bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1013: Angiopoietin 1/2 inhibition impairs tumor growth in an orthotopic model of renal cell carcinoma
Authors: Orillion Ashley , Miles Kiersten Marie , Shen Li , Adelaiye Remi , Ciamporcero Eric , Ramakrishnan Swathi , Ku Sheng Yu , Elbanna May , Chintala Sreenivasulu , Pili Roberto
Published: May 2015

Background: Clear cell renal cell carcinoma (ccRCC), the most common form of renal cell carcinoma, is characterized VEGF driven tumor vascularization. However, in patients treated with anti-VEGF therapies inevitably tumor progression occurs, likely driven by up-regulation of alternative proangiogenic pathways including angiopoietins and c-MET. We hypothesized that angiopoietin inhibition has anti-tumor effect on RCC and may affect the development of resistance to anti-VEGF therapies. In the current study we utilized the dual angiopoietin inhibitor AMG 386 in murine models of RCC, and assessed the preclinical impact of this strategy. Methods: Seven week old female Balb/c mice were injected with 250,000 RENCA cells (luciferase expressing), one week later these mice were randomized, using bioluminescence imaging into two treatment groups: vehicle or Amgen 386 (5.6mg/kg/day, twice a week, subQ injection). Body weight measurements, tumor growth rate, and end point tumor weight measurements were used to evaluate possible side effects and the progression of RENCA tumors. Four weeks of treatment culminated in collection of tumor tissues for immunohistochemistry analysis of Ki67 and CD31. In addition we have ongoing studies utilizing patient derived xenografts (PDX) of ccRCC developed in our lab. Results: In the RENCA model AMG 386 induced a significant decrease of tumor weight by 44.0 percent (p = 0.0123). In addition, histological analysis of tumor sections revealed a high proportion of necrosis and decreased blood vessel density. Studies are ongoing with sunitinib sensitive PDXs and matched sunitinib resistant PDXs treated with AMG 386 in combination with a selective c-MET inhibitor. Results from these models will be available at the time of the meeting. Conclusions: Our preliminary results suggest that AMG 386 as a single agent is inhibiting both vascularization and tumor growth in an orthotopic renal cell carcinoma model. Ongoing studies with AMG 386 in combination with a selective c-MET inhibitor in sunitinib sensitive and resistant PDX models will provide guidance for the optimal clinical development of angiopoietin inhibitors in patients with ccRCC.

Citation Format: Ashley Orillion, Kiersten Marie Miles, Li Shen, Remi Adelaiye, Eric Ciamporcero, Swathi Ramakrishnan, Sheng Yu Ku, May Elbanna, Sreenivasulu Chintala, Roberto Pili. Angiopoietin 1/2 inhibition impairs tumor growth in an orthotopic model of renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1013. doi:10.1158/1538-7445.AM2014-1013
Abstract 1019: Salmonella break tumor immune tolerance by downregulation tumor indoleamine 2, 3-dioxygenase 1 expression
Authors: KUAN YU-DIAO , Lee Che-Hsin
Published: May 2015

Over past decades, bacteria have been proven the capability of inhibiting tumor growth. It can specifically target tumor and due to its facultative anaerobe property, can be more penetrative than other drug therapies. However, the molecular mechanism that bacteria inhibit tumor growth is still incompletely known. Here we used Salmonella choleraesuis to investigate the mechanism. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at tumor site and T helper 1-type immune response. Many tumors have been proven high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine thus causing immunotolerance within tumor microenvironment. Our previous studies elucidated that Salmonella can also upregulate connexin 43 (CX43) to inhibit tumor progression and even enhance tumor chemosensitivity. Herein, we demonstrated that Salmonella can inhibit IDO expression through upregulation of CX43 in melanoma cells. Furthermore, we can also find the same phenomena in CX43 knock-down 4T1 and B16F10 cells. In brief, we point out that Salmonella-induced CX43 expression is a modulator of IDO expression and may play a crucial role in anti-tumor therapy, which combined with immunotherapy and Salmonella may be a prospective anti-tumor strategy.

Citation Format: YU-DIAO KUAN, Che-Hsin Lee. Salmonella break tumor immune tolerance by downregulation tumor indoleamine 2, 3-dioxygenase 1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1019. doi:10.1158/1538-7445.AM2014-1019
Abstract 1020: Trefoil factor 3 promotes angiogenesis in mammary carcinoma
Authors: Lau Wai-Hoe , Pandey Vijay , Kong Xiangjun , Banerjee Arindam , Zhu Tao , Lobie Peter E.
Published: May 2015

Mammary carcinoma cells produce various growth factors that stimulate endothelial cells to promote angiogenesis in mammary carcinoma. Trefoil Factor 3 (TFF3) is a small secreted protein which is involved in protection of the gastrointestinal tract against mucosal damage. Our laboratory has also previously identified TFF3 as an orthotopically expressed oncogene in mammary carcinoma and observed that TFF3 stimulated oncogenicity and invasiveness of mammary carcinoma cells. Several studies have reported a significant positive association between TFF3 protein expression and microvessel density. However, the potential functional role of TFF3 in mammary carcinoma angiogenesis has not been determined. Herein, we defined the functional effect of TFF3 secreted by mammary carcinoma cells on the behavior of endothelial cells in de novo tumor angiogenesis. We utilized MCF-7 and T47D cells as our in vitro models by stable forced expression of TFF3 or siRNA mediated depletion of TFF3 in these cell lines. The effect of TFF3 secreted from mammary carcinoma cells on human umbilical vein endothelial cells (HUVEC) was determined by using a co-culture transwell system, whereby HUVEC cells were co-cultured with mammary carcinoma cells. We demonstrated that TFF3 secreted from mammary carcinoma cells promoted HUVEC cells monolayer cell cycle progression and proliferation, survival, migration, invasion, and in vitro tubule formation. In a xenograft model, mammary carcinoma cells with forced expression of TFF3 produced tumors with increased microvessel density (CD31 and CD34) compared to tumors formed by control cells. Depletion of TFF3 in mammary carcinoma cells by siRNA or inhibition with anti-TFF3 polyclonal antibody significantly decreased HUVEC cells monolayer cell cycle progression and proliferation, migration, invasion, and in vitro tubule formation. Mechanistically, we observed that TFF3 increased IL-8 expression in mammary carcinoma cells with forced expression of TFF3. Depletion of IL-8 in mammary carcinoma cells by siRNA or inhibition with anti-IL8 monoclonal antibody significantly decreased migration, invasion, and in vitro tubule formation of HUVEC cells promoted by TFF3. Hence, TFF3 is a promoter of tumor angiogenesis, which may co-coordinate with the growth promoting and metastatic actions of TFF3 in mammary carcinoma to enhance tumor progression.

Citation Format: Wai-Hoe Lau, Vijay Pandey, Xiangjun Kong, Arindam Banerjee, Tao Zhu, Peter E. Lobie. Trefoil factor 3 promotes angiogenesis in mammary carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1020. doi:10.1158/1538-7445.AM2014-1020
Abstract 1023: NOV C-ter: A novel preclinical anti-angiogenic agent
Authors: Luo Junfeng , Teng Yong , Li Minghui , Johnson Theodore S. , Cuttitta Franck , Chu Zhengtao , Qi Xiaoyang , Cowell John K. , Rixe Olivier
Published: May 2015

NOV (Nephroblastoma OVerexpressed) C-ter (NOV C-ter) is the carboxy-terminal sequence (170aa) of NOV/CCN3 protein. NOV/CCN3 (357aa), a member of CCN family, is secreted by quiescent endothelial cells and is involved in the regulation of various cellular functions including angiogenesis, proliferation, differentiation, survival, adhesion and migration. Although NOV C-ter does not have a direct cytotoxic effect when tested on a large panel of human cell lines including the NCI 60 cell lines (NCI Drug Screening Program), our data demonstrates significant anti-angiogenic and anti-tumor effects in a variety of experimental models. NOV C-ter inhibits in vitro endothelial cell tube formation in a dose-dependent fashion, using endothelial cells derived from porcine aorta, human microvasculature, or human umbilical vein (HUVECs). In addition, NOV C-ter impairs HUVEC proliferation, migration, invasion and adhesion. Interestingly, inhibition of HUVEC proliferation occurs via interference with growth-promoting signals involving Apelin-13 and adenomedullin. Furthermore, NOV C-ter specifically reduced phosphorylation of PI3K/Akt in endothelial cells, with no significant effect on the p44/42 (ERK1/2) pathway. In vivo, NOV C-ter had significant anti-tumor effect as a single agent in a murine glioblastoma xenograft model using human X-12 culture-adapted cells and in a nude mouse xenograft model using human non-small-cell lung cancer cells (A549). In these two systems, NOV C-ter provided superior survival benefit compared to treatment with bevacizumab or vehicle (PBS). Similarly, NOV C-ter significantly increased overall survival versus vehicle (PBS), an effect which was comparable to temozolomide treatment, in a murine syngeneic orthotopic glioblastoma model using the GL-261 cell line. To directly study the effect of NOV C-ter on angiogenesis, we treated zebrafish embryos prior to the onset of angiogenesis, and found a considerable reduction in vessel formation compared to bevacizumab-treated or untreated zebrafish. NOV C-ter also reduced tumor neovascularization in human breast cancer (T47D cell line) implanted in zebrafish. In summary, NOV C-ter demonstrates anti-angiogenic and tumoricidal effects via a novel mechanism of action. Mechanistic studies of NOV C-ter are underway to evaluate its precise effects on key signaling pathways in endothelial cells. Preclinical pharmacology and toxicology studies are also underway with a plan to advance to early-phase clinical trials.

Citation Format: Junfeng Luo, Yong Teng, Minghui Li, Theodore S. Johnson, Franck Cuttitta, Zhengtao Chu, Xiaoyang Qi, John K. Cowell, Olivier Rixe. NOV C-ter: A novel preclinical anti-angiogenic agent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2014-1023
Abstract 1031: Blockade of radiation-induced neuropilin-1 in glioblastoma cells impairs migration of endothelial cells
Authors: Maddirela Dilip Rajasekhar , Kesanakurti Divya , Gogineni Venkateswara R. , Chetty Chandramu
Published: May 2015

Neuropilin-1 (NRP-1) is an isoform specific receptor for vascular endothelial growth factor (VEGF), that regulates the development of the nervous system and also promotes angiogenesis in endothelial cells. Glioblastoma multiforme (GBM) is a malignant tumor characterized by robust neoangiogenesis with an aberrant elevation of VEGF. Recent evidence suggests that the majority of GBMs after radiation therapy (IR) exhibit an angiogenic regeneration process, which contributes phenotypic plasticity and therapeutic resistance. A high expression of NRP-1 in GBM correlates with disease progression. In this study, we show that the IR-induced NRP-1 role in the VEGFR-2 mediated signaling cascade promotes migration of endothelial cells. We observed that IR (8Gy) significantly elevated levels of VEGF and NRP-1 expression in 4910 and 5310 human GBM xenograft cells. Endothelial cells cultured on tumor- conditioned media from IR induced xenograft cells showed a significant increase in migration of endothelial cells; whereas, conditioned medium (CM) from NRP-1 knockdown xenograft cells inhibited IR-induced migration effects in endothelial cells. Further, CM from NRP-1 inhibited cells downregulated IR-induced expression of VEGFR-2 and its downstream molecules Sema3A, Plexin-A1 and E-cadherin in endothelial cells. Immunoflourescence co-localization studies revealed that the interaction of NRP-1 and VEGFR-2 were significantly decreased in endothelial cells cultured on CM from NRP-1-siRNA-transfected xenograft cells compared to mock- and SV-transfected cells. Collectively, our data indicate that NRP-1 plays a significant role in the regulation of VEGF/VEGFR-2 signaling and migration in endothelial cells, and thereby suggests NRP-1 as an important therapeutic target for glioma patients receiving radiation therapy.

Citation Format: Dilip Rajasekhar Maddirela, Divya Kesanakurti, Venkateswara R. Gogineni, Chandramu Chetty. Blockade of radiation-induced neuropilin-1 in glioblastoma cells impairs migration of endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2014-1031
Abstract 1033: Inhibiting bladder cancer metastasis with a dietary supplement
Authors: Hoye Kelly T. , Yates Travis , Alonzo David , Lokeshwar Vinata B.
Published: May 2015

INTRODUCTION AND OBJECTIVE: Hyaluronic acid (HA) and HYAL-1 hyaluronidase (HAase) family molecules are potential markers for BCa diagnosis and prognosis and promote tumor growth and metastasis. 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. Epithelial-to-mesenchymal transition (EMT) is the hallmark of invasion and metastasis. Since HA promotes tumor metastasis and EMT, we evaluated the expression of EMT markers in bladder tissues and antitumor effects of 4-MU in BCa models.

METHODS: Quantitative PCR was used to measure mRNA expression of EMT genes (β-catenin, Twist, and Snail) in 66 bladder tissue specimens (27 normal; 39 tumor); follow-up: 26±4.3 months; median 20 months. The effect of 4-MU (40 - 120 µg/ml) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors and EMT genes were examined in BCa cell lines, 253J-Lung and HT1376. Effect of 4-MU on tumor growth was analyzed in subcutaneous xenografts.

RESULTS: Among the EMT genes, Snail and Twist were differentially expressed in BCa tissues when compared to normal bladder (P<0.001). Furthermore, elevated Twist expression significantly correlated with metastasis (2-fold increase; P=0.028). β-catenin expression negatively correlated with survival (chi square=4.3, p=0.038). At IC50 for HA synthesis, 4-MU inhibited proliferation (∼ 2.5-fold) and induced apoptosis (3-fold) in BCa cells. 4-MU induced caspase-8, -9 and -3 activation and up-regulation of Fas and FADD. 4-MU caused 4-10-fold downregulation of HA-receptors (CD44, RHAMM), and EMT promoters - β-catenin, Snail and Twist, but increased E-cadherin expression by 2-fold. In xenograft studies, 4-MU significantly decreased tumor growth (> 3-fold) when treatment was started either on the day of tumor cell injection or after tumors became palpable. No weight loss or serum or organ toxicity was observed in treated mice. Tumors showed reduced microvessel density (∼3-fold) and HA expression but increased TURNEL positive cells.

CONCLUSION: This study shows that expression of EMT determinants correlates with clinical outcome. Furthermore, the non-toxic dietary supplement 4-MU has potent antitumor activity and reverses EMT.

Support: R01 CA 123063-04 (VBL); R01 CA 72821-11 (VBL).

Citation Format: Kelly T. Hoye, Travis Yates, David Alonzo, Vinata B. Lokeshwar. Inhibiting bladder cancer metastasis with a dietary supplement. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2014-1033
Abstract 1037: WISP3/CCN6 regulates epithelial-mesenchymal transition and tumor initiating cell properties in triple negative breast cancer cells through Slug
Authors: Huang Wei , Toy Kathy A. , Kleer Celina G.
Published: May 2015

Background: Although triple-negative breast cancers (TNBCs) constitute 10%-20% of all breast cancers, they are aggressive tumors with higher recurrence and poorer prognosis than other breast cancer subtypes. TNBCs frequently exhibit features of epithelial to mesenchymal transition (EMT) and are enriched in breast tumor initiating cells (BTICs). Recent studies have demonstrated that aberrant activation of the EMT program induces stem cell features in breast cells, suggesting that blocking the cross-talk between EMT and stemness may be an effective treatment for TNBCs. WISP3/CCN6, a secreted matrix associated protein, is expressed in normal breast tissues and is decreased in aggressive breast cancer samples. We have shown that CCN6 knockdown in nontumorigenic breast cells triggers EMT and invasion. We hypothesize that CCN6 regulates the cross-talk between EMT and BTICs pathways in TNBCs.

Methods: We overexpressed CCN6 in the TNBC cell lines, MDA-MB-231, SUM-159 and MDA-MB-436, which are aggressive and exhibit a mesenchymal-like phenotype and low endogenous CCN6 expression. We tested the effect of CCN6 overexpression in these cell lines using motility and invasion assays in vitro. Stem cell features were investigated by mammosphere assays, and the ALDEFLUOR assay by flow cytometry. We developed mammary xenografts in nude mice using serial dilutions to investigate the role of CCN6 in tumor initiation and metastasis.

Results: Overexpression of CCN6 inhibits cell motility and invasion, and leads to phenotypic changes of mesenchymal-epithelial transition (MET), with downregulation of the mesenchymal cell marker vimentin, increased epithelial marker cytokeratin, and decreased protein levels of the EMT transcription factor Slug. CCN6 overexpression significantly decreased the ALDEFLUOR-positive population and mammosphere formation of TNBC cell lines. In vivo, CCN6 overexpression delayed tumor initiation and reduced metastasis in human breast cancer xenografts. Slug reconstitution was sufficient to rescue the effects of CCN6 on MET and stem cell properties of TNBCs.

Conclusions: These data demonstrate that CCN6 regulates EMT and tumor initiating cell features in TNBC at least in part through Slug. Further, our study suggests that CCN6 may be a novel regulator of the EMT-stem cell cross talk in TNBC, with possible clinical application.

Citation Format: Wei Huang, Kathy A. Toy, Celina G. Kleer. WISP3/CCN6 regulates epithelial-mesenchymal transition and tumor initiating cell properties in triple negative breast cancer cells through Slug. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2014-1037
Abstract 1038: ZEB1 plays a pivotal role in hypoxia-mediated increase of in vitro invasion of glioblastoma-derived cell cultures and represents a novel neural stem cell marker in early development
Authors: Kahlert Ulf D. , Raabe Eric E. , Siebzehnrubl Florian A. , Bar Eli E. , Maciaczyk Jaroslaw , Eberhart Charles G.
Published: May 2015

Objective

Hypoxia is thought to induce migration in many neoplasms, often by promoting epithelial-to-mesenchymal transition (EMT), but the mechanisms by which it affects brain tumor invasion is less well understood. In particular, little is known about the possible role of EMT-associated factors in hypoxia-mediated induction of migratory phenotype in gliomas.

Methods

Migration and invasion of GBM-derived cell lines (n=3) cultivated as spheres in serum-free media under atmospheric oxygen (21% O2) and hypoxia (1% O2) were assessed in Boyden Chamber-based in vitro assays. Digoxin was used to inhibit the HIF-1-mediated hypoxic response. The importance of the EMT-factor ZEB1 in our model was investigated by inhibiting its expression using sh-RNA delivered by lentivirus.

Results

Hypoxia led to a significant increase of in vitro invasion in all three neurosphere lines tested (GBM1 up to 6fold, 622 up to 3fold and over 10fold for 821; p=0.001). This was accompanied by induction of EMT effectors including ZEB1, SNAI1 and TWIST1 as compared to normoxic cultures. Digoxin-mediated inhibition of HIF1a transcription resulted in abolition of this phenomenon. Additionally, histological analysis of patient-derived GBM samples revealed an abundance of ZEB1 positive cells in hypoxic, peri-necrotic tumor areas. Strikingly, ZEB1-knockdown significantly reduced the invasive behavior of the cells. Interestingly, we also identified numerous ZEB1-postive cells in fetal neurogenic regions and migrating neuroblasts in both human and rodent brains. Furthermore, analyses of human fetal neural progenitors showed that ZEB1 expression is correlated with stem cell phenotype and established neural stem- and progenitor markers including SOX2 and NESTIN. Finally, analysis of TCGA and Rembrandt datasets identified up-regulation of EMT factors such as TWIST1, SNAI1 and SNAI2 was found in the aggressive, mesenchymal subgroup of GBM.

Conclusion

While malignant gliomas are not epithelial tumors, hypoxia increases the invasion capacity of GBM cells at least in part by inducing expression of EMT markers such as ZEB1. ZEB1 is also most highly expressed in the developing fetal brain in migrating stem and progenitor cells, suggesting it may play a similar role in normal development.

Citation Format: Ulf D. Kahlert, Eric E. Raabe, Florian A. Siebzehnrubl, Eli E. Bar, Jaroslaw Maciaczyk, Charles G. Eberhart. ZEB1 plays a pivotal role in hypoxia-mediated increase of in vitro invasion of glioblastoma-derived cell cultures and represents a novel neural stem cell marker in early development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1038. doi:10.1158/1538-7445.AM2014-1038
Abstract 1044: Pyruvate kinase M2 stimulates the epithelial-mesenchymal transition by genetically controlling E-cadherin expression in colon cancer
Authors: Konno Masamitsu , Hamabe Atsushi , Doki Yuichiro , Mori Masaki , Ishii Hideshi
Published: May 2015

Pyruvate kinase M2 (PKM2) maintains aerobic glycolysis in cancer cells and stimulates cell proliferation by controlling cancer metabolism (Warburg effect)(Nature, 2008). In addition, PKM2 translocates to nucleus in response to EGFR signaling and up-regulates Cyclin D1 expression by controlling its transcription (Nature, 2011). PKM2 confers malignant potential by controlling both metabolism and nuclear function. In this study, we investigated the correlation between PKM2 and invasion and metastasis of colorectal cancer in terms of nuclear function, which has yet to be investigated. Tumor invasion is initiated with the change of epithelial-mesenchymal transition (EMT) in cancer cell, and we induced EMT by adding TGF-β1 and EGF to SW 480. After EGF induction, in parallel with E-cadherin down-regulation and Vimentin up-regulation, the expression of PKM2 was increased both in transcriptional and translational level and PKM2 translocated into nucleus. PKM2 knock-down by siRNA suppressed E-cadherin decrease and invasion capacity in invasion assay. We created sh pyruvate kinase SW 480 with mouse PKM1 or PKM2 was overexpressed both of which have DNA sequence resistant to sh pyruvate kinase. PKM2 overexpression stimulated EMT induction by enhancing E-cadherin decrease. The protein X was identified which interacted with PKM2 in nucleus in response to EMT induction, which was considered to have a key role in controlling EMT induction. We performed immunohistochemical staining with specific antibody to PKM2 using clinical samples of colorectal cancers. Clinicopathological analysis showed that PKM2 positivity significantly correlated with lymph node metastasis and distant organ metastasis. In conclusion, our data suggested that PKM2 nuclear function had a crucial role in controlling invasion and metastasis.

Citation Format: Masamitsu Konno, Atsushi Hamabe, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Pyruvate kinase M2 stimulates the epithelial-mesenchymal transition by genetically controlling E-cadherin expression in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1044. doi:10.1158/1538-7445.AM2014-1044
Abstract 1045: Differentiated tumor cells secrete stem cell factor (SCF) to promote maintenance of cancer stem cells and induce EMT in colorectal tumors
Authors: Fatrai Szabolcs , Schelven Susanne J. van , Rinkes Inne H.M. Borel , Kranenburg Onno
Published: May 2015

Colorectal tumors are hierarchically organized in which cancer stem cells (CSCs) are at the top of the hierarchy and give rise to non-tumorigenic differentiated tumor cells (DTC). The maintenance of normal intestinal stem cells requires ‘niche’ signals that are provided, at least in the small intestine, by neighboring differentiated Paneth cells. CSC maintenance may also depend on niche signals although little is known about the nature of such signals and their potential source.

For the present study we tested the hypothesis that DTCs could provide CSC-maintaining signals. We used CSC-enriched colonosphere cultures that were established from human colorectal tumors and CSC-derived stably differentiated offspring that was derived from them. We found that DTCs established from multiple colorectal tumors greatly increased the clone-forming capacity of CSCs. This effect was reproduced by DTC-conditioned medium, suggesting the involvement of a secreted factor. By using a cytokine array we identified stem cell factor (SCF) as a major DTC-secreted factor. While SCF was mainly produced by DTCs, its receptor, c-Kit, was mainly expressed by CSCs. Indeed, recombinant SCF enhanced clone-forming capacity to a similar extent as DTC-conditioned medium. Furthermore, basal and DTC-stimulated clone-forming capacity was strongly reduced in the presence of an SCF-neutralizing antibody or in the presence of the cKit inhibitors Imatinib or ISCK03. qPCR and western blot analysis showed that inhibition of cKit caused a strong reduction in the expression of stem cell genes, including LGR5, CD133, OLMF4 and SOX2. Furthermore, DTCs induce the E-to N-cadherin switch in CSCs and to a strong increase in the expression of EMT markers such as vimentin, ZEB1, ZEB2, SNAIL1 and fibronectin. cKIT inhibitors prevented EMT.

Our results identify a critical role for non-tumorigenic DTCs in the support of colorectal CSCs. DTCs induce EMT and EMT-associated stem cell characteristics at least in part via paracrine SCF-cKit signaling. We conclude that cKit inhibitors that are widely used in the clinic to treat gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML), may have therapeutic value in the treatment of (a subgroup of) colorectal tumors.

Citation Format: Szabolcs Fatrai, Susanne J. van Schelven, Inne H.M. Borel Rinkes, Onno Kranenburg. Differentiated tumor cells secrete stem cell factor (SCF) to promote maintenance of cancer stem cells and induce EMT in colorectal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2014-1045
Abstract 1046: Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer
Authors: Gupta Parul , Srivastava Sanjay K.
Published: May 2015

Breast cancer is a second most lethal form of cancer. Metastasis is responsible for about 90% of cancer- related deaths. Metastasis is a multi-step process and epithelial to mesenchymal transition (EMT) is the initiating step. HER2 is overexpressed in about 25-30% of breast cancer patients and responsible for poor prognosis. Our results show a novel role of HER2 in inducing EMT in breast cancer cells. To delineate the mechanism of EMT and role of HER2, we used MDA-MB-231, MCF-7 breast cancer cells with their syngeneic variants having stable HER2 overexpression (referred to as high HER2 or HH cells) and 4T-1 cells. The Boyden's chamber assay showed that HH cells were more invasive than the parent cells. The higher invasive potential of cells was accompanied with increased expression of mesenchymal markers like N-cadherin, along with suppression of epithelial markers like Cytokeratin-18. HER2 overexpression was also associated with enhanced expression of transcription factors like SNAIL and ZEB-1 that orchestrate the process of EMT. It is known that TGFβ is a master regulator of EMT process in cancer cells. Interestingly, HH cells exhibited higher levels of TGFβ protein and mRNA levels along with increased expression of SMADs relative to the parent cell lines. SMADs are the classical downstream effector molecules for TGFβ. Hence these results indicate that HER2 overexpression modulates TGFβ signaling to promote the process of EMT. In addition, induction of SNAIL, TGFβ and SMAD4 by heregulin treatment further established the role of HER2 in EMT process. To confirm the requirement of HER2 in the maintenance of mesenchymal traits, we silenced HER2 in MDA-MB-231 and MDA-MB-231 (HH) cells. HER2 silencing suppressed the invasion and mesenchymal markers indicating a direct connection of EMT with HER2 in breast cancer cells. Taken together our results for the first time provides a molecular evidence for the regulation of EMT by HER2 through TGFβ signaling. Further mechanistic and pre-clinical in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: Parul Gupta, Sanjay K. Srivastava. Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2014-1046
Abstract 1052: Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition
Authors: Hoang Van T. , Elliott Steven , Martin Elizabeth C. , Rhodes Lyndsay V. , Segar Henry C. , Burks Hope , Chakrabarty Suravi , Monlish Darlene , Phamduy Theresa B. , Chrisey Doug , Cavanaugh Jane E. , Flaherty Patrick , Collins-Burow Bridgette M. , Burow Matthew E.
Published: May 2015

The mitogen-activated protein kinase (MAPK) pathway has well-established roles in cellular processes including proliferation, differentiation, and regulation of cell fate, namely survival and apoptosis. In breast cancer, constitutive activation of the MAPK/extracellular signal-regulated kinases (ERK) pathways have been linked to chemoresistance and metastatic progression through distinct mechanisms including the activation of epithelial-to-mesenchymal transition (EMT). Our previous studies have shown that overexpression of MEK5 promotes EMT markers and induces the progression to a mesenchymal phenotype. Here, we tested the effects of a novel MEK1/2 and MEK5 inhibitor, SC-1-151, and other known MAPK signaling inhibitors (PD184,352 (MEK1/2), AZD6244 (MEK1/2), BIRB796 (p38)) on a panel of mesenchymal and highly metastatic breast cancer cell lines. While the MEK1/2 and p38 inhibitors decreased cell viability across cell lines, only the dual inhibition of MEK1/2 and MEK5 though the use of SC-1-151 demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET). Furthermore, the cells exhibited a significant decrease in migration potential following SC-1-151 treatment.

Further analysis of the effects of SC-1-151 in the triple-negative breast cancer cell lines revealed an alteration of the genes associated with EMT, notably a decrease in expression of Fra-1, a transcription factor downstream of MAPK. Immuno-compromised mice inoculated with the MDA-MD-231 cell line and treated with SC-1-151 demonstrated decreased tumor volumes compared to vehicle-treated animals at day 30 post cell injection, implicating the role of MEK inhibition on tumorigenesis. These data demonstrate the need for a better understanding of the dual role of MEK1/2 and MEK5 signaling in breast cancer, and suggest that inhibition of the MEK1/2 and MEK5 signaling pathways leads to a decrease in EMT and cell migration.

Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Henry C. Segar, Hope Burks, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Doug Chrisey, Jane E. Cavanaugh, Patrick Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2014-1052
Abstract 1057: LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes
Authors: Ha Ji Hee , Ward Jeremy , Dhanasekaran Danny N.
Published: May 2015

Ovarian cancer is currently the most fatal gynecological cancer with a 5-year survival rate of only 45%. Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the genesis and the progression of ovarian cancer. In order to identify transcription factors that are induced by LPA, a commercial pathway reporter array was used. Hypoxia induced factor 1 (HIF-1α) induction was the most prominent response showing more than 150 fold transcriptional activation compared with control. LPA treatment under normoxic and hypoxic (1% O2) conditions increased HIF-1α protein level, which leads to epithelial-to-mesenchymal transition (EMT) in the ovarian cancer cell line OVCA432. We also found that stimulation of the OVCA432 cells with LPA at normoxic and hypoxic conditions led to a significant increase of the mRNA and protein expression of the EMT transcription factor Twist. Immunofluorescent imaging confirmed that Twist levels were significantly up-regulated and that Twist was translocated to the nucleus as early as 4 hours after stimulation with LPA in both a normoxic and hypoxic environment. Additionally, immunofluorescent imaging showed that expression of E-cadherin on the periphery of the cell as well as in the whole cell was greatly reduced when cells were stimulated with LPA in normoxic and hypoxic conditions. Finally, our study demonstrated that LPA-mediated induction of EMT required both gip2 and gep oncogenes. Overall, our study indicated that the downstream components regulated by oncogenic G-proteins could potentially be targeted in the future to prevent EMT in ovarian cancer.

Citation Format: Ji Hee Ha, Jeremy Ward, Danny N. Dhanasekaran. LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2014-1057
Abstract 106: A requirement for FADD and its phosphorylation for KRAS-driven oncogenesis
Authors: Bowman Brittany M. , Galban Stefanie , Hoff Benjamin A. , Heist Kevin A. , Boes Jennifer L. , Galban Craig J. , Patel Rajiv M. , Zhang Jianke , Ross Brian D. , Rehemtulla Alnawaz
Published: May 2015

Lung cancer accounts for 27% of all cancer deaths worldwide. 80% of these deaths are due to a specific subtype Non-small cell lung cancer (NSCLC). Almost half of all NSCLC patients carry gatekeeper mutations in the EGFR/KRAS pathway. Although KRAS mutations are the most prevalent mutations in human cancers, therapies targeting KRAS have fallen short. Tyrosine kinase inhibitors have been successful in NSCLC patients with mutant or amplified EGFR, however resistance to these inhibitors is common. Thus therapies targeting mutant KRAS or its downstream effectors are desperately needed for the treatment of NSCLC.

FAS Associated Death Domain (FADD) is a well characterized adaptor protein involved in death receptor mediated apoptosis, but more recently has been implicated in the regulation of other cellular functions such as cell cycle and proliferation. Increasing evidence suggests that it is FADD's phosphorylated state which regulates these non-apoptotic signaling pathways. We and others have previously identified phosphorylated FADD as a predictor of aggressiveness and clinical stage in lung and head and neck cancer patients, respectively.

Using non-invasive bioluminescence and µCT imaging in a Kras driven mouse model of NSCLC, we demonstrate the requirement for FADD in tumor progression. Lung-specific activation of KrasG12D and simultaneous FADD deletion resulted in reduced lung tumor burden. Furthermore lung lesions of KrasG12D FADD null mice were significantly smaller with a decrease in proliferative index. Similar findings were obtained in MEFs, where siRNA mediated Kras knockdown or the use of MEK inhibitors resulted in a Kras dependent decrease in FADD phosphorylation, decrease in cell cycle progression and cell proliferation. These data propound the phosphorylation of FADD as a downstream event of Kras signaling and demonstrate its requirement for Kras dependent neoplasia in a mouse model for NSCLC.

In summary our findings demonstrate that elevated FADD and phosphorylated FADD levels are predictive of poor survival in an animal model of NSCLC agreeing with clinical findings. Furthermore, it implicates phosphorylation of FADD downstream of KRAS activation and thus suggests FADD phosphorylation as a valid target in the treatment of NSCLC patients.

Citation Format: Brittany M. Bowman, Stefanie Galban, Benjamin A. Hoff, Kevin A. Heist, Jennifer L. Boes, Craig J. Galban, Rajiv M. Patel, Jianke Zhang, Brian D. Ross, Alnawaz Rehemtulla. A requirement for FADD and its phosphorylation for KRAS-driven oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 106. doi:10.1158/1538-7445.AM2014-106
Abstract 1066: MicroRNA-125b mediated par2 activation-induced cell migration
Authors: YANG LAN
Published: May 2015

Backgrounds: Tumor microenvironment plays a promoting role in cancer metastasis. As important component of the microenvironment, some proteinases regulate tumorigenesis through selective activation of proteinase activated receptor 2 (PAR2). Our previous study demonstrated that overexpression of PAR2 was significantly correlated with lymphatic metastasis in colorectal cancers.

Aims: To identify whether and by what mechanisms PAR2 promotes cell migration.

Methods and Results: Activation of PAR2 with activating peptide (PAR2-AP, 100μM) dramatically increased cell migration in different cell lines which was measured by transwell assay. In addition, miR-125b was reduced at 6 and 12 hours, while the migration was observed at 12 hours after PAR2-AP treatment. Consistent with previous report, inhibitor of miR-125b dramatically prompted cell migration in A549 cells. To test whether the downregulation of miR-125b mediates PAR2-induced migration, miR-125b mimic was transfected into A549 cells to recover the reduction of miR-125b induced by PAR2 activation. It was showed that miR-125b mimic also successfully blocked PAR2-induced cell migration. Furthermore, pretreatment with 5-aza-CdR (5umol/L) for 24h dramatically prevented PAR2-induced downregulation of miR-125b in A549 cells. It suggests that methylation of miR125b promoter may be the mechanism by which PAR2 repressed the expression of miR-125b. On the other hand, the data showed that Lin28B was increased dramatically by miR-125b inhibitor, which indicated that Lin28B may be the downstream target gene of miR-125b in A549 cells. Most importantly, we found that upregulation of PAR2 and downregulation of miR-125b were significantly correlated with lymphatic metastasis in human colorectal cancer specimens.

Conclusion: Our study demonstrated that activation of PAR2 promotes cell migration through miR-125b, which may contribute to PAR2-related lymphatic metastasis in CRC.

Citation Format: LAN YANG. MicroRNA-125b mediated par2 activation-induced cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1066. doi:10.1158/1538-7445.AM2014-1066
Abstract 1069: M1 macrophages suppress tumorigenesis via accumulated p53 and upregulated STAT1 in lung cancer
Authors: Hsiao Yi-Jing , Su Kang-Yi , Chen Jian-Wei , Yu Sung-Liang
Published: May 2015

Purpose

Macrophages involved in innate immunity and possess cytotoxicity via NO, ROS and pro-inflammatory cytokines, such as TNF-α and IFN-γ. But long-term effects of M1 macrophages on cancer cells, especially their tumoricidal activity, remain unclear. In our previous study, conditioned medium (CM) from M1 macrophages can induce A549 apoptosis and senescence, but detail mechanism is unexplored.

Materials & Methods

Lung cancer cell lines, A549 and H460, are cells with wild-type p53, but H1299 is p53-null. Cytokine and cDNA microarray were used to evaluate the content of M1 macrophages and their effects on A549 cell. Flow cytometry was assessed cell apoptosis; real-time PCR and Western blot were assessed the expression of identified genes and proteins. Clinical statistical analysis for predicting survival of lung cancer patients was applied by a published clinical microarray dataset.

Results

According to our previous results of cDNA microarray, several significantly differential genes were the down-stream of p53, the important initiator for apoptosis. After M1 CM treatment, A549 and H460 cells went apoptosis and p53 was abundantly accumulated. But cell death was not observed in H1299 cells, unless exogenous wt-p53 was expressed and M1 CM treated. Another transcription factor STAT1 was also up-expressed at translational and transcriptional levels after M1 CM stimulation. STAT1 may interact and assist with p53 to cause cell death. IFN-γ which is a well known cytokine for STAT1 activation highly expresses in M1 macrophages, but antibody neutralization can not reverse cell apoptosis. M1 macrophages from human PBMCs also had tumoricidal activity for A549. Moreover, the M1/M2 gene signature from A549 cDNA microarray can predict the patients' survival.

Conclusion Accumulated data show that p53 and STAT1 are the main tumoricidal regulators induced by M1 macrophages in long-term culture and that is IFNs independent signaling pathway. Those data imply that M1 macrophage plays an important role in the immune surveillance for cancer progression. That may be a potential immunol target therapy.

Citation Format: Yi-Jing Hsiao, Kang-Yi Su, Jian-Wei Chen, Sung-Liang Yu. M1 macrophages suppress tumorigenesis via accumulated p53 and upregulated STAT1 in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2014-1069
Abstract 1074: Tumor-associated microglia secrete paracrine factors that promote Nf1-deficient optic glial cell growth
Authors: Solga Anne C. , Pong Winnie W. , Cimino Patrick J. , Kim Keun Y. , Walker Jason , Wylie Todd , Magrini Vincent , Rubin Joshua B. , Piwnica-Worms David , Ellisman Mark H. , Mardis Elaine R. , Gutmann David H.
Published: May 2015

Previous studies have demonstrated that brain tumors are composed of heterogeneous mixtures of neoplastic and non-neoplastic (stromal) brain cells. However, current brain tumor therapies are primarily focused on inhibiting the growth of the cancerous cells, largely ignoring the contribution of the surrounding stromal elements. One of these stromal cell types is an immune system-like cell (microglia), which constitutes as many as half of the cells present in human brain tumors (gliomas). The tumor microenvironment is considered to play an important role in tumor formation and maintenance by providing signals that both negatively and positively influence tumor cell growth.

Since children with the neurofibromatosis type 1 (NF1) inherited cancer syndrome are prone to the development of gliomas involving the optic nerve (optic gliomas), we have employed Nf1 genetically-engineered mice as an experimental model system to examine the role of microglia in optic glioma formation and maintenance. We leveraged RNA-sequencing data from FACS-isolated, mouse optic glioma-associated microglia to identify specific paracrine factors that are expressed in tumor-associated, but not normal, microglia. These factors were validated using RNA fluorescent in situ hybridization and investigated for their ability to increase the growth of Nf1-deficient optic nerve astroglial cells in vitro. Current studies are ongoing to determine whether (1) inhibition of microglia function in Nf1 genetically engineered mice reduces the expression of these paracrine factors and (2) inhibiting these factors attenuates optic glioma growth in vivo. Together, these studies identify a novel set of paracrine factors secreted by a key glioma-associated stromal cell type (microglia) relevant to the pathogenesis of NF1-associated tumors, raising the possibility that future therapies might be directed against tumor microenvironment paracrine factors.

Citation Format: Anne C. Solga, Winnie W. Pong, Patrick J. Cimino, Keun Y. Kim, Jason Walker, Todd Wylie, Vincent Magrini, Joshua B. Rubin, David Piwnica-Worms, Mark H. Ellisman, Elaine R. Mardis, David H. Gutmann. Tumor-associated microglia secrete paracrine factors that promote Nf1-deficient optic glial cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2014-1074