bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-275: Epithelial-mesenchymal transition rewires mechanisms upstream of PI3K-dependent growth.
Authors: Salt Megan B. , McCormick Frank
Published: August 2014

Tumors that show evidence of epithelial to mesenchymal transition (EMT) have been associated with metastasis, drug resistance, and poor prognosis. EMT may alter the molecular requirements for growth and survival in different contexts, but the underlying mechanisms remain incomplete. Given the heterogeneity along the EMT spectrum between and within tumors it is important to define the requirements for growth and survival in cells with an epithelial or mesenchymal phenotype to maximize therapeutic efficacy. We have established an inducible cell line model in which a tamoxifen regulatable Twist-ER fusion protein is stably expressed in the H358 non-small cell lung cancer cell line. Upon tamoxifen addition, cells undergo EMT and provide a system in which we can compare the growth and survival requirements directly related to EMT, removing confounding factors present when comparing different cell lines. Using this system we have found that while cells require PI3K for growth before and after the transition, the mechanisms through which they can activate the signal are altered. Specifically, before EMT basal PI3K is maintained by autocrine activation of ErbB3 signaling, despite the presence of an activating Ras mutation. Loss of ErbB3 ablates proliferation in pre-EMT cells suggesting that oncogenic KRas is not sufficient to drive growth in this context. After induction of EMT ErbB3 expression is downregulated along with downstream PI3K signal, leading to a loss of cell autonomous proliferation. Restoration of PI3K signaling by either re-expression of ErbB3, activated p110, or through stimulation with various growth factors is able to restore growth in post-EMT cells. These data have implications for therapeutic targeting of different RTKs, suggesting that within a heterogeneous tumor environment, only a subset of cells may respond to such treatments. Furthering the understanding of how EMT alters signaling through specific pathways will facilitate the design of more efficacious treatment strategies to target all cells along this phenotypic spectrum.

Citation Format: Megan B. Salt, Frank McCormick. Epithelial-mesenchymal transition rewires mechanisms upstream of PI3K-dependent 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 LB-275. doi:10.1158/1538-7445.AM2013-LB-275
Abstract LB-280: Inhibition of SHP2 in basal-like and triple-negative breast cancer cells induces basal-to-luminal transition, suppresses tumorigenesis, and blocks metastasis.
Authors: Matalka Fatimah , Zhao Hua , Agazie Yehenew M.
Published: August 2014

One of the factors that contribute to the disproportionately high mortalities in basal-like and triple-negative breast cancer (BTBC) is the lack of targeted therapies. Identifying and characterizing potential targets is therefore critical for future development of such therapies. In the current report, we show that silencing the expression of the Src homology phosphotyrosyl phosphatase 2 (SHP2) in BTBC cell lines (MDA-MB231 and MDA-MB468) suppresses cell proliferation, reverses transformation, and blocks invasiveness. Unexpectedly, we have discovered that inhibition of SHP2 induces basal-to-luminal transition (BLT) in BTBC cells as evidenced by a decrease in the expression of mesenchymal markers and/or drivers (vimentin, ZEB1, Snail, and alpha smooth muscle actin) and an increase in the expression of the luminal marker cytokeratin 18. Furthermore, we have discovered that inhibition of SHP2 induces the expression of the estrogen receptor alpha in otherwise triple-negative breast cancer cells. Intramammary transplantation studies in the NOD/SCID mice demonstrated that inhibition of SHP2 suppresses tumorigenesis and blocks metastasis. Analyses of total cell lysates prepared from cells grown in 3D matrigel cultures demonstrated that SHP2 mediates the activation of the Ras-ERK, the PI3K, and the β-catenin signaling pathways to promote the tumorigenic, invasive, and metastatic potential of BTBC cells. By and large, the results presented in this report suggest that SHP2 plays an essential role in the biology of BTBC.

Citation Format: Fatimah Matalka, Hua Zhao, Yehenew M. Agazie. Inhibition of SHP2 in basal-like and triple-negative breast cancer cells induces basal-to-luminal transition, suppresses tumorigenesis, and blocks metastasis. [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 LB-280. doi:10.1158/1538-7445.AM2013-LB-280
Abstract LB-285: Pro-tumor N2 neutrophils contribute to the pre-metastatic niche.
Authors: Meyers Jered Cope , Pittman H. Keith , Story Hunter , Imes Dare M. , Howard George A. , Verbanac Kathryn M.
Published: August 2014

Metastasis is the primary cause of cancer-related mortalities and the tissue microenvironment that supports tumor cell attachment and growth at metastatic sites has become an active area of investigation. Here we present evidence that pro-tumor N2 neutrophils are one of the bone marrow-derived cell types that establish this pre-metastatic niche. In the murine Lewis Lung adenocarcinoma (LLC) in vivo metastasis model, tumor cells metastasize to the lungs with increasing primary tumor growth over time. In these studies, immunocompetent mice injected subcutaneously with syngeneic LLC cells are sacrificed sequentially 10-32 days later, before and after established lung metastases. Neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2 are significantly elevated in pre-metastatic tissue compared to normal lungs of healthy contemporary controls. MCP-1/CCL2, a marker of pro-tumor N2 neutrophils, increases steadily over time in pre-metastatic lung tissue of study mice and is significantly elevated in pre-metastatic tissue and in metastases compared to normal lung tissue. Isolated lung-infiltrating neutrophils also express N2 markers. In contrast, TNF-alpha, a marker for anti-tumor N1 cells, remains low or undetectable in lung tissue throughout metastatic tumor development. Neither active nor total TGF-beta, the putative control switch for a pro-tumor phenotype, is elevated in pre-metastatic tissue. Notably, levels of all cytokines are low or undetectable in normal lungs from naïve mice and in other organs of tumor-bearing mice (non-metastatic sites). Ly6G+ neutrophils were detected at a higher density in the lungs of pre-metastatic and metastatic mice compared to normal lungs, although the density of myeloperoxidase (MPO)-stained cells was similar in all groups. These data are consistent with recent reports that MPO activity is low or absent from tumor-associated neutrophils, although high in MDSC and naïve neutrophils. Neutrophil distribution in metastatic lung tissue is most concentrated around the tumor foci and tumor vasculature. Finally, studies of lung tissue from mice bearing LLC variants with different metastatic capacities are also consistent with our premise that neutrophils help establish the pre-metastatic niche. Understanding the factors that regulate the development and growth of malignant cells at metastatic sites is critical for improved clinical strategies in the prevention, diagnosis and treatment of metastases.

Citation Format: Jered Cope Meyers, H. Keith Pittman, Hunter Story, Dare M. Imes, George A. Howard, Kathryn M. Verbanac. Pro-tumor N2 neutrophils contribute to the pre-metastatic niche. [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 LB-285. doi:10.1158/1538-7445.AM2013-LB-285
Abstract LB-3: Fully synthetic self-assembling tumor-targeted virus-like nanoparticles .
Authors: Tarasova Nadya , Chen Yuhong , Dyba Marzena , Tarasov Sergey G.
Published: August 2014

Virus particles have evolved as highly effective delivery systems. They are able to deliver whole proteins and large nucleic acid molecules to certain types of cells. Genetically engineered virus-like particles (VLPs) have been explored as drug delivery and imaging agents. However, generation of fully synthetic VLPs has not been achieved. We have found that properly derivatized synthetic analogs of transmembrane domains not only inhibit the function of the corresponding receptor, but can self-assemble into virus-like particles with high precision (Proc. Natl. Acad. Sci. U. S. A 108: 9798 (2011). The described particles have intrinsic biological activity of the peptide they are composed of and fuse with cell spontaneously. Particles composed of CXCR4 antagonist fuse spontaneously with cell membrane, inhibit CXCR4 function in vitro and CXCR4-mediated metastasis in vivo. However, the fusion is non-selective. We have now developed the first fully synthetic VLPs that fuse with cells like real viruses, in receptor-mediated manner. Prevention of non-selective fusion was achieved by derivatization of self-assembling peptides with polyethylene glycol. Addition of ligands recognizing receptors over expressed on prostate tumor cells allowed for the particles that fuse selectively with receptor-positive cells. Particles targeting gastrin-releasing peptide receptor (GRPR), luteinizing hormone-releasing hormone (LHRH) receptor and prostate-specific membrane antigen (PSMA) are remarkably stable, more than 99 % homogeneous in size and undergo receptor-mediated fusion with tumor cells expressing corresponding receptor. Whole- animal fluorescence tomography of nude mice injected with fluorescent nanoparticles targeted to PSMA and GRPR revealed that particles rapidly accumulate in tumors and allow for tumor detection as early as two hours after the injection. Development of fully synthetic VLPs establishes a foundation for generation of delivery systems with investigator-defined biophysical properties and targeting ability for a wide range of biomedical applications. Tumor-targeted self-assembling particles represent a new class of agents with dual and triple biological activity for the therapy and detection of tumors.

Citation Format: Nadya Tarasova, Yuhong Chen, Marzena Dyba, Sergey G. Tarasov. Fully synthetic self-assembling tumor-targeted virus-like nanoparticles . [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 LB-3. doi:10.1158/1538-7445.AM2013-LB-3
Abstract LB-300: Development of an orally-administrative MELK (maternal embryonic leucine zipper kinase)-targeting small molecule inhibitor that suppresses the growth of various types of human cancer.
Authors: Chung Suyoun , Suzuki Hanae , Miyamoto Takashi , Takamatsu Naofumi , Tatsuguchi Ayako , Ueda Koji , Kijima Kyoko , Matsuo Yo , Nakamura Yusuke
Published: August 2014

Breast cancer is the most common malignancy among women worldwide. Treatment for breast cancer acting on molecular targets in critical signaling pathways in cancer cells have successfully reduced the motility rate in a subset of breast cancer patients, but a significant portion of patients expect to have little benefit from these treatments. Therefore, development of new therapeutics for those patients is urgently required. We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was highly upregulated in multiple types of human cancer including breast, prostate and lung cancers, and plays indispensable roles in cancer cell survival, particularly in the maintenance of tumor-initiating cells. Although several substrates of MELK were reported, we still do not understood fully how MELK contributes to breast cancer development and/or progression. To elucidate the MELK signaling pathway in breast cancer cells, we screened and identified two novel MELK substrates, DBNL and PSMA1, which were also found to play critical roles in human carcinogenesis and mammary tumor-initiating cells. Furthermore, we conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC50 of 0.41 nM. OTSSP167 inhibited the phosphorylation of these two substrates by MELK as well as mammosphere formation of breast cancer cells, and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. Here, we report the biological function of novel substrates of MELK and the first MELK inhibitor OTSSP167 that is a promising compound to treat various types of human cancer.

Citation Format: Suyoun Chung, Hanae Suzuki, Takashi Miyamoto, Naofumi Takamatsu, Ayako Tatsuguchi, Koji Ueda, Kyoko Kijima, Yo Matsuo, Yusuke Nakamura. Development of an orally-administrative MELK (maternal embryonic leucine zipper kinase)-targeting small molecule inhibitor that suppresses the growth of various types of human 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 LB-300. doi:10.1158/1538-7445.AM2013-LB-300
Abstract LB-301: Copper chelation therapy suppresses tumor growth by inhibiting mitochondrial ATP production in tumors.
Authors: Ishida Seiko , Andreux Pénélope , Auwerx Johan , Hanahan Douglas
Published: August 2014

Through our yeast genetic screen, we have previously identified the copper transporter Ctr1 as a major mediator of cisplatin uptake in mammals (1), and have subsequently shown in a transgenic mouse model of human cervical cancer that reducing systemic copper levels with a copper chelator, which is clinically used to treat patients with copper disorders, results in enhanced uptake of cisplatin selectively in tumors but not in normal, non-cancerous tissues (2), suggesting that tumors are particularly sensitive to changes in copper levels. In fact, environmental copper exposure has been associated with cancer risk in humans, although the nature of its effects on tumourigenesis remains unclear. Using a transgenic mouse model of pancreatic islet carcinoma, here we demonstrate that chronic exposure to elevated levels of copper in drinking water, corresponding to the maximum level allowed in our drinking water, accelerate tumor growth by stimulating proliferation of cancer cells. Conversely, reducing systemic copper with a chelating drug resulted in inhibition of tumor growth and cancer cell proliferation. Under copper limitation, tumors displayed decreased activity of the copper-binding mitochondrial enzyme cytochrome c oxidase and reduced ATP levels, despite enhanced glycolysis. The anti-proliferative effect of copper chelation was not observed in cells deficient in cytochrome c oxidase activity, but was enhanced when combined with inhibitors of glycolysis. Interestingly, larger tumors contained less copper than smaller tumors, and exhibited lower activity of cytochrome c oxidase and higher glucose uptake, suggesting that a shift towards glycolytic metabolism observed in tumors even in the presence of ample oxygen (Warburg effect) may in part reflect insufficient copper bioavailability in the tumour microenvironment. Our work identifies copper as a rate-limiting element for tumor growth and mitochondrial ATP production, and provides experimental evidence that explains the association between copper intake levels and cancer implicated in previous epidemiological and clinical studies. These findings offer a novel concept of limiting intake levels of this environmental factor as a strategy for cancer prevention and treatment.

Citation Format: Seiko Ishida, Pénélope Andreux, Johan Auwerx, Douglas Hanahan. Copper chelation therapy suppresses tumor growth by inhibiting mitochondrial ATP production in 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 LB-301. doi:10.1158/1538-7445.AM2013-LB-301
Abstract LB-310: Small molecule inhibitors of IKBKE with antitumor activity in cancer cells overexpressing IKBKE .
Authors: Xin Yu , Guo Jianping , Cheng Jin Q.
Published: August 2014

The serine/threonine kinase IKBKE is frequently overexpressed/activated in human cancer. Ectopic expression of IKBKE induces malignant transformation, cell migration, invasion and chemoresistance. Thus, IKBKE is a new attractive target for anti-cancer drug discovery. By screening of NCI Diversity Set and Clinical Collection I and II compound libraries using cell-based assay, we identified several IKBKE inhibitors, 3 of which directly inhibited IKBKE kinase activity in vitro and in vivo and the others only reduced phospho-IKBKE in vivo. One of the inhibitors, MCCK1, was further characterized and was found to directly bind to IKBKE. It inhibits IKBKE kinase as well as its downstream targets such as IkappaB, p65 and IRF3. MCCK1 is selective for IKBKE with moderate effect on TBK1 but does not inhibit the activation of IKKalpha/beta, STAT3, Erk-1/2, p38 or JNK. The inhibition of IKBKE by MCCK1 results in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor aberrant expression IKBKE. Further, MCCK1 inhibits tumor growth in nude mice of human cancer cells in which IKBKE is elevated but not of those cancer cells in which it is not. These data indicate that MCCK1 is an IKBKE inhibitor with anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express elevated IKBKE.

Citation Format: Yu Xin, Jianping Guo, Jin Q. Small molecule inhibitors of IKBKE with antitumor activity in cancer cells overexpressing IKBKE . [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 LB-310. doi:10.1158/1538-7445.AM2013-LB-310
Abstract LB-326: Zeb1 regulates inflammation-induced invasion and self-renewal of colon cancer cells.
Authors: Wang Lei , Li Yijing , Beckley Amy , Pappan Lorretta , Shi Jishu
Published: August 2014

Up-regulation of inflammatory cytokines such as IL-1β in tumor microenvironment has been associated with progression of human colorectal cancer, although the exact molecular mechanisms are still unclear. To understand how IL-1β influences colon cancer cell behaviors, we used a human colon cancer cell line HCT-116 as well as primary human colon cancer cells derived from a patient, which more closely reflect the properties of the primary tumor than established cell lines.

Here, we demonstrate that IL-1β promotes epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) development in colon cancer. IL-1β treatment resulted in loss of E-cadherin and gain of a mesenchymal phenotype in both HCT-116 cells and primary colon cancer cells. Among E-cadherin suppressors, Zeb1 was up-regulated in IL-1β-induced EMT cells. Consistent with EMT properties, IL-1β-treated HCT-116 cells exhibited higher cell migratory ability. Moreover, IL-1β induced expression of stem-cell marker Bmi-1 in both HCT-116 cells and primary colon cancer cells. IL-1β increased these cells’ self-renewal ability to form spheres in serum-free conditions and chemo-resistance; properties associated with colon CSCs. Taken together, these results indicate that IL-1β can induce EMT and promote generation of CSCs in colon cancer.

More importantly, we found that ZEB1, but not other E-cadherin transcriptional repressors (such as Zeb2, Snail and Twist), was consistently up-regulated in IL-1β-induced EMT cells and sphere cells. This suggests that Zeb1 links IL-1β-induced CSC self-renewal and EMT in colon cancer cells. Moreover, knockdown of Zeb1 in HCT-116 cells using shRNAs reversed the IL-1β effects on these two processes, further indicating that Zeb1 is essential for IL-1β-induced stem cell and EMT phenotypes in colon cancer cells. Our finding indicates that IL-1β promotes colon tumor growth and invasion through activation of CSC self-renewal and EMT, while Zeb1 plays a critical role in activation of these two processes. Thus, targeting tumor microenvironment cytokines or Zeb1 might form the basis of a promising treatment for colon cancer.

Citation Format: Lei Wang, Yijing Li, Amy Beckley, Lorretta Pappan, Jishu Shi. Zeb1 regulates inflammation-induced invasion and self-renewal of colon 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 LB-326. doi:10.1158/1538-7445.AM2013-LB-326

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract LB-327: Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D.
Authors: Kashima Kenji , Watanabe Miho , Satoh Yasuko , Hata Junichi , Ishii Nobuya , Aoki Yuko
Published: August 2014

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor called vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors, and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment.

Citation Format: Kenji Kashima, Miho Watanabe, Yasuko Satoh, Junichi Hata, Nobuya Ishii, Yuko Aoki. Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D. [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 LB-327. doi:10.1158/1538-7445.AM2013-LB-327
Abstract LB-331: Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast cancer.
Authors: Chen Xin , Yang Yuan , Zhou Qiong , Howard O.M.Zack , McPherson John , Wakefield Lalage , Oppenheim Joost J.
Published: August 2014

Elimination of CD4+FoxP3+ regulatory T cells (Tregs) has become an important strategy of cancer immunotherapy. It was reported that TGFβ is responsible for accumulation of Tregs in tumor. Thus, we treated mouse 4T1 mammary carcinoma model with 1D11 (0.1 mg/mouse), a neutralizing antibody of TGFβ(1,2,3). The treatment delayed growth of primary tumor, however, unexpectedly increased the proportion of FoxP3+ Tregs present in the intratumoral CD4 cells. In order to enhance the anti-tumor effect, 1D11 was administered with a single dose of cyclophosphamide (4 mg/mouse), a chemotherapeutic agent which was previously shown to selectively eliminate intratumoral TNFR2+ Tregs. This combination, but not the therapeutics by themselves, resulted in tumor-free long term survival of up to 80% of mice, and those tumor free mice were more resistant to re-challenge with 4T1 tumor. In order to examine the phenotype of tumor infiltrating immune cells, 4T1 tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide (2 mg/mouse). This treatment markedly inhibited tumor growth, accompanied by a massive infiltration by IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic MDSCs, and increased their expression levels of MHC II and CD80. Taken together, our data show that anti-TGFβ Ab and cyclophosphamide represents a novel chemoimmunotherapeutic combination which may prove useful in the treatment of human cancer patients.(Funded by NCI Contract HHSN261200800001E)

Citation Format: Xin Chen, Yuan Yang, Qiong Zhou, O.M.Zack Howard, John McPherson, Lalage Wakefield, Joost J. Oppenheim. Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast 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 LB-331. doi:10.1158/1538-7445.AM2013-LB-331

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract LB-40: Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion.
Authors: Murray David W. , Didier Sebastien , Paulino Vincent M. , Chan Amanda , Tran Nhan L. , Byrne Annette T. , Symons Marc H.
Published: August 2014

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. It is an incurable disease with an average life expectancy of just 14 months following diagnosis. GBM morbidity is primarily due to its rapid growth, neovascularization and invasion throughout the brain. The angiogenesis inhibitor, bevacizumab, now approved for GBM treatment, has been shown to cause enhanced tumor cell invasion in part via c-MET (HGF-receptor) signaling. We have previously shown that several members of the Rho family of small GTPases, including Rac1, Rac3, RhoG and Cdc42, play central roles in GBM tumor cell invasion. Rho GTPases are activated by guanine nucleotide exchange factors (Rho GEFs). Herein we focus on identifying GEFs that act on Rac subfamily members and which are of functional importance in GBM.

We found that Dock7 and Ect2, two GEFs that can activate Rac proteins, show elevated mRNA and protein expression in human GBM tissue in comparison with non-neoplastic brain. RNAi-mediated depletion of these GEFs in GBM cell lines resulted in decreased HGF- and serum-induced tumor cell invasion in 3-dimensional extracellular matrix in vitro and organotypic ex vivo brain slice invasion assays. GEF activity of Dock7 and Ect2 were shown to be increased by HGF using a pull-down with Rac1G15A, a nucleotide binding-deficient mutant of Rac1. Furthermore, Dock7 and Ect2 were shown to be required for HGF-induced Rac1 activation, lamellipodia formation and cell spreading. Dock7, but not Ect2, was shown to co-immunoprecipitate with c-MET and this interaction was HGF-dependent and required the c-MET adaptor protein GAB1. Dock7 and GAB1 were furthermore shown to co-immunoprecipitate in an HGF-dependent manner. GAB1 was shown to be required for HGF-induced Dock7 and Ect2 Rac1 GEF activities, Rac1 activation and HGF-induced GBM cell invasion.

In summary, we have identified two GEFs that are of functional and clinical relevance in the development of the GBM pathologic phenotype. Dock7 and Ect 2 represent potential new drug targets for the management of GBM, especially in combination with an anti-angiogenic approach.

Citation Format: David W. Murray, Sebastien Didier, Vincent M. Paulino, Amanda Chan, Nhan L. Tran, Annette T. Byrne, Marc H. Symons. Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion. [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 LB-40. doi:10.1158/1538-7445.AM2013-LB-40
Abstract LB-43: MiR-147 is a novel tumor suppressor that reverses EMT and native resistance to EGFR inhibitors by inactivating Akt and activating Stat3.
Authors: Lee Chang Gong , Gruidl Michael , Timmee Cindy R. , McCarthy Susan , Yeatman Timothy J.
Published: August 2014

Background: The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis, and resistance to chemotherapy. The EGFRi class of reagents are thought to target tumors with the epithelial phenotype.

Hypothesis: We hypothesized that reversing this biological process through a mesenchymal to epithelial transition (MET), using a recently identified miR-147, might also lead to a reversal of resistance to EGFR inhibitors (EGFRi).

Methods/Results: Following a screen of > 400 miRs, we recently identified miR-147 as highly correlated with an “intrinsic” and prognostic EMT gene expression signature derived from an unsupervised analysis of >2000 colorectal cancers. This suggested miR-147 might regulate the EMT program. We have shown that miR-147 is capable of reversing the EMT phenotype in vitro and converting EGFRi-resistant cells to sensitive cells. To further understand the mechanism underpinning this observation, we analyzed the genes affected by miR-147 over-expression in colon cancer cells using a global gene expression survey (Affymetrix). Among significantly altered genes, Akt expression was down-regulated (>2 fold) and Stat3 expression was up-regulated (>2 fold) by miR-147.

Subsequent knock down of Akt with siRNA did induce MET and significantly increased EGFRi sensitivity. Western analysis showed increased Stat3 protein levels and total Stat3 phosphorylation with miR-147 transfection. Interestingly, knockdown of Stat3 inhibited the capacity of miR147 to reverse native EGFRi resistance.

Conclusion: miR-147 induced MET and reversed resistance to EGFRi. This observation appears to be related to both the inhibition of Akt and to the induction of Stat3 activity. Understanding the precise mechanisms by which miRs regulate chemosensitivity and resistance will be important in designing more effective EGFRi therapies.

Citation Format: Chang Gong Lee, Michael Gruidl, Cindy R. Timmee, Susan McCarthy, Timothy J. Yeatman. MiR-147 is a novel tumor suppressor that reverses EMT and native resistance to EGFR inhibitors by inactivating Akt and activating Stat3. [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 LB-43. doi:10.1158/1538-7445.AM2013-LB-43
Abstract LB-50: Identification and functional analysis of p53MRA as a novel p53 target gene.
Authors: Funauchi Yuki , Tanikawa Chizu , Lo Paulisally Hau Yi , Nakamura Yusuke , Matsuda Koichi
Published: August 2014

Identification and characterization of p53-target genes are critical step for the understanding of tumor suppression mechanisms. So far, we have isolated a number of p53 target genes such as p53AIP1, p53R2, p53RDL1, XEDAR and PADI4, and revealed molecular mechanisms by which p53 regulate human carcinogenesis. Here we identified p53MRA (p53-inducible mitochondrial regulator of apoptosis) as a novel p53 target gene that play an important role in an apoptotic pathway through a cDNA microarray analysis using mRNAs isolated from U373MG cells ectopically expressed p53.

p53MRA mRNA and protein expression was remarkably induced by ectopic expression of p53 but not by that of LacZ. We also found that adriamycin treatment remarkably induced p53MRA expression in HCT116 colorectal cancer cells in a p53 dependent manner. Subsequently, we surveyed the genomic sequence of the p53MRA gene and found putative p53-binding region within the first intron. Luciferase assay using reporter vector containing this putative p53-binding region revealed that co-transfection of wild-type p53 expressing plasmid enhanced the luciferase activity more than 10-fold. On the other hand, substitution of nucleotides within putative p53-binding region completely diminished the luciferase activity, indicating that p53 regulates p53MRA expression through this p53-binding region.

To further investigate the role of p53MRA on tumor cell growth, we treated HCT116 cells with adriamycin 36h after transfection with sip53MRA or siEGFP. As a result, treatment with sip53MRA remarkably increased cell viability to the extent similar to that with sip53. Taken together, p53MRA is novel p53 target gene which plays an important role in the p53-mediated apoptotic pathway.

Citation Format: Yuki Funauchi, Chizu Tanikawa, Paulisally Hau Yi Lo, Yusuke Nakamura, Koichi Matsuda. Identification and functional analysis of p53MRA as a novel p53 target gene. [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 LB-50. doi:10.1158/1538-7445.AM2013-LB-50
Abstract LB-51: p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress.
Authors: Farkas Carlos , Martins Carla , Escobar David , Castro Ariel , Donner David , Pincheira Roxana
Published: August 2014

SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggest it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin receptors and showed that it plays a role in neuronal function, which does not necessarily explain why or how SALL2 is deregulated in cancer. Previous evidences indicate that SALL2 gene is regulated by the WT1 and AP4 transcription factors. Here, we identified SALL2 as a novel downstream target of the P53 tumor suppressor protein. Bioinformatic analysis of the SALL2 gene revealed several putative P53 half sites along the promoter region. Either overexpression of wild-type P53 or induction of the endogenous P53 by the genotoxic agent doxorubicin repressed SALL2 promoter activity in various cell lines. However, Q143A, R175H and R248W P53 mutants, which are frequently found in the tumors of cancer patients, were unable to repress SALL2 promoter activity. Chromatin immunoprecipitation analysis using a specific P53 antibody further supported the binding to, and regulation of the SALL2 promoter by P53. Importantly, by using a p53ERTAM knockin model expressing a variant of P53 that is completely dependent on 4-hydroxy-tamoxifen for its activity, we show that P53 activation diminished SALL2 RNA and protein levels during genotoxic cellular stress in primary mouse embryo fibroblasts (MEFs) and radiosentive tissues in vivo. Thus, our finding indicates that P53 represses SALL2 expression in a context-specific manner, adding knowledge to understanding SALL2 gene regulation, and a potential mechanism for its deregulation in cancer

Citation Format: Carlos Farkas, Carla Martins, David Escobar, Ariel Castro, David Donner, Roxana Pincheira. p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress. [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 LB-51. doi:10.1158/1538-7445.AM2013-LB-51
Abstract LB-52: Increased incidence of cancer in the transgenic mice with 8-Lipoxygenase knocked out.
Authors: Al-Fardan Rana , Karki Rabindra , Guo Changxiong , Nie Daotai
Published: August 2014

15-Lipoxygenase-2 (ALOX15B), an oxidoreductase in the metabolism of arachidonic acid, is a functional tumor suppressor whose expression is reduced in a variety of human cancers. To determine the roles of ALOX15B in carcinogenesis, we generated transgenic mice with ALOX8, the murine homolog of ALOX15B, knocked out. ALOX8 expression at mRNA level was abolished in homozygous knockout mice and reduced to half of wild type in heterozygous knockout mice. It's observed that homozygous female ALOX8 knockout mice are infertile but male ALOX8 knockout mice are fertile. Increased incidence of tumor as uni or multimass was found in the lung, prostate and in the mesentery region of homozygous and heterozygous ALOX8 knockout mice, when compared with little mate wild type mice. Histological evaluations showed an increase in secondary tumors and inflammation in different tissues like lung and large intestine in mice with ALOX8 knocked out. The transgenic mice could be a good model to study the role of 8-lipoxygenase in carcinogenesis and may provide insights into the tumor suppressing activities of human ALOX15B.

Citation Format: Rana Al-Fardan, Rabindra Karki, Changxiong Guo, Daotai Nie. Increased incidence of cancer in the transgenic mice with 8-Lipoxygenase knocked out. [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 LB-52. doi:10.1158/1538-7445.AM2013-LB-52
Abstract LB-59: ERG induces taxane resistance in castration-resistant prostate cancer.
Authors: Galletti Giuseppe , Beltran Himisha , Matov Alexandre , Fontugne Jacqueline , Mosquera Juan Miguel , Fazli Ladan , Tagawa Scott , Nanus David , Gleave Martin , Rubin Mark , Giannakakou Paraskevi , Rickman David S.
Published: August 2014

Taxanes are the only chemotherapies to prolong survival for patients with metastatic castration-resistant prostate cancer (CRPC), and both docetaxel (doc) and cabazitaxel (cab) are FDA approved for this indication. Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study we show that ERG over-expression, which occurs in roughly 50% of prostate cancers, is associated with both doc and cab resistance using in vitro and in vivo models of CRPC. Using either tetracycline-inducible or constitutively active expression systems in multiple prostate cancer cell lines, we found that ERG over-expression leads to at least a 10 fold increase in IC50 doses of doc or cab based on cell viability assays and inhibition of taxane-induced apoptosis. Similarly, ERG over-expressing prostate cancer xenografts were reproducibly more resistant to cab compared to the cab-sensitive control xenografts. Based on a biochemical and cell biological characterization, we observed that ERG binds directly to tubulin in the cytoplasm of prostate cancer cells, as well as in circulating tumor cells from patients treated with doc, and prevents taxane-induced microtubule stabilization. ERG over-expression affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of doc or cab with tubulin, either directly via its tubulin interaction or indirectly by affecting transcription of genes that modulate microtubule homeostasis. In addition, ERG induces and binds to clusterin, a molecular chaperone also associated with taxane resistance. Clusterin knock-down with siRNA abrogates ERG-induced taxane resistance. Altogether, this data suggests that ERG, an oncogenic transcription factor, plays a novel role beyond regulating gene expression and functions outside the nucleus to cooperate with clusterin and tubulin in promoting taxane resistance. Determining ERG status in patient tumors may aid in patient selection for doc or cab therapy and/or influence co-targeting approaches using the clusterin antisense inhibitor OGX-011.

Citation Format: Giuseppe Galletti, Himisha Beltran, Alexandre Matov, Jacqueline Fontugne, Juan Miguel Mosquera, Ladan Fazli, Scott Tagawa, David Nanus, Martin Gleave, Mark Rubin, Paraskevi Giannakakou, David S. Rickman. ERG induces taxane resistance in castration-resistant 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 LB-59. doi:10.1158/1538-7445.AM2013-LB-59
Abstract LB-75: Blood-based molecular detection of acquired resistance to anti-EGFR therapies in colorectal cancer patients.
Authors: Siravegna Giulia , Bianchi Andrea Sartore , Cassingena Andrea , Bencardino Katia , Diaz Luis A. , Sausen Mark , Velculescu Victor E. , Nicolantonio Federica Di , Siena Salvatore , Bardelli Alberto
Published: August 2014

The anti EGFR monoclonal antibodies cetuximab and panitumumab are used to treat metastatic colorectal cancer patients. The clinical efficacy of EGFR inhibitors is limited by the development of acquired resistance, which typically occurs within 3-12 months since starting of therapy. We recently reported that secondary KRAS mutations are responsible for acquired resistance in approximately 50% of patients (Misale et al.; Diaz et al., Nature 2012). We reasoned that the detection of KRAS mutant alleles in the plasma of cetuximab treated patients may enable the early identification of secondary resistance to anti EGFR blockade before radiographic documentation of relapse. Using the highly sensitive BEAMing approach we have examined cell-free circulating tumor DNA (ctDNA), a form of "liquid biopsy". KRAS mutant alleles emerged in the circulation of approximately 50% of patients treated with anti-EGFR antibodies months before radiographic documentation of disease progression. The liquid biopsy approach was then used to select patients in which KRAS mutations did not emerge during anti-EGFR therapy. Exome sequencing identified amplification of the MET proto-oncogene in biopsies from patients who did not develop KRAS mutations. Using an innovative approach, we find that MET amplification can be identified in a non-invasive manner through the examination of ctDNA months before relapse becomes clinically manifest. As multiple anti-MET therapeutic strategies are available, these findings offer immediate novel opportunities to design clinical studies.

Citation Format: Giulia Siravegna, Andrea Sartore Bianchi, Andrea Cassingena, Katia Bencardino, Luis A. Diaz, Mark Sausen, Victor E. Velculescu, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Blood-based molecular detection of acquired resistance to anti-EGFR therapies in colorectal 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 LB-75. doi:10.1158/1538-7445.AM2013-LB-75
Abstract LB-80: ATRX loss refines the classification of anaplastic glioma and is a favorable prognostic marker.
Authors: Wiestler Benedikt , Capper David , Holland-Letz Tim , von Deimling Andreas , Pfister Stefan M. , Platten Michael , Weller Michael , Wick Wolfgang
Published: August 2014

Mutation / loss of ATRX expression has been described in anaplastic gliomas. The present study explored the role for ATRX status in the molecular classification of anaplastic gliomas and the impact of ATRX status on survival in the biomarker cohort of the NOA-04 trial.

Patients (n=123) of the NOA-04 trial were analyzed for ATRX expression using immunohistochemistry. ATRX status was correlated with age, reference histology, IDH, 1p/19q and MGMT status, and the efficacy endpoints of the NOA-04 trial.

Loss of ATRX expression was detected in 42% of anaplastic astrocytomas, 24% of anaplastic oligoastrocytomas and 10% of anaplastic oligodendrogliomas. It was restricted to IDH mutated tumors and almost mutually exclusive with 1p/19q co-deletion. Anaplastic oligoastrocytoma harboring ATRX loss shared a similar clinical course with anaplastic astrocytoma. Survival analysis showed a separation of IDH mutant tumors into two groups based on ATRX status: Tumors with ATRX loss have a significantly better prognosis (median time to treatment failure 1758 days [95% CI 1316 days - to not reached] vs. 968 days [95% CI 496 days - to not reached], log rank test, p = 0.0044). Independent of histological subtype, patients whose tumors harbored ATRX loss had a longer overall survival when initially treated with radiotherapy rather than with chemotherapy (p = 0.03).

ATRX status helps to re-classify oligoastrocytomas, since ATRX loss is a hallmark of astrocytic tumors. Furthermore, ATRX loss defines a subgroup of tumors with a favorable prognosis and predicts benefit from radiotherapy.

Citation Format: Benedikt Wiestler, David Capper, Tim Holland-Letz, Andreas von Deimling, Stefan M. Pfister, Michael Platten, Michael Weller, Wolfgang Wick. ATRX loss refines the classification of anaplastic glioma and is a favorable prognostic marker. [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 LB-80. doi:10.1158/1538-7445.AM2013-LB-80
Abstract SY05-03: Mechanisms regulating stemness in skin cancers.
Authors: Blanpain Cedric
Published: August 2014

Cancer stem cells (CSCs) have been described in various cancers by their ability to reform secondary tumours upon transplantation into immunodeficient mice.

Using different approaches in mice, we have recently shown that squamous skin tumors contain stem cells that present the ability to reform the parental tumor upon transplantation (Lapouge et al. EMBO J 2012) or by their ability to fuel tumor growth using lineage tracing experiments (Driessens et al Nature 2012).

To define the mechanisms that regulate skin cancer stem cells, we transcriptionally profiled cutaneous CSCs at different stages of tumor progression. We uncovered a set of genes, which are enriched in CSCs irrespective of the stages of tumor progression and another set of genes, which are upregulated in CSCs only during malignant progression. We used genetic gain and loss of function in mice to unravel how some of these genes regulate tumor stemness and stimulate malignant transition in vivo within their natural environment. I will discuss how the combination of extrinsic factors such as the vascular niche (Beck et al. Nature 2011) and intrinsic factors regulate the renewal and invasive properties of CSCs during skin cancer progression.

This work is supported by WELBIO, the FNRS, the European Research Council (ERC) and the EMBO Young Investigator Program.

Citation Format: Cedric Blanpain. Mechanisms regulating stemness in skin 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 SY05-03. doi:10.1158/1538-7445.AM2013-SY05-03