bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2611: The developmental patterning gene HOXA9 promotes ovarian cancer growth via paracrine effects on mesenchymal stem cells and peritoneal fibroblasts.
Authors: Ko Song Yi , Barengo Nicolas , Ladanyi Andras , Lee Ju-Seog , Marini Frank , Lengyel Ernst , Naora Honami
Published: August 2014

As is the case in normal organogenesis, it is increasingly recognized that tumor growth is regulated by cross-talk between epithelial and stromal cells. Whereas the ability of cancer-associated fibroblasts (CAFs) to stimulate tumor cell growth has been extensively studied, it is not clear whether CAFs are reciprocally controlled by developmental programs that are activated in tumor cells. Epithelial ovarian cancers (EOCs) frequently colonize peritoneal surfaces that overlie connective and adipose tissues. We found that expression of HOXA9, a homeobox gene that is normally expressed in the developing female reproductive tract, is strongly associated with poor outcomes in EOC patients and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. Expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts and adipose mesenchymal stem cells (MSCs) to express CAF markers and to stimulate growth of EOC and endothelial cells. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and adipose MSCs to induce CXCL12, IL-6 and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Citation Format: Song Yi Ko, Nicolas Barengo, Andras Ladanyi, Ju-Seog Lee, Frank Marini, Ernst Lengyel, Honami Naora. The developmental patterning gene HOXA9 promotes ovarian cancer growth via paracrine effects on mesenchymal stem cells and peritoneal fibroblasts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2611. doi:10.1158/1538-7445.AM2013-2611
Abstract 2612: Tumor associated microvesicles confer neoplastic transformation of patient derived adipose stem cells .
Authors: Abd Elmageed Zakaria Y. , Yang Yijun , Thomas Raju , Moparty Krishnarao , Ranjan Manish , Mondal Debasis , Moroz Krzysztof , Sartor Oliver , Abdel-Mageed Asim B.
Published: August 2014

Background: Unraveling the underlying mechanisms of prostate tumor growth and metastasis is critical to developing curative therapies currently unavailable for castration-resistant prostate cancer (CRPC). Obesity has been linked to increased risks of PC progression. Emerging evidence suggests that adipose-derived stem cells (ASCs) are often recruited to and promote tumor growth via unknown mechanisms.

Methods: Patient derived ASCs (pASCs) with tumor homing potential were enriched and treated with condition media (CM) or microvesicles (MVs) of PC (C4-2B, PC-3) or normal prostate epithelial (RWPE1) cells. CM or MV-primed pASCs were examined for their ability to form prostate tumors in vivo. Tumor formation was validated by histopathologic analysis, expression of neoplastic and vasculogenic markers, karyotyping and PC specific markers. LC/MS-MS, miRNA array, miRNA mimics and qRT-PCR analyses were employed to decipher the underling mechanistic roles of oncogenic RNAs and proteins of PC cell associated MVs in pASC tumor formation.

Results: Herein, we report that PC cell microenvironment induces genotypic and phenotypic changes in pASCs and subverts them to undergo neoplastic transformation. Unlike normal counterparts, the pASCs primed with PC cell CM form neoplastic lesions in vivo and reproduce aggressive tumors in secondary recipients. The pASC tumors acquire cytogenetic aberrations and express epithelial, neoplastic and vasculogenic markers reminiscent of molecular features of PC tumor xenografts and mesenchymal-to-epithelial transition (MET). MVs derived from PC cell CM recapitulated prostate tumorigenic mimicry by pASCs in vivo. The oncogenic ‘reprogramming’ of pASCs is associated with PC cell-derived MVs transfer of oncomiRNAs (miR-125b, miR-130b, miR-155), H-Ras and K-Ras transcripts and Ras-related oncoproteins (RAB1A, RAB2A, RAB1B, RAB11A) and down-regulation of tumor suppressors Lats2 and PDCD4.

Conclusion: Our findings establish previously uncharacterized mechanisms for tumor-derived MV-pASC axis in tumor formation, phenotypic variations in mesenchymal and epithelial states in tumor microenvironment, and in promoting tumor burden and metastasis in cancer patients.

Citation Format: Zakaria Y. Abd Elmageed, Yijun Yang, Raju Thomas, Krishnarao Moparty, Manish Ranjan, Debasis Mondal, Krzysztof Moroz, Oliver Sartor, Asim B. Abdel-Mageed. Tumor associated microvesicles confer neoplastic transformation of patient derived adipose stem cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2013-2612
Abstract 2613: Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors.
Authors: Jachetti Elena , Mazzoleni Stefania , Grioni Matteo , Ricupito Alessia , Brambillasca Chiara , Generoso Luca , Calcinotto Arianna , Freschi Massimo , Mondino Anna , Galli Rossella , Bellone Matteo
Published: August 2014

Purpose. Objectives of this study were to investigate if prostate cancer stem/initiating cells (CSC) obtained from autochthonous tumors possess the molecular characteristics that allow their in vitro and in vivo recognition by cells of the innate and/or adaptive arms of the immune system, and if CSC are source of antigens for the induction of tumor-specific immune responses.

Material and Methods. CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were assessed in vitro for the expression of tumor-associated antigens and susceptibility to NK and T cell killing. We also investigated NK and T cells immune surveillance against CSC by measuring the frequency of CSC-induced tumors in immunocompetent and selectively immunodeficient mice. Finally, we assessed if vaccination with dendritic cells pulsed with CSC induced a tumor-specific immune response able to delay the growth of both transplantable and autochthonous prostate tumors.

Results. CSC expressed prostate cancer associated antigens, MHC I and MHC II molecules and ligands for natural killer (NK) cells. Indeed, CSC were targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with dendritic cells pulsed with apoptotic CSC induced a tumor-specific immune response that delayed tumor growth in mice challenged with prostate CSC, and caused tumor regression in TRAMP mice.

Conclusions. CSC are targets of both innate and adaptive immune responses and could be exploited for the design of novel immunotherapeutic approaches against cancer.

Citation Format: Elena Jachetti, Stefania Mazzoleni, Matteo Grioni, Alessia Ricupito, Chiara Brambillasca, Luca Generoso, Arianna Calcinotto, Massimo Freschi, Anna Mondino, Rossella Galli, Matteo Bellone. Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2613. doi:10.1158/1538-7445.AM2013-2613
Abstract 262: The evolution of diverse cancer stem cells in human liver cancer.
Authors: Yamashita Taro , Honda Masao , Nio Kouki , Hara Yasumasa , Hayashi Takehiro , Oishi Naoki , Sunakozaka Hajime , Takatori Hajime , Kaneko Shuichi
Published: August 2014

Cancer stem cells (CSCs) are considered a pivotal target for the eradication of liver cancer. CSCs have been identified by the use of various stem cell markers; however, it remains unclear how individual CSC markers are expressed in individual tumors. We hypothesized that the distinct marker-positive CSCs show unique features of tumorigenicity/metastasis and chemosensitivity which may reflect the heterogeneous nature of hepatocellular carcinoma (HCC). Gene expression profiling and immunohistochemistry analyses were used to analyze 364 tumor specimens. Fluorescence-activated cell sorting (FACS) was used to isolate marker-positive HCC cells, which were tested for hepatic stem/progenitor cell properties. CSC markers EpCAM and CD90 are independently expressed in primary liver cancer. EpCAM+ cells share features with tumorigenic epithelial stem cells, whereas CD90+ cells share those of metastatic vascular endothelial cells with expression of c-Kit. CD90+ cells facilitate metastasis of EpCAM+ cells through activation of TGF-beta signaling, and this effect is abolished by c-Kit inhibitor imatinib mesylate. These distinct tumorigenic/metastatic CSCs can evolve from marker-negative cells de novo, and their frequencies vary dramatically according to the genomic alterations and genotoxic stress. Our results suggest that liver CSCs are not a single, static entity but rather exist heterogeneously and undergo dynamic changes through genomic alteration and genotoxic stress. The evolution of CSCs explains the enrichment of CSCs in advanced cancer and the limitation of targeting marker-positive CSCs alone for tumor eradication.

Citation Format: Taro Yamashita, Masao Honda, Kouki Nio, Yasumasa Hara, Takehiro Hayashi, Naoki Oishi, Hajime Sunakozaka, Hajime Takatori, Shuichi Kaneko. The evolution of diverse cancer stem cells in human liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 262. doi:10.1158/1538-7445.AM2013-262
Abstract 2620: Progressive expression of n-cadherin and epidermal growth factor receptor in colorectal adenocarcinoma.
Authors: Katkoori Venkat R. , Manne Upender , Basson Marc D. , Bumpers Harvey L.
Published: August 2014

Purpose: N-cadherin (NCAD) and epidermal growth factor receptor (EGFR) have been found to be inducers of epithelial-to-mesenchymal transition that are known to play a role in invasion and metastasis of cancer. Over expression of NCAD and EGFR are associated with increased tumor invasiveness. However, their progressive expression in colorectal cancer (CRC) has not been studied. Therefore, in the present study, we investigated expression status of NCAD and EGFR in paired lesions of CRC. Experimental Design: We examined the expression of NCAD and EGFR in paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft by immunofluorescence (IF). Images were acquired using the Olympus FluoView FV1000 (OFVFV1000) Confocal Microscope. The average FITC fluorescence intensity (aFFI) was measured by OFVFV1000 software. Western blot analysis was performed to validate results obtained from IF method.

Results: Differential expression pattern of NCAD was observed in paired normal, primary and metastatic lesions of hCRC and hmCRC xenograft tissues. For NCAD, it was mainly localized in cytoplasm (diffused), nucleus (moderate levels), and membrane or cell junctions of CRC. For EGFR, normal colon tissue or primary CRC showing membranous EGFR-low positive staining. In contrast corresponding liver metastasis showing membranous EGFR-high positive staining. The NCAD and EGFR expressions were correlated with metastasis phenotype of hCRC. The expressions of NCAD ((aFFI; 812 vs. 261) and EGFR (aFFI 421 vs.131) were each found to be 3 times higher in liver metastasis of hCRC as compared to primary tumor. It has been observed that the expression of NCAD was substantially increased in metastatic lesion of hmCRC xenograft as compared to primary lesion of hmCRC xenograft.

Conclusion: This study revealed that upregulation of NCAD and EGFR expressions is a mechanism by which CRC lesions acquire a more aggressive phenotype and, thereby, progress to a more advanced stage of CRC. Furthermore, our findings are likely to have implications in the design of clinical trials targeting the NCAD and EGFR proteins in CRC.

Citation Format: Venkat R. Katkoori, Upender Manne, Marc D. Basson, Harvey L. Bumpers. Progressive expression of n-cadherin and epidermal growth factor receptor in colorectal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2013-2620
Abstract 2622: TMEFF2 inhibits human prostate cancer cell migration through its G protein activating domain.
Authors: Chen Xiaofei , Justus C. , Yang L , Ruiz-Echevarria M.j.
Published: August 2014

The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate. Expression of TMEFF2 is deregulated in a significant fraction of primary and metastatic prostate, suggesting a role in this disease. In fact, we have previously shown that TMEFF2 can function as a tumor suppressor in vitro, inhibiting monolayer and anchorage-independent cellular growth in HEK293T cells and sarcosine-induced cell migration and invasion in benign prostate epithelial RWPE-1 cells. However, the molecular mechanisms involved in the tumor suppressor phenotype of TMEFF2 are not clear. TMEFF2 has several biologically important features. The extracellular domain contains two follistatin domains and an epidermal growth factor-like (EGF) domain, while the transmembrane domain and short cytoplasmic tail have features resembling a G protein activating domain, suggesting that it may signal through a G protein. We hypothesize that the tumor suppressor activity of TMEFF2 is, at least in part, due to signaling mediated by the putative G protein activating domain. In order to test this hypothesis, here we investigate the role of TMEFF2 and a mutant lacking the cytoplasmic tail on cellular migration of prostate cancer cells, and begin analyzing the molecular mechanisms involved in this effect.

Our results demonstrate that overexpression of TMEFF2 in human prostate cancer cell line RWPE-2 causes about 60% decrease in cellular migration as assessed by wound-healing assays. Interestingly, we found that RWPE-2 cells overexpressing TMEFF2 have a defect in cell spreading on culture dishes (∼20% decrease) as well as on vitronectin-coated coverslips (∼30% decrease) and that this defect is accompanied by abolished focal adhesion and stress fiber formation. Moreover, the inhibition of TMEFF2 on cellular migration is dependent on its G protein activating domain as deletion of this domain rescues migration, spreading on vitronectin, and focal adhesion formation. The specific G protein pathway(s) involved in the anti-migratory effect of TMEFF2 is currently under investigation.

In summary, the data presented here indicate, for the first time, that the anti-migratory effect of TMEFF2 in human prostate cancer cells is mediated by its G protein activating domain. These results raise the possibility that TMEFF2 agonists may be used as therapeutic agents to mediate tumor suppression via this pathway.

Citation Format: Xiaofei Chen, C. Justus, L Yang, M.j. Ruiz-Echevarria. TMEFF2 inhibits human prostate cancer cell migration through its G protein activating domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2622. doi:10.1158/1538-7445.AM2013-2622
Abstract 2623: Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells.
Authors: Randle Diandra D. , Marah Valerie
Published: August 2014

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -β (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Androgen-dependent LNCaP and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. Superarray analysis revealed an upregulation in uPAR expression in Snail-transfected cells as compared to Neo control. Next, the protein expression levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Also, uPAR was transiently silenced in ARCaP Snail-overexpressing cells using uPAR siRNA. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, and uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP Snail-overexpressing cells, as compared to Neo controls. Additionally, the silencing of uPAR decreased cell invasion in ARCaP Snail-overexpressing cells. Finally, MAPK inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human prostate cancer cells may occur via the regulation of the MAPK and uPA/ uPAR signaling pathway.

Acknowledgements: These studies were supported by the: NIH/NCRR/RCMI grant G12RR03062 and NIH P20 grant 1P20MD002285.

Citation Format: Diandra D. Randle, Valerie Marah. Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2013-2623
Abstract 2625: Knockdown of death receptor 5 promotes cancer cell invasion through activation of the ERK and JNK signaling pathways.
Authors: Oh You-Take , Yue Ping , Khuri Fadlo R. , Sun Shi-Yong
Published: August 2014

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor and can trigger apoptosis upon overexpression or ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Despite of the primary function of DR5 in induction of apoptosis, accumulating evidence suggest that DR5 may also have non-apoptotic function in regulation of cancer development and progression, which is the focus of this study. Both transient and stable knockdown of DR5 did not affect the growth of cancer cells, but significantly promoted the invasive ability of cancer cells in an in vitro matrigel invasion assay. In contrast, DR5 activation by low doses of the agonistic antibody, AMG655, suppressed cancer cell invasion. Similar results could not generated by knocking down DR4 expression. Thus, DR5 negatively regulates invasion of cancer cells. Knockdown of DR5 expression was accompanied by activation of both ERK and JNK signaling pathways as evidenced by elevate levels of p-ERK, p-JNK and p-c-Jun as well as their downstream proteins c-Jun and Fra-1. Chemical (e.g., with small molecule inhibitors) or genetic (e.g., with siRNA) inhibition of either ERK or JNK blocked DR5 knockdown-induced enhancement of cell invasion. In agreement, knockdown of c-Jun and Fra-1 had similar impact on blockage of cell invasion induced by DR5 knockdown. Hence, DR5 knockdown promotes cancer cell invasion through activation of the ERK and JNK signaling pathways and subsequent activation of AP-1. Our findings highlight a novel non-apoptotic function of DR5 as a negative regulator of cancer cell invasion and provide preliminary mechanisms to account for this function.

(This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH/NCI SPORE P50 grant CA128613 and Winship Cancer Institute Robbins Scholar award. FR Khuri and SY Sun are Georgia Cancer Coalition Distinguished Cancer Scholars. YT Oh is a recipient of Winship Cancer Institute Robbins Scholar award)

Citation Format: You-Take Oh, Ping Yue, Fadlo R. Khuri, Shi-Yong Sun. Knockdown of death receptor 5 promotes cancer cell invasion through activation of the ERK and JNK signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2013-2625
Abstract 2639: MicroRNA miR-99a exerts anti-tumorigiceffect through decreasing myotubularin related protein 3 expression in oralcancer.
Authors: Wu Li-Wha
Published: August 2014

Head and neck cancer is the six most common carcinomas worldwide. Oral cancer, one head and neck cancer subtype, is the fourth leading causes of male cancer death in Taiwan.

The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. MicroRNAs are noncoding RNA molecules in a length of 19∼25 nucleotides, which can mediate gene expression by base pairing to the 3’untralslated region of target mRNAs. Using human microRNA microarray analysis, we found that miR-99a was most down-regulated among 1146 miRs in oral cancer cells when compared to normal oral keratinocytes. Real-time

PCR analysis further confirmed the down-regulation of miR-99a both in both tested oral cancer cell lines and human clinical specimens. Transient and stable expression of miR-99a inhibited the migration and invasion of oral cancer cells while having differential effects on cell proliferation depending on the cell types. The introduction of anti-miR-99a, silencing the effect of miR-99a, led to the increase of cell migration and invasion. Luciferase reporter assays and site-directed mutagenesis identified MTMR3, a member of myotubularin-related genes encoding phosphatidylinositol 3-phosphate 3-phosphatases, to be a downstream target of miR-99a. Knocking down MTMR3 expression in high

MTMR3-expressing oral cancer cells significantly impaired the ability of oral cancer cells to proliferate, migrate or invade Matrigel, suggesting an oncogenic role of MTMR3 in oral cancer cells. Together, our findings suggest that miR-99a plays a repressive role in cell migration and invasion partly through regulating MTMR3 expression in oral cancer cells.

Citation Format: Li-Wha Wu. MicroRNA miR-99a exerts anti-tumorigiceffect through decreasing myotubularin related protein 3 expression in oralcancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2013-2639
Abstract 2642: Multiple roles of p53-related pathways in somatic cell reprogramming and stem cell differentiation.
Authors: Yi Lan , Lu Chiwei , Hu Wenwei , Levine Arnold J.
Published: August 2014

The inactivation of p53 functions enhances the efficiency and decreases the latency of producing induced pluripotent stem cells (iPSC) in culture. The formation of iPSCs in culture starts with a rapid set of cell divisions followed by an epigenetic reprogramming of the DNA and chromatin. The mechanisms by which the p53 protein inhibits the formation of iPSCs are largely unknown. Employing a temperature sensitive mutant of the p53 (Trp53) gene, we examined the impact of the temporal expression of wild type p53 in preventing stem cell induction from somatic cells. We also explored how different p53 mutant alleles affect the reprogramming process. We found that little or no p53 activity favors the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs, but also induced newly formed stem cells to differentiate. Among p53 regulated genes, p21 (Cdkn1a), but not Puma (Bbc3) played a partial role in iPSCs formation probably by slowing cell division. Activation of p53 functions in iPS cells induced senescence and differentiation in stem cell populations. High rate of birth defects and increases in DNA methylation at the IGF2-H19 loci in female offspring of p53 knockout mice suggested that the absence of p53 may give rise to epigenetic instability in a stochastic fashion. Consistently, selected p53 missense mutations showed differential effects on the stem cell reprogramming efficiency in a c-Myc dependent manner. The absence of p53 activity and functions also contributed to an enhanced efficiency of iPSC production from cancer cells. The production of iPSCs in culture from normal and cancer cells, while different from each other in several ways, both responded to the inhibition of reprogramming by the p53 protein.

Citation Format: Lan Yi, Chiwei Lu, Wenwei Hu, Arnold J. Levine. Multiple roles of p53-related pathways in somatic cell reprogramming and stem cell differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2013-2642
Abstract 2644: In vivo and in vitro stem cell models suggest a role for LGR5 in regulating intestinal epithelial lineage specification.
Authors: Williams Courtney M. , Harper Jessica L. , Huang Hui , Boland Pat , Zhang Qing , Yancopoulos George D. , Li Zhe , Thurston O. Gavin
Published: August 2014

The stem cell marker Leucine Rich Repeat Containing G-protein Coupled Receptor 5 (LGR5) is frequently upregulated in human colon cancer. Numerous observations implicate LGR5 in stem cell maintenance during intestinal development, tissue homeostasis and cancer, however the precise role of LGR5 in these processes is still unclear. To explore the role of LGR5, we generated mouse embryonic stem cells (ESCs) in which one or both alleles of Lgr5 were deleted and replaced with either β-galactosidase or green fluorescent protein (gfp) reporter genes, or the human LGR5 gene. These cell lines were used in three complimentary systems to differentiate and/or isolate cell lineages. The first system was an in vivo model in which subcutaneous xenografts of murine ESCs grew and formed tumors (teratomas) containing cellular structures from all three germ cell layers. Immunostaining showed that a subset of E-cadherin-positive epithelial cells also expressed Lgr5 reporter genes. Teratomas derived from Lgr5 null ESCs showed an increase in E-cadherin-positive epithelial structures, and gene expression profiling of Lgr5 null teratomas showed an upregulation of epithelial genes including Cdh1, Epcam, Cldn7, Krt5, and Krt14 compared with control teratomas. In contrast, ESCs constitutively overexpressing a secreted form of LGR5 ectodomain formed teratomas with fewer epithelial structures and reduced expression of epithelial genes compared to control teratomas. The second system used in vitro differentiation of ESCs towards an intestinal epithelial lineage, followed by 3D culturing, to derive intestinal organoids. Lgr5+/+ (WT), Lgr5lacZ/+ (Het), or Lgr5lacZ/gfp (Null) ESCs are being examined for their ability to adopt an intestinal fate and form organoids in vitro. A third system used ex vivo culture of murine intestinal crypts isolated from humanized LGR5 heterozygous mice to form intestinal organoids. In summary, the morphological and gene expression changes observed in teratomas implicate a role of LGR5 in regulating epithelial tissue homeostasis. The ability to grow and manipulate organoid cultures ex vivo/in vitro will allow us to use various soluble reagents and detailed microscopy to further assess the role of the Lgr5 pathway in intestinal cell lineage specification as well as tumorigenesis.

Citation Format: Courtney M. Williams, Jessica L. Harper, Hui Huang, Pat Boland, Qing Zhang, George D. Yancopoulos, Zhe Li, O. Gavin Thurston. In vivo and in vitro stem cell models suggest a role for LGR5 in regulating intestinal epithelial lineage specification. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2013-2644
Abstract 2645: Role of embryonic signaling pathways in lung cancer.
Authors: Singh Samer , Robbins David , Capobianco Anthony J. , Nguyen Dao M.
Published: August 2014

Background: Embryonic signaling pathways that play an important role during embryo development, later on support the maintenance of adult stem cells and are also found activated in various cancers. The activated embryonic signaling mainly Hedgehog (HH), WNT and NOTCH have been shown to be activated in a number of cancers and demonstrated to promote growth, provide survival advantage and resistance to various drugs. The role of these embryonic signaling pathways in the development of lung cancer is not well defined. Identification of particular pathways active in cancer and elucidation of their mechanism of action in cancer development could provide the basis of devising a successful therapeutic intervention.

Objective: Define the role of embryonic signaling pathways in cancer cells.

Methodology: The presence and functional interaction of various embryonic signaling pathways in lung cancer was evaluated using human non-small cell lung carcinoma cell lines (e.g. HOP62, A549, H23, H522) as model system. The signaling pathways were inhibited using specific small molecule inhibitors and shRNAs mediated knockdown of the pathway components. The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. The effect on the target gene expression (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), cell migration (scratch/wound assay) and anchorage independent growth (soft agar assay), was evaluated. Currently, the activation of pathways in cancer cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH).

Preliminary Results and Conclusions: Attenuation of individual embryonic signaling pathways using specific small molecule inhibitors and shRNAs, indicated crosstalk and hierarchy of these pathways in controlling various processes in tested cancer cells. Our results suggest the predominance of HH signaling in non-small cell lung carcinoma. We are in the process of expanding our observation to more cell lines and human cancer samples. Our work will establish the role of embryonic signaling pathways in lung cancer and would further help in establishing a framework for future therapeutic intervention.

Citation Format: Samer Singh, David Robbins, Anthony J. Capobianco, Dao M. Nguyen. Role of embryonic signaling pathways in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2645. doi:10.1158/1538-7445.AM2013-2645

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2647: In vitro niche created by cancer stem-like cells derived from mouse induced pluripotent stem cell.
Authors: Mizutani Akifumi , Matsuda Shuichi , Yan Tin , Sota Tatsuyuki , Kasai Tomonari , Kudoh Takayuki , Murakami Hiroshi , Seno Masaharu
Published: August 2014

Cancer stem cell (CSC) is a cell within tumor, which is considered to possess the capacity of self-renewal and differentiation for providing heterogeneous lineages of cancer cells in the tumor. Previously, we converted mouse iPS (miPS) cells into CSCs by treatment with conditioned medium prepared from Lewis Lung Carcinoma culture (miPS-LLCcm)1. RT-PCR and FACS analysis revealed that miPS-LLCcm could give rise to endothelial cells. When miPS-LLCcm were cultured on MatrigelTM, they exhibited the tubular structure containing CD31 positive cells. In contrast, undifferentiated cells enriched population, which were resistant to puromycin (puro) as parental miPS cell2, failed to form tubes. After puro was removed to allow the differentiate cells to survive, the tube formation was observed again, indicating that the differentiated cells were likely to contribute to tube formation. To confirm the stability of this tube formation capacity, we repeated further two cycles of puro selection and releasing differentiation. As the result, the tube formation ability of the differentiated cells decreased. In parallel, we added the conditioned medium of miPS-LLCcm culture into sphere-forming assay, observed the increase of size and number of spheroids. This effect was partly depended on Notch signaling pathway. These results raise the possibility that the differentiated cells from miPS-LLCcm including endothelial cells or their progenitor cells should be required for the maintenance of miPS-LLCcm to differentiate towards vascular endothelial linage. Collectively, we concluded that the differentiated cells from miPS-LLCcm create a niche in vitro for the self-renewal of CSC in the aspect of not only proliferation but also differentiation lineage.

1. Chen, L., et al. PLoS ONE 7, e33544 (2012).

2. Okita, K., et al. Nature 448, 313-317 (2007).

Citation Format: Akifumi Mizutani, Shuichi Matsuda, Tin Yan, Tatsuyuki Sota, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Masaharu Seno. In vitro niche created by cancer stem-like cells derived from mouse induced pluripotent stem cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2013-2647
Abstract 265: Mechanisms of Met-dependent tumorigenesis in models of triple negative breast cancer.
Authors: Sung Vanessa Y.C. , Knight Jennifer F. , Park Morag
Published: August 2014

Human breast cancer is a heterogeneous disease encompassing multiple subtypes. The basal-like and claudin-low subtypes, collectively known as triple negative (TN) breast cancer, are predominantly negative for the expression of therapeutic targets (Her2 and estrogen receptors) and have poor prognosis. In human breast cancer, elevated levels of MET are correlated with TN subtypes and poor outcome, but the reasons for this are relatively unknown. The Met receptor tyrosine kinase (RTK) is a cell surface protein that is activated in response to the Hepatocyte Growth Factor (HGF). Upon stimulation, Met signaling pathways initiate a program of cell survival, migration, and invasive growth. To investigate the role of Met in tumourigenesis, we generated transgenic mice with mammary specific expression of an oncogenic variant of Met (MMTV-Metmut). These mice develop tumours with features of TN breast cancer.

Tumour-initiating cells (TICs) have emerged as a key concept in prevailing models of tumour development. In breast and other solid tumours, where the bulk of the tumour mass consists of differentiated non-tumourigenic cells, TICs constitute a cell fraction of less differentiated tumourigenic cells, and have been shown to be highly resistant to radiation and chemotherapy. TICs are thus a key therapeutic target, yet little is known about TICs in TN breast cancer. Using tumoursphere assays, we have established the presence of TICs in tumours derived from MMTV-Metmut transgenic mice. MMTV-Metmut TICs have constitutively active Met. Interestingly, treatment with a small molecule inhibitor targeting Met can diminish both tumoursphere formation and proliferation. Thus, in TN breast cancers, elevated levels of Met signaling may promote TIC tumourigenicity. Here, mechanisms of Met-dependent TIC propagation and tumourigenicity will be addressed and supporting data presented.

The molecular events that lead to human basal-like and claudin-low breast cancers are poorly understood. Validating a role for Met in TICs could identify key pathways involved in the pathogenesis of TN breast cancer, providing information for new therapeutic targets.

Citation Format: Vanessa Y.C. Sung, Jennifer F. Knight, Morag Park. Mechanisms of Met-dependent tumorigenesis in models of triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 265. doi:10.1158/1538-7445.AM2013-265
Abstract 2661: C11 Acetate and 18F FDG PET/CT imaging of MDR2-/- mouse model of hepatocellular carcinoma.
Authors: Skill Nicholas J. , Territo Paul R. , Riley Amanda A. , McCarthy Brian P. , Maluccio Mary A.
Published: August 2014

Our previous research reveals a link between the enzyme autotaxin (ATX) and its product lysophosphatidic acid (LPA) in hepatocellular carcinoma (HCC). We have shown a shift in ATX levels and LPA variant biosynthesis associated with HCC in patients that was corrected by liver transplantation. Moreover we have demonstrated a reduction in hepatic tumor burden in the MDR2-/- mouse model of HCC by treatment with commercial ATX inhibitors. The purpose of this study was to establish the relevance of FDG PET versus 11C acetate imaging to monitor response to ATX inhibition in the in-vivo MDR2 /- mouse model of HCC whereby we would be able to test the impact of novel inhibitors on both the initiation and progression of HCC. Methods: Dynamic high-resolution Positron Emission Tomography (PET) images of 12 month MDR-/- mice and FVB controls were acquired using 18F-FDG or 11C-Acetate (N=3 per group). At the end of the PET imaging session whole body CT images were acquired. Image segmentation for discrete volumes of interests (VOI) was manually constructed from parametric images for the liver for 18F-FDG images while for 11C-Acetate VOIs were submitted to kinetic modeling, using a 3-compartment, 4-parameter model. Results. MDR2-/- mice had significant hepatic tumors at 12 month (7.9±1.8mm) whereas no tumors were observed in FVB controls. MDR2-/- mice had significantly higher hepatic 11C-Acetate uptake and metabolic rates when compared to FVB controls (0.6±0.04 vs 0.49±0.02ml/g/min P=0.038). In contrast there was no significant difference in 18F-FDG metabolism or uptake in the livers of MDR2-/- mice when compared to wild type FVB controls (1.1±0.23 v 1.32±0.2 and 3.61±0.97 vs. 4.3±0.6SUV respectively p=ns). Conclusion: The biology of HCC formation in MDR2-/- is better suited to 11C acetate PET/CT imaging than 18F FDG. 11C acetate is the most effective imaging method available to test response to treatment in an in-vivo model of HCC.

Citation Format: Nicholas J. Skill, Paul R. Territo, Amanda A. Riley, Brian P. McCarthy, Mary A. Maluccio. C11 Acetate and 18F FDG PET/CT imaging of MDR2-/- mouse model of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2013-2661

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2666: Radiation increases permeability and retention of PEG-conjugates in solid tumors.
Authors: Fong Jerry , Ferraro Daniel J. , Hunn Jeremy , Roberts Stefan , Berezin Mikhail Y. , Rogers Buck E. , Hallahan Dennis E.
Published: August 2014

Poly-ethylene glycol (PEG), a hydrophobic polymer used to improve the circulation and biocompatibility of biomolecules, accumulates in solid tumors in a size-dependent manner that has been attributed to the enhanced permeability and retention (EPR) effect. Irradiation of cancer disrupts cancer microvasculature, resulting in enhanced EPR effect. In the present study, we found that radiation increases the retention of PEG in solid tumors. The accumulation of PEG in solid tumors was observed with in vivo imaging. Nude mice were prepared with heterotopic unilateral murine and human glioma (GL261, U87) and lung carcinoma (LLC, A549) tumors. A near-infrared (NIR) dye conjugated to PEG was then administered by IV to the mice which were imaged by NIR for seven days. There was increased retention of PEG-dye in tumors that had been irradiated. To further elucidate the mechanism of this increased retention, a similar in vivo imaging experiment was conducted in bilateral heterotopic tumor models (GL261, U87, LLC, A549) for seven days. The irradiated tumors demonstrated increased accumulation of PEG-dye, suggesting that the response to irradiation influenced the accumulation of PEG-dye. To observe the distribution of PEG-dye in the solid tumors, after the in vivo imaging was complete, the tumors were harvested and then sectioned for microscopy and histological staining. PEG-dye was observed in both the necrotic and non-necrotic regions of the tumor. The cancer microvasculature environment differs depending on where the tumor is located; to determine if the increased uptake was limited to only the hind limb, in vivo imaging was conducted in nude mice bearing orthotopic brain (GL261) and lung (LLC) tumors. PEG-dye was also observed to accumulate in these irradiated tumor models. Radiation induced EPR led to cancer specific delivery of PEG conjugates. Collectively, our data suggest that irradiation increases the retention of PEG-dye in solid tumors. This effect appears in more than one tumor type suggesting that the mechanism may arise from an increase in EPR following irradiation of cancer. If so, the concurrent administration of radiation therapy with polymer-conjugated therapeutic complexes may improve targeted drug delivery to cancer.

Citation Format: Jerry Fong, Daniel J. Ferraro, Jeremy Hunn, Stefan Roberts, Mikhail Y. Berezin, Buck E. Rogers, Dennis E. Hallahan. Radiation increases permeability and retention of PEG-conjugates in solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2666. doi:10.1158/1538-7445.AM2013-2666
Abstract 2669: Predicting glioblastoma recurrence using novel analysis of perfusion MRI.
Authors: Macyszyn Luke , Akbari Hamed , Da Xiao , Verma Ragini , Wolf Ronald , Bilello Michel , Melhem Elias , O'Rourke Donald , Davatzikos Christos
Published: August 2014

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in humans. Patients diagnosed with GBM have a poor prognosis, with less than 25% of patients surviving more than two years. Despite intensive, multimodality treatment with extensive surgical resection, radiotherapy, and chemotherapy, recurrence of GBM is inevitable. Thus far GBM research has focused mainly on classification of tumor subtypes, segmentation of enhancing and non-enhancing tumor tissue on MRI, and distinguishing treatment-related effects from recurrence. The purpose of this research is to utilize MR perfusion imaging along with advanced image analysis methods to predict specific areas of future recurrence in GBM.

Forty patients with glioblastoma (WHO Grade IV), who subsequently experience recurrence, were utilized for this study. T1, T1CE, T2, FLAIR, and perfusion images were co-registered and regions of interest (ROIs) were drawn for each subject on imaging obtained prior to surgery in white matter, gray matter, CSF, edema, enhancing tumor, non-enhancing tumor and necrosis. Principal component analysis (PCA) was then employed to extract the uncorrelated variables that reflect the temporal dynamics of perfusion. Leave-one-out cross-validation was used when building the PCA model from a training set and testing it on new patients. The results demonstrate marked separation between edematous peritumoral regions and peritumoral regions that later recurred. Hence, this study indicates that imaging biomarkers predictive of tumor recurrence can be constructed using advanced imaging and analysis methods, potentially leading to the creation of a novel, and important, clinical tool.

Citation Format: Luke Macyszyn, Hamed Akbari, Xiao Da, Ragini Verma, Ronald Wolf, Michel Bilello, Elias Melhem, Donald O'Rourke, Christos Davatzikos. Predicting glioblastoma recurrence using novel analysis of perfusion MRI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2669. doi:10.1158/1538-7445.AM2013-2669

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2672: Early evaluation of response to Vemurafenib treatment with FDG PET/CT in patients with metastatic melanoma.
Authors: Aloj Luigi , Caracò Corradina , Di Gennaro Francesca , Grimaldi Antonio M. , Simeone Ester , Curvietto Marcello , Caracò Corrado , Mozzillo Nicola , Torri Valter , Ascierto Paolo A. , Lastoria Secondo
Published: August 2014

Targeted therapy against the V600 mutated form of BRAF is a proven effective therapy for melanoma. Reduction of FDG accumulation on PET/CT studies occurs very rapidly in responding patients. This exploratory study aims to correlate early changes in FDG uptake patterns with treatment outcome in patients undergoing Vemurafenib treatment. Patients treated at our institution were evaluated with PET-CT at baseline and at day 7, 14 and 28 from the start of therapy. Maximal Standardized Uptake Value (SUVmax) and Total Lesion Glycolysis (TLG, mean SUV x lesion volume) were recorded and compared for single lesions in the 4 studies. Patients were then monitored for clinical, laboratory and imaging signs of progression and results correlated to the early PET/CT findings. Fifteen patients have been studied. A total of 87 lesions are available for comparative evaluation. A greater than 30% reduction in SUVmax compared to baseline was seen in 69 lesions at day 7, 76 at day 14 and 75 at day 28. Nineteen lesions showed a greater than 20% increase in FDG uptake between day 14 and 28. Similar results were found when comparing TLG values. Quantitative and visual analysis of PET/CT studies identified 5 patients showing signs of metabolic progression between day 14 and 28 likely indicating early signs of treatment resistance. Three of these patients showed signs of disease progression by other means within 4 months from the beginning of treatment while 2 are still in treatment with 3 months follow-up. Of the 10 patients not showing metabolic signs of progression at day 28, four have progressed between 2 and 9 months from the beginning of treatment, while the remaining 6 are still in treatment and progression free with 2 to 12 months of follow-up. In conclusion, our preliminary results suggest that PET/CT may be a powerful surrogate marker of treatment response to vemurafenib. Sequential monitoring of targeted therapy with PET/CT may be useful in early identification of treatment resistance, aid in tailoring alternative strategies and deserves further investigation.

Citation Format: Luigi Aloj, Corradina Caracò, Francesca Di Gennaro, Antonio M. Grimaldi, Ester Simeone, Marcello Curvietto, Corrado Caracò, Nicola Mozzillo, Valter Torri, Paolo A. Ascierto, Secondo Lastoria. Early evaluation of response to Vemurafenib treatment with FDG PET/CT in patients with metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2013-2672