bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 39: FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner
Authors: Benitez Anaid , Yuan Fenghua , Nakajima Satoshi , Wei Leizhen , Qian Liangyue , Myers Richard , Hu Jennifer J. , Lan Li , Zhang Yanbin
Published: May 2015

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand crosslinks (ICL). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, interstrand crosslink repair protein, FANCA, greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.

Citation Format: Anaid Benitez, Fenghua Yuan, Satoshi Nakajima, Leizhen Wei, Liangyue Qian, Richard Myers, Jennifer J. Hu, Li Lan, Yanbin Zhang. FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 39. doi:10.1158/1538-7445.CANSUSC14-39
Abstract IA03: DICER1: From ontogenesis to oncogenesis
Authors: Foulkes William D. , Wu Mona , Kock Leanne De , Witkowski Leora , Sabbaghian Nelly , Weber Evan , Hamel Nancy , Priest John R.
Published: May 2015

The DICER1 syndrome, also known as the pleuropulmonary blastoma familial tumor dysplasia syndrome (PPB-FTDS) (OMIM #601200), is a recently described entity comprising a number of rare to ultra-rare tumors arising mainly in childhood or adolescence. The most frequent and characteristic disorders are pleuropulmonary blastoma, cystic nephroma and ovarian Sertoli-Leydig cell tumors. Some aspects of the syndrome were identified in the 1970s and 1990s, but discovery in 2009 by Hill et al of heterozygous disease-associated germ-line DICER1 mutations in affected kindred brought the syndrome into focus. Several studies since 2009 have extended the phenotypes to include more common conditions such as multinodular goiter and Wilms tumor, as well as much rarer entities such as cervical embryonal rhabdomyosarcoma, pineoblastoma and pituitary blastoma. The critical molecular defect appears to be impairment of DICER1's RNase III endonuclease function, which normally would cleave precursor microRNAs to their final mature length. These microRNAs function by targeted silencing and/or degradation of specific messenger RNAs. DICER1 may be considered an unusual type of tumor suppressor gene, in that the first inherited “hit” usually cripples one allele completely, whereas a second somatic “hit” is nearly always limited to the RNase III domains (and is in fact often even more focused on the metal-binding domains of RNase IIIb). These second hits are most commonly a single base substitution leading to an amino acid change, which functionally impairs the protein without overall protein loss. In this presentation I will summarize the current knowledge on the role of DICER1 mutations in cancer and will describe the edges of the known associated phenotypes.

Citation Format: William D. Foulkes, Mona Wu, Leanne De Kock, Leora Witkowski, Nelly Sabbaghian, Evan Weber, Nancy Hamel, John R. Priest. DICER1: From ontogenesis to oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA03. doi:10.1158/1538-7445.CANSUSC14-IA03
Abstract IA11: New insights into DNA double-strand break responses
Authors: Kastan Michael B.
Published: May 2015

Repair of damaged DNA in cells is complicated by the necessity to repair the damage in the context of complex chromatin structures. Elucidation of molecular mechanisms of DNA repair in intact cells has been challenging. We developed a system that can rapidly induce DNA double strand breaks (DSBs) at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs in viable cells. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Using chromatin immunoprecipitation, we found that nucleosomes are partially disassembled around DSBs during non-homologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its RGG domain and is rapidly recruited to DSBs in an MRE11-NBS1-RAD50 complex dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and repair of the DSB, demonstrating for the first time the functional importance of nucleosome disruption in DSB repair and identifying a chromatin remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for RPA recruitment to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair. Since inhibition of nucleolin function blunts the DSB repair process, it has become a potential therapeutic target for radiosensitization of tumors. Similarly, small molecule inhibitors of PIKK kinases like ATM and DNA-PK have been developed and are potent radiosensitizers. The potential clinical use of such compounds will be discussed.

Citation Format: Michael B. Kastan. New insights into DNA double-strand break responses. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA11. doi:10.1158/1538-7445.CANSUSC14-IA11
Abstract IA12: Regulation of the RNF168-dependent response to DNA double-strand breaks
Authors: Orthwein Alexandre , Durocher Daniel
Published: May 2015

The chromatin-based response to DNA double-strand breaks (DSBs) is characterized by the accumulation of DNA repair factors and DNA damage signaling molecules within a large domain that surrounds the lesion. This response is largely dependent on ATM-dependent phosphorylation but also on chromatin ubiquitylation. Indeed, the RNF8 and RNF168 E3 ubiquitin ligases are necessary for the accumulation of proteins such as 53BP1, BRCA1 and RAD18 at sites of DNA damage. The critical importance of this pathway is manifested by the observation that biallelic mutations in the RNF168 gene results in an ataxia-telangiectasia-like syndrome referred to as RIDDLE. We are interested in understanding how this pathway is regulated and how ubiquitin-dependent protein interactions orchestrate the hierarchy of protein recruitment at sites of DNA lesions. I will present our current understanding on the regulation and organization of this signaling cascade and in particular, I will focus my presentation on addressing how the pathway underpinned by RNF168 is regulated during the course of the cell cycle.

Citation Format: Alexandre Orthwein, Daniel Durocher. Regulation of the RNF168-dependent response to DNA double-strand breaks. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA12. doi:10.1158/1538-7445.CANSUSC14-IA12
Abstract IA15: Amelioration of ionizing irradiation damage in Fanconi anemia (Fancd2-/- mice) by GS-nitroxide (JP4-039)
Authors: Greenberger Joel S.
Published: May 2015

Fancd2-/- mice derived from both the C57BL/6 and FEV/B background demonstrate bone marrow stromal cell radiosensitivity and paradoxical hematopoietic cell radioresistance. Fancd2-/- (B6) mouse long-term bone marrow cultures showed reduced longevity of hematopoiesis consistent with decreased capacity to handle oxidative stress. FA patients demonstrate an increased risk for hematopoietic and epithelial cancers, which are difficult to treat with radiotherapy due to intrinsic normal tissue radiosensitivity. Intraoral administration of GS-nitroxide (JP4-039) in F15 emulsion prior to head and neck irradiation significantly reduces the increased susceptibility of Fancd2-/- mice to oral cavity ulceration. Baseline biomarkers for oxidative stress related proteins in oral cavity and bone marrow of Fancd2-/- mice compared to heterozygote and normal littermates (B6) and FVB/N were exacerbated by oral cavity irradiation. Suppression of femur marrow hematopoiesis by head and neck irradiation, was also increased in Fancd2-/- mice, but was ameliorated by JP4-039/F15 intraoral administration, as were gene transcripts, proteins, and DNA promoter demethylation. Amelioration of normal tissue radiosensitivity by organ specific radioprotective mitochondrial targeted antioxidants is effective in both DNA repair defective and wild type murine epithelial tissues.

Citation Format: Joel S. Greenberger. Amelioration of ionizing irradiation damage in Fanconi anemia (Fancd2-/- mice) by GS-nitroxide (JP4-039). [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA15. doi:10.1158/1538-7445.CANSUSC14-IA15
Abstract IA20: A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest
Authors: Hayashi Makoto T. , Karlseder Jan
Published: May 2015

Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres in human cells. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome cohesion resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 (Telomere Repeat binding Factor 2) from telomeres, telomeric 3′-overhang degradation and ATM (Ataxia Telangiectasia Mutated) activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration monitoring system that reacts to improper progression through mitosis and will discuss the molecular details and implications of this pathway.

Citation Format: Makoto T. Hayashi, Jan Karlseder. A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA20. doi:10.1158/1538-7445.CANSUSC14-IA20
Abstract A11: Cyclosporin A promotes tumor angiogenesis in a calcineurin-independent manner by increasing mitochondrial reactive oxygen species
Authors: Zhou Alice Yao , Ryeom Sandra W.
Published: May 2015

The commonly used immunosuppressant cyclosporin A (CsA), a potent calcineurin inhibitor, significantly increases the incidence of cancer in organ transplant patients. Calcineurin signaling is an important mediator of VEGF signaling in endothelial cells. Negative regulation of calcineurin by its endogenous inhibitor, Down Syndrome Candidate Region-1 (DSCR1), suppresses tumor growth and angiogenesis, in contrast to the effect observed after long-term CsA treatment. Despite the significance of calcineurin signaling in endothelial cells, the consequences of CsA on tumor angiogenesis has not been investigated. Using an in vivo model of skin carcinogenesis, we show that long-term CsA treatment promotes tumor growth and angiogenesis. Treatment of endothelial cells in vitro with CsA increases proliferation and migration in a calcineurin-independent manner and is associated with increased mitochondrial reactive oxygen species. Co-treatment with antioxidants significantly abrogated CsA-induced endothelial cell activation. Furthermore, mice treated with antioxidants were protected against CsA-mediated tumor progression. Taken together, these findings show that CsA functions independent of calcineurin to potentiate tumor growth by promoting tumor angiogenesis via mitochondrial reactive oxygen species production. This work identifies a previously undescribed mechanism underlying a significantly adverse off-target effect of CsA and suggests that co-treatment with antioxidants may inhibit the tumor promoting effects of CsA.

Citation Format: Alice Yao Zhou, Sandra W. Ryeom. Cyclosporin A promotes tumor angiogenesis in a calcineurin-independent manner by increasing mitochondrial reactive oxygen species. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A11. doi:10.1158/1538-7445.CHTME14-A11
Abstract A13: Hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis by regulating VEGF and S100A8 production
Authors: Ahn G-One , Kim Young-Eun , Hong Beom-Ju , Bok Seoyeon , Lee Chan-Ju , Kim Hak Jae , Kim Il Han , Seita Jun , Weissman Irving , Brown J Martin
Published: May 2015

Myeloid cells (cells that give rise to monocytes and macrophages) are a critical component in the solid tumor microenvironment, promoting angiogenesis and tumor recurrence to therapies. Recent literature have extensively demonstrated that hypoxia-inducible factor (HIF) is a key transcription factor in myeloid cells in responding to hypoxic stimuli including solid tumors and inflammation by secreting various cytokines and growth factors.

Although a number of studies have reported that hypoxia exposure to primary macrophages induces HIF-1 activation and subsequent vascular endothelial growth factor (VEGF) production, there had been no mouse model to demonstrate this phenomenon. To better understand the role of transcriptional activation of HIF in pathological macrophages, we have created a new strain of myeloid-specific knockout (KO) mice targeting HIF pathways using hS100A8 as the myeloid promoter. S100A8 is an intracellular calcium binding protein and its expression has been heavily detected in pathological macrophages of many diseases including solid tumors, inflammatory bowel disease, obesity, and rheumatoid arthritis.

Upon generating mice deficient for von Hippel Lindau (pVHL) tumor suppressor, the negative regulator of HIF, in myeloid cells, we observed erythema and increased VEGF expression in the bone marrow lysate. Moreover, these mice exhibited an enhanced angiogenesis in the subcutanouesly implanted matrigel plugs, which was accompanied by increased VEGF-VEGFR2 signaling in matrigel. We further found that these phenotypes were dependent on transcriptional activation of HIF-1 as a pharmacological or genetic inhibition of HIF-1α completely suppressed the phenotypes in mice deficient for pVHL in myeloid cells. Importantly, we found that HIF-1 activation in myeloid cells regulate not only VEGF but also S100A8 production and identified that monocytes were the major effector driving angiogenesis.

Together these results suggest that transcriptional activation of HIF-1 in myeloid cells plays a critical role in promoting angiogenesis. We are currently investigating how HIF-1 in myeloid cells regulates tumor microenvironment thereby affecting tumor progression.

Citation Format: G-One Ahn, Young-Eun Kim, Beom-Ju Hong, Seoyeon Bok, Chan-Ju Lee, Hak Jae Kim, Il Han Kim, Jun Seita, Irving Weissman, J Martin Brown. Hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis by regulating VEGF and S100A8 production. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A13. doi:10.1158/1538-7445.CHTME14-A13
Abstract A21: Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation
Authors: Lee Jisook , Borboa Alexandra , Baird Andrew , Eliceiri Brian
Published: May 2015

Myeloid cells regulate tumor angiogenesis and promote tumor invasion, yet few strategies exist to exploit their presence to reverse the overall immunosuppressive tumor microenvironment. Based on bioinformatic mining of sentinel genes encoding biologically active peptides, we have recently identified Ecrg4 (Esophageal Cancer-Related Gene 4), as a novel secreted pro-inflammatory mediator of macrophage activation. While higher Ecrg4 expression levels correlate with improved survival in cancers of the esophagus, prostate and breast, indicating a role as a candidate tumor suppressor gene, we have demonstrated that the biological activity of Ecrg4 in cancer functions through a paracrine mechanism, in which Ecrg4 regulates myeloid cells in brain tumors. Moreover, mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro, demonstrating its role as a novel chemoattractant. Here we establish Ecrg4 as a thrombin-processed chemoattractant using subcutaneous tumor models. Ecrg4 gene delivery or Ecrg4 (133-148) peptide administration to tumor reduced melanoma progression through mobilization and activation of myeloid cells to tumor site. Furthermore, in Ecrg4-knockout mice, myeloid cell recruitment is impaired, leading to increased tumor burden. Together, these results identify Ecrg4 as a paracrine factor that is chemotactic for monocytes, with translational potential as an anti-tumor therapeutic.

Citation Format: Jisook Lee, Alexandra Borboa, Andrew Baird, Brian Eliceiri. Thrombin-processed Ecrg4 recruits myeloid cells and induces anti-tumorigenic inflammation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A21. doi:10.1158/1538-7445.CHTME14-A21
Abstract A25: SOCS3 deficiency in myeloid cells promotes prostate tumor development
Authors: Yu Hao , Liu Yudong , McFarland Braden C. , Benveniste Etty N. , Qin Hongwei
Published: May 2015

Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3.

Citation Format: Hao Yu, Yudong Liu, Braden C. McFarland, Etty N. Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A25. doi:10.1158/1538-7445.CHTME14-A25
Abstract A55: Tumor-associated fibroblasts: A new target for cancer treatment
Authors: Hamilton Chase , Shelton Shary , Kakarala Kiran , Thomas Sufi Mary
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a 50% rate of local failure with current standard of care. We recently reported that patient-derived tumor associated fibroblasts (TAF) facilitate HNSCC proliferation and metastasis to the cervical lymph node and lung of mice. Although the paracrine molecular interactions between HNSCC and TAFs have not been fully described, we demonstrated that TAFs (and not HNSCC) secrete hepatocyte growth factor (HGF), a ligand for the c-Met receptor on HNSCC cells. Further, activation of the HGF-c-Met pathway increased HNSCC growth and invasion. In addition, we demonstrated that HNSCC cells induce the expression of HGF from TAFs. We hypothesized that inhibition of TAF-secreted HGF would reduce HNSCC proliferation, invasion and migration. We tested the efficacy of ficlatuzumab (AV-299) a humanized HGF inhibitory immunoglobulin G1 (IgG1) monoclonal antibody in mitigating TAF-induced HNSCC progression. . Ficlatuzumab exhibits antitumor effects in xenograft models as a single agent and has been tested in patients with non-small cell lung cancer in a phase I setting. The antitumor efficacy of HGF neutralization has not been tested in the context of TAFs in HNSCC. Our data demonstrate that ficlatuzumab is effective in mitigating TAF-induced HNSCC proliferation, migration and invasion through inhibition of the c-met pathway in HNSCC.

Citation Format: Chase Hamilton, Shary Shelton, Kiran Kakarala, Sufi Mary Thomas. Tumor-associated fibroblasts: A new target for cancer treatment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A55. doi:10.1158/1538-7445.CHTME14-A55
Abstract A59: A novel bispecific FAP-DR5 antibody inducing potent and tumor-specific death receptor 5 (DR5) activation by fibroblast activation protein (FAP) dependent crosslinking
Authors: Wartha Katharina , Weiser Barbara , Friess Thomas , Majety Meher , Runza Valeria , Herting Frank , Weber Thomas , Scheuer Werner , Harring Suzana Vega , Sade Hadassah , Niu Huifeng
Published: May 2015

Background: Activation of the extrinsic apoptosis pathway in tumor cells through agonistic death receptor 5 (DR5) antibodies has been evaluated in the clinic with limited success so far. In this context, several reports show that DR5 activation is strongly dependent on receptor hyperclustering on the cell surface. Therefore a therapeutic principle that induces DR5 hyperclustering specifically at the tumor site may provide superior efficacy, potency and safety compared to conventional DR5 agonistic antibodies. Fibroblast activation protein (FAP) is a marker for activated fibroblasts and abundantly expressed in cancer associated fibroblasts of various epithelial tumor indications and as a tumor antigen on tumors of mesenchymal origin. Due to its relative absence from normal tissues, FAP can be used as a tumor targeting antigen. Here, we are using the broad expression of FAP in tumor stroma for crosslinking of DR5 by a bispecific antibody.

Aim: In order to achieve superior tumor targeting and tumor located DR5 hyperclustering we have generated a bispecific antibody, RG7386, comprised of an agonistic DR5 binder and a FAP targeting moiety.

Results: RG7386 shows potent and selective binding to FAP and DR5 and can simultaneously bind to both targets. In in vitro co-culture assays, using human DLD1 colon tumor cells and FAP expressing fibroblasts, RG7386 induces potent, FAP dependent DR5 hyperclustering and apoptosis induction in DR5 positive tumor cells (IC50: 0.05 nM). In preclinical in vivo models with co-injection of DLD-1 tumor cells and fibroblasts as well as patient-derived colorectal cancer models, RG7386 shows FAP dependent efficacy and apoptosis induction superior to conventional DR5 antibodies. Furthermore the superior induction of apoptosis could be confirmed by in vivo and ex vivo analysis of cleaved Caspase-3 with imaging, Luminex and histopathology.

Conclusion: RG7386 is a promising novel therapeutic entity for the treatment of solid tumors with FAP positive tumor stroma inducing DR5 activation by FAP dependent DR5 hypercrosslinking which results in potent anti-tumor activity.

Citation Format: Katharina Wartha, Barbara Weiser, Thomas Friess, Meher Majety, Valeria Runza, Frank Herting, Thomas Weber, Werner Scheuer, Suzana Vega Harring, Hadassah Sade, Huifeng Niu. A novel bispecific FAP-DR5 antibody inducing potent and tumor-specific death receptor 5 (DR5) activation by fibroblast activation protein (FAP) dependent crosslinking. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A59. doi:10.1158/1538-7445.CHTME14-A59
Abstract A62: TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.
Authors: Zou An , Lambert Diana , Yeh Henry , Fang Wei Bin , Cheng Nikki
Published: May 2015

CXCL1 is a chemokine secreted by macrophages, neutrophils, epithelial cells and fibroblasts, and plays a role in inflammation and wound healing. Enhanced expression of CXCL1 in tumor epithelium is associated with cell invasion, angiogenesis in melanoma, bladder, ovarian and breast cancer. However, little is known about CXCL1 expression in tumor stroma. Through analysis of gene expression data, our studies reveal that high RNA levels of CXCL1 in breast tumor stroma is associated with increased rate of recurrence and shorter relapse-free survival. We noticed decreased secretion levels of TGF-β and increased secretion of CXCL1 in tumor derived fibroblasts. Increased CXCL1 expression was also observed in mammary fibroblasts in which the TGF-β type II receptor was knocked out. By immunohistochemistry analysis we found that expression of CXCL1 inversely correlated with expression of TGF-β, phosphorylated-Smad2 and phosphorylated-Smad3 in breast tumor stroma. We hypothesized that TGF-β suppresses CXCL1 expression in breast fibroblasts. Our goal for this project was to determine the molecular mechanisms through which TGF-β suppresses CXCL1 expression using mammary fibroblasts derived from MMTV-PyVmT mammary tumor model. Treatment of fibroblasts with recombinant TGF-β decreased CXCL1 transcription and secretion by up to 85% over time, as determined by RT-PCR and ELISA. SiRNA silencing of TGF-β signaling transducers, Smad2 and Smad3, increased CXCL1 transcription and secretion in mammary fibroblasts. In addition, TGF-β suppressed expression of HGF, a factor that enhanced CXCL1 expression in mammary fibroblasts. Knockdown of HGF or treatment of fibroblasts with c-Met inhibitors reduced CXCL1 expression. These data indicate TGF-β suppresses CXCL1 expression through Smad2/3-dependent mechanisms. As a secondary mechanism, TGF-β suppresses CXCL1 expression through down-regulation of HGF/c-Met signaling. This is the first report to characterize the mechanisms of CXCL1 expression in mammary fibroblasts and demonstrate a clinical relevance for CXCL1 expression in breast cancer stroma.

Citation Format: An Zou, Diana Lambert, Henry Yeh, Wei Bin Fang, Nikki Cheng. TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A62. doi:10.1158/1538-7445.CHTME14-A62
Abstract A73: AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer
Authors: Lin Yu-Chin Lin , Wu Meng-Hsuan , Wei Tzu-Tang , Lin Yun-Chieh , Chen Ching-Chow
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a Bcr-abl and Src in hibitor, has been approved clinically for CML and shown activities against solid tumors in vitro. In our recent work, degradation of epidermal growth factor receptor (EGFR) plays a role in dasatinib-induced apoptosis in HNSCC cells. We further explored the mechanism of this event. ER stress was induced by dasatinib and played a role in dasatinib-EGFR degradation. C-cbl activation was induced by ER stress and shown to mediate ER-stress induced EGFR degradation. In addition, AMPK activation was induced by dasatinib, which was proved to be involved in dasatinib-induced ER stress and EGFR degradation. Activation of AMPK with metformin not only enhanced dasatinib-induced growth inhibition and apoptosis but also sensitized dasatinib-resistant cells. Furthermore, xenograft model showed that metformin potentiated anti-cancer effect of dasatinib through activation of AMPK and ER stress. Our results disclose that AMPK-dependent ER stress plays a crucial role in the anti-cancer effect of dasatinib in HNSCC and further activation of AMPK by metformin might enhance dasatinib efficacy.

Citation Format: Yu-Chin Lin Lin, Meng-Hsuan Wu, Tzu-Tang Wei, Yun-Chieh Lin, Ching-Chow Chen. AMPK activation mediates dasatinib-induced EGFR degradation and apoptosis in head and neck cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A73. doi:10.1158/1538-7445.CHTME14-A73
Abstract B09: Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer
Authors: Beach Jessica A. , Aspuria Paul-Joseph , Cheon Dong-Joo , Gozo Maricel C. , Karlan Beth Y. , Orsulic Sandra
Published: May 2015

Sphingosine kinase-1 (SPHK1) is an enzyme that catalyzes the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the proapoptotic lipid sphingosine. The balance between sphingosine and S1P forms a sphingolipid rheostat that is primed for cell death when the balance shifts towards sphingosine/ceramide or to cell survival when S1P levels are increased. Elevated levels of S1P have been observed in several cancers, including ovarian cancer where it is elevated in both the ascites and serum of patients. Correspondingly, high expression of SPHK1 has also been described in multiple cancer types and has been linked to disease progression. Here we report that SPHK1 is overexpressed in a subset of epithelial ovarian cancers (EOC) and correlates with poor progression-free survival. Overexpression and knockdown of SPHK1 in multiple human EOC cell lines modulates in vitro cell proliferation, anchorage-independent growth, and chemosensitivity. In mouse xenograft studies, intraperitoneal administration of a SPHK1-specific inhibitor (SKI-5c) decreases tumor size, indicating that SPHK1 may be a potential therapeutic target in EOC. S1P is a secreted factor that has been shown to influence the tumor microenvironment of other cancers; however its role in EOC has not been fully established. Using a co-culture model, we show that overexpression of SPHK1 in EOC cells stimulates the transition of normal ovarian stromal fibroblasts to myofibroblasts, and enhances stromal SPHK1 expression. In a reciprocal manner, overexpression of SPHK1 in stromal fibroblast results in increased expression of MMP2 and MMP9 in EOC cells, which may contribute to a more invasive phenotype. Interestingly, stromal SPHK1 expression also enhanced expression of SPHK1 and the S1P receptor (S1PR1) in the EOC cells. Biostatistical analysis suggests a significant correlation between SPHK1 expression in EOC cells and the expression of several extracellular matrix genes (FN1, POSTN, VCAN), which have been shown to have role in the EOC tumor microenvironment. Overall, these results suggest that SPHK1 is a critical regulator of ovarian tumor cell proliferation and survival, and a mediator of tumor-stroma interaction.

Citation Format: Jessica A. Beach, Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel C. Gozo, Beth Y. Karlan, Sandra Orsulic. Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B09. doi:10.1158/1538-7445.CHTME14-B09
Abstract B14: Activin A regulates cell interactions in the microenvironment of oral squamous cell carcinomas
Authors: Coletta Ricardo D. , Bufalino Andreia , Sobral Lays M. , Rodrigues Priscila C. , Graner Edgard , Kowalski Luis P. , Salo Tuula
Published: May 2015

Although the molecular mechanisms by which carcinoma-associated fibroblasts (CAFs) influence oral squamous cell carcinomas (OSCC) remain largely unknown, previous studies demonstrated that presence of CAFs in the stroma of OSCCs is an important risk factor of patient's shortened survival. Here we showed that activin A is produced in higher levels by CAFs compared to normal oral fibroblasts, and that activin A released by CAFs significantly increases OSCC cell proliferation and xenograft tumor volume, whereas down-regulation of activin A decreases OSCC proliferation. Furthermore, activin A reduced OSCC cell death even when challenged by staurosporine, and promoted migration, invasion and the expression of epithelial-mesenchymal transition (EMT) markers (decreases E-cadherin and increases vimentin and N-cadherin) of OSCC cells. Immunohistochemical analysis of 115 OSCC samples revealed that increased activin A expression is significantly correlated with presence of regional metastasis (N stage, p=0.034), poorly differentiated tumors (p=0.013) and shown to be predictive of a shortened disease-free survival (HR: 1.74, 95% CI: 1.39-2.97, p=0.016). Importantly, immunohistochemical expression of activin A was significantly associated with presence of occult lymph node metastasis (p=0.006), which has an important impact on clinical management and prognostication of patients with OSCCs. In conclusion, our data demonstrate that activin A is an important molecule for OSCCs, and its pleiotropic effects on proliferation and invasion are important on control of the aggressiveness of OSCCs.

Citation Format: Ricardo D. Coletta, Andreia Bufalino, Lays M. Sobral, Priscila C. Rodrigues, Edgard Graner, Luis P. Kowalski, Tuula Salo. Activin A regulates cell interactions in the microenvironment of oral squamous cell carcinomas. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B14. doi:10.1158/1538-7445.CHTME14-B14
Abstract B15: Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells
Authors: Chang Megan , Morgado Micaela , Patel Viral , Gwede Michael , Farach-Carson Mary Cindy , Delk Nikki
Published: May 2015

It is well established that prostate cancer (PCa) cells hone to, adapt, and thrive in the bone. Importantly, bone metastases are found in over 80% of PCa deaths. Therefore, it is imperative to elucidate and characterize the molecular mechanisms that enable PCa cells to thrive in the bone and resist conventional PCa therapies, such as hormone ablation or anti-androgens. We found that the HS-5 bone marrow stromal cell (BMSC) line, a secretory BMSC line that can support hematopoietic stem cell growth, secretes paracrine factors that induce apoptosis in PCa cells. However a subpopulation of the PCa cells can survive the BMSC-induced death. The surviving PCa subpopulation differentiates into a neuronal morphology and looses androgen receptor (AR) expression, reminiscent of treatment-resistant neuroendocrine PCa. Ideally, the loss of AR would induce PCa cell death; however we observe a concomitant increase in the autophagy-related cell survival protein, p62/SQSTM1, and induction of cytoprotective autophagy. Furthermore, we find that interleukin-1 beta (IL-1β) and IL-6, inflammatory cytokine secreted by HS-5 BMSCs, are sufficient to promote PCa neuronal morphology, reduce AR accumulation, upregulate p62/SQSTM1 levels, and induce autophagy. In addition, genetic silencing of p62/SQSTM1 causes PCa cell death and pharmacological inhibition of cytokine or autophagy signaling attenuates PCa neuroendocrine differentiation. Thus, our PCa-bone marrow stromal cell model suggests that PCa cells can co-opt the immune system in the bone microenvironment leading to PCa androgen independence, treatment resistance, and survival in the bone. Furthermore, our data suggests that inhibiting inflammatory cytokine signaling and/or the autophagy process are rational strategies to combat PCa bone metastasis.

Citation Format: Megan Chang, Micaela Morgado, Viral Patel, Michael Gwede, Mary Cindy Farach-Carson, Nikki Delk. Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B15. doi:10.1158/1538-7445.CHTME14-B15
Abstract B17: Exploiting evolution to develop better personalized therapeutic strategies
Authors: Gallaher Jill A. , Anderson Alexander R. A.
Published: May 2015

Targeted drugs have been emerging as the future direction for personalized medicine. These drugs focus on blocking specific pathways identified by genetic alterations, but often a single biopsy reveals only a portion of the genetic heterogeneity. Even upon complete characterization of a particular tumor, successful treatment relies on targeting all of the major driver mutations, which furthermore have a possibility to fail due to resistance. With a very heterogeneous disease, this is a point-and-shoot approach with many moving targets.

While the genotypes become more heterogeneous over time, the phenotypes tend to converge as the environment selects for the most fit subtypes of cells. And while genetic mutation happens by chance, phenotypic adaptation in response to environmental changes are more predictable. By focusing on phenotypic traits, we are targeting the highest level of functional cell behavior. But even at this scale there is complexity. For example, how should a phenotype propagate - do all clones share the same phenotype, or is there some plasticity? The answer affects how the right timing and sequencing of drugs could shape the population into a more targetable one instead of into a mutationally resistant one.

To explore this tangled web of genotypic and phenotypic progression and selection, we build an agent based model. This approach allows us to investigate interactions of single cells and evolution of the collective population in an environment with spatial competition. Utilizing this agent based approach we investigate how both sequence and timing of different therapeutic methods alter the intratumor heterogeneity both at the genotypic and phenotypic scales.

Citation Format: Jill A. Gallaher, Alexander R. A. Anderson. Exploiting evolution to develop better personalized therapeutic strategies. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B17. doi:10.1158/1538-7445.CHTME14-B17
Abstract B26: Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.
Authors: Khialeeva Elvira , Chou Joan W. , Carpenter Ellen M.
Published: May 2015

We have investigated the role of reelin signaling in the microenvironment of breast cancer and found that reelin is essential for progression of mammary tumors to metastasis. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate, while mouse mammary carcinoma 4T1 cells lose this response, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into wild type Balb/C mice resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. However, when these cells were injected into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice), we saw a dramatic reduction in lung metastases despite the presence of primary tumors comparable in size to the tumors formed in wild type mice. We show that this phenotype is dependent upon loss of reelin in the host environment, since tumor cells isolated from reeler mutant mice produce primary tumors and lung metastases when injected into Balb/C mice. Using soft agar colony assays and Western blotting, we show that the inability of 4T1 cells to metastasize in mutant animals might be due to a reduced capacity of mammary carcinoma cells to undergo epithelial to mesenchymal transition (EMT) in an environment deficient for reelin signaling. In addition, tumors from reeler and Dab1 mutant mice appear to have more differentiated cells as shown by anti-α smooth muscle Actin and anti-K14 immunostaining. Ki-67 staining showed no differences in proliferation between tumors from wild type, reeler or Dab1 mutant mice. Finally, tail vein injections of 4T1 cells into reeler mutant mice also fail to produce metastases, indicating that reelin signaling in the host environment might have important functions at several steps of the metastatic process. Our results implicate reelin signaling as an essential component of transition from the primary tumor to a metastatic carcinoma.

Citation Format: Elvira Khialeeva, Joan W. Chou, Ellen M. Carpenter. Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B26. doi:10.1158/1538-7445.CHTME14-B26
Abstract B35: The role of tumor associated macrophages (TAMs) in triple-negative breast cancer (TNBC) invasion revealed by species-specific RNA sequencing
Authors: Rabe Daniel C. , Frankenberger Casey A. , Bainer Russell , Sankarasharma Devipriya , Chada Kiran , Krausz Thomas , Gilad Yoav , Rosner Marsha Rich
Published: May 2015

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. While five-year survival rates have reached 98% in patients treated with anti-ER or anti-HER2 therapies, patients with TNBC have a five-year survival rate of only 24%. Currently, the only form of therapy for these patients is surgery and platinum based chemotherapy. However, patient outcome is generally poor. Additionally, this disease disproportionately affects African-American women and lower income women, with rates seen approximately three times higher in African-American women compared to the rest of the population. An alternative strategy for treating TNBC patients involves targeting the tumor stroma. To better understand interaction between the tumor and stroma necessary for metastasis and invasion, we employed a triple-negative breast cancer (TNBC) model in which the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) controls primary tumor invasiveness. RKIP expression, which converts invasive tumors to non-invasive tumors, dramatically inhibits macrophage infiltration. The mechanism, which is dependent on Let-7 suppression of HMGA2, involves decreased expression of the chemokine CCL5. Furthermore, overexpression of CCL5 partially rescued the infiltration of macrophages into the tumor and intravasation of tumor cells into the blood stream. The relationships of the genes in the RKIP, HMGA2, CCL5, and macrophage pathways were observed in multiple sets of expression array data from breast cancer patients. Regulation of macrophage infiltration was observed in tumors from an HMGA2 knockout mouse model. These results show that RKIP regulates macrophage recruitment by tumors and demonstrate for the first time that metastasis suppressor genes can regulate the tumor microenvironment.

Citation Format: Daniel C. Rabe, Casey A. Frankenberger, Russell Bainer, Devipriya Sankarasharma, Kiran Chada, Thomas Krausz, Yoav Gilad, Marsha Rich Rosner. The role of tumor associated macrophages (TAMs) in triple-negative breast cancer (TNBC) invasion revealed by species-specific RNA sequencing. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B35. doi:10.1158/1538-7445.CHTME14-B35