bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1023: Regulation of cell proliferation by GPR55/cannabinoid receptors using (R,R’)-4’-methoxy-1-naphthylfenoterol in rat C6 glioma cell line.
Authors: Paul Rajib K. , Wnorowski Artur , Indig Fred E. , Bernier Michel , Wainer Irving W.
Published: August 2014

(R,R’)-4’-Methoxy-1-naphthylfenoterol (MNF) is a potent inhibitor of the proliferation of rat C6 glioma cells with an IC(50) of 0.45 nM and initial in vivo data indicate that the compound may be potentially useful in the treatment of malignant gliomas. MNF is an agonist of the β(2)-adrenergic receptor and an antagonist of the GPR55 receptor. Previous studies have demonstrated that MNF inhibits proliferation in 1321N1 astrocytoma cells via the β(2)-adrenergic receptor and in HepG2 hetapocallular carcinoma cells via GPR55. In this study, the mechanism of the observed MNF effect in C6 cells was investigated using [(3)H]-thymidine incorporation, cell motility assay, and internalization of the fluorescent GPR55 ligand, Tocrifluor 1117 (T1117). The anti-mitogenic response of MNF was not affected by the β(2)-AR antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551). Cell treatment with synthetic agonists of cannabinoid receptors 1 and 2 (WIN-55212,2 and CP55940) led to growth inhibition, whereas pretreatment with AM251 (0.1-5 μM) dose-dependently blocked the anti-mitogenic responses of MNF. Both AM251 and the atypical cannabinoid O-1602 increased cell motility, which was blocked by MNF. Finally, there was marked inhibition in the cellular accumulation of T1117 in MNF-treated C6 glioma cells. These results indicate that MNF inhibits proliferation of C6 cells via GPR55/cannabinoid receptors and not via the β(2)-adrenergic receptor. This work was supported entirely by the Intramural Research Program of the NIA, NIH.

Citation Format: Rajib K. Paul, Artur Wnorowski, Fred E. Indig, Michel Bernier, Irving W. Wainer. Regulation of cell proliferation by GPR55/cannabinoid receptors using (R,R’)-4’-methoxy-1-naphthylfenoterol in rat C6 glioma cell line. [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 1023. doi:10.1158/1538-7445.AM2013-1023
Abstract 1037: Agents that target the PI3K/mTOR pathway decrease oxygen consumption, reduce tumor hypoxia and improve radiation response in solid tumors.
Authors: Cerniglia George J. , Daurio Natalie , Gallagher-Colombo Shannon M. , Busch Theresa M. , Tuttle Stephen W. , Koch Cameron J. , Lin Alexander , Koumenis Constantinos , Maity Amit
Published: August 2014

Hypoxia/anoxia is a well-characterized component of the solid tumor microenvironment and has profound consequences for patient outcome. In particular, clinical data indicate that patients with certain types of tumors treated with radiotherapy have a significantly better outcome if the tumors are less hypoxic. Therefore, we are interested in finding agents that can improve tumor oxygenation. In this study, we investigated the effects of two drugs on tumor oxygenation, NVP-BEZ235 (Novartis), a dual PI3K/mTOR inhibitor, and nelfinavir, an HIV protease inhibitor that inhibits PI3K signaling. Treatment of mice bearing SQ20B head and neck squamous cell carcinoma xenografts with either drug led to a decrease in tumor hypoxia as measured by binding of the 2-nitroimidazole EF3. In vitro O2 consumption measurements of cells grown in tissue culture using a Clark electrode showed that both drugs led to decreased O2 consumption. This result was confirmed using a XF Extracellular Flux Analyzer (Seahorse Bioscience). Using this technique, we found that incubation with NVP-BEZ235 caused a 30- 50% decrease in the oxygen consumption rate (OCR). O2 consumption was stimulated by addition of the uncoupling agent CCCP in both controls and NVP-BEZ235-treated cells, which indicates that they were not killed by NVP-BEZ235 treatment. Similar results were obtained with nelfinavir. In order to identify whether components of the PI3K/mTOR pathway could contribute to the decrease in O2 consumption, we used siRNA directed against either mTOR or the p110alpha subunit of PI3K. Knockdown of either protein led to a decrease in O2 consumption. Studies are ongoing to determine whether the mitochondria are a direct target of NVP-BEZ235 or nelfinavir and if so, which mitochondrial respiratory complex might be affected. In summary, we show that NVP-BEZ235 and nelfinavir, both of which inhibit PI3K signaling, decrease O2 consumption and ameliorate tumor hypoxia. We have found both of these agents to increase radiation response in vivo in animal models, and nelfinavir is currently being used in combination with radiotherapy in clinical trials. These findings offer insight as to how these agents act as radiation response modifiers.

Citation Format: George J. Cerniglia, Natalie Daurio, Shannon M. Gallagher-Colombo, Theresa M. Busch, Stephen W. Tuttle, Cameron J. Koch, Alexander Lin, Constantinos Koumenis, Amit Maity. Agents that target the PI3K/mTOR pathway decrease oxygen consumption, reduce tumor hypoxia and improve radiation response in solid tumors. [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 1037. doi:10.1158/1538-7445.AM2013-1037
Abstract 1040: The effect of Irinotecan in k-Ras wild and mutant type colon cancer cell lines.
Authors: Park Joo Sung , Jeon Jung Guk , Kang Myoung Hee , Jang You Jin , Lee Sun Il , Byun Jae Ho , Kim Jun Suk , Oh Sang Cheul
Published: August 2014

Irinotecan(IRI) prevents DNA from unwinding by inhibition of topoisomerase1. Its main use is in colon cancer, in particular, in combination with other chemotherapy agents. Recent studies, those with

Irinotecan treatment has a synergistic effect appears. However, there is no study on the effect of the Irinotecan mono-treatment. In addition their roles and the underlying mechanism in anti-tumorigenic function of solid tumor, especially kras wild and mutant type colon cancer, are not yet known. Bax protein is a key mediator of apoptosis and might be related to chemosensitivity. In this work, we evaluated the anti-tumorigenic action of these Irinotecan anti-cancer agent with especially k-ras wild and mutant type colon cancer cell lines, and investigated their apoptosis-inducing effects as the potential mechanism in this process.

The results show that the growth inhibitory effects of 5-fuorouracil (5-FU), Oxaliplatin(OX) produced a dose- and time dependent fashion in k-ras wild(HT29, Colo205) and mutant type(HCT116, DLD-1) colon cancer cell lines. Otherwise, Irinotecan(IRI) produced a cell growth of k-ras wild type cell lines were resistant versus that shown in the sensitive k-ras mutant type cell lines. Cell cycle analysis by FACS indicated that IRI-treated cell lines showed an increase in the proportion of cells in sub-G1 phase, compared to untreated cells. Exposure of colon cancer cells to IRI also resulted in the increase in the percentage of annexin V- positive and PI-negative cells. The results by multiple in vitro experimental model, showed that IRI induce these k-ras mutant type colon cancer cells to undergo apoptosis with increased the Bax protein and a release of cytochrome C from mitochondria, the activation of caspase9, the cleavage of PARP. But Bax protein was inactivation in k-ras wild type colon cancer cell lines. Importantly, inhibition of ERK kinase activity demonstrated that IRI-induced apoptotic pathway was regulated by the ERK signaling cascade. These data indicate that Anti-cancer agent, Irinotecan, promotes Bax mediate apoptosis of colon cancer cell lines by sequential activation of the ERK signaling pathway followed by the up-regulation of intrinsic pathway of caspase 9.

Citation Format: Joo Sung Park, Jung Guk Jeon, Myoung Hee Kang, You Jin Jang, Sun Il Lee, Jae Ho Byun, Jun Suk Kim, Sang Cheul Oh. The effect of Irinotecan in k-Ras wild and mutant type colon 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 1040. doi:10.1158/1538-7445.AM2013-1040
Abstract 1047: AKT inhibitor MK-2206 induces necrotic cell death of uterine leiomyoma cells in vitro and reduces tumor volume in vivo.
Authors: Sefton Elizabeth , Qiang Wenan , Serna Vanida , Kurita Takeshi , Kim Julie
Published: August 2014

Uterine leiomyomas (UL), benign tumors of the myometrium, are the number one indication for hysterectomies in the U.S. due to a lack of effective therapy. ULs show increased phosphorylation of the pro-survival protein AKT compared to normal myometrium indicating that AKT signaling is increased in UL. However, substantial data directly linking AKT to UL cell survival is lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable drug target for inhibiting UL growth. We utilized the AKT inhibitor MK-2206 (MK), currently in phase II trials, on cultured primary human UL cells. MK-2206 effectively reduced phosphorylation of AKT and PRAS40, an AKT substrate. MK-2206 did not affect cell proliferation but caused cell death. We observed a lack of apoptosis in dead UL cells by Annexin V staining. Additionally, MK-2206 was able to induce UL cell death in the presence of pan-caspase inhibitor, Z-VAD-FMK, indicating that MK-2206 mediated cell death is caspase independent and non-apoptotic. Given that caspases did not modulate cell death, we explored necrosis. MK-2206 induced HMGB1 cytoplasmic localization and mitochondrial disruption indicating possible leiomyoma necrosis. We next explored programmed necrosis, a form of non-apoptotic cell death mediated by RIP kinases (RIPK). However, MK-2206 was able to induce cell death in the presence of RIPK inhibitors, necrostatin-1 and necrosulfonamide, indicating that programmed necrosis is not the mechanism of MK-2206 mediated UL cell death. We are actively exploring alternate necrotic cell death pathways as the mechanisms of MK-2206 induced UL cell death. Lastly, in-vivo MK-2206 treatments reduced tumor volume of human UL cells implanted under the kidney capsule of NSG mice. Xenograft sections from mice treated with MK-2206-2206 contained dying cells in the absence of cleaved caspase 3 supporting the in-vitro results. This is the first report of non-apoptotic cell death in UL and the first time that AKT has been targeted in-vivo for UL therapy. Our data provides molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option for the future treatment of UL. This work was funded by NIH P01HD057877.

Citation Format: Elizabeth Sefton, Wenan Qiang, Vanida Serna, Takeshi Kurita, Julie Kim. AKT inhibitor MK-2206 induces necrotic cell death of uterine leiomyoma cells in vitro and reduces tumor volume in vivo. [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 1047. doi:10.1158/1538-7445.AM2013-1047
Abstract 1065: IL-6 and IGF-1 receptor signal in stemness expression of HBV-related hepatocellular carcinoma .
Authors: Chang Te-Sheng , Ling Thai-Yen , Wu Yu-Chih , Su Wei-Chi , Wu Jaw-Ching , Chi Ching-Chi , Chang Pey-Jium , Chang Jui-Hung , Yang Ya-Ting , Lee Kam-Fai , Liu Jun-Jen , Tung Shui-Yi , Huang Yen-Hua , Kuo Liang-Mou , Ho Hong-Nerng , Huang Yen-Hua
Published: August 2014

Expression of cancer stemness in hepatocellular carcinoma (HCC) is linked to drug resistance and tumor relapse which challenge the current therapy. However, mechanisms underlying the regulation of cancer stemness in HCC remain unclear. Effects of interleukin (IL)-6 and insulin-like growth factor I (IGF-I) on stemness expression in HCC were examined using OCT4/NANOG promoter luciferase reporter assays, RNA interference, secondary sphere formation, side population assay, and a xenograft animal model. Western blot analysis and immunohistochemical staining were used to detect the expression of OCT4/NANOG and phospho-IGF-I receptor (p-IGF-IR) in tissues. Levels of OCT4/NANOG and IGF-IR expressions were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) and their association with tumor recurrence of hepatectomized HCC patients (n = 119) was determined using a Kaplan-Meier survival analysis. A high positive correlation between OCT4/NANOG and IGF-IR/p-IGF-IR expressions in human HCC tissues was observed. The concurrent expression of OCT4/NANOG/IGF-IR was mostly confined to HBV-related HCC (HBV-HCC) and was significantly associated with early tumor recurrence. Investigation of the underlying mechanism showed that IL-6 stimulated the autocrinal IGF-I/IGF-IR expression in a STAT dependent manner, and IGF-I/IGF-IR activation promoted the expression of stemness-related properties of Hep3B cells in both cell lines and xenograft murine models. IL-6 stimulated the expression of stemness-related properties in Hep3B cells, and inhibition of IGF-IR activation significantly suppressed the IL-6’s effects both in vitro and in vivo. Conclusions: Expression of OCT4/NANOG is regulated by IL-6-mediated IGF/IGF-IR signaling in HBV-HCC and is associated with the early recurrence of HCC.

Citation Format: Te-Sheng Chang, Thai-Yen Ling, Yu-Chih Wu, Wei-Chi Su, Jaw-Ching Wu, Ching-Chi Chi, Pey-Jium Chang, Jui-Hung Chang, Ya-Ting Yang, Kam-Fai Lee, Jun-Jen Liu, Shui-Yi Tung, Yen-Hua Huang, Liang-Mou Kuo, Hong-Nerng Ho, Yen-Hua Huang. IL-6 and IGF-1 receptor signal in stemness expression of HBV-related hepatocellular 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 1065. doi:10.1158/1538-7445.AM2013-1065
Abstract 108: Coffee prevents early events in tamoxifen-treated breast cancer patients and modulates hormone receptor status.
Authors: Simonsson Maria , Söderlind Viktoria , Henningson Maria , Hietala Maria , Rose Carsten , Ingvar Christian , Jernström Helena C.
Published: August 2014

Whether coffee modulates response to endocrine therapy in breast cancer patients is currently unknown. The CYP1A2 and CYP2C8 enzymes contribute to tamoxifen and caffeine metabolism. The aim was to investigate the impact of coffee consumption on tumor characteristics and disease-free survival in relation to breast cancer treatment and CYP1A2 and CYP2C8 genotypes. Questionnaires regarding lifestyle were completed preoperatively by 592 patients in Southern Sweden. CYP1A2*1F and CYP2C8*3 were genotyped. Clinical data and tumor characteristics were obtained from patients’ charts, population registries, and pathology reports. Coffee consumption was categorized as low (0-1 cups/day), moderate (2-4 cups/day), or high (5+cups/day). The proportion of estrogen receptor negative (ER-) tumors increased with increasing coffee consumption (Ptrend=0.042). Low consumption was associated with higher frequency of discordant receptor status (ER+PgR-) OR 2.61 (1.59-4.29) compared to higher consumption. Median follow-up time was 4.92 years. Tamoxifen-treated patients with low consumption had significantly increased risk for early events compared to patients who consumed two or more cups/day, adjusted HR 2.50 (1.20-5.26). Low coffee consumption was also associated with higher risk for distant metastases in tamoxifen-treated patients with ER+ tumors, adjusted HR 2.43 (1.00-5.88). Low consumption combined with at least one CYP1A2*1F C-allele or CYP2C8*3 was associated with a high risk for early events in tamoxifen-treated patients, adjusted HRs 3.49 (1.54-7.90) and 6.14 (2.46-15.34), respectively. In conclusion, a moderate to high coffee consumption of caffeinated coffee was associated with significantly lower risk for early breast cancer events in tamoxifen-treated patients. This may be due to modification of the hormone receptor status or altered CYP enzyme activity. If confirmed, new recommendations regarding coffee consumption during tamoxifen-treatment may be warranted.

Citation Format: Maria Simonsson, Viktoria Söderlind, Maria Henningson, Maria Hietala, Carsten Rose, Christian Ingvar, Helena C. Jernström. Coffee prevents early events in tamoxifen-treated breast cancer patients and modulates hormone receptor status. [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 108. doi:10.1158/1538-7445.AM2013-108
Abstract 1132: Identification of synthetic lethal genes to MYCN-amplified neuroblastoma.
Authors: Zhang Shuobo , Yu J , Wei N , Silva J , Kandel J J. , Yamashiro D J.
Published: August 2014

Purpose. Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed, in part due to the inherent difficulty in designing therapeutic molecules that can target transcription factors. An alternative approach is to identify genes that are essential for the growth of MYCN-amplified neuroblastoma.

Methods. We have screened for MYCN synthetic lethal genes using a highly efficient microRNA-short-hairpin RNA (miRNA-shRNA) library that contains more than 200,000 shRNAs targeting almost all predicted genes in the human genome. If a shRNA leads to cellular death, this shRNA will diminish in the cell population. The level of each shRNA was measured both by hybridizing genomic PCR products to microarrays that contain complementary sequence to the shRNA region and by direct high-throughput sequencing of the shRNA region. Selected candidates were further validated in MYCN amplified and non-amplified neuroblastoma cell lines.

Results. We have performed an shRNA screen utilizing the SHEP-21N cell line, which expresses MYCN under a tetracycline controlled promoter. 396 significantly (P<0.01) depleted or enriched genes by both best shRNA and integrated gene level were identified. Gene set enrichment analysis was performed utilizing the Pathway Commons data set. 25 potential synthetic lethal genes were examined by secondary screening utilizing Dharmacon SMARTpool siRNAs. The potential synthetic lethal gene transcription factor AP-4/activating enhancer binding protein (TFAP4) was further validated in three MYCN amplified cell lines and three MYCN non-amplified cell lines. Knocking down TFAP4 by siRNA inhibited cell growth in MYCN amplified cell lines (NGP, SKNDZ, and Be(2)c) by 30%-50%, 96 hours after transfection, while knocking down TFAP4 in MYCN non-amplified lines (CHP212, SKNSH, and SH-SY5Y) did not affect cell growth. Utilizing a tetracycline inducible shRNA system, knocking down TFAP4 significantly reduced colony formation of NGP by70% and Be(2)c by 25%.

Conclusions. By the use of a genome wide RNAi screen, we identified potential synthetic lethal genes to MYCN amplification. The top candidate TFAP4 was validated to inhibit neuroblastoma cell growth and colony formation in a MYCN dependent manner. This may provide novel targets for the treatment of neuroblastoma.

Citation Format: Shuobo Zhang, J Yu, N Wei, J Silva, J J. Kandel, D J. Yamashiro. Identification of synthetic lethal genes to MYCN-amplified neuroblastoma. [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 1132. doi:10.1158/1538-7445.AM2013-1132
Abstract 1161: The relationship between the 14-3-3zeta expression pattern and the clinicopathological variables of the 192 gastric cancer patients.
Authors: Aizawa Tetsuya , Tsukamoto Yoshiyuki , Moriyama Masatsugu , Noguchi Tsuyoshi
Published: August 2014

Gastric cancer remains one of the most common malignant diseases, despite its steady declining trend worldwide. Overall, gastric cancer mortality is estimated to be 700000 cases annually (10.4% of all cancer related death), ranking second only after lung cancer. 14-3-3zeta, a member of 14-3-3 proteins, have important roles in a wide range of biological processes through the interactions with >100 key proteins, such as FOXO3a, Bad and CCND1. Recent studies have revealed that up-regulation of 14-3-3zeta contribute to the transformation phenotype of breast and lung cancer. However, the relevance of 14-3-3zeta expression in gastric carcinogenesis remained poorly understood.

In this study, we aimed to clarify the clinicopathological significance of 14-3-3zeta expression in patients with gastric cancer. A total of 192 gastric cancer specimens were obtained from patients undergoing curative surgery at Oita University Hospital. The expression levels of 14-3-3zeta were investigated by immunohistochemistry. The relationship between the 14-3-3zeta expression pattern and the clinicopathological variables of the gastric cancer patients were analyzed.

14-3-3zeta was strongly expressed in 102of the 192 cases. Strong expression of 14-3-3zeta was correlated with Lymph node metastasis, Histological subtype and Tumor stage (Fisher's exact test, p<0.05). These results suggest that up-regulation of 14-3-3zeta is associated with the clinically malignant features of gastric cancer.

Citation Format: Tetsuya Aizawa, Yoshiyuki Tsukamoto, Masatsugu Moriyama, Tsuyoshi Noguchi. The relationship between the 14-3-3zeta expression pattern and the clinicopathological variables of the 192 gastric cancer patients. [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 1161. doi:10.1158/1538-7445.AM2013-1161
Abstract 1166: Upregulation of microRNAs in colorectal tumors and association with disease stage.
Authors: Makar Karen W. , Wurscher Michelle A. , Habermann Nina , Grady William M. , Ulrich Cornelia M.
Published: August 2014

Introduction

MicroRNAs (miRNAs) are small, noncoding RNAs that function as regulators of many critical cellular processes. Increased expression of specific microRNAs in tumor tissue may be indicative of a patient's prognosis. The purpose of this study was to assess miRNA upregulation in fresh frozen colorectal cancer (CRC) tumors compared to paired normal colorectal tissue in a prospective cohort of CRC patients.

Methods

Tumor and normal tissue were collected at the time of surgery from 40 stage I-IV CRC patients participating in the ColoCare Study in Seattle, WA. The Qiagen miRNeasy kit was used to isolate miRNAs for analysis. Expression of candidate miRNAs (e.g., miR-141, miR-31, miR-29a, mir-92a, miR-203) was measured using Taqman qRT-PCR with miRNA-specific stem loop primers. For each miRNA, copy number was determined via standard curves and normalized to miR-16 expression for each sample. Expression in tumor was compared to expression in paired normal tissue for each participant. In addition, we profiled two stage III CRC tumor-normal pairs using the miCURY LNA human panel I qRT-PCR array from Exiqon. Relative expression from the Exiqon panel was calculated using GenEx software and endogenous control assays. The Wilcoxon two sample test was used to compare normalized miRNA upregulation between groups.

Results

Expression of miR-31 was greater than 10-fold higher in tumor compared to normal tissue for 13 of 38 tumor-normal pairs with Taqman data available. Overexpression of miR-31 in tumor tissue was significantly correlated with stage III-IV CRC when compared to stage I-II CRC (p<0.01). The Taqman and miCURY platforms both showed miR-31 upregulation in the two stage III CRC tumors, but the extent of upregulation varied between platforms.

Conclusion

In this study, upregulation of miR-31 in CRC tumor tissue was associated with more advanced disease stage in CRC patients. Future studies will investigate additional miRNAs, and associations with clinicopathological features, patient outcomes, and circulating miRNA levels.

Citation Format: Karen W. Makar, Michelle A. Wurscher, Nina Habermann, William M. Grady, Cornelia M. Ulrich. Upregulation of microRNAs in colorectal tumors and association with disease stage. [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 1166. doi:10.1158/1538-7445.AM2013-1166
Abstract 1209: Next generation sequencing demonstrates multiple gene amplifications and mutations in 3 patients with estrogen receptor-positive breast cancer with responses to treatment with combination aromatase and PI3K/AKT/mTOR pathway inhibition.
Authors: Zinner Ralph , Atkins Johnique T. , Janku Filip , Moulder Stacy , Stephens Phil , Yelensky Roman , Wolff Robert , Kurzrock Razelle , Wheler Jennifer J.
Published: August 2014

Background:

Gene amplifications in breast cancer may indicate poor prognosis disease, particularly for patients with multiple amplifications. Endocrine response and resistance in breast cancer maybe revealed to PI3K/AKT/mTOR pathway activation.

Patients and Methods:

We analyzed tissue with next generation sequencing (NGS) from 3 patients with estrogen receptor (ER)-positive breast cancer treated on a dose escalation study with anastrozole, an aromatase inhibitor, and everolimus, an mTOR inhibitor. Genomic libraries were captured for 3230 exons in 182 cancer related genes plus 37 introns from 14 genes, often rearranged in cancer (Foundation Medicine, Cambridge, MA).

Results:

Twelve of 50 evaluable patients with ER-positive breast and gynecologic tumors treated with anastrozole (1mg PO daily) and everolimus (either 5mg PO daily or 10mg PO daily) had SD ≥ 6 months/PR/CR. NGS analysis on 3 of the 12 patients tumors demonstrated multiple amplifications, including: CCND1, CCNE1, FGFR1, MYC, IRS2 and MCL1. One patient had a complete response (CR) and two patients achieved partial responses (PR) with an 80% decrease in disease in one patient and a 38% decrease in disease in the other. All 3 patients had ER-positive breast cancer and previously experienced progression of disease while receiving hormone therapy. These amplifications seen have all been linked to hormone therapy resistance in ER-positive breast cancers. Two of these patients also had direct alterations in the PI3K/AKT/mTOR pathway: PIK3CA and PIK3R1 mutations in one patient and PTEN loss in another.

Conclusions:

Molecular profiling of 3 patients with ER-positive breast cancer with PR/CR on treatment with anastrozole/everolimus revealed multiple genetic alterations. Among these alterations were amplifications that may be associated with resistance to hormone therapies. Further exploration of combination hormonal therapies and correlative molecular profiling is warranted.

Citation Format: Ralph Zinner, Johnique T. Atkins, Filip Janku, Stacy Moulder, Phil Stephens, Roman Yelensky, Robert Wolff, Razelle Kurzrock, Jennifer J. Wheler. Next generation sequencing demonstrates multiple gene amplifications and mutations in 3 patients with estrogen receptor-positive breast cancer with responses to treatment with combination aromatase and PI3K/AKT/mTOR pathway inhibition. [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 1209. doi:10.1158/1538-7445.AM2013-1209
Abstract 1213: Clinicopathological significance of ALK alterations in colorectal carcinoma.
Authors: Jehan Zeenath , Prabhakaran Sarita Esther , Bu Rong , Al-Sanea Nasser , Abduljabbar Alaa , Ashari Luai , Al-Homoud Samar , Al Dayel Fouad , Uddin Shahab , Bavi Prashant , Al-Kuraya Khawla Sami
Published: August 2014

ALK has been shown to be a putative oncogene in lung cancer and neuroblastoma that can be potentially targeted with selective inhibitors. Ongoing clinical trials using ALK inhibitors such as Crizotinib have shown promising results. ALK gene copy gain number in colorectal cancer (CRC) patients have been shown to be associated with tumors that have a more aggressive phenotype. We investigated the alterations in ALK gene by FISH analysis, immunohistochemistry and screened for ALK mutations by sequencing the ALK gene in 737 CRC. We screened for mutations in ALK by sequencing the tyrosine kinase domain that covers exons 20 -28. ALK alterations were correlated with tumor pathological features including TNM staging, MSI status, KRAS and BRAF mutations, as well as clinical outcome. Overall survival was analyzed using Kaplan Meyer plots. On FISH analysis in colorectal cancers, ALK amplifications were observed in 2.6%. Amplifications and gain with increase in gene copy number(4 to 6 copies) were seen in 3.4%. No translocations were seen in our study. There were no associations with nodal metastasis and other clinical or molecular parameters. However in Stage III & IV, ALK amplification was associated with KRAS mutations. CRC subgroup with ALK amplifications showed a poor overall survival overall survival and ALK gene amplifications was an independent prognostic marker in multivariate Cox proportional Hazards model. IHC showed cytoplasmic overexpression in 14.7% CRCs and no associations with any clinical or molecular parameters. We are screening for ALK mutations in 100 CRC samples and results are awaited. The insights gained from our current study and future clinical trials are likely to directly benefit subgroups of CRC patients whose tumors are driven by ALK and will pave the way to more targeted approaches to cancer treatment.

Citation Format: Zeenath Jehan, Sarita Esther Prabhakaran, Rong Bu, Nasser Al-Sanea, Alaa Abduljabbar, Luai Ashari, Samar Al-Homoud, Fouad Al Dayel, Shahab Uddin, Prashant Bavi, Khawla Sami Al-Kuraya. Clinicopathological significance of ALK alterations in colorectal 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 1213. doi:10.1158/1538-7445.AM2013-1213
Abstract 1219: Identification of synthetic lethal interactions with the BRAF oncogene in colorectal cancer.
Authors: Vecchione Loredana , Gambino Valentina , D'Ario Giovanni , Sun Tian , Simon Iris , Popovici Vlad , Delorenzi Mauro , Bernards Rene’ , Tejpar Sabine
Published: August 2014

Background

Approximately 8-15% of colorectal (CRC) patients carry an activating mutation in BRAF. This CRC subtype is associated with poor outcome and with resistance, both to chemotherapeutic treatments and to tailored drugs.

We recently showed that BRAF (V600E) colon cancers (CCs) have a characteristic gene expression signature (1, 2) which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred as “BRAF-like," have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation.

By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes and pathways necessary for survival and growth of BRAF mutant CC. Such studies may reveal additional targets for therapy and potentially provide new biomarkers for patient stratification.

Method

We identified 363 genes that are selectively overexpressed in BRAF mutant tumors as compared to WT2 type tumors, based on gene expression profiles of the PETACC3 (1) and Agendia (1) datasets.

The TRC human genome-wide shRNA collection (TRC-Hs1.0) was used to generate a 1815 hairpins sub-library targeting those identified genes (BRAF library). BRAF(V600E) CC cell lines were infected with the BRAF library and screened for shRNAs that cause lethality. LIM1215 CC cell line (WT2) was used as a control. Cells stably expressing the shRNA library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the specific depletion of shRNA in BRAF(V600E) cells as compared to LIM1215 cells.

Results

Candidate genes were identified by using following filtering criteria: depletion in BRAF(V600E) cells by at least 50% and depletion in BRAF(V600E) cells 1,5-fold higher than in control cells with the corresponding p-value to be ≤ 0.1. A total of 34 genes met our criteria of which 6 genes were presented with more than one hairpin and were concordant across the cell lines selected for validation.

Conclusion

We identified candidate synthetic lethal genes in BRAF mutant CC cell lines. Functional analysis is ongoing. Data will be presented.

Citation Format: Loredana Vecchione, Valentina Gambino, Giovanni D'Ario, Tian Sun, Iris Simon, Vlad Popovici, Mauro Delorenzi, Rene’ Bernards, Sabine Tejpar. Identification of synthetic lethal interactions with the BRAF oncogene in colorectal cancer. [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 1219. doi:10.1158/1538-7445.AM2013-1219
Abstract 1222: Simultaneous detection of 30 cytokines in human sera using barcoded magnetic bead technology.
Authors: Zhang Aiguo , Chuang Rachel , Chen Lily , Zhu XiNing , Diaz Rachel , Tsao Dean
Published: August 2014

Cytokines mediate a wide range of physiological responses including immunity, inflammation, and hematopoiesis, which are important circulating proteins. It has been well demonstrated that cytokines are associated with a spectrum of diseases ranging from tumor growth to infection and to Parkinson. Traditionally, cytokines are measured by immunoblot or immunoassay, which are both time consuming and not cost effective. There is an increasing need for multiplexing assays in this field. With the development of bead array technology, which uses fluorescent color-coated microspheres analyzed with a dedicated reader, simultaneous detection of multiple analytes becomes possible in a single well of a microtiter plate. Although the bead array technology becomes more and more popular and dominated in the multiplexing assay market, there are still some limitations and potential issues from this technology, such as photobleaching of the fluorescent color-coated beads and expensive laser detector. Recently, a new generation of the reader has been developed, which replaces laser detector with CCD camera. However, it still uses fluorescent color-coated beads, which makes the assay sensitive to light and unstable. Here we present a new Barcoded Magnetic Bead technology, which employs a permanent bar code on magnetic beads with high resolution CCD camera for scanning. This technology has successfully solved the problems from fluorescent color-coated bead and makes the multiplex assay much more stable and reliable. We can detect 30 cytokines from human sera with limit of detection less than 5 pg/ml, while inter- and intra assay CV less than 10%. The results are correlated very well with the bead array technology and traditional ELISA. In addition, our multiplex protein assays have unique internal control in each sample well, which makes the assay much more reliable and robust. This multiplex technology can be used in both life science research and clinical fields.

Citation Format: Aiguo Zhang, Rachel Chuang, Lily Chen, XiNing Zhu, Rachel Diaz, Dean Tsao. Simultaneous detection of 30 cytokines in human sera using barcoded magnetic bead technology. [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 1222. doi:10.1158/1538-7445.AM2013-1222
Abstract 1223: Antitumor immunotherapeutic and toxic properties of an HDL-conjugated chimeric IL-15 fusion protein.
Authors: Ochoa Maria Carmen , Berraondo Pedro , Melero Ignacio
Published: August 2014

Interleukin 15 (IL-15) effects on CD8 T and NK lymphocytes hold promise to treat cancer. Fusion proteins have been engineered to provide IL-15 receptor alpha (IL-15Rα) mediated trans-presentation to lymphocytes and extend the plasma half-life of the cytokine. In this study, we report on a triple fusion protein combining apolipoprotein A-I, IL-15 and IL-15Rα’s sushi domain. Apolipoprotein A-I conveys IL-15 to high-density lipoproteins (HDL), from which the cytokine is trans-presented by the IL-15Rα's sushi domain. Such a construction was tested by hydrodynamic gene transfer to the liver of mice. Lethal toxicity was observed upon injection of 10μg of the expression plasmid. Mice died from an acute lymphocytic pneumonitis in which T and NK cells dominate a severe inflammatory infiltrate. Importantly, mice devoid of NK cells were not susceptible to such toxicity and mice lacking granzymes A and B also survived the otherwise lethal gene transfer. Lower plasmid doses (<2.5μg) were tolerated and dramatically increased the numbers of NK and memory CD8 T lymphocytes in the liver, spleen and lungs, to the point of rescuing the deficiency of such lymphocyte subsets in IL-15Rα-/- mice. Doses of plasmid within the therapeutic window successfully treated metastatic tumor models, including B16OVA lung metastasis of melanoma and MC38 colon cancer liver metastasis. Sushi-IL15-Apo as a recombinant protein was also bioactive in vivo, became conjugated to HDL and displayed immuno-therapeutic effects against metastatic disease.

Citation Format: Maria Carmen Ochoa, Pedro Berraondo, Ignacio Melero. Antitumor immunotherapeutic and toxic properties of an HDL-conjugated chimeric IL-15 fusion protein. [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 1223. doi:10.1158/1538-7445.AM2013-1223
Abstract 1228: AV-203, a fully humanized ERBB3 inhibitory antibody, reverses ERBB3-induced resistance to targeted therapies.
Authors: Tyler Steven M. , Vincent Sylvie , Fleet Christina , Bottega Steve , McIntosh Donna , Meetze Kristan , Gyuris Jeno
Published: August 2014

Targeted therapies have changed the treatment paradigm of cancer. Specifically, the use of tyrosine kinase inhibitors (TKIs) has improved therapeutic responses in a number of tumor types. However, initial response to TKIs is often followed by rapid relapse due to the acquisition of molecular features that lead to resistance. ERBB3, a receptor of the epidermal growth factor receptor (EGFR/ERBB1) family, has been linked to the development of resistance to multiple ERBB targeting TKIs. A proposed potential mechanism is the recruitment and activation of ERBB3 as a bypass mechanism to activate PI3K dependent pro-survival pathways. We investigated whether the inhibition of ERBB3 could reverse TKI resistance linked to ERBB3 activation. AV-203 is a humanized IgG1 antibody directed against ERBB3. AV-203 potently inhibits both ligand dependent and ligand independent activation of ERBB3. AV-203 was shown to inhibit the binding of NRG1 to ERBB3 and promote ERBB3 degradation. The tumor growth inhibitory activity of AV-203 was demonstrated in numerous xenograft models representing major human carcinomas (breast, lung, ovarian, kidney and pancreas). Here, we investigated the ability of AV-203 to restore TKI sensitivity in models with activated ERBB3. First, we demonstrated that ligand mediated (NRG1) activation of ERBB3 leads to acquired resistance to the EGFR TKI erlotinib in a lung cancer model bearing EGFR TKI sensitizing mutation, to the ERBB2/HER2 inhibitor lapatinib in a HER2 amplified breast cancer model, and to the EGFR inhibitory antibody cetuximab in head and neck cancer models. AV-203 was able to restore sensitivity to these RTK inhibitors. This data suggest that the combination of AV-203, with EGFR/HER2 targeted therapies could potentially extend the efficacy of these agents by preventing the emergence of ERBB3 mediated resistance. This hypothesis will be tested in future clinical trials. AV-203 is currently in Phase I development for the treatment of solid tumors.

Citation Format: Steven M. Tyler, Sylvie Vincent, Christina Fleet, Steve Bottega, Donna McIntosh, Kristan Meetze, Jeno Gyuris. AV-203, a fully humanized ERBB3 inhibitory antibody, reverses ERBB3-induced resistance to targeted therapies. [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 1228. doi:10.1158/1538-7445.AM2013-1228
Abstract 123: Hormonal and genetic risk factors of soft tissue sarcoma in Sweden.
Authors: Wagner Philippe , Magnusson Susanne , Olsson Håkan
Published: August 2014

BACKGROUND AND METHODS. The aetiology of soft tissue sarcoma (STS) is largely unknown. Merely a handful putative risk factors such as certain genetic syndromes, a positive family history of cancer/STS, specific chemical exposures and exposure to ionizing radiation have been identified. With respect to the genetic aetiology, most STS patients do not have a clear family history of cancer. Only in rare cases heritable or germline genetic abnormalities associated with cancer predisposition syndromes that manifest as STS have been identified. We study STS and cancer morbidity in first degree relatives (FDR) together with hormone related factors in the Swedish population in the years between the years 1988 and 2009 using a population-based case-control design. RESULTS. Our study is the largest on this topic to date, including 855 cases and 2024 controls. It identified protective effects of being shorter than your peers at age eleven with a relative risk (RR) of 0.5 (0.4 - 0.7). Having used oral contraceptives (OC), RR 0.6 (0.4 - 1.0), and high parity, RR 0.6 (0.3 - 0.9) comparing three or more children to none, also appeared to reduce the risk for STS. The reduction increased further comparing use for eleven years or more to use for three years or less, RR 0.2 (0.1 - 0.6). Those with an FDR with STS and with those with an FDR with cancer had increased risks, RR 5.16 (1.03 - 25.9) and 1.31 (1.05 - 1.65), respectively. The latter varied with sex and histological subtype. In men, the familial risk was significant for malignant fibrous histiocytoma/undifferentiated pleomorphic sarcomas (MFH), RR 2.21 (1.23 - 3.97) and was higher in younger men (P=0.015). In women, there was a significant overall familial risk when stratifying on OC use, RR 1.55 (1.06 - 2.26). This risk seemed to derive from a group of rare and unclassified tumours present mainly in retroperitoneum and visceral organs, RR 6.19 (0.54 - 71.2). CONCLUSIONS. STS are a heterogeneous group of tumours with possibly very different aetiologies. However, the genesis of sarcoma seems to be influenced by both sex and growth hormones as well as genetic factors. Studies are on the way of the molecular genetic background of these associations.

Citation Format: Philippe Wagner, Susanne Magnusson, Håkan Olsson. Hormonal and genetic risk factors of soft tissue sarcoma in Sweden. [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 123. doi:10.1158/1538-7445.AM2013-123
Abstract 1235: Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer.
Authors: Thiele Michael , Freissmuth Michael , Hussain Filza , Douillard Patrice , Völkel Dirk , Ehrlich Hartmut , Scheiflinger Friedrich , Kerschbaumer Randolf
Published: August 2014

Macrophage migration inhibitory factor (MIF) was found to be upregulated in many cancers and to act in para- and autocrine loops within the tumor microenvironment. Thereby, MIF contributes to the modulation of cytokine patterns and triggers anti-apoptotic and pro-proliferative cell signaling events. MIF was also described as a pro-angiogenic factor promoting neoangiogenesis and vascularization. In prostate cancer, MIF mRNA and protein levels were shown to be increased and to contribute to tumor development. We have discovered a novel, previously unrecognized conformational isoform of MIF (designated ‘active MIF’) that is produced in diseases and cannot be detected in healthy subjects. A set of phage display derived human monoclonal anti-MIF antibodies neutralizing MIF biological activity in vitro and in vivo specifically recognize active MIF. In vitro, these antibodies elicited both, a growth inhibitory effect and a pro-apoptotic response on PC3 and DU145 prostate cancer cells by interfering with ERK1/2 and Akt/PKB signaling pathways and by promoting activation of caspase-3. Furthermore, the antibodies inhibited tumor cell invasion of PC3 cells in a transwell assay in a dose-dependent manner. In vivo, treatment with human antibodies specific for active MIF dose-dependently inhibited the growth of the human prostate cancer cell line PC3 xenografted into MF1 nude mice. New ELISA methods have been established to allow the detection of active MIF as well as non-active MIF in plasma samples. In the plasma of PC3 xenografted nude mice, we observed a reduction of active MIF upon antibody treatment. In addition we were able to prove the occurrence of active MIF in the plasma of prostate cancer patients (median 2445 pg/mL, p < 0.002 versus controls), whereas active MIF was not detectable in plasma from healthy donors. These studies demonstrate that active MIF represents a new target in prostate cancer, as well as a promising marker for disease activity or disease progression. The data further suggest that our fully human anti-MIF antibodies have a good potential for clinical use in oncology.

Citation Format: Michael Thiele, Michael Freissmuth, Filza Hussain, Patrice Douillard, Dirk Völkel, Hartmut Ehrlich, Friedrich Scheiflinger, Randolf Kerschbaumer. Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer. [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 1235. doi:10.1158/1538-7445.AM2013-1235