bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1589: LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines .
Authors: Yang Chao , Giri Uma , Cortez Maria Angelica , Gudikote Jayanthi , Fan YouHong , Diao Lixia , Byers Lauren A. , Heymach John V.
Published: August 2014

Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC).In general, DNA repair pathway activation leads cellular resistant to DNA damaging agents. The loss of LKB1 leads NSCLC to be more invasive and resistant to DNA damage based therapies. We hypothesize that loss of LKB1 may contribute to DNA damage response. We investigated LKB1- mediated signaling pathways by stably overexpressing LKB1 in its mutated NSCLC cell lines, A549 and H460. Methods: Expression of 139 proteins was analyzed by reverse phase protein array (RPPA) in LKB1 overexpressing cell lines along with the appropriate vector control lines. Differences in protein expression at baseline in clones with LKB1 versus clones with vector control were assessed by unpaired T-test with Welch's correction. Ionizing radiation sensitivity (reproductive cell death) was assessed by clonogenic cell survival assay (CSA). Results: LKB1 overexpression in A549 and H460 showed a significant downregulation of genes involved in DNA damage and repair pathway, such as RAD50 (1.4-fold relative expression, p< 0.05 and 1.5-fold relative expression p<0.02 respectively), XRCC1 (1.2-fold relative expression p<0.005 and 1.1-fold relative expression p<0.05, respectively), and DNA-PKCS (1.2-fold relative expression p≈0.067 and 1.2-fold relative expression p<0.01. Also, CSA showed that A549 with LKB1 overexpression led to enhanced sensitization of cells to radiation (DER = 2) which is in consistent with the downregulation of RAD50, XRCC1 and DNA-PKCS. Furthermore, expression of CHK2 and phosphorylation of CHK1 are downregulated, H460 with LKB1 overexpression showed a significant decreasing of CHK1 phosphorylation level (p<0.05). Conclusion: Our results suggest that LKB1 overexpression modulates DNA repair capacity through downregulation of RAD50, XRCC1, DNA-PKCS, CHK1 and CHK2. LKB1 loss may lead to the resistance to DNA damaging induced cell death.

Citation Format: Chao Yang, Uma Giri, Maria Angelica Cortez, Jayanthi Gudikote, YouHong Fan, Lixia Diao, Lauren A. Byers, John V. Heymach. LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2013-1589
Abstract 160: Modulation of the serum metabolomic profiles of breast cancer patients after receiving limonene intervention.
Authors: Miller Jessica A. , Pappan Kirk , Hu Chengcheng , Want Elizabeth J. , Keun Hector , Lang Julie E. , Chow H-H Sherry
Published: August 2014

Background. Limonene is a bioactive food component found in citrus peel oil that has demonstrated chemopreventive and chemotherapeutic activities in preclinical studies. To better characterize its mechanism of action in the clinical setting, we measured the serum metabolomic profiles of breast cancer patients before and after receiving a short-term limonene intervention.

Methods. Forty women with stage 0 - II breast cancer received 2 g QD limonene daily for 2 - 6 weeks (21.5 ± 8.8 days) before surgical resection of their tumor. Pre/post intervention blood draws were taken to assess serum metabolomic profiles. Metabolomic analysis was performed on the gas chromatography mass spectrometry and liquid chromatography mass spectrometry platforms established at Metabolon, Inc.

Results. Of the 397 identified biochemicals, 71 changed significantly (p<0.05) from baseline to post limonene intervention. Pathway analysis revealed several key metabolic changes. Significant increases were noted in eight bile acid conjugates following limonene intervention, including a 3-fold elevation in taurocholate, taurodeoxycholate, and glycoursodeoxycholate. There were also significant decreases in a number of steroid hormones including dehydroisoandrosterone sulfate (DHEA-S), 4-androsten-3beta, 17beta-diol disulfate, epiandrosterone sulfate, androsterone sulfate, and pregnen-diol disulfate. In addition, markers of collagen remodeling or degradation were elevated following limonene intervention. Increased circulating vitamin C, improved insulin sensitivity and glycemic control, and reduced inflammatory potential were also noted following limonene treatment. An increase in plasma levels of ten different glycerophosphocholines and four carnitines were all significantly correlated to a decrease in cyclinD1 (all p's<0.01 and Rˆ2>0.4) in an exploratory analysis.

Conclusions. This global metabolomic profiling study revealed several novel mechanistic insights into the potential clinical activities of limonene. Efforts are ongoing to correlate these metabolic changes to carcinogenesis, clinical characteristics, and risk markers measured in breast tissue and serum.

Citation Format: Jessica A. Miller, Kirk Pappan, Chengcheng Hu, Elizabeth J. Want, Hector Keun, Julie E. Lang, H-H Sherry Chow. Modulation of the serum metabolomic profiles of breast cancer patients after receiving limonene intervention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2013-160
Abstract 1604: ABT-869 enhances radiosensitivity of head and neck squamous cell carcinoma cells.
Authors: Hsu Heng-Wei
Published: August 2014

Purpose: Novel targeted therapeutic strategies to overcome radio-resistance of cancer cells traditionally treated with radiation may improve patient survival with the added benefit of reduced systemic toxicity. Herein, we tested the feasibility of ABT-869 (Linifanib), a multi-receptor tyrosine kinase inhibitor of members of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families, on radio-sensitization of head and neck squamous cell carcinoma (HNSCC).

Materials and Methods: UMSCC-22A and UMSCC-22B cells were treated with ABT-869 and γ-radiation response was determined. Cell viability, cytotoxicity, apoptosis induction and cell cycle distribution were examined by MTT assay, colony formation assay and flow cytometry. In addition, expression of STAT3 and downstream signaling proteins were assessed using western immunoblotting.

Results: Treatment with ABT-869 resulted in cell growth inhibition, induction of apoptosis, G2/M cell cycle arrest, reduced phosphorylation of STAT3, which has been linked to radio-resistance, lower expression of cyclin D1, survivin and increase PARP cleavage. In addition, ABT-869 overcame the radio-resistance of the cell lines and significantly enhanced radiation-induced cytotoxicity (p < 0.05).

Conclusions: These data suggest the possibility of combining targeted therapeutic such as ABT-869 with radiation to enhance inhibition of cell growth and apoptosis in HNSCC cells. Thus, it may provide a novel therapeutic strategy and improve efficacy of radiation against HNSCC in the future.

Citation Format: Heng-Wei Hsu. ABT-869 enhances radiosensitivity of head and neck squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1604. doi:10.1158/1538-7445.AM2013-1604
Abstract 1623: PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells.
Authors: Xue Yuan , Lim Sharon , Yang Yunlong , Wang Zongwei , Jensen Lasse Dahl Ejby , Hedlund Eva–Maria , Andersson Patrik , Sasahara Masakiyo , Larsson Ola , Galter Dagmar , Cao Renhai , Hosaka Kayoko , Cao Yihai
Published: August 2014

PDGF signaling system contributes to tumor angiogenesis and vascular remodeling. Here, we show PDGF-BB markedly induces erythropoietin (EPO) mRNA and protein expression by targeting the PDGFR-β+ stromal and perivascular compartments. In mouse tumor models, PDGF-BB-induced EPO promotes tumor growth via two mechanisms: 1) paracrine stimulation of tumor angiogenesis by directly inducing endothelial cell proliferation, migration, sprouting and tube formation; and 2) endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia. Similarly, delivery of an adenovirus-PDGF-BB to tumor-free mice markedly increases EPO production and hematopoietic parameters. An EPO-specific antibody attenuates PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis. At the molecular level, we show that the PDGF-BB-PDGFR-β signaling system activates EPO promoter via in part transcriptional regulation of ATF3 by possible association with c-Jun and SP1. These findings uncover a novel mechanism of PDGF-BB-induced tumor growth, angiogenesis and hematopoiesis.

Citation Format: Yuan Xue, Sharon Lim, Yunlong Yang, Zongwei Wang, Lasse Dahl Ejby Jensen, Eva–Maria Hedlund, Patrik Andersson, Masakiyo Sasahara, Ola Larsson, Dagmar Galter, Renhai Cao, Kayoko Hosaka, Yihai Cao. PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1623. doi:10.1158/1538-7445.AM2013-1623
Abstract 1632: Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease.
Authors: Lubik Amy A. , Adomat Hans , Guns Emma S. , Buttyan Ralph
Published: August 2014

Androgen deprivation therapy (ADT) is frontline treatment for metastatic prostate cancer (PCa). ADT initially blocks disease progression but eventually allows the outgrowth of castration resistant disease (CRPC) that is refractory to the diminished systemic androgen levels in ADT-treated patients. CRPC is associated with acquired intratumoral steroidogenesis (IS), a process that increases the androgen levels in the local microenvironment of prostate tumors and enables further tumor growth and progression. IS has mainly been attributed to a steroidogenic process linked to CRPC cells and here we present evidence that benign tumor-associated prostate stromal cells (PrSCs) can also contribute to IS through a paracrine Hedgehog (HH) tumor microenvironment associated with increased production and release ofHedgehog ligands (Hhs) from prostate cancer cells in response to ADT (Chen, et al., Mol Cancer, 9: 89, 2010). Primary human PrSCs upregulate expression of a large repertoire of steroidogenic genes upon treatment with HH chemical agonists, SAG (100 nM) or Hg 1.5 (1 nM). These effects are Gli2-dependent (Levina, et al., Prostate, 72: 817, 2012) and they can be inhibited by the HH antagonist, KAAD-cyclopamine. Previously we showed that this upregulation significantly increased output of testosterone (T) in PrSCs fed DHEA precursor. More comprehensive analysis (via LC/MS/MS) of androgen outputs from PrSCs fed 22 OH-cholesterol showed that Hg1.5 significantly increased the outputs of Progesterone, DHEA, androsterone, T and dihydrotestosterone (DHT) showing that HH signaling activity effects outputs throughout the androgen biosynthetic pathway. Upregulation of the RDH5 gene by Hg1.5 suggests that the effects on production of DHT from PrSCs might be generated by increased synthesis through both classical and back-door pathways. On a weight-to-weight basis, Hg1.5-stimulated PrSCs produce higher T and DHT outputs than LNCaP cells or an androgen growth-independent variant, LNCaP-AI. Finally, we found that treatment of Sonic Hh-producing LNCaP cells with simvastatin reduced the ability of conditioned medium from these cells to induce steroidogenic gene expression from PrSCs. In summary, this work provides evidence for a stromal cell contribution to IS in CRPC mediated by a paracrine-active HH signaling tumor microenvironment induced by ADT-treatment and identifies potential treatment strategies to interfere with this process.

Citation Format: Amy A. Lubik, Hans Adomat, Emma S. Guns, Ralph Buttyan. Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2013-1632
Abstract 1636: Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth.
Authors: Shao Hongwei , Moller Mecker G. , Velazquez Omaida C. , Liu Zhao-Jun
Published: August 2014

Objectives. Cancer-associated fibroblasts (CAF) play critical roles in determining tumor growth, invasion, metastasis, drug response and tumor angiogenesis. We have previously demonstrated that activation of the Notch1 pathway confers normal human dermal fibroblasts a suppressive phenotype to melanoma growth. Here, we further investigated whether manipulation of Notch signaling in melanoma-associated fibroblasts (MAF) alter their regulatory phenotype by which affects melanoma growth.

Methods. MAF were isolated from human metastatic melanoma and characterized. Notch pathway activity in MAF versus normal human dermal fibroblasts was examined using Notch pathway RT2-PCRArray and immunoblotting. Enforced activation of Notch pathway was achieved by transducing MAF with lentiviral vector encoding active form of Notch1 (NIC). The effect of active Notch1-engineered MAF on melanoma growth was tested by in vitro co-culture and in vivo co-xenografting animal model.

Results. Isolated MAF are α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) positive. MAF exhibited a relatively lower Notch activity compared to normal human dermal fibroblasts. Enforced activation of Notch1 pathway suppressed cellular activities of MAF. Active Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro and retarded xenografted human melanoma cell growth on mouse skin.

Conclusions. Notch pathway activity is lower in MAF. Constitutive activation of Notch pathway confers MAF to a suppressive phenotype in regulating melanoma growth. Our study demonstrated that Notch signaling functions as a critical molecular switch in determining the regulatory role of tumor stromal fibroblasts and provided a novel approach to target tumor microenvironment through modulating stromal fibroblasts by manipulation of Notch signaling.

Citation Format: Hongwei Shao, Mecker G. Moller, Omaida C. Velazquez, Zhao-Jun Liu. Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1636. doi:10.1158/1538-7445.AM2013-1636
Abstract 1641: Color-coded imaging of interaction between RFP-expressing blood vessels and αv integrin-GFP expressing osteosarcoma cells.
Authors: Uehara Fuminori , Tome Yasunori , Maehara Hiroki , Kanaya Fuminori , Hoffman Robert M.
Published: August 2014

We report here imaging the interaction between αv integrin tagged to GFP in human osteosarcoma cells and blood vessels growing on Gelfoam@ implanted subcutaneously in mice. 143B osteosarcoma cells expressing αv integrin-GFP were generated by transfection of an αv integrin-GFP vector. Gelfoam@ (5×5 mm) was treated with basic fibroblast growth factor (bFGF) and transplanted subcutaneously in RFP transgenic nude mice. Skin-flap imaging windows were made at days 7, 14, 21, 28 after transplantation of Gelfoam@. Angiogenesis was observed in the Gelfoam@ using fluorescence imaging via the skin flap windows. Frozen sections were made at day 28 after Gelfoam@ transplantation. RFP-expressing blood vessels were imaged growing into the Gelfoam@. Immunohistochemical (IHC) staining showed that CD31 and RFP fluorescence co-localized in the newly formed RFP-expressing blood vessels in the Gelfoam@. To observe the interaction of tumor cells and RFP blood vessels, 143B cells (5×105),expressing αv integrin-GFP, were injected into the Gelfoam@ 7 days after transplantation. Seven days after cancer-cell injection, the cancer cells were observed in the Gelfoam@ by confocal microscopy via skin-flap windows. 143B cells expressing αv integrin-GFP proliferated into the Gelfoam@ as observed by skin-flap-window imaging. Strong expression of αv integrin-GFP cells was observed near RFP vessels in the Gelfoam@ in the RFP mice 28 days after cancer cell injection. The results show that Gelfoam@ is a useful tool to observe angiogensis in vivo and the interactions of αv integrin-GFP with blood vessels which will allow further understanding of its role in angiogenesis and tumor progression.

Citation Format: Fuminori Uehara, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Robert M. Hoffman. Color-coded imaging of interaction between RFP-expressing blood vessels and αv integrin-GFP expressing osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2013-1641
Abstract 1655: Synthetic lethal interaction of CDK inhibition and autophagy inhibition in human solid cancer cell lines.
Authors: Okada Yoshinari , Kato Shunsuke , Sakamoto Yasuhiro , Oishi Takayuki , Ishioka Chikashi
Published: August 2014

Autophagy is a stress-induced cellular reaction in cancer cells. Recent studies showed that cyclin dependent kinase inhibitor proteins, such as p27 or p16, also induced cytoprotective autophagy in cancer cells. However, it is not clear whether small molecule CDK inhibitors also induce autophagy in solid cancer cells. In this study, we revealed that CDK4 inhibitor and a broad-range CDK inhibitor (flavopiridol) induced autophagy in some solid cancer cell lines, but not all. The autophagy induced by CDK4 inhibitors was observed in BT474, MDA-MB435S, SKBr3 (derived from breast cancer), A431 (derived from lung cancer) and SW480 (derived from colorectal cancer), While it was not observed in MCF7, MDA-MB231 (derived from breast cancer), NCI-N87 (derived from gastric cancer) and KMST-6 (derived from fibroblast). In the cell lines which were induced autophagy by CDK inhibitors, the combination of a CDK4 inhibitor and an autophagy inhibition, by either chloroquine or knockdown of ATG5 or BECN1, induced apoptosis. However it did not induce apoptosis in the cell lines which were not induced autophagy by CDK4 inhibitor. From these results, the autophagy induced by CDK4 inhibitor mimic the stress-induced autophagy in some solid cancer cell lines. The combination of a small-molecule CDK inhibitor and an autophagy inhibitor is a synthetic lethal interaction and could become a new antitumor strategy in solid tumors which are induced cytoprotective autophagy by CDK inhibitors.

Citation Format: Yoshinari Okada, Shunsuke Kato, Yasuhiro Sakamoto, Takayuki Oishi, Chikashi Ishioka. Synthetic lethal interaction of CDK inhibition and autophagy inhibition in human solid cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2013-1655
Abstract 166: Elevated levels of serum fatty acid synthase (FAS) in patients with gastric carcinoma.
Authors: Ito Tomoaki , Sato Koichi , Maekawa Hiroshi , Tada Takashi , Sakurada Mutsumi , Orita Hajime , Shimada Kazunori , Wada Ryo , Abe Masaaki , Hino Okio , Kajiyama Yoshiaki
Published: August 2014

Aim: Gastric cancer is the second leading cause of cancer mortality in the world. In East Asia especially, in countries such as China, Japan, and Korea, more than one million new cases are diagnosed each year. The need remains for biomarkers to detect early stage gastric cancer. Fatty acid synthase (FAS) is highly expressed in many human cancers, but the potential utility of measuring FAS for detection of gastric cancer has not been previously investigated. The aim of this study was to provide a preliminary assessment of serum FAS as a marker of gastric carcinoma.

Materials and methods: Our study included 47 patients with gastric cancer (37 men and 10 women, aged 33-89 years) and 150 healthy subjects (113 men and 37 women, aged 33-83 years). Serum

FAS levels were measured by ELISA and compared across the two groups of patients. Blood samples were collected from each cancer patient before treatment, and additionally, post-therapeutic samples were obtained from eleven of the patients with gastric cancer. Measurement data were analyzed using the

Mann-Whitney test. P<0.05 was considered statistically significant. The receiver operator characteristic (ROC) curve was used to determine sensitivity and specificity values.

Results: We found significantly higher levels of serum FAS in gastric cancer patients (95% confidence interval,

95% CI=30.37-52.46) than in healthy controls (95% CI=1.331-2.131), with elevated levels even in patients with early stage tumors. Using 5.5 ng/ml as the cut-off value, the sum of sensitivity (95.74%) and specificity (91.33%) was the highest, and the positive predictive values and negative predictive values were 77.59% and 98.56%, respectively (Area Under the Curve=0.9845,

Standard Error=0.0084, 95% CI=0.9681-1.001). The pre-therapeutic FAS levels were greater than 5.5 ng/ml, and in 8 of 11 patients, the post- therapeutic serum FAS levels decreased to less than 5.5 ng/ml.

Conclusion: Our results suggest that measuring serum FAS levels has strong potential to provide a biomarker for detection of gastric cancer with high sensitivity and high specificity.

Citation Format: Tomoaki Ito, Koichi Sato, Hiroshi Maekawa, Takashi Tada, Mutsumi Sakurada, Hajime Orita, Kazunori Shimada, Ryo Wada, Masaaki Abe, Okio Hino, Yoshiaki Kajiyama. Elevated levels of serum fatty acid synthase (FAS) in patients with gastric carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 166. doi:10.1158/1538-7445.AM2013-166
Abstract 1660: Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract.
Authors: Jingjing Gong , Kumar Addanki P.
Published: August 2014

5-year survival for pancreatic cancer (PanCA) continuous to be lower than any other solid tumor due to late stage diagnosis combined with lack of effective therapeutic options. Therefore developing novel strategies possibly based on molecular targets is critical for successful management of PanCA. Recent studies from our laboratory show potential anti-pancreatic cancer activity for NexrutineR (Nx), a bark extract isolated from traditional Chinese medicine Phellodendron amurense. These studies also show that Nx reduces pancreatic fibrosis in preclinical animal model through targeting two critical oncogenic transcription factors including NF-κB and Stat3. Nonetheless, the precise molecular mechanism associated with the observed biological activity is unclear. We investigated whether Nx inhibits pancreatic cell proliferation and pancreatic fibrosis via reactive oxygen species (ROS)-mediated activation of autophagy. Our data show that Nx can induce autophagy at earlier time points (4h) but inhibit autophagy at later time points (24h) in human pancreatic cancer cell lines Capan-2 and BxPC-3 as evidenced by autophagosome formation, alteration in the expression and levels of various autophagy related genes. Interestingly Nx also induced apoptosis at 24h time point but not at 4h suggesting that autophagy precedes apoptosis. Further under these experimental conditions, treatment of pancreatic cancer cells with Nx enhanced intracellular production of ROS implicating a cross talk between Nx-induced ROS, autophagy and apoptosis. Overall, our findings divulge an important role for ROS/autophagy/apoptosis in Nx-mediated anti-pancreatic cancer effects. Supported by NCCAM (APK).

Citation Format: Gong Jingjing, Addanki P. Kumar. Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2013-1660
Abstract 1678: Metastatic breast cancer cell phenotype is regulated by autophagy.
Authors: Li Yi , Lewis Monica J. , Liu Jianzhong , Cody James J. , Hurst Douglas R.
Published: August 2014

Autophagy is a catabolic process that is tightly regulated during normal cell growth, development, and homeostasis. Although it may be death inducing, it is also an important survival mechanism for cells in stressful environments, including hypoxia, nutrient deprivation, chemical or physical pressure, or detachment from colonies. Considering the stress associated with the process of metastasis, we hypothesized that autophagy may play a role in the ability of breast cancer cells to survive and metastasize. To test this, we first compared cell proliferation after autophagic induction between metastatic breast cancer cell lines (MDA-MB-231 and -435), a non-metastatic breast cancer cell line (MDA-MB-436), and a ‘normal’ breast epithelial cell line (MCF10A). Both metastatic cell lines continued to proliferate following induction of autophagy; however, the non-metastatic and normal cell lines were growth-inhibited. Autophagy was also more rapidly induced by rapamycin in the metastatic cell lines as demonstrated by an increase in LC3II expression. To test the different survival capabilities between metastatic and normal cells, we selected cells through five rounds of starvation with Earle's balanced salt solution (EBSS) and compared their ability to induce autophagy. Although both the normal and metastatic cells maintained their ability to proliferate under normal conditions, the MDA-MB-231 cells that survived starvation (231-EB5) proliferated more rapidly than the parental population following induction of autophagy. In contrast, both MCF10A and MCF10A-EB5 cells were growth-inhibited. The 231-EB5 cells induced autophagy more rapidly than the parental population as demonstrated by increased expression of LC3II (analyzed by immunoblot) and increased LC3 puncta (analyzed by immunofluorescence). Migration of parental 231 cells was inhibited by rapamycin; however, there was no change in the ability of 231-EB5 cells to migrate after treatment with rapamycin. Growth in three dimensions also was significantly altered. MCF10A-EB5 cells were more extended compared to the spherical growth of the parental MCF10A cells. Protrusions emitted from the 231-EB5 cells were more rounded compared to the extended spikes of the parental 231 cells. Altogether, these results demonstrate that select populations derived from metastatic breast cancer cells have different abilities to survive, proliferate, and migrate following induction of autophagy, suggesting that autophagy may be a key mechanism for tumor progression and metastasis.

Citation Format: Yi Li, Monica J. Lewis, Jianzhong Liu, James J. Cody, Douglas R. Hurst. Metastatic breast cancer cell phenotype is regulated by autophagy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2013-1678
Abstract 1693: Intracellular hypoxia is dominantly determined by mitochondrial respiratory activity.
Authors: Prior Sara , Kim Ara , Tobita Seiji , Takeuchi Toshiyuki , Higuchi Masahiro
Published: August 2014

Intracellular oxygen concentration ([O2]i.c.) regulates key biological functions such as HIF-1α activation, glucose transport, potassium pumps, intracellular calcium concentration, P450 family enzymes and HMGR expression. However, the mechanisms regulating [O2]i.c remain to be defined. Here, we provide direct evidence of the mitochondrion's role in [O2]i.c. regulation. We used bis(2-(2′-benzothienyl)-pyridianto-N,C3’irridium(acetylacetonate) (BTP) to measure [O2]i.c. in living cells via microscopy.5 Well-differentiated cancers, LNCaP and MCF-7 showed intracellular hypoxia without exogenous hypoxia. In contrast, poorly-differentiated cancers exhibited intracellular normoxia, and exogenous hypoxia could induce hypoxia. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content increased in [O2]i.c,, indicating that [O2]i.c. is regulated by mitochondrial respiratory activity. [O2]i.c. is additionally regulated by glucose, but the dose-dependent relationship is parabolic. The no glucose condition induced normoxia, and the low concentration glucose condition (0.045mg/ml) induced strong hypoxia. [O2]i.c. was found to be additionally determined by the presence of FCS in the growth medium, and this effect was mimicked by an androgen analog, R1881, suggesting that androgen, glucose concentration and respiratory function are important regulators of [O2]i.c..

Citation Format: Sara Prior, Ara Kim, Seiji Tobita, Toshiyuki Takeuchi, Masahiro Higuchi. Intracellular hypoxia is dominantly determined by mitochondrial respiratory activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2013-1693
Abstract 1694: Warburg effect increases intracellular oxygen concentration and determines membrane localization of Ras and other prenylated proteins.
Authors: Kim Ara , Higuchi Masahiro
Published: August 2014

The Warburg effect is a famous cancer-related phenomenon which describes a metabolic shift from oxidative phosphorylation to glycolysis and fermentation in a cancer cell. Many studies suggest that the Warburg effect is in response to changes (mutation, deletion, and depletion) within the mitochondrial genome. We previously showed that poorly differentiated prostate cancer cells have reduced mitochondrial genome content and are more influenced by the Warburg effect than well differentiated prostate cancer cells. The reduction of mitochondrial genome content induced overexpression and activation of proto-oncogenic Ras, which is responsible for the activation of AKT and ERK, leading to advanced phenotypes of prostate cancer. Farnesylation (membrane localization) of Ras was required for Ras activation and was induced by the overexpression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), the rate-limiting enzyme of the mevalonate pathway, through inhibition of hypoxia-dependent degradation of HMGR. We hypothesize that increased intracellular oxygen concentration due to the Warburg effect responding to the reduction of mitochondrial genome content determines membrane localization of Ras via induction of farnesylation mediated by overexpression of HMGR. In the poorly differentiated prostate cancer cells such as LNCaP (intracellularly normoxic cells), Ras localized in both the plasma membrane and Golgi, whereas in well differentiated prostate cancer cells such as PC-3 (intracellularly hypoxic cells), Ras is prominent in the cytosol. Exogenous hypoxic conditions eliminated Ras localization in the membrane in PC-3, possibly through inhibition of the farnesylation of Ras. In contrast, exogenous hyperoxic condition increased membrane localization of Ras in LNCaP. Next, we demonstrated the localization of other prenylated proteins, such as Rab5a and prelamin A. In the PC-3, Rab5a and prelamin A were localized in the early endosomes and the nuclear membrane, respectively. In LNCaP, they were present throughout the cytosol and inside of the nucleus. Similar to Ras, the localization of Rab5a and prelamin A was reversible with changes in exogenous oxygen concentration. Our results revealed a coherent mechanism that directly links changes in intracellular oxygen concentration, determined by the mitochondrial genome, with the progression of prostate cancer.

Citation Format: Ara Kim, Masahiro Higuchi. Warburg effect increases intracellular oxygen concentration and determines membrane localization of Ras and other prenylated proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2013-1694
Abstract 1697: Pharmacological inhibition of ATM results in mitochondrial biogenesis in AMPK independent manner.
Authors: Khalil Hilal S. , Tummala Hemanth , DeCaris Laura , Zhelev Nikolai
Published: August 2014

The gene mutated in Ataxia telangiectasia termed Ataxia Telangiectasia Mutated (ATM) encodes for a Serine/Threonine protein kinase which functions at the core of signalling network responsible for DNA damage recognition and repair. Recent evidence has supported the role of ATM beyond DNA repair and has been shown to function in metabolic regulation and cellular homeostasis. Here, we show that ATM regulates mitochondrial biogenesis in MCF-7 and A549 cancer cell lines. Inhibition of ATM kinase activity with its specific inhibitor KU55933 (KU) both with and without DNA damage by Doxorubicin or Bleomycin resulted in evoked cytosolic Ca2+ ions leading to rapid, but sustained induction of mitochondrial biogenesis in these cell lines. This induction was independent of AMPK, a kinase previously shown to be involved in mitochondrial biogenesis as KU treatment resulted in downregulation of phospho AMPK. Moreover siRNA mediated knockdown of AMPK could not prevent KU dependent induction of mitochondrial biogenesis. Interestingly, KU treatment resulted in induction of Extracellular signal-regulated kinase(ERK) and CREB phosphorylation. Inhibition of ERK signalling by using MEK inhibitor U0126 disrupted KU dependent induction of mitochondrial biogenesis demonstrating the involvement of ERK signalling cascade in mitochondrial biogenesis upon ATM or AMPK inhibition. These results demonstrate that ATM maintains cellular homeostasis by regulating AMPK mediated mitochondrial biogenesis and that perturbation in this pathway by KU treatment causes a switch in mitochondrial biogenesis in ERK dependent manner. This study indicates cancer cell's fail-safe mechanism in maintaining its homeostasis by up-regulating mitochondrial biogenesis upon inhibition of ATM.

Citation Format: Hilal S. Khalil, Hemanth Tummala, Laura DeCaris, Nikolai Zhelev. Pharmacological inhibition of ATM results in mitochondrial biogenesis in AMPK independent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1697. doi:10.1158/1538-7445.AM2013-1697
Abstract 1702: Metabolic heterogeneity of HER2-positive breast cancers.
Authors: Dennison Jennifer B. , Mills Gordon B.
Published: August 2014

Breast cancers are at least five molecularly distinct diseases with reported metabolic differences. Basal-like breast cancers are typically more glycolytic as compared to luminal-like breast cancers. However, HER2-positive breast tumors have variable FDG-PET maximum uptake values. We hypothesized that HER2-positive breast cancer cell lines would reflect the metabolic heterogeneity reported in breast cancer tumors. In the current study, a panel of HER2-positive breast cancer cell lines (N = 8) was profiled by their metabolic properties. To evaluate glycolytic dependency for ATP production, ATP-synthase dependent oxygen consumption rates were normalized to extracellular acidification rates using the Seahorse Bioscience XF analyzer. The glycolytic dependency was highly variable (12-fold range); ER-negative lines including AU565 and HCC1954 were the most glycolytic. Consistent with these results, HER2-positive/ER-negative cell lines produced more lactate compared to HER2-positive/ER-positive cell lines (1.5 versus 1.2 lactate produced/glucose consumed, p = 0.02). The glycolytic phenotypes of HER2-positive/ER-negative breast cancer cell lines were similar to that of basal-like breast cancer cell lines. For the HER2-positive lines, the highest rates of glutamine consumption were in ER-positive lines BT474 and HCC1419. Differential expression of mitochondrial glutaminase 2 (GLS2, p = 0.02), but not cytosolic glutaminase (GLS) or glutamine synthetase (GLUL), may contribute to higher rates of glutamine consumption by HER2-positive/ER-positive breast cancers. Consistent with higher rates of mitochondrial respiration, ER-positive/HER2-positive cells were also associated with higher expression of carbonic anhydrase 12 (CA12) and lower expression of the lactate transporter MCT1 (SLC16A1). Metabolic heterogeneity of HER2-positive cells was only evident in normoxic conditions. Per the “Pasteur effect,” all cell lines cultured under hypoxia (1% O2) were equally glycolytic (average = 1.8 lactate production/glucose consumption ratio) with correspondingly reduced rates of glutamine uptake. We conclude that the heterogeneity observed in FDG-PET may be influenced by fundamental metabolic differences between HER2-positive breast cancers, in particular, for well-oxygenated regions of the tumor.

Citation Format: Jennifer B. Dennison, Gordon B. Mills. Metabolic heterogeneity of HER2-positive breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1702. doi:10.1158/1538-7445.AM2013-1702
Abstract 1705: Bioenergetic changes in glycolysis, Kreb's cycle, and glutaminolysis in pancreatic cells treated with mitochondria-targeted cationic drugs.
Authors: Cheng Gang , Zielonka Jacek , Joseph Joy , Kalyanaraman Balaraman
Published: August 2014

Human pancreatic ductal adenocarcinoma (PDAC) is an incurable and highly aggressive human cancer. Current chemotherapy provides a slight increase in survival. New therapeutic strategies to treat PDAC are desperately needed. In this study we used a relatively non-toxic mitochondria-targeted cationic nitroxide (Mito-CP) in combination with antiglycolytic agents (2-deoxyglucose, 2-DG) to selectively inhibit pancreatic cancer cell proliferation. We used high-throughput XF24 extracellular flux analyzer and mass spectrometry-based metabolomics approach to investigate cellular bioenergetics changes in pancreatic cancer cells (MiaPaCa-2 and Panc-1 cells). Results indicate that at nanomolar concentrations, Mito-CP alone potently inhibited proliferation of MiaPaCa-2 cells and synergized with 2-DG in inhibiting intracellular ATP levels. In the presence of glycolytic inhibitors, i.e. 2-DG or 3-bromopyruvate, Mito-CP exhibited enhanced toxicity in both pancreatic cell lines. LC-MS-based metabolomics studies indicate that Mito-CP inhibits Kreb's cycle metabolites (citric acid, cis-aconitic and isocitric acids) but significantly enhances intracellular glutamine, alpha-ketoglutarate, succinate, fumarate, and malate. This enhancement is abolished in glutamine-free media. We conclude that strategies to modulate bioenergetic metabolism with relatively non-toxic mitochondria-targeted nitroxides in combination with glycolysis or glutaminolysis inhibitors may provide a novel route for treating patients with PDAC with minimal toxic side effects.

Citation Format: Gang Cheng, Jacek Zielonka, Joy Joseph, Balaraman Kalyanaraman. Bioenergetic changes in glycolysis, Kreb's cycle, and glutaminolysis in pancreatic cells treated with mitochondria-targeted cationic drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2013-1705
Abstract 1715: Estrogen receptor β induces apoptosis through PUMA independent of p53 in prostate cancer cells.
Authors: Dey Prasenjit , Gustafsson Jan-Ake , Strom Anders M.
Published: August 2014

Estrogen receptor β1’s (ERβ1) expression is reduced during the progression of prostate cancer. In agreement with several studies indicating a tumor suppressive effect of ERβ, we find that expression of ERβ increases apoptosis in the prostate cancer cells. Cells expressing ERβ showed an increase in apoptotic cell population by TUNEL assay and flow cytometric analysis. In addition, cells expressing ERβ showed an increase in sub G1 cell population compared to the control cells. Also, ERβ decreases the expression of anti-apoptotic factor Bcl-2 and increases the expression of the pro-apoptotic factor PUMA. Regulation of PUMA was found to be independent of p53. Although phosphorylation of AKT was decreased by ERβ expression we could not see any reduction of phosphorylated FOXO3A, a known regulator of PUMA. On the other hand total mRNA and protein of FOXO3A increased indicating a direct regulation of FOXO3A by ERβ. Using non-transfected PC3 and LNCaP cells we could observe induction of FOXO3A and PUMA as well as apoptosis in response to added 3β-adiol, DPN or 8β-VE2 indicating that the small amount of ERβ expressed in these cell lines are sufficient to induce apoptosis in response to ERβ specific ligands. Overall, our study indicates an apoptotic action of ERβ and suggests that it can be advantageous to treat prostate cancer with ERβ agonist.

Citation Format: Prasenjit Dey, Jan-Ake Gustafsson, Anders M. Strom. Estrogen receptor β induces apoptosis through PUMA independent of p53 in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1715. doi:10.1158/1538-7445.AM2013-1715
Abstract 1725: HDAC inhibition induces Bim expression and apoptosis in breast cancer cells undergoing paclitaxel or antiestrogen treatment.
Authors: Berning Aric , Eason Alexander , Gilley Nathan , Takhar Suchreet , ElShafey Sally , Thangaraju Muthusamy , Schoenlein Patricia V.
Published: August 2014

Paclitaxel functions by preventing microtubule degradation, leading to mitotic arrest and apoptotic death. Of particular interest, paclitaxel-induced death in breast cancer cells is dependent, in part, on the levels of BimEL, a pro-apoptotic member of the Bcl-2 family of proteins. In addition, our recent studies demonstrated that BimEL is required for 4-hydroxytamoxifen-induced apoptosis of estrogen receptor positive (ER+) MCF-7 breast cancer cells [Breast Cancer Res. 2012 Mar 19;14(2):R52]. In contrast, we demonstrated low-level BimEL expression in ER+ T47D breast cancer cells that do not undergo antiestrogen-induced apoptosis. Thus, low-level BimEL expression in ER+ breast cancer may predict a poor apoptotic threshold which ultimately would facilitate the development of acqured resistance to paclitaxel, as well as antiestrogen therapy. Based on the ability of HDAC inhibitors to increase the transcription of pro-apoptotic genes, we hypothesized that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would increase BimEL expression in T47D breast cancer cells and induce a robust apoptotic response to paclitaxel chemotherapy and antiestrogen treatment. In this study, we now demonstrate that SAHA does significantly up-regulate BimEL expression in T47D cells, as well as in MCF-7 cells. Concomitant with BimEL upregulation, SAHA sensitizes T47D and MCF-7 cells to paclitaxel-induced apoptosis. Similarly, SAHA sensitizes T-47D cells to antiestrogen-induced apoptosis, while augmenting the level of antiestrogen-induced apoptosis in MCF-7 cells. These studies indicate that the pro-apoptotic protein BimEL is required for SAHA-induced sensitization of breast cancer cells to paclitaxel and/or antiestrogen-induced apoptosis. Currently, siRNA studies are being conducted to determine if BimEL is a key death effector in response to SAHA treatment and if the increased death from SAHA and paclitaxel or SAHA and antiestrogens is synergistic or additive. Our results provide strong support for the use of HDAC inhibitors when designing novel combination therapies to reduce the emergence of acquired resistance in breast cancer cells undergoing chemo- or antihormonal therapy.

Acknowledgement: this work was supported by teh MCG foundation and NIHRO1 CA121438 to P.V.S.

Citation Format: Aric Berning, Alexander Eason, Nathan Gilley, Suchreet Takhar, Sally ElShafey, Muthusamy Thangaraju, Patricia V. Schoenlein. HDAC inhibition induces Bim expression and apoptosis in breast cancer cells undergoing paclitaxel or antiestrogen treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2013-1725
Abstract 1733: p62/sequestosome 1 regulates caspase-8 activation by the Bcl-2 antagonist ABT-263 to enable crosstalk between autophagy and apoptosis.
Authors: Huang Shengbing , Park Jae Myung , Yu Chunrong , Sinicrope Frank A.
Published: August 2014

Background: Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, the functional relationship between these processes is unclear. During autophagy, the multifunctional p62/sequestosome 1 protein shuttles ubiquitinated proteins to the lysosome. Previously, we found that ectopic p62 can promote chemotherapy-induced apoptosis. Since Bcl-2 can regulate both autophagy and apoptosis, we determined whether ABT-263-induced apoptosis is regulated by p62.

Methods: Human colorectal cancer cell lines (HCT116, DLD1) were incubated with ABT-263 (0-2 μM) and caspase cleavage (immunoblotting), caspase-8 activation (luminescence), apoptosis (annexin V labeling), and autophagy (localization of GFP-LC3 by confocal microscopy) were analyzed. Caspase-8 was suppressed by siRNA or using caspase-8-deficient Jurkat cells. P62 mutants were generated by site-directed mutagenesis. Caspase-8 self aggregation and colocalization with p62 were studied in cells with lentiviral mCherry-p62, transfected with a non-cleavable caspase-8 fused with fluorescent Venus proteins (BiFC).

Results: We demonstrate that ABT-263 induces autophagy and a caspase-8-mediated apoptosis, shown using caspase-8 siRNA or caspase-8-deficient cells. Regulation of this process by p62 was demonstrated by the ability of ectopic p62 expression to enhance apoptosis and caspase-8 activation that were disrupted by mutations at p62 functional domains. In contrast, p62 shRNA was shown to attenuate ABT-263-induced apoptosis. ABT-263 treatment enhanced binding between caspase-8 and p62, and triggered caspase-8 self aggregation that colocalized with p62, shown by Biomolecular Fluorescence Complementation (BiFC) assay using confocal microscopy. Consistent with ectopic p62, accumulation of p62 by the lysosomal autophagy inhibitors, chloroquine or bafilomycin A1, enhanced ABT-263-mediated caspase cleavage.

Conclusion: p62 can regulate caspase-8 self aggregation/activation by ABT-263 to enable crosstalk between autophagy and apoptosis.

Citation Format: Shengbing Huang, Jae Myung Park, Chunrong Yu, Frank A. Sinicrope. p62/sequestosome 1 regulates caspase-8 activation by the Bcl-2 antagonist ABT-263 to enable crosstalk between autophagy and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2013-1733