: Anaïs Belledant, Hélène Hovington, Hervé Brisson, Bernard Têtu, Yves Fradet, Louis Lacombe, Patrick Caron, Chantal Guillemette, Eric Lévesque. Overexpression of the steroid inactivating UGT2B28 enzyme is associated with high circulating androgens, tumor aggressiveness and adverse prostate cancer outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2015-3448
Abstract 3452: Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer
Authors: Li Zhenfei , Sharifi Nima
Published: August 2015

Castration resistant prostate cancer (CRPC) still relies on androgens for progression. Abiraterone, an FDA approved CYP17A inhibitor, blocks androgen synthesis and prolongs survival. Abiraterone metabolites are generally thought to be inactive. We have identified a novel abiraterone metabolite, D4A, which inhibits three steroidogenic enzymes and androgen receptor (AR) itself. After abiraterone acetate administration, plasma from mice and patients were analyzed with liquid chromatography-mass spectrometry (LC-MS). The LC-MS results indicate that abiraterone is converted to D4A at detectable concentrations in both mice and patients. To test the function of D4A, the androgen synthesis pathway was analyzed in cell lines and xenografts. After incubation with [3H] labeled androgens, metabolites from cell lines and xenografts were analyzed by HPLC. We found that D4A inhibits activity of the steroidogenic enzymes CYP17A, 3β-hydroxysteroid dehydrogenase (3[[Unsupported Character - Symbol Font ]]HSD) and 5α-reductase. Furthermore D4A competes with the AR agonist R1881 and binds to AR directly. D4A inhibits DHT-induced AR occupancy on the PSA enhancer as detected by chromatin immunoprecipitation and reduces PSA expression. Our findings suggest that D4A is an abiraterone metabolite and a potent inhibitor of the androgen pathway.

Citation Format: Zhenfei Li, Nima Sharifi. Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2015-3452
Abstract 3466: Regulation of iodine uptake by SENP2 in thyroid cancer
Authors: Lee Eun Kyung , Park Min Ji , Lee You Jin
Published: August 2015

Background: About 10∼20% of thyroid cancer patients does not respond to radioactive iodine(RAI) therapy, since the expression or membrane trafficking of sodium iodine symporter(NIS) was ameliorated. We investigated whether iodine uptake and expression of NIS were regulated by the balance of SUMOylation in thyroid cancer.

Methods: We analyzed the expression of NIS mRNA using fresh frozen tissues of papillary thyroid cancer. After overexpression of desumoylating enzyme SENP2 in human anaplastic thyroid cancer cell line (FRO) and silencing SENP2 in human NIS-overexpressed HeLa cell line (HeLa-hNIS), we evaluated the expression of NIS mRNA and iodine uptake. We selected 30 patients of different expression level of SENP2 (low and normal SENP2) and constructed tissue microarray for immunohistochemistry stain. And the patients’ clinico-pathologic features were reviewed using medical records.

Results: Twenty six percent of papillary thyroid cancer tissue showed reduced expression of SENP2 and NIS mRNA. Silencing SENP2 in HeLa-hNIS, NIS mRNA and iodine uptake was reduced. By co-immunoprecipitation, the interaction between SUMO2/3 and C/EBPβ was detected. After overexpression of SENP2, NIS mRNA and iodine uptake was restored. By comparison of response of RAI among two groups, reduction of serum thyroglobulin level after RAI was blunted in the group of low SENP2, compared to that in the group of normal SENP2.

Conclusion: Overexpression of SENP2 can induce NIS expression and iodine uptake. This may be a novel therapeutic target for RAI-refractory thyroid cancer patients.

Citation Format: Eun Kyung Lee, Min Ji Park, You Jin Lee. Regulation of iodine uptake by SENP2 in thyroid cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2015-3466
Abstract 3478: A multi-orthogonal strategy for targeting EGFRvIII expressing glioblastomas
Authors: Shen Ying , Li Jie , Furnari Frank B , Desai Arshad , Chen Hong-Zhuan , Chen Clark
Published: August 2015

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a recurrent oncogenic EGFR isoform found in 25-64% of glioblastomas, the most common form of primary brain cancer. While EGFRvIII expressing glioblastomas are initially sensitive to EGFR inhibition, emergence of resistance clones is universal. We previously demonstrated that EGFRvIII expressing glioblastoma can be ablated through inhibition of compensatory mechanisms that allow cancer cells to adapt to EGFRvIII induced oncogenic stress. Here we propose that this strategy can be combined with direct inhibition of EGFR signaling.

A synthetic lethal RNAi screen revealed synthetic lethality between Polo-Like Kinase 1 (PLK1) inhibition and EGFRvIII expression in glioblastoma cells. PLK1 mediated glioblastoma adaptation to excessive DNA damage associated with EGFRvIII expression through activation of Rad51, a protein essential for homologous recombination (HR). PLK1 inhibition (by BI2536) enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastoma. This TMZ-sensitizing effect is 3-5 fold more pronounced in a formed Ink4a/Arf(-/-) EGFRvIII GEMM (genetically engineerd murine model)-derived glioblastoma model relative to an Ink4a/Arf(-/-) PDGF-β GEMM-derived glioblastoma model and correlated to the endogenous level of DNA damage accumulation in these models.

Importantly, the tumoricidal and TMZ-sensitizing effects of BI2536 were uniformly observed across Ink4a/Arf(-/-) EGFRvIII glioblastoma clones that acquired independent resistance mechanisms to EGFR inhibitors, Gefitinib and Erlotinib, suggesting these resistant clones retain oncogenic stress that required PLK1 compensation. Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, complete ablation was not achieved until TMZ was added. Such synergistic effect was not observed in the Ink4a/Arf(-/-) PDGF-β model.

Our study revealed a novel synthetic lethal interaction between PLK1 inhibition and EGFRvIII expression. Our results suggest that optimal therapeutic effect against EGFRvIII expressing glioblastomas requires “multi-orthogonal” integration of oncogenic inhibition, oncogenic stress, and conventional chemotherapy, tailored to the molecular physiology associated with the target cancer genome.

Citation Format: Ying Shen, Jie Li, Frank B Furnari, Arshad Desai, Hong-Zhuan Chen, Clark Chen. A multi-orthogonal strategy for targeting EGFRvIII expressing glioblastomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3478. doi:10.1158/1538-7445.AM2015-3478
Abstract 3498: Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells
Authors: Kim Ji-Won , Xu Wendan , Yoon Sung-Soo
Published: August 2015

Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas, although its clinical efficacy was not satisfactory, due possibly to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive anaplastic large cell lymphoma (ALCL). We treated ALK-positive ALCL cell lines, K-299 and SU-DHL-1, with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (0.5 nM, 1.0 nM, and 2.0 nM for everolimus, and 20 nM, 40 nM, and 80 nM for crizotinib, respectively). After 72 hours, the combination index (CI) values were less than 1 (0.596, 0.583, and 0.763 in K-299 cells, and 0.326, 0.616, and 0.271 in SU-DHL-1 cells, respectively) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. Western blot analysis demonstrated that everolimus up-regulated phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited mTOR Ser2448 phosphorylation, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased mTOR Ser2481 phosphorylation, a marker for mTORC2, as well. Cell-cycle analysis showed that the combination treatment induced G1 arrest. PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib. In summary, everolimus in combination with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis. Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL.

Citation Format: Ji-Won Kim, Wendan Xu, Sung-Soo Yoon. Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2015-3498
Abstract 3512: Measuring intracellular target engagement and drug residence time with nanoBRET
Authors: Robers Matthew B. , Dart Melanie , Zimprich Chad , Kirkland Thomas , Levin Sergiy , Machleidt Thomas , Hartnett Jim , Zimmerman Kris , Ohana Rachel , Daniels Danette , Cong Mei , Fan Frank , Wood Keith
Published: August 2015

We present a biophysical method to directly measure target engagement within intact mammalian cells using bioluminescence energy transfer (BRET). Compound interactions with intracellular targets can be detected with complete specificity by their ability to compete with energy transfer complexes introduced into the cells. These complexes can be detected at physiologically relevant levels by exploiting an extraordinarily bright luciferase (NanoLuc), together with fluorescent tracers optimized for cell-permeability and spectral resolution from the luciferase. We demonstrate applications of the technology for target engagement among key drug target classes, including; kinases, histone deacetylases (HDACs), bromodomains, and the methyltransferase EZH2. Intracellular selectivity and affinity profiles of various reference compounds and approved drugs will be presented. For a panel of HDAC inhibitors, affinity profiles for specific HDAC isozymes strongly correlate with phenotypic potencies (e.g. cell viability). Furthermore, the luminescent output of the energy transfer complex enables a technique to monitor ligand occupancy in real-time. Association and dissociation rates can be derived from the kinetic measurements, providing a means to quantify drug residence time on select targets within intact cell populations. This novel application of intracellular BRET should significantly advance target engagement work flows, and allow for intracellular target affinities to be coupled to phenotypic outcomes.

Citation Format: Matthew B. Robers, Melanie Dart, Chad Zimprich, Thomas Kirkland, Sergiy Levin, Thomas Machleidt, Jim Hartnett, Kris Zimmerman, Rachel Ohana, Danette Daniels, Mei Cong, Frank Fan, Keith Wood. Measuring intracellular target engagement and drug residence time with nanoBRET. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2015-3512
Abstract 354: Resistance to TGF-beta-induced apoptosis is not associated with the de-differentiation in hepatocellular carcinoma
Authors: Kogiso Tomomi , Hashimoto Etsuko , Kodama Kazuhisa , Ikarashi Yuichi , Torii Nobuyuki , Taniai Makiko , Tokushige Katsutoshi , Shiratori Keiko
Published: August 2015

Aim: Transforming growth factor (TGF)-beta is a cytokine which induces apoptosis of hepatocellular carcinoma (HCC) cells. However, some HCC cells show resistance to the induction of apoptosis by TGF-beta. The analyses of the molecular mechanism underlying these phenomena may be related with the mechanisms of acquirement of drug-resistance. Thus, we examined molecularly the apoptosis-resistant HCC cells which we previously established, especially emphasizing on the analyses of the stem-cell marker expression in this study.

Methods: Previously established HuH7R cells, which acquired a resistance to TGF-beta-induced apoptosis after a long-term culture under small amount of TGF-beta, and control parental HuH7 cells, were used. Apoptosis rate and cell cycle state of these cells after TGF-beta treatment were determined by a flow-cytometry. The expression levels of stem-cell surface markers, such as epithelial cell adhesion molecule (EpCAM) and CD133, and those of transcriptional factors: Oct4, HNF3alfa, HNF4beta, and C/EBPbeta, were analyzed by fluorescent labeling using specific antibodies, at 24hrs and 48hrs after TGF-beta treatment.

Results: 1) HuH7 cells showed apoptosis after 48hrs of TGF-beta treatment, whereas HuH7R did not show apoptosis but entered cell-cycle arrest. 2) The expression levels of CD133 were not affected at both 24hrs and 48hrs after TGF-beta treatment, while EpCAM expression levels increased from 24hrs, in both HuH7 and HuH7R cells. 3) The expression levels of Oct4, HNF3alfa, HNF4beta, and C/EBPbeta were also increased in HCC cells. However, those expression profiles were almost comparable in between HuH7 and HuH7R cells.

Conclusions: The acquisition of the resistance to TGF-beta-induced apoptosis in HCCs were not related with the expression changes of stem-cell markers. The acquisition of drug-resistance in HCC cells was considered through mechanisms other than the changes of the cellular differentiation status of HCC cells.

Citation Format: Tomomi Kogiso, Etsuko Hashimoto, Kazuhisa Kodama, Yuichi Ikarashi, Nobuyuki Torii, Makiko Taniai, Katsutoshi Tokushige, Keiko Shiratori. Resistance to TGF-beta-induced apoptosis is not associated with the de-differentiation in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 354. doi:10.1158/1538-7445.AM2015-354