bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4986: Distinct temporal regulation of RET isoform internalization: Roles of clathrin and AP2
Authors: Crupi Mathieu Joseph François , Yoganathan Piriya , Bone Leslie N. , Lian Eric , Fetz Andrew , Antonescu Costin N. , Mulligan Lois M.
Published: August 2015

The RET receptor tyrosine kinase is an important contributor to kidney and enteric nervous system development. In normal development, RET signaling regulates cell proliferation, migration, survival and differentiation, but RET is also implicated in growth and spread of several human cancers. Activating mutations of RET are drivers of inherited and sporadic thyroid cancers, while expression of wildtype RET receptors in breast and pancreatic cancers has been associated with enhanced tumor invasion, metastasis and poor disease prognosis. RET is expressed as two major isoforms, RET9 and RET51, that have unique 9 or 51 amino acid C-terminal tails, respectively. These isoforms induce unique phosphorylation patterns on intracellular tyrosine residues, differentially bind downstream signalling proteins, and possess different intrinsic abilities to cause cellular transformation. We have previously shown that RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line derived neurotrophic factor (GDNF) ligand stimulation, but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence microscopy to demonstrate that RET internalization occurs through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin, at sites of CCPs. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated pathways but that RET9 and RET51 have distinct internalization kinetics that may contribute to the functional differences between RET isoforms which are critical to regulating RET's contribution to tumorigenesis and normal development. These data may also have clinical importance, as they characterize key regulatory events for an important oncogene that may in future indicate biologically relevant targets for therapeutic interventions in RET-associated tumors.

Citation Format: Mathieu Joseph François Crupi, Piriya Yoganathan, Leslie N. Bone, Eric Lian, Andrew Fetz, Costin N. Antonescu, Lois M. Mulligan. Distinct temporal regulation of RET isoform internalization: Roles of clathrin and AP2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2015-4986
Abstract 4990: The RET receptor Y791F variant activates the kinase but diminishes ligand responsiveness
Authors: Fetz Andrew , Crupi Mathieu J.F. , Lian Eric , Hyndman Brandy D. , Mulligan Lois M.
Published: August 2015

The RET receptor tyrosine kinase is essential for normal development of the kidneys and enteric nervous system, and is also implicated in several human pathologies. Gain-of-function mutations in RET are associated with the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), where single amino acid substitutions lead to constitutive RET activation in the absence of its ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Conversely, loss-of-function RET mutations are associated with Hirschsprung disease, a congenital abnormality of the enteric nervous system. Previous studies have identified a specific substitution variant of a phenylalanine for tyrosine at amino acid 791 in the RET kinase domain in both cancer and Hirschsprung disease patients. Thus, the functional implications of this variant and its contributions to these diverse phenotypes are not clear. Here, we have explored the role of MEN 2-associated RET mutations in RET-mediated cell invasion and migration. We showed that GDNF-stimulation promotes RET-mediated cell migration and that a GDNF-chemotactic gradient significantly increased invasion of cells expressing wild type RET or a MEN2B (M918T) mutant RET form. However, we found that a GDNF gradient did not enhance invasion of cells expressing RET-Y791F. Consistent with this, we showed that MEN 2-associated RET mutants M918T and Y791F were phosphorylated in the absence of GDNF and stimulated downstream signaling pathways. GDNF treatment further increased phosphorylation of the RET-M918T but not RET-Y791F proteins, suggesting that RET-Y791F has reduced responsiveness to GDNF ligand. Our results suggest that despite constitutive activation of RET signaling, cells expressing the Y791F variant may possess a diminished capacity to recognize and respond to GDNF in the cell microenvironment. This may be linked to a disturbance in the directional migration of cells expressing the mutant receptor thus contributing to the phenotypic variability observed.

Citation Format: Andrew Fetz, Mathieu J.F. Crupi, Eric Lian, Brandy D. Hyndman, Lois M. Mulligan. The RET receptor Y791F variant activates the kinase but diminishes ligand responsiveness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2015-4990
Abstract 4991: RET and integrins cooperate in cell-microenvironment response
Authors: Lian Eric , Crupi Mathieu J.F. , Cockburn Jessica G. , Wagner Simona M. , Goel Anirudh , Mulligan Lois M.
Published: August 2015

The RET receptor tyrosine kinase is expressed in neural crest-derived cell types, where it is activated by soluble ligands of the Glial Cell-derived Neurotrophic Factor (GDNF) family and signals through multiple downstream pathways, contributing to cell growth, survival, and migration during embryonic development. In addition to its normal developmental roles, oncogenic mutations or aberrant expression of RET are also linked to tumour spread and metastasis in multiple human tumour types. Thus, a consistent feature of RET involvement in these disease processes is its contribution to directional cell motility. We have previously shown that RET-mediated cell-adhesion and migration requires multiple members of the integrin family of cell adhesion molecules, including integrin β1 (ITGB1) and β3 (ITGB3) subunits, however the regulation of cell migration and invasion by RET and integrins has not been explored.

In this study, we showed that GDNF-mediated RET activation induced rapid ITGB1 and ITGB3 activation with transiently increased cell adhesion, followed by enhanced cell protrusion formation and reduced adhesion typical of more migratory cells. In addition, we showed that directional cell migration in response to GDNF requires the presence of both extracellular matrix and a GDNF concentration gradient. Our data also suggest that ITGB1 and ITGB3 play complementary but non-compensatory roles in the initiation of RET-mediated cell migration and invasion, and that the inhibition of either ITGB1 or ITGB3 resulted in impaired migration and invasion in both 2D and 3D culture conditions. We have also shown that multiple signaling pathways downstream of RET are involved in these processes and that these pathways contribute differently to migration in 2D vs 3D microenvironments. These data highlight the ability of tumour cells to leverage different signaling pathways to mediate cell migration and invasion downstream of RET, and reveal ways in which cooperation between RET and integrins might affect aspects of tumour progression. Our data, together with other studies which have shown combination therapy targeting integrins and RTKs to be more effective than monotherapy, suggest that adjuvant treatment with agents targeting integrin function may further enhance therapeutic options for tumours where RET is expressed.

Citation Format: Eric Lian, Mathieu J.F. Crupi, Jessica G. Cockburn, Simona M. Wagner, Anirudh Goel, Lois M. Mulligan. RET and integrins cooperate in cell-microenvironment response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2015-4991
Abstract 4992: Nicotinic acetylcholine receptor α5 mediates low-dose nicotine-dependent regulation of tumorigenesis in lung adenocarcinoma cells
Authors: Wang Mong-Lien Lotus , Hsu Yi-Fan , Liu Chih-Hsuan , Wu Cheng-Wen
Published: August 2015

The concept that lung adenocarcinoma is less related to smoking behavior than lung squamous cell carcinoma may be due to the fact that a considerable number of LAC patients do not have smoking habit. However, this argument may mislead the effect of tobacco smoke on LAC because it ignores the potential effects of environmental smoking that cause second hand smoking in non-smokers. In Taiwan, 70% of female LAC patients would encounter heavy smokers either at home or in working place, suggesting that environmental smoke may have substantial effects than we thought. Transcriptional deregulation at the chromosome 15q25 locus revealed an association between the gene encoding CHRNA5 with lung adenocarcinoma risk. It has been showed that overexpression of the genes encoded in this locus in mice increased the sensitivity to nicotine. In this report, we found that nAChR α5, but neither nAChR α7 or nAChR α9 that have been respectively shown to mediate tumorigenic alterations in lung SCC and breast cancer cells, was differentially expressed between tumor and non-tumor specimens of LAC patient specimens. Knockdown of nAChR α5 in LAC cell lines resulted in decreased cell proliferation rate as well as cell migrating and invasive capabilities. In these nAChR α5-knockdown cells, low dose (10 nM) nicotine was not able to significantly increase cell proliferation and motility, while low dose nicotine enhanced both abilities of cells when nAChR α5 expressed. Colony formation assay indicated that nAChR α5 maintained cell survival and prevent contact inhibition in LAC cells when encountering low serum stress condition. Through molecular analysis, we showed that nAChR α5 positively regulates epithelial-mesenchymal transition factors such as Snail and Slug, and negatively regulates E-cadherin. Animal experiment showed a severely suppressed tumor growth in mice transplanted with LAC cells with nAChR α5 being knocked down, in comparison to mice transplanted with parental cells. These data demonstrated a crucial role of nAChR α5 in low-dose nicotine-dependent regulation of tumorigenesis in LAC. It is, for the first time, a study of mechanism in response to low dose nicotine that implies the regulatory pathway underlying the effect of environmental nicotine.

Citation Format: Mong-Lien Lotus Wang, Yi-Fan Hsu, Chih-Hsuan Liu, Cheng-Wen Wu. Nicotinic acetylcholine receptor α5 mediates low-dose nicotine-dependent regulation of tumorigenesis in lung adenocarcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4992. doi:10.1158/1538-7445.AM2015-4992
Abstract 5001: High dose IL-2 (HDIL-2) results in increased PD-1 expression on CD45RA-CCR7-CD8+ and CD45RA-CCR7-CD4+ T cells in melanoma patients
Authors: Diller Maggie L. , Maio Susan , Eysmans Cabell E. , Lawson David , Delman Keith A. , Kudchadkar Ragini R. , Ford Mandy L.
Published: August 2015

Introduction: HDIL-2 is associated with long-term remissions in Stage IV melanoma in less than 10% of patients. We hypothesized that HDIL-2 alters the expression of PD-1 on T cell subsets in melanoma patients.

Methods: Samples were collected after written consent under an IRB approved protocol. Peripheral blood was collected at baseline and serially post-treatment at 24, 48, 72, and 96 hours from 6 patients with advanced stage melanoma undergoing HDIL-2. All patients were treatment naïve with regards to anti-PD-1 and anti-CTLA4 therapies. PBMCs were isolated and underwent intracellular cytokine and extracellular receptor staining. Co-signaling molecule expression was analyzed via flow cytometry. Statistical analysis was performed using paired t tests via Prism 6.0e software.

Results: HDIL-2 was associated with a preferential loss of effector memory (TEM; CD45RA-CCR7-) CD4+ and CD8+ T cells and a relative enrichment of naïve (CD45RA+CCR7+) CD4+ and CD8+ T cells in the blood during the first 3 days of treatment. On day 4, frequencies of CD4+ and CD8+ TEM had returned to baseline. Importantly, PD-1 expression was significantly increased on day 4 post-treatment on CD8+ TEM relative to baseline (22.1% +/- 13.2% on day 4 compared to 13.4% +/- 7.4% at baseline; p = 0.04). This finding was also true for CD4+ TEM (11.4% +/- 6.1% on day 4 compared to 6.1% +/- 3.7% at baseline, p = 0.04). PD-1 expression was not statistically different within the other CD4+ and CD8+ memory T cell subsets including: naïve, central memory (CD45RA-CCR7+), or TEMRA (CD45RA+CCR7-) lymphocytes.

Conclusion: These results demonstrate that HDIL-2 is associated with an increase in expression of PD-1 on TEM cells in patients with advanced melanoma suggesting a possible mechanism contributing to the limited efficacy of HDIL-2 in most patients. It is known that TEM cells function by migrating to peripheral tissues, and that CD8+ TEM cells are found specifically in metastatic melanoma lesions. Importantly, our findings illustrate a potential synergistic effect between HDIL-2 and PD-1 blockade. Upregulation of PD-1 and the chronologic pattern in which its expression changes under the effects of HDIL-2 provide a mechanistic rationale for testing the combination of HDIL-2 and anti-PD-1 therapy in patients with advanced melanoma.

Citation Format: Maggie L. Diller, Susan Maio, Cabell E. Eysmans, David Lawson, Keith A. Delman, Ragini R. Kudchadkar, Mandy L. Ford. High dose IL-2 (HDIL-2) results in increased PD-1 expression on CD45RA-CCR7-CD8+ and CD45RA-CCR7-CD4+ T cells in melanoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5001. doi:10.1158/1538-7445.AM2015-5001
Abstract 5006: Elucidating the mechanism of bile acid-induced activation of estrogen receptor β in colorectal adenocarcinoma
Authors: Trujillo Jesse
Published: August 2015

In previous studies, Ursodeoxycholic Acid (UDCA) was investigated as a chemopreventive drug for the prevention of colorectal adenoma recurrence. These studies revealed that the efficacy of UDCA was gender-specific. The use of UDCA in men significantly lowered the odds of advanced lesions in men, but significantly increased the odds of advanced lesions in women, more specifically, in females who were younger, obese, or whose diet was high in fat. The gender specific role of UDCA led us to investigate this bile acids’, along with deoxycholic acid's (DCA), ability to activate the estrogen receptor in colon cancer.

Estrogen receptor β (ERβ) is the predominant estrogen receptor in both normal and malignant human colon tissue, and loss of ERβ has been shown to be critical for tumor cell differentiation. Elevated concentrations of bile acids have previously been shown to promote colon cancer and growing evidence indicates that these molecules play a vital role in regulating cell signaling and gene expression. In our current studies, through western blot and qRT-PCR analysis, we have shown that DCA-treated HT-29 colorectal adenocarcinoma cells significantly induce the phosphorylation of ERβ, as well as increase gene expression of the pS2 estrogen response element (ERE). UDCA was also shown to induce phosphorylation of ERβ, but much later than DCA. However, UDCA increases gene expression of pS2 at much earlier time points than DCA. In addition, we have also shown that when pre-treating HT-29 cells with the MEK 1/2 inhibitor, PD98059, bile acid-induced phosphorylation of ERβ is significantly reduced, along with ERE gene up-regulation. Collectively, this data suggests that bile acids are inducing the activation of ERβ through a ligand-independent mechanism via the MAPK signaling cascade.

Citation Format: Jesse Trujillo. Elucidating the mechanism of bile acid-induced activation of estrogen receptor β in colorectal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5006. doi:10.1158/1538-7445.AM2015-5006
Abstract 5010: A versatile tool to study immune checkpoint therapeutics: Syngenic tumor mouse models in vivo
Authors: Schaefer-Obodozie Cynthia , Jantscheff Peter , Moor Sandra , Hoffmann Steffen , Saso Tihomir , Schaechtele Christoph , Weber Holger
Published: August 2015

Checkpoint inhibitor treatment is a common therapy of various cancer types, however, clinical data indicate that patients only partially respond to this regiment due to attenuated anti-tumor immune response. Thus, it has been recognized as important to consider the immune response already during preclinical drug development in animal experiments to anticipate such clinical drawbacks. Although immune-humanized models using human leukocytes in immuno-compromised mice may provide a starting point, syngenic mice models appear to be the better method of choice, reflecting the whole spectrum of a functional immune system. Here, we report on the establishment of syngenic mouse models for breast, colon, kidney and skin cancer both for subcutaneous and orthotopic implantation. Analyzing untreated as well as checkpoint inhibitor treated mice, we will present data on tumor growth kinetics as well on the distribution of immune cells such as Treg cells, M1/M2 macrophages and M/PMN-MDSCs in tumor and spleen tissue based on multi-color flow-cytometric analysis. These data may help to select a suitable model for testing new drug candidates and define a sensitive combination therapy to support the anti-tumor immune defense in addition to checkpoint inhibitor treatment.

Citation Format: Cynthia Schaefer-Obodozie, Peter Jantscheff, Sandra Moor, Steffen Hoffmann, Tihomir Saso, Christoph Schaechtele, Holger Weber. A versatile tool to study immune checkpoint therapeutics: Syngenic tumor mouse models in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2015-5010
Abstract 5026: Tumor associated targeting with Manocept: HIV associated Kaposi's sarcoma as a model system
Authors: Zhang Rongzhen , Cope Frederick O. , Bracci Paige M. , McGrath Michael S.
Published: August 2015

Introduction

Inflammation appears to play a critical role in tumor development of HIV-associated Kaposi's sarcoma (KS). Specifically, emerging data show that KS tumor cells that co-express various macrophage (MO) antigens become resistant to current anti-viral therapies used to treat KS and AIDS. MOs also are a known source of KS tumor cell growth factors and substantial evidence suggests that tumor associated macrophages (TAMs) may represent a reservoir for HIV and its evolved retroviral variants. The MO pool driving these two pathological pathways share a common element rooted in the MOs, the CD206 human macrophage mannose receptor. Manocept, a molecular targeting agent, binds and enters MOs via pinocytosis of holo-CD206, providing a cell portal for the evaluation of Manocept as a MO and KS targeting agent.

Methods

Manocept was prepared as a synthetic molecule with a dextran backbone, and 12-20 mannose moieties. The reporter molecule (Cy3) was included as a fluorescent tracer for locating Manocept on and in MOs and KS tumor cells.

Binding and localization Cy3-Manocept was assessed through intracellular visualization and flow cytometric quantitation of Cy3-Manocept binding and uptake using in vitro generation of monocyte-derived CD206+ MOs. The fresh HIV+ KS tumor tissue culture followed by immunofluorescence staining and confocal imaging was performed to confirm Cy3-Manocept uptake in KS tumor cells and TAMs.

Results

In vitro culture showed that Cy3-Manocept binds avidly to CD206 expressing MOs. Time course studies of increasing Cy3-Manocept concentrations confirmed continuous and ongoing uptake of Manocept into CD206+ MOs at 37°C. Fresh HIV+ KS tissue culture studies showed both Manocept uptake and CD206 staining of TAMs as well as KS tumor spindle cells (HHV8+ cells). Cy3-Manocept co-localized with CD206 in nearly all KS-associated cells expressing HHV8 and/or CD68 (Tissue MO marker), confirming that CD206 acts both as a target and Manocept concentrating receptor for TAMs and KS tumor cells.

Conclusions

Our results support the potential use of CD206 macrophage mannose receptor-targeted Manocept for imaging KS tumor cells, TAMs, and more importantly, for delivery of therapeutic/diagnostic agents capable of targeting all KS-associated cells including a potential MO reservoir for HIV.

Citation Format: Rongzhen Zhang, Frederick O. Cope, Paige M. Bracci, Michael S. McGrath. Tumor associated targeting with Manocept: HIV associated Kaposi's sarcoma as a model system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5026. doi:10.1158/1538-7445.AM2015-5026
Abstract 5027: Interleukin-17B promotes chemoresistance of breast tumors through ERK1/2 anti-apoptotic pathway
Authors: Laprevotte Emilie , Bastid Jeremy , Cochaud Stéphanie , Giustiniani Jerôme , Philippe Marion , Frewer Kathryn A. , Sanders Andrew J. , Jiang Wen G. , Bensussan Armand , Alberici Gilles , Eliaou Jean-François , Bonnefoy Nathalie
Published: August 2015

Interleukin-17B (IL-17B) is a pro-inflammatory cytokine that belongs to a family encompassing 6 interleukins (IL-17A to F) and binds to the IL-17 receptor B (IL-17RB). Little is known about the potential role of IL-17B/IL-17RB signaling pathway in cancer. Both IL-17B and IL-17RB are up-regulated in breast tumors and are associated with a poor prognosis in patients. We thus focused on their implication in breast cancer. We demonstrated that recombinant IL-17B induces resistance to conventional chemotherapeutic agents such as taxol, an effect that is totally abrogated by disrupting IL-17RB signaling. To further understand the molecular events involved in IL-17B-induced chemoresistance, we focused on signaling pathways activated under stimulation of human breast cancer cell lines by recombinant IL-17B. We observed that IL-17B induces the activation of both ERK1/2 and NFkB pathways, leading to an up-regulation of anti-apoptotic proteins of the Bcl-2 family. Interestingly we showed that the inhibition of ERK1/2 pathway totally prevents IL-17B-induced chemoresistance of breast cancer cell lines. Relevance of these data is currently under investigation in tumor mouse models. Altogether our results demonstrate the implication of the IL-17B/IL-17RB signaling pathway in breast tumors and identify IL-17B and its receptor as potential therapeutic targets for cancer.

Citation Format: Emilie Laprevotte, Jeremy Bastid, Stéphanie Cochaud, Jerôme Giustiniani, Marion Philippe, Kathryn A. Frewer, Andrew J. Sanders, Wen G. Jiang, Armand Bensussan, Gilles Alberici, Jean-François Eliaou, Nathalie Bonnefoy. Interleukin-17B promotes chemoresistance of breast tumors through ERK1/2 anti-apoptotic pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5027. doi:10.1158/1538-7445.AM2015-5027
Abstract 5032: Acute phase liver graft injury mobilizes regulating B cells after transplantation for HCC patients through TLR4/CXCL10/CXCR3 signaling
Authors: Man Kwan , Shao Yan , Li Chang Xian , Lo Chung Mau
Published: August 2015

We aim to investigate the impact of acute-phase small-for-size graft injury on mobilization of circulating regulating B cells (Bregs) in HCC patients after liver transplantation and to explore the molecular mechanism therein.

Methods

From May 2000 to November 2009, 115 HCC recipients were included. The intragraft gene expression profile and Bregs infiltration of the grafts greater (Group 1) and less than 60% (Group 2) of standard liver weight (SLW) were detected by RT-PCR and immunostaining. Circulating Bregs (CD19+CD24hiCD38hi) were compared by FACS analysis after transplantation. Clinical-pathological data including the incidence of tumor recurrence and metastasis were compared.

The direct roles of TLR4, CXCL10 and CXCR3 on circulating Bregs mobilization were investigated in TLR4-/-, CXCL10-/- and CXCR3-/- mice models, respectively.

The association of intragraft Bregs infiltration and tumor invasiveness were also examined in a rat liver transplantation for liver cancer model. The role of Bregs on liver tumor growth and invasiveness were further studied in a series of in vitro and in vivo functional experiments.

Results

The patients were grouped into Group 1 (> = 60% SLW, n = 37) and Group 2 (<60% SLW, n = 78). Much more patients in Group 2 developed tumor recurrence and lung metastasis [19/78(24.4%) vs 3/37(8%), p = 0.04]. Level of circulating Bregs was significantly higher in Group 2 (Week1: 7.02 vs 1.31/10^5PBMC, p = 0.03; month3: 5.7 vs 1.3/10^5PBMC, p = 0.03). There was more intragraft Bregs infiltration in group 2 indicated by CD20/IL10 staining. Intragraft gene expression of TLR4, CXCL10 and CXCR3 were significantly higher in Group 2 at early phase after transplantation. In rat liver transplantation model, more

Bregs infiltration at early phase after transplantation correlated with late phase invasive tumor growth. Levels of circulating Bregs were significantly lower in the mice model with major hepatectomy and hepatic I/R injury using TLR4-/-, CXCL10-/- and CXCR3-/- mice, respectively. Bregs also promoted liver cancer cell proliferation and migration in vitro, and tumor growth in vivo.

Conclusion

A significantly higher population of circulating Bregs, which are mobilized by small-for-size graft injury, may lead to a higher incidence of tumor recurrence and metastasis after LDLT.

TLR4/CXCL10/CXCR3 signaling may play important roles on Bregs mobilization.

Citation Format: Kwan Man, Yan Shao, Chang Xian Li, Chung Mau Lo. Acute phase liver graft injury mobilizes regulating B cells after transplantation for HCC patients through TLR4/CXCL10/CXCR3 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2015-5032
Abstract 5038: Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone
Authors: McFall Thomas B. , Patki Mugdha , Rosati Rayna , Ratnam Manohar
Published: August 2015

Introduction: High expression of the progesterone receptor (PR) isoform A, relative to PR isoform B, is clinically associated with progression to invasiveness in estrogen receptor (ER)-positive breast cancer and lower disease free survival. This is apparently at odds with in vitro studies implicating PR-B in mediating progestin-induced invasiveness. As the in vitro studies were conducted in the absence of estrogen (E2) signaling, we considered the possibility that modulation of E2 action may comprise a distinct aspect of the regulation of invasiveness by progestins.

Methods: Standard ER+/PR+ and ER+/PR+/HER2+ breast cancer cell line models were studied, as well as recombinant cells expressing a single PR isoform. For the progestin hyper-sensitization studies, PR-A was over-expressed by lentiviral infection. Hormonal influence on invasiveness was studied using the Boyden chamber assay. Gene expression was examined by Illumina DNA microarray analysis and by Taq-Man real time RT-PCR assays.

Results: E2 strongly suppressed invasiveness of ER+ breast cancer cells at concentrations below 0.01nM. At low (< 1 nM) concentrations, progesterone, R5020 and medroxyprogesterone acetate completely abrogated the inhibition of invasiveness by E2. It was only in a higher (5nM - 50 nM) concentration range that progestins induced invasiveness in the absence of E2. The ability of progestins to rescue invasiveness from E2 regulation was exclusively mediated by PR-A whereas PR-B mediated the E2-independent component of progestin-induced invasiveness. Overexpression of PR-A in PR-A+/PR-B+ cells lowered the progestin concentration needed to completely rescue invasiveness (to < 0.2 nM). Progesterone opposed E2 regulation of a distinctive set of genes, exclusively through PR-A.

Conclusion: At relatively low concentrations, progesterone influences breast cancer cell invasiveness by rescuing it from E2 regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of E2 signaling, through PR-B. The findings establish a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue estrogen and progesterone levels.

Citation Format: Thomas B. McFall, Mugdha Patki, Rayna Rosati, Manohar Ratnam. Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5038. doi:10.1158/1538-7445.AM2015-5038
Abstract 5043: ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 gain-of-function
Authors: Bado Igor , Nikolos Fotis , Rajapaksa Gayani , Gustafsson Jan-Ake , Thomas Christoforos
Published: August 2015

Triple-negative breast cancers (TNBCs) display distinct metastatic potential and a poor prognosis despite a relative chemosensitivity. Most (80%) of these highly aggressive tumors express mutant p53 proteins that are known to elicit oncogenic activities that contribute to metastasis and chemoresistance. Despite the lack of estrogen receptor α (ERα), TNBCs express wild-type ERβ (ERβ1). We previously showed that ERβ1 impedes epithelial to mesenchymal transition (EMT) in TNBC cells by repressing epidermal growth factor receptor (EGFR) signaling. Our recent findings indicate that ERβ1 regulates EGFR expression and decreases the invasiveness of TNBC cells by interacting with mutant p53 and inhibiting its function. Suppression of a mutant p53 transcriptional program and induction of p63 activity are essential for the anti-migratory activity of ERβ1 in TNBC cells. These results describe an ERβ1/mutant p53 interaction that disturbs oncogenic signals to inhibit aggressiveness in TNBC.

Citation Format: Igor Bado, Fotis Nikolos, Gayani Rajapaksa, Jan-Ake Gustafsson, Christoforos Thomas. ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 gain-of-function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2015-5043
Abstract 5046: Amphiregulin is a key effector of estrogen receptor in breast cancer
Authors: Peterson Esther A. , Chandiramani Natasha , Aranda Jaque Evelyn J. , Jenkins E. Charles , Kenny Paraic A.
Published: August 2015

Estrogen is an essential hormone for mammary gland development and is a key driver of proliferation during the development of estrogen receptor positive breast tumors. The actions of estrogen are primarily mediated by its receptor, the ERα transcription factor, which regulates several hundred estrogen-responsive genes in breast cancer cells. However, despite much investigation, the identity of the key effectors of this proliferative signal in breast cancer remains unclear. Amphiregulin (AREG) is a ligand of the Epidermal Growth Factor Receptor. It is transcriptionally regulated by estrogen and is required for estrogen-dependent mouse mammary gland development. In this study, we have tested whether Amphiregulin is a key effector of estrogen in breast cancer.

shRNA-mediated knockdown of Amphiregulin in MCF7 cells strongly attenuated ER+ breast cancer cell line growth in vivo. Conversely, expression of Amphiregulin from an estrogen-insensitive promoter allowed MCF7 xenografts to escape the therapeutic effects of tamoxifen. Thus, Amphiregulin expression is both necessary and sufficient for the proliferative response to estrogen in MCF7 cells in vivo, and downregulation of Amphiregulin in response to tamoxifen is required for a therapeutic benefit.

After initially responding to endocrine therapy, many ER+ tumors progress to resistance to these therapies. Our model predicts that breast tumors which either (1) express AREG in an estrogen-insensitive manner or (2) have elevated levels of alterative ERBB family ligands, will be endocrine resistant. We present experimental and clinical evidence demonstrating that these features contribute to at least some examples of endocrine resistance. Firstly, in MCF7 cells selected for resistance to fulvestrant, we show that AREG expression is no longer regulated by estrogen or its antagonists, but the cells remain AREG-dependent. Secondly, in a series of 52 ER+ breast cancer patients treated with neo-adjuvant letrozole, we show that the downregulation of Amphiregulin in response to endocrine therapy observed in our mouse studies is recapitulated in human breast cancer patients, and that tumors in which alternate ERBB ligands (TGFalpha, Epiregulin, and Neuregulins 1 & 2) are expressed at high levels are least likely to respond to letrozole.

In summary, these data underline the importance of ERBB signaling in estrogen receptor positive breast cancer and implicate Amphiregulin as the primary ERBB ligand in mediating the effects of estrogen in these tumors.

Citation Format: Esther A. Peterson, Natasha Chandiramani, Evelyn J. Aranda Jaque, E. Charles Jenkins, Paraic A. Kenny. Amphiregulin is a key effector of estrogen receptor in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5046. doi:10.1158/1538-7445.AM2015-5046
Abstract 5049: Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth
Authors: Ta Huy Q. , Ivey Melissa L. , Frierson Henry F. , Conaway Mark R. , Dziegielewski Jaroslaw , Larner James M. , Gioeli Daniel
Published: August 2015

Prostate cancer (PCa) is the second leading cause of cancer death in American men, and the cure for metastatic disease remains a significant challenge. While nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), virtually every patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling pathways continue to play a crucial role in CRPC progression. Previous studies have shown that signal transduction pathways can stimulate AR activation, suggesting that the ability of signaling cascades to influence AR function may have a significant role in CRPC progression, and that CRPC may not be effectively treated by ligand-directed therapy alone. A high-throughput RNAi screen identifying signaling pathways that regulate PCa cell growth led to our discovery that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased PCa proliferation in the presence and absence of androgen. Furthermore, CHK2 depletion hypersensitized cells to castrate androgen levels. These CHK2-mediated effects on growth were dependent on the downstream signaling proteins CDC25C and CDK1 and could be blocked by the AR antagonist MDV3100. Immunohistochemical analysis of CHK2 in patient samples demonstrated that reduced CHK2 expression significantly correlated with increased Gleason score indicating the clinical relevance of CHK2 in PCa. Moreover, CHK2 expression is lower in castration-resistant C4-2 and CWR22Rv1 cells compared to androgen-sensitive LNCaP cells consistent with loss of CHK2 expression during PCa progression. CHK2 depletion increased AR transcriptional activity on both androgen-activated and androgen-repressed genes, substantiating that CHK2 affects PCa growth through the AR. Remarkably, quantitative PCR, chromatin immunoprecipitation, and western blot analyses revealed that CHK2 is a novel AR-repressed gene, suggesting a negative feedback loop between CHK2 and the AR. Furthermore, we show that CHK2 physically associates with the AR, and that cellular stress, such as DNA damage and serum starvation, increases this association. Based on these data, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth. Thus, alterations to CHK2 signaling may sensitize CRPC to ADT and radiation.

Citation Format: Huy Q. Ta, Melissa L. Ivey, Henry F. Frierson, Mark R. Conaway, Jaroslaw Dziegielewski, James M. Larner, Daniel Gioeli. Checkpoint kinase 2 is a novel regulator of prostate cancer cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5049. doi:10.1158/1538-7445.AM2015-5049
Abstract 5052: The association between estrogen receptor alpha and platinum sensitivity in ovarian cancer cells
Authors: Matsumura Sohei , Ohta Tsuyoshi , Takahashi Toshifumi , Takahashi Kazuhiro , Nagase Satoru
Published: August 2015

[Objective] The objective of this study was to examine whether estrogen receptor alpha (ERα) is associated with the sensitivity to cisplatin in ovarian cancer cells.

[Methods] Using two human ovarian cancer cell lines (Caov-3, Ovcar-3) which express ERα, we investigated the effect of cisplatin on ERα activation. We evaluated the association between ERα activation and Erk cascade in the cells treated with cisplatin. We next examined the antitumor effects of estradiol (E2) with cisplatin by using MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and Western blot assay. Then, ERα expression was down-regulated in Caov-3 and Ovcar-3 by using lentiviral shRNA against ERα, and we generated batch clonal lines. The nontarget shRNA (NTS) served as control. Finally, we determined the effect of ERα down-regulation on the sensitivity to cisplatin.

[Results] Cisplatin induced the phosphorylation of ERα at serine 118 via Erk cascade. Pretreatment with E2 antagonized cisplatin-induced cytotoxicity, and decreased the expression of cleaved PARP induced by cisplatin. Pretreatment of E2 followed by cisplatin increased the expression of Bcl-2, which is anti-apoptotic protein. The shRNA- mediated down-regulation of ERα increased the sensitivity to cisplatin compared with NTS.

[Conclusions] These results indicate that E2 promotes resistance to cisplatin via ERα, and down-regulation of ERα may increase efficacy of cisplatin in ovarian cancer.

Citation Format: Sohei Matsumura, Tsuyoshi Ohta, Toshifumi Takahashi, Kazuhiro Takahashi, Satoru Nagase. The association between estrogen receptor alpha and platinum sensitivity in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5052. doi:10.1158/1538-7445.AM2015-5052
Abstract 5055: Role of insulin-like growth factor 1 receptor in pancreatic cancer pathogenesis
Authors: Subramani Reddy Ramadevi , Arumugam Arunkumar , Nandy Sushmita Bose , Gonzalez Elizabeth , Gonzalez Viviana , Bonkoungou Sandrine , Ortega Andrew , Lakshmanaswamy Rajkumar
Published: August 2015

Pancreatic cancer incidence and the associated mortality are increasing worldwide. This indicates that there is a clear lack of understanding about pancreatic cancer pathogenesis. This can be attributed to the lack of specific symptoms to diagnose the disease at an early stage. Various growth factor receptors are known to play a vital role in the development of pancreatic cancer. In the present investigation, we have attempted to target insulin-like growth factor-1 receptor (IGF-1R) due to its well-established role in progression of pancreatic cancer. RNA interference (small interfering RNA [siRNAs]) approach targeted to IGF-IR was used to study the effect of IGF-1R silencing on pancreatic cancer growth and metastasis. For this we used HPAC and PANC-1 pancreatic cancer cell lines and primary pancreatic cancer tissue array. Our results demonstrated that silencing IGF-1R remarkably inhibited pancreatic cancer growth and metastasis. Silencing IGF-1R significantly altered key signaling molecules such as PI3K, AKT, MAPK, ERK1/2, mTOR, PTEN, p70S6K, JAK2 and STAT3. Further IGF-1R silencing also reduced epithelial to mesenchymal transition (N-Cad, Notch, Snail, Slug and Vimentin). In addition, silencing IGF-1R inhibited cell viability, invasive and migratory capabilities of PANC-1 and HPAC pancreatic cancer cell lines. Colony forming ability was also drastically reduced in IGF-1R silenced cells. Flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R decreased the expression of Kras, which is altered in almost 95% of pancreatic cancers. Overall, our data clearly demonstrates the significance of IGF-1R in pancreatic cancer pathogenesis and also identifies IGF-1R as a potential target for pancreatic cancer treatment and therapy.

Citation Format: Ramadevi Subramani Reddy, Arunkumar Arumugam, Sushmita Bose Nandy, Elizabeth Gonzalez, Viviana Gonzalez, Sandrine Bonkoungou, Andrew Ortega, Rajkumar Lakshmanaswamy. Role of insulin-like growth factor 1 receptor in pancreatic cancer pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5055. doi:10.1158/1538-7445.AM2015-5055
Abstract 5067: Preadipocyte exosomes promote early stage breast cancer formation by enhancing cancer stem cell renewal signaling
Authors: Wolfson Benjamin , Gernapudi Ramkishore , Duru Nadire , Zhou Qun
Published: August 2015

The tumor microenvironment plays a critical role in regulating breast cancer progression. Adipocytes differentiate from preadipocytes, which possess mesenchymal stem cell properties. Preadipocytes and adipocytes are essential components of the tumor microenvironment, and signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are also important components of the cancer stem cell niche. The primary goal of our studies is to examine how preadipocyte-derived exosomes can regulate early-stage breast cancer formation via regulating stem cell renewal. We use human and mouse preadipocytes and early-stage breast cancer cells (ductal carcinoma in situ, DCIS), as well as xenografts in immunodeficient mice to determine the role of preadipocyte secreted exosomes in DCIS stem cell behavior and tumor formation. By using protein arrays, we reveal CD81 and FLOT-1 as key exosomal protein markers and SOX2 and SOX9 as exosomal secretion of stem cell renewal regulators. We identify a key miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound shikonin can suppress preadipocyte signaling, inhibiting nearby DCIS cells. Through co-culture experiments we show that shikonin treated preadipocytes secrete exosomes containing high levels of miR-140 that can impact nearby DCIS cells through targeting SOX9 signaling. We demonstrate that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Finally, our data also show that targeting the tumor microenvironment may assist in blocking tumor formation.

Citation Format: Benjamin Wolfson, Ramkishore Gernapudi, Nadire Duru, Qun Zhou. Preadipocyte exosomes promote early stage breast cancer formation by enhancing cancer stem cell renewal signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2015-5067
Abstract 5071: Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models
Authors: Thompson Ben A. , Messenger Kate J. , Linnane Emily , Coupland Sarah E. , Calcraft Peter J.
Published: August 2015

Aberrant activation of the pro-survival Wnt signalling pathway is a key determinant in numerous cancers (Nusse and Varmus, 2012) and therefore constituents of this pathway are potentially promising therapeutic targets.

The membrane-bound o-acyltransferase porcupine (PORCN) is required for post-translational modification of all Wnts, and is vital for Wnt secretion (Herr et. al., 2012). Due to the complex and varied oncogenic roles of the known Wnt ligands, prevention of active ligand secretion provides an effective mechanism for inhibiting canonical and non-canonical Wnt signalling in both paracrine and autocrine systems.

Here we show that a novel inhibitor of porcupine potently inhibits both autocrine and paracrine models of Wnt/β-catenin signalling. Treatment of the pancreatic cancer cell line CAPAN-2 with REDX05562 resulted in a significant decrease of axin 2 mRNA expression by qPCR (10.4 fold, 10nM). This reduction in expression was partially reversed by the addition of recombinant human Wnt-3A (100ng.ml−1). The decrease in axin 2 expression was concomitant with an inhibition of CAPAN-2 proliferation (IC50 1.4nM +/- 0.8nM).

Conversely treatment of MCF-7 mammospheres with REDX05562 did not inhibit sphere forming efficiency (SFE). However, when stimulated with recombinant Wnt-3A an increase in anoikis resistance was observed suggesting a potential paracrine influence. In vivo the presence of cancer associated fibroblasts (CAFs) are a likely source of paracrine Wnt (Green et. al., 2013). Indeed, conditioned media taken from primary CAFs (isolated from fresh mastectomies) increased anoikis resistance in MCF-7 cells without any discernible effect on CAF proliferation. This effect was significantly inhibited when MCF-7 cells were incubated with conditioned media taken from primary CAFs treated with REDX05562. Moreover, immunofluorescent labelling of PORCN revealed that PORCN expression is markedly higher in CAFs than in MCF-7s. This is consistent with the hypothesis that different mechanisms underpin Wnt-signalling in breast and pancreatic cancer.

The above data show that the novel PORCN inhibitor REDX05562 effectively reduces the impact of pro-survival Wnt ligand-mediated signalling in both paracrine and autocrine models. This further emphasises the broad therapeutic potential of PORCN inhibition in Wnt-dependent cancers.

Citation Format: Ben A. Thompson, Kate J. Messenger, Emily Linnane, Sarah E. Coupland, Peter J. Calcraft. Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2015-5071