bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4100: Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones
Authors: Steinman Richard , Huang Jingjing , Woods Paul , Normolle Daniel , Goff Julie , Stehle Christine
Published: July 2016

Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. Bone marrow is the most common site for luminal breast cancer recurrence. We explored whether the HS5 bone marrow stromal cell line that is known to arrest co-cultured breast cancer cells could suppress estrogen receptor alpha (ER) expression during arrest, and whether it could prime the emergence of estrogen-independent breast cancer clones. We demonstrated that paracrine signaling from HS5 cells downregulated ER at the mRNA level and also decreased ER protein stability in T47D and MCF7 breast cancer cells. Conditioned medium (CM) from HS5 arrested the cells in G0/G1 in part through interleukin-1 (IL1), inhibiting mammosphere formation and 2D-growth despite activation of proliferative pathways (Erk and AKT) by the CM. Reversal of stromal-induced growth arrest by blockade of signaling by IL1 in HS5-CM enabled the outgrowth of ER-negative breast cancer cells that were fulvestrant-resistant and estrogen-independent. Outgrowth depended on AKT signaling. Our results describe pro- and anti-proliferative paracrine signals arising from a bone marrow stromal cell line. Blockade of the growth suppressive signaling unmasked stromal facilitation of ER conversion and anti-estrogen resistance in breast cancer cells.

Citation Format: Richard Steinman, Jingjing Huang, Paul Woods, Daniel Normolle, Julie Goff, Christine Stehle. Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4100.
Abstract 411: Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay
Authors: Liu Xiaoyun , Loredo Leticia , Dai Houquan , Castro Aaron , Zhao Yuewei , Kim Sung Kun , Dinkel Austin , Castro Miguel
Published: July 2016

BRAF is a human gene encoding a serine/threonine protein kinase in the RAS-RAF-MEK-ERK signal pathway, which is important in regulating cellular responses to extracellular signals. Frequent mutations on BRAF gene have been detected in various types of human cancer, including malignant melanoma, non-small cell lung cancer, colorectal cancer, papillary carcinoma of the thyroid, ovarian cancer and hairy cell leukemia. Among all, a single point 1799T>A (V600E) transversion, the most common mutation on BRAF, results in increased kinase activity. It serves as a driver mutation in several types of cancer and confers increased sensitivity to tyrosine kinase inhibitors. Thus, detecting the BRAF V600E mutation may offer valuable information for cancer diagnosis, treatment selection, and prognosis. Here we report a rapid, highly sensitive method for the detection of BRAF V600 mutation using a bridged nucleic acid (BNA) clamp technology associated with quantitative PCR (qPCR). Due to the feature of enhanced affinity for hybridization template, BNA clamp is able to selectively block PCR reactions on the perfect match DNA sequence, while leaves the mismatch template unaffected. In this study several BNA clamps that bind to BRAF V600 wild-type (WT) template was designed, synthesized, and characterized. As compared to DNA clamp counterparts, these BNA clamps increased Tm values for the BRAF V600 WT by greater than 30°C. In addition, BNA clamps efficiently distinguished BRAF WT (perfect match) from V600E (mismatch) templates (deltaTm >12°C). In qPCR assays BNA clamps could suppress amplification of WT by >1,000-fold, while had little or no effects on the V600E mutant. Such BNA clamps allowed us to detect 1.0% or lower levels for the BRAF V600E mutant in a wild-type background. These results indicate that BNA clamp-based qPCR technology offers a new tool for rapid, sensitive detection of BRAF V600E mutation. The technology may be adapted for detection of mutations of a variety of biomarker genes.

Citation Format: Xiaoyun Liu, Leticia Loredo, Houquan Dai, Aaron Castro, Yuewei Zhao, Sung Kun Kim, Austin Dinkel, Miguel Castro. Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 411.
Abstract 4111: Imaging of colon cancer-cell mitosis within liver metastasis in nude mice
Authors: Hasegawa Kosuke , Suetsugu Atsushi , Nakamura Miki , Matsumoto Takuro , Aoki Hitomi , Kunisada Takahiro , Shimizu Masahito , Hoffman Robert M.
Published: July 2016

Colon cancer frequently results in metastasis to the liver, when it becomes the main cause of death. The tumor cell cycle in primary tumors and metastases are poorly understood. We developed a liver metastasis mouse model using a human colon cancer cell line (HCT-116), which expresses GFP in the nucleus and RFP in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP (2.0 × 106) cells were injected in the spleen of a BALB/c nude mouse. The cells subsequently formed tumor colonies in the liver and retroperitoneum 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, mitosis of the dual-colored colon cancer cells could be clearly imaged in the liver and other metastases. Multi-nucleated cancer cells, in addition to a mononuclear cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nuclear cells were also observed in cell culture of HCT-116 GFP-RFP cells. Multi-nuclear HCT-116 cells may participate in the tumor growth in vivo. A significant difference in mitotic cells count in the primary tumor and metastasis was not observed.

Citation Format: Kosuke Hasegawa, Atsushi Suetsugu, Miki Nakamura, Takuro Matsumoto, Hitomi Aoki, Takahiro Kunisada, Masahito Shimizu, Robert M. Hoffman. Imaging of colon cancer-cell mitosis within liver metastasis in nude mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4111.
Abstract 4119: Inhibition of PORCN prolongs metastasis free survival in a mouse model of Ewing sarcoma
Authors: Hayashi Masanori , Baker Alissa C. , Goldstein Seth D. , Albert Catherine M. , Jackson Kyle W. , Loeb David M.
Published: July 2016

Metastatic disease is the most important factor in determining the survival of patients with Ewing sarcoma (ES). Although intensive cytotoxic chemotherapy has improved the survival of patients with localized disease, the survival rate for patients with metastatic disease remains dismal, and the most pressing unmet need in ES is to prevent and treat metastasis better. The Wnt signaling pathway is a key regulator of a number of cellular functions associated with metastasis, including proliferation, motility, and stem cell self-renewal. A single enzyme, Porcupine (Porcn), mediates post-translational palmitoylation of Wnt ligands, and it's activity is required for secretion of all 17 Wnt ligands, making it an attractive target for a pan-Wnt inhibitor. We have studied the effect of LGK974, a potent, selective, Porcn inhibitor, on Ewing sarcoma metastasis. In vitro, we have observed that LGK974 does not affect proliferation or sarcosphere formation, but inhibits migration of ES cell lines through the suppression of expression of multiple transcriptional regulators of Epithelial-Mesenchymal Transition (EMT). In our orthotopic implantation/amputation mouse model, single agent LGK974 treatment leads to significant delay in formation of lung metastasis following amputation of the affected leg, without a significant effect on primary tumor growth. This delay leads to a significant post-amputation survival benefit in multiple xenografts. In LGK974 treated xenografts, the expression of multiple EMT related transcription factors were observed to be suppressed. Furthermore, in tail vein injection models, LGK974 did not produce a survival benefit, suggesting that LGK974 effects Ewing sarcoma metastasis by inhibiting early steps in the metastatic cascade, such as migration and invasion. Our findings strongly implicate Wnt signaling in the early steps of ES metastasis and demonstrate that LGK974 has the potential to significantly improve the survival of ES patients through the specific inhibition of metastasis.

Citation Format: Masanori Hayashi, Alissa C. Baker, Seth D. Goldstein, Catherine M. Albert, Kyle W. Jackson, David M. Loeb. Inhibition of PORCN prolongs metastasis free survival in a mouse model of Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4119.
Abstract 4120: c-Met inhibition blocks MEK-induced tumor cell invasion in uveal melanoma
Authors: Surriga Oliver , Smith Bryan D. , Ambrosini Grazia , Flynn Daniel L. , Carvajal Richard D. , Schwartz Gary K.
Published: July 2016

Uveal melanoma is the most common primary intraocular malignant tumor in adults. About half of patients with uveal melanoma will develop metastatic disease to the liver and the lung. These tumors are characterized by mutations in G-proteins (GNAQ and GNA11), activation of MAPK, and over-expression of c-Met. We have previously reported that c-Met inhibition inhibits tumor cell invasion and metastasis formation in uveal melanoma. Altiratinib is a selective inhibitor for c-Met as well as TIE2, VEGFR2 and TRK kinases. We found that this agent had no effect on inhibiting cell growth, consistent with our previous findings with crizotinib. However, it did inhibit invasion of uveal melanoma cells through matrigel in a concentration dependent fashion (25 nM to 250 nM) with inhibition of phospho-Met noted at concentrations as low as 25 nM. Selumetinib, a MEK inhibitor, is currently in clinical trial in patients with this disease. We found that treatment with 250 nM selumetinib inhibited cell proliferation but unexpectedly induced a marked increase in cell invasion of GNAQ and GNA11 mutant cell lines. This was associated with an increase in c-Met RNA and protein expression, as well as receptor phosphorylation, after 24 hours of selumetinib treatment. Combining selumetinib with altiratinib inhibited cell invasion to the level of altiratinib alone and continued to inhibit cell proliferation to the level of selumetinib alone. This effect was recapitulated by the knockdown of c-Met by siRNA prior to treatment with selumetinib. In a uveal melanoma xenograft model, the combination treatment of 15 mg/kg altiratinib and 25 mg/kg selumetinib significantly delayed tumor growth compared to vehicle control, altiratinib and selumetinib alone. Western blot analysis of tumor tissue confirmed target inhibition of p-Met and p-ERK in animals treated with altiratinib and selumetinib, respectively. Furthermore, tumor metastasis was inhibited by altiratinib, selumetinib and combination treatments in a uveal melanoma mouse model. These results indicate that the combined inhibition of MEK and c-Met by selumetinib and altiratinib, respectively, may be sufficient to suppress uveal melanoma tumor growth and metastasis. This strategy can potentially be used as therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease.

Citation Format: Oliver Surriga, Bryan D. Smith, Grazia Ambrosini, Daniel L. Flynn, Richard D. Carvajal, Gary K. Schwartz. c-Met inhibition blocks MEK-induced tumor cell invasion in uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4120.
Abstract 4128: Anti-angiogenic and anti-metastatic activities of juglone in pancreatic cancer cells
Authors: Karki Namrata , Greenway Frank , Hansel William , Aggarwal Sita , Losso Jack N.
Published: July 2016

Pancreatic cancer is the fourth leading cause of cancer related deaths in the US. The late diagnosis, early metastasis and poor survival rate make this disease lethal. This current study was designed to examine the effect of juglone, 5 hydroxy-1, 4-naphthoquinone in modulating the angiogenic and metastatic potential of pancreatic cancer cells in vitro, using the MIA Paca-2 human pancreatic cancer cell line. Juglone is a natural quinoid abundantly found abundantly in plants of Juglandacea family. To understand the anti-angiogenic and anti-metastatic properties of juglone, expression of markers related to angiogenesis were measured by Western blot or ELISA and the effect on ability of cancer cells to migrate and invade after treatment was analyzed by transwell invasion and migration assay, wound healing assay, and in vitro tube formation assay in human umbilical vein endothelial cells. The Akt pathway is highly upregulated and is responsible for the regulation of various biological processes in pancreatic cancer cells including proliferation, growth, survival and angiogenesis. Akt activation is also associated with regulation of key angiogenic markers such as, VEGF (Vascular Endothelial Growth Factor) and HIF-1 (Hypoxia Inducible Factor). Upon treatment of pancreatic cancer cells with juglone, reduced expression of serine-phosphorylated Akt expression was observed. Juglone inhibited metastasis of pancreatic cancer cells as indicated by the results of cell migration, invasion and wound healing assay. Juglone treatment also downregulated the levels of VEGF and HIF-1α. Significantly, it was found that juglone inhibited pancreatic cancer cell motility, migratory and invading capabilities. These findings suggest that Akt inhibitors such as juglone possess ability to attenuate the aggressiveness of pancreatic cancer cells and can be used as a novel anti-cancer agent to prevent metastasis of highly invasive pancreatic adenocarcinomas.

Citation Format: Namrata Karki, Frank Greenway, William Hansel, Sita Aggarwal, Jack N. Losso. Anti-angiogenic and anti-metastatic activities of juglone in pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4128.
Abstract 4138: Immunological profile of metastatic or recurrent breast cancer patients
Authors: Imoto Shigeru , Ueno Takayuki , Isaka Hirotsugu , Ito Hiroki , Miyamoto Kaisuke , Chiba Tomohiro , Kamma Hiroshi
Published: July 2016

Background: We previously reported about immune suppression in breast cancer patients at this meeting. In brief, tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were analyzed in 50 early or advanced breast cancer (BC) patients. To compare between 38 early and 12 advanced BC cases, CD163-positive tumor cells and CCR4-positive tumor cells were detected more frequently in advanced cases than in early cases. Regulatory T (Treg) cells in PBMC significantly increased in percentage of the population in 37 BC patients than in 21 healthy volunteers (AACR 2011). In addition, several cytokines were examined in that cohort and plasma IL-17A had significantly higher levels in early BC than in advanced BC (AACR 2013). Then, we examined their prognosis and immunological profile.

Patients and methods: Treg cells were examined by counting CD4+CD25highCD127low/-cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, CD163 and CCR4. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/-, ++, +++). Human IL-2, IL-4, IL-6, IL-10, TNF, INFγ and IL-17A were measured using cytometric beads array system. Most patients received chemotherapy, hormonal therapy, and/or anti-HER2 therapy on the basis of intrinsic subtype and breast irradiation after breast-conserving surgery.

Results and discussions: At the median follow-up of 7 years after blood sample collection, only 2 operable patients relapsed and 3 patients including 2 cases of stage IV died of disease. Of 5 cases of stage IV or recurrent BC, CD163 and/or CCR4 were strongly stained positive in tumor cells. There were significant differences of staining intensity in CD163 and CCR4-positive tumor cells between those cases and the rest 45 cases (p<0.01 at Chi-square test). However, other immune cell profiles, Treg cells in PBMC and cytokines in plasma had no trend between them. Several reports demonstrated that cancer patients with CD163-positive tumor-infiltrating macrophages and CD163-positive tumor cells had poor prognosis due to tumor-associated macrophage. Phenotypic macrophage traits in cancer cells, like CD163 expression, may be explained by heterotypic cell fusion between monocytes/macrophages and cancer cells.

Conclusion: Targeted therapy against M2 macrophage or CCR4 is considered as a promising strategy of advanced breast cancer.

Citation Format: Shigeru Imoto, Takayuki Ueno, Hirotsugu Isaka, Hiroki Ito, Kaisuke Miyamoto, Tomohiro Chiba, Hiroshi Kamma. Immunological profile of metastatic or recurrent breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4138.
Abstract 4154: Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer
Authors: Kawaguchi Kosuke , SUzuki Eiji , Inoue Masashi , Kii Isao , Karaoke Tatsuki R. , Hiram Masahiro , Iwaisako Keiko , Fengling Pu , Niche Mariko , Yamaguchi Ayane , Haga Hironori , Hagiwara Masatoshi , Toi Masakazu
Published: July 2016

Tumor-infiltrating immune cells (TIIs) in HER2+ breast cancer (BC) play an important role in treatment with anti-HER2 therapy. However, the precise mechanisms of how TIIs exert anti-tumor activity remain unclear. Neuropilin-1 (NRP-1) on macrophages regulates immune functions in various cancers and thus we explored the role of NRP-1 expressing macrophages in anti-tumor activity in HER2+ BC. We show that NRP-1 on macrophages regulated the migration of and chemokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model showed that NRP-1 knockdown of macrophages in adoptively transferring peripheral blood mononuclear cells (PBMCs) suppressed anti-tumor activity and infiltration of CD45+ immune cells into tumors. Interestingly, NRP-1 expressing TIIs were mainly CD4+ T cells, despite little expression of NRP-1 on CD4+ T cells in PBMCs. We found that NRP-1 expression on CD4+ T cells was induced by NRP-1 transfer from macrophages to T cells. In HER2+ BC patients, NRP-1 expressing TIIs correlated with better clinical outcomes. These results demonstrate that NRP-1 expressing macrophages are key subsets of immune cells in trastuzumab-mediated anti-tumor activity and may predict better outcomes for HER2+ BC patients. In conclusion, NRP-1 expression is required for differentiation and activation of macrophages. NRP-1 expressing macrophages promote NRP-1 expression on CD4+ T cells by direct interaction and contribute to anti-tumor immune responses in HER2+ BC.

Citation Format: Kosuke Kawaguchi, Eiji SUzuki, Masashi Inoue, Isao Kii, Tatsuki R. Karaoke, Masahiro Hiram, Keiko Iwaisako, Pu Fengling, Mariko Niche, Ayane Yamaguchi, Hironori Haga, Masatoshi Hagiwara, Masakazu Toi. Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4154.
Abstract 4161: Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk
Authors: Renrick Ariana N. , Thounaojam Menaka C. , Dudimah Duafalia F. , Thomas Portia , Pellom Samuel T. , Uzhachenko Roman V. , Shanker Anil
Published: July 2016

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NF-κB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that miR-155 plays a role in bortezomib-induced regulation of T cell function. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions.

Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Duafalia F. Dudimah, Portia Thomas, Samuel T. Pellom, Roman V. Uzhachenko, Anil Shanker. Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4161.
Abstract 4179: Expression profiling of zebrafish rb1- neuroectodermal-like brain tumors: A new model of CNS PNETs
Authors: McGrail Maura A. , Solin Staci L. , Haltom Jeffrey A. , Schultz Laura E. , Essner Jeffrey J. , Shive Heather R.
Published: July 2016

We examined the gene expression signature of zebrafish brain tumors induced by somatic inactivation of the retinoblastoma tumor suppressor rb1 to determine the similarity with human CNS primitive neuroectodermal tumors (PNETs). Childhood CNS PNETs are aggressive tumors characterized by undifferentiated cells with features of the embryonic neuroepithelium. Previously our histological analysis showed the majority of zebrafish rb1- lesions were proliferative neuroectodermal-like tumors composed of neoplastic cells with small hyperchromatic nuclei that form rosettes. To obtain the transcription profile of the zebrafish rb1- tumors, RNA-Seq was performed on 10 tumors from fish ranging in age from 3.5 to 10 months of age. Duplicate RNA-Seq libraries were synthesized for each tumor and multiplex sequenced on an Illumina HiSeq NextGen Sequencer at the Genome Sequencing Core, Kansas University. Our initial gene expression profiling of the zebrafish rb1- brain tumors revealed a molecular signature consistent with an undifferentiated tumor phenotype. In each zebrafish tumor sample, standard markers of neuronal differentiation (sypa, sypb, neurod1/2, neurog1/2) and glial differentiation (GFAP, S100β) were not significantly expressed. In contrast, the neural stem/progenitor marker sox2 was highly expressed in all tumors, consistent with the primitive neuroectodermal-like tumor histology. Comparison with human childhood CNS-PNET gene expression analysis revealed a similar profile. Four of the nine genes that define the human oligoneural (group 2) molecular subgroup (olig2, bcan, sox8b, sox10 and ascl1) described by Picard et al, 2012 were highly expressed in the zebrafish tumors. Together, the data indicate the zebrafish brain tumors induced by somatic inactivation of rb1 model human CNS PNETs. Additional gene expression, pathway, and cross-species comparative analyses are in progress. The zebrafish rb1- tumor model will be useful for investigating the molecular pathways driving the aggressive and undifferentiated features of CNS PNETs.

Citation Format: Maura A. McGrail, Staci L. Solin, Jeffrey A. Haltom, Laura E. Schultz, Jeffrey J. Essner, Heather R. Shive. Expression profiling of zebrafish rb1- neuroectodermal-like brain tumors: A new model of CNS PNETs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4179.
Abstract 4183: Targeting mTOR signaling during gliomagenesis
Authors: Torres Daniela , Mela Angelina , Peter Canoll D.
Published: July 2016

Diffusely infiltrating gliomas are the most common type of primary brain tumor seen in adults. In a subset of patients, these tumors present as low grade lesions that progress to high grade glioma (HGG). Conventional treatment for gliomas includes surgical resection followed by radiation and temozolomide. Glioma cells invade peripheral brain tissue making complete surgical resection impossible and despite aggressive treatment tumor recurrence is inevitable. Current research has focused on identifying new therapeutic targets, mainly in HGG. However, investigating the therapeutic targets at early stages of gliomagenesis could provide a novel approach for the treatment of low grade gliomas (LGG). Gliomas have been characterized by gene expression profiles and classified into distinct subtypes including proneural, classical and mesenchymal. Proneural gliomas, which account for the majority of LGG, show high expression of PDGFRα and frequently harbor p53 mutations. The resulting alterations in PDGFRα signaling and inactivation of p53 both induce an upregulation in mTOR signaling, which contributes to gliomagenesis via its regulation of cell growth and proliferation. Thus, inhibiting mTOR signaling should have a robust effect on glioma growth. However, inhibiting mTOR signaling in HGG has had limited success. This may be due to the accumulation of genetic alterations that occur during glioma progression resulting in loss of sensitivity to the anti-tumor effects of mTOR inhibitors. We hypothesize that inhibiting mTOR signaling at early stages of gliomagenesis will be more effective. To test this hypothesis we are characterizing the effects of mTOR inhibition at different stages of gliomagenesis in our proneural glioma mouse model. We assessed the effects of inhibiting mTOR with the ATP-competitive mTOR inhibitor (AZD8055) in vitro and in vivo. Our preliminary data show that AZD8055 inhibits mTOR activity and reduces glioma cell viability in vitro. Inhibition of mTOR in our proneural glioma model showed that AZD8055 crosses the blood brain barrier and effectively inhibits mTOR in glioma cells within the tumor, as well as in the surrounding brain tissue. In addition, we have characterized mTOR activity at different time points in glioma formation and show upregulation of mTOR activity during early stages of gliomagenesis. Our findings support the role of mTOR in early stages of gliomagenesis and motivate our ongoing studies to test the effects of AZD8055 for the treatment of LGG.

Citation Format: Daniela Torres, Angelina Mela, Canoll D. Peter. Targeting mTOR signaling during gliomagenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4183.
Abstract 42: A new type of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and ATP-drug competition
Authors: Wang Xuan , Li Yunsheng , Qian Yanrong , Cao Yanyang , Chen Xiaozhuo
Published: July 2016

Drug resistance is a major problem in cancer therapies and responsible for most relapse, one of the major causes of death in cancer. Several cancer drug resistance mechanisms are known, including genetic mutations in protooncogenes or tumor suppressor genes, cancer stem cells, and multi-drug resistance (MDR). Among these mechanisms, MDR is ATP-dependent. Intratumoral (extracellular) ATP levels of various cancer types are 10⁁3 to 10⁁4 times higher than those in their corresponding normal tissues but the biological significance of the high intratumoral ATP concentrations is unknown. We recently reported that extracellular ATP is internalized by cancer cells via macropinocytosis and enhances their growth and survival (1). Based on these results, we hypothesized that the internalized ATP also promotes drug resistance. Here we report that extracellular ATP, at concentrations in or below the range of reported intratumoral ATP levels, substantially increased intracellular ATP levels and promoted cancer cell drug resistance to tyrosine kinase inhibitor (TKI) sunitinib in human NSCLC A549 cells. The ATP increase and the drug resistance were mediated, at least in part, by macropinocytosis, clathrin- and caveolae-mediated endocytoses. The elevated intracellular ATP induced upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR-mediated signaling pathways, such as Akt, mTOR, Raf and MEK, resulting in augmented cell growth and reduced apoptosis induced by sunitinib. The upregulated phosphorylation was not mediated by purinergic receptor signaling. Furthermore, in vitro, ex vivo, and in vivo, extracellular ATP partially restored phosphorylation levels of PDGRF and its downstream proteins/enzymes inhibited by sunitinib, a PDGFR inhibitor (TKI). These results strongly suggest that the extracellular ATP-increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitors for the ATP-binding site of PDGFR, enhancing the Akt-mTOR and Raf-MEK signaling pathways and promoting cancer cell survival. All these findings significantly expand our understanding of the roles of extracellular ATP in the Warburg effect (2) and drug resistance in cancer and indentify a new anti-drug resistance target.

1. Qian Y, Wang X, et al., Cancer Lett. 351(2014): 242-5.

2. Chen X, Qian Y, Wu S. Free. Radic. Biol. Med. 79(2015): 253-263.

Citation Format: Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. A new type of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and ATP-drug competition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 42.
Abstract 4207: microPET/CT imaging of the mobilization of CD11b+ cells
Authors: Cao Qizhen , Huang Qian , Li Chun
Published: July 2016

Objectives: Mobilization of immune cells is often associated with prognosis and treatment response for many diseases. The objective of this study was to test whether mobilization of CD11b+ myeloid cells could be noninvasively imaged by PET/CT. Methods: Anti-mouse CD11b antibody was labeled with 64Cu. Immunocompetent mice with 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in the ears, lipopolysaccharide (LPS)-induced inflammation in the lung, mice bearing 3 different syngeneic murine tumors (4T1 mammary carcinoma, CMS4 sarcoma, and CT26 colon carcinoma), and mice bearing ID8 ovarian cancer undergoing photothermal therapy were imaged with [64Cu]-labeled anti-CD11b. microPET-CT imaging and biodistribution were obtained at 24 h after intravenous injection of the radiotracer. Subpopulations of bone marrow cells, cells from spleen, and cells from the disease sites were analyzed by flow cytometry and/or immunohistochemistry (IHC) staining. Results: 64Cu-labeled anti-CD11b bound specifically to CD11b+ myeloid cells. microPET-CT images showed significantly higher uptake of 64Cu-DOTA-anti-CD11b antibody at the inflammatory sites and tumors undergoing photothermal therapy. In addition, microPET-CT images also revealed mobilization of CD11b+ cells from bone marrow to secondary lymphoid organs (spleen and/or lymph nodes). Interestingly, mice bearing 4T1 carcinoma displayed a markedly different imaging/distribution pattern compared to mice bearing CMS4 and CT26 tumors, characterized by a significant increase in blood pool and liver activities with corresponding reduction in the uptake of the radiotracer in the bone marrow and the spleen in different stages of tumor development. These findings were evaluated in the context of flow cytometry and IHC analyses. Conclusions: The differential distribution pattern of 64Cu-labeled anti-CD11b in different disease models could be attributed to mobilization of CD11b+ cells. PET/CT may be used to image systems response of immune cells to disease development and treatments. (Supported in part by the John S. Dunn Foundation)

Citation Format: Qizhen Cao, Qian Huang, Chun Li. microPET/CT imaging of the mobilization of CD11b+ cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4207.
Abstract 4224: Multiplexing single cell time lapse imaging of intra-cellular oxygen in cancer cells
Authors: Düssmann Heiko , Perez Sergio , Kumar Ujval Anil , Papkovsky Dmitri B. , Prehn Jochen HM
Published: July 2016

Oxygen plays an important role in aerobic energy metabolism and signal transduction. The detection of intracellular or subcellular molecular oxygen (O2) levels is important for the understanding of cell physiology and mitochondrial energy metabolism, and the alterations correlated with cancer. Detection of O2 has recently been improved significantly with the development of live cell time lapse microscopy compatible oxygen sensitive probes.

We here utilized nanoparticle probe MitoImageTM-MM2 with embedded poly(9,9-dioctylfluorene) (PFO) as an O2-independent component and Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) to perform time lapse ratiometric measurements of intra-cellular oxygen levels. Loading of HeLa cervical cancer cells with MM2 enables us to image kinetics of intracellular O2 concentration in response to the mitochondrial respiratory chain inhibitors Antimycin A (complex III) or sodium azide (complex IV). These changes were detected in respiration-supporting medium equilibrated with 5% (53 μM) and 20% (225 μM)O2. Also exposing cells to varying extracellular oxygen concentrations (21 - 225 μM) showed strong dependence of the intracellular oxygen levels. The mitochondrial membrane potential was monitored by simultaneous imaging of Tetra-Methyl-Rhodamine-Methyl ester (TMRM) in the same sample. Multiplexing with GFP finally allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilisation (MOMP) in HeLa expressing cytochrome-c-eGFP and stained with TMRM.

In conclusion, the MitoImageTM-MM2 nanoparticle probe enables single-cell, time-lapse imaging of alterations in intracellular oxygen levels in cancer cells, and can be employed in multiplex assays to investigate alterations in intracellular oxygen levels relative to protein dynamics and mitochondrial function.

Keywords: Intracellular oxygen; phosphorescent oxygen-sensitive nanoparticles; HeLa cells; MitoImage ratiometric probes; confocal microscopy.

Citation Format: Heiko Düssmann, Sergio Perez, Ujval Anil Kumar, Dmitri B. Papkovsky, Jochen HM Prehn. Multiplexing single cell time lapse imaging of intra-cellular oxygen in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4224.
Abstract 4236: SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides
Authors: Kelderhouse Lindsay E. , Hardy Amanda , Pisaneschi Federica , Gray Joshua P. , Gammon Seth , Roberts Richard W. , Takahashi Terry T. , Fiacco Steve , Millward Steven W.
Published: July 2016

In recent years, patients with Her2-positive breast cancer have benefitted greatly from targeted therapies such as Trastuzumab and Pertuzumab. However, there are currently no FDA-approved Her2-targeted imaging agents to diagnose and monitor Her2-positive breast cancer leaving physicians to rely on biopsies to determine Her2 status. Whole-body visualization of Her2 would allow the noninvasive identification of all Her2 primary and metastatic lesions as well as serve as a powerful tool to monitor the effectiveness of Her2-targeted therapeutics. We recently described the generation of Her2-targeted SUPR (Scanning Unnatural Protease Resistant) peptides that selectively bind the Her2 receptor in vitro with low nanomolar affinity. Cy5-labeled SUPR peptides showed rapid and Her2-specific tumor uptake and minimal retention in non-tumor tissues after 24 hours of washout. In this study, we describe 18F radiolabeling of Her2-targeted SUPR peptides and their evaluation as PET radiotracers to visualize Her2 expression in vivo. The lead compound, SUPR4, was labeled with 18F-fluoroethylazide in high radiochemical yield and specific activity on an automated radiochemical synthesis platform. The resulting radiotracer (SUPR-18F) showed rapid and Her2-selective tumor uptake between 30-60 minutes post-injection with minimal liver uptake. The majority of the tracer was cleared by the kidneys at 2 hours post-injection although some activity was observed in the GI tract suggesting hepatobiliary excretion. No significant uptake was observed in the brain. 90 minute dynamic PET scans were performed to estimate the rate of tumor uptake and clearance in major organ systems and the biodistribution quantified by autoradiography post-mortem. Pre-injection of unlabeled SUPR peptide, Trastuzumab, and Pertuzumab followed by PET imaging with SUPR-18F was used to confirm tumor uptake as a function of specific binding to the Her2 receptor. Having established the utility of SUPR-18F in pre-clinical mouse models, we anticipate that this class of PET tracers could be employed in same-day imaging procedures throughout the administration of Her2-targeted therapy.

Citation Format: Lindsay E. Kelderhouse, Amanda Hardy, Federica Pisaneschi, Joshua P. Gray, Seth Gammon, Richard W. Roberts, Terry T. Takahashi, Steve Fiacco, Steven W. Millward. SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4236.
Abstract 4274: Porcine ovarian matrix supports maintenance of human intestinal organoid cultures
Authors: George Jay , De Gese Sol , Benton Gabriel
Published: July 2016

Currently, extracellular matrix hydrogels derived from the Engelbreth-Holmes-Swarm murine tumor (Cultrex BME, Matrigel) are used for stem cell maintenance and differentiation of both normal and tumor organoid culture models. While this matrix is suitable for many in vitro applications, there is increasing demand for a 3D organoid culture platform that is more suitable for clinical applications. Here, we have developed a soluble extracellular matrix derived from normal porcine ovaries that forms a hydrogel polymer at physiological temperature and that supports maintenance of human small intestine organoids in culture. This matrix supports expansion of LGR5+ human stem cell populations using Wnt, EGF, Noggin, and R-Spondin 1 cell culture medium. The matrix also supports differentiation of stem cells into mini organs by removing Wnt which is characterized by the formation of intestinal crypts which exhibit immunostaining for lineage specific markers. This new extracellular matrix hydrogel represents a non-tumor sourced, normal extracellular matrix for evaluating both development and cancer progression.

Citation Format: Jay George, Sol De Gese, Gabriel Benton. Porcine ovarian matrix supports maintenance of human intestinal organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4274.
Abstract 4284: Exploratory plasma proteomic analysis in a randomized cross-over trial of aspirin among healthy individuals
Authors: Wang Xiaoliang , Zhang Yuzheng , Shojaie Ali , Lampe Paul D. , Levy Lisa , Peters Ulrike , Potter John D. , White Emily , Lampe Johanna W.
Published: July 2016

Background: Long-term use of aspirin is associated with lower colorectal cancer (CRC) incidence; however, the mechanism of the chemopreventive effect of aspirin is not fully understood. Animal studies suggest that COX-2, NFκB signaling and Wnt/β-catenin pathways may play a role, but no clinical trials have systematically evaluated the biological response to aspirin in healthy humans. Methods: We assessed the difference in plasma protein levels after 60 days of regular dose aspirin (325 mg/day) compared to placebo in a randomized, double-blinded, placebo-controlled, cross-over trial of 44 healthy non-smoking men and women, aged 21-45 years. Plasma proteomics was analyzed on an antibody microarray with ∼3,000 full-length antibodies, printed in triplicate. Moderated paired t-test was performed on individual antibodies, and gene set analyses were performed for KEGG and GO pathways. Results: Among the 3,387 antibodies, significant differences in plasma protein levels were observed for 267 antibodies (p<0.05), the most significant protein being a transcription-factor regulator belonging to a steroid receptor family and found to be differentially expressed in colon cancer cells. Other significant proteins are involved in multiple oncogenic pathways related to colon tumorigenesis. In the pathway analysis, 4 KEGG (among 138) and 69 GO (among 1,089) pathways were found to be significant (p<0.05), including natural killer (NK) cell-mediated cytotoxicity, butanoate metabolism and Wnt signaling pathways. Pathways that modulate cellular protein binding to steroid receptors were also significantly different between aspirin treatment and placebo (p<0.05). None of the results remained statistically significant after correction for multiple testing. Conclusion: Several proteins and pathways, which have previously been reported as playing a role in colorectal carcinogenesis in vitro were found to be differentially expressed after aspirin treatment vs. placebo in healthy human subjects. This study suggests several chemopreventive mechanisms of aspirin; however, larger, confirmatory studies are needed.

Citation Format: Xiaoliang Wang, Yuzheng Zhang, Ali Shojaie, Paul D. Lampe, Lisa Levy, Ulrike Peters, John D. Potter, Emily White, Johanna W. Lampe. Exploratory plasma proteomic analysis in a randomized cross-over trial of aspirin among healthy individuals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4284.
Abstract 4304: Prevalence and triggers of drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in a cancer patient cohort
Authors: Gillis Nancy K. , Bell Gillian C. , McLeod Howard L. , Brandt Amy J.
Published: July 2016

SJS and TEN are extremely rare (approximately 2-7 cases per million per year) hypersensitivity reactions most commonly attributed to medications. The life-threatening nature of SJS/TEN necessitates early diagnosis and immediate identification and withdrawal of causative agents. Some of the most commonly associated culprits include sulfonamide antibiotics, aromatic anticonvulsants (phenytoin, carbamazepine, lamotrigine), -oxicam NSAIDs (e.g., meloxicam), allopurinol, and nevirapine. Individual case reports of SJS/TEN have been associated with anti-cancer agents, but a comprehensive assessment has not been performed. A large-scale retrospective analysis of the prevalence of SJS/TEN in cancer patients treated at Moffitt Cancer Center (MCC) between Jan 1, 2002 - Dec 31, 2014 was conducted. A total of 104,917 cancer patients were screened for SJS/TEN-related ICD-9 codes in the MCC health research informatics database. SJS/TEN diagnoses were identified in 121 patients. Manual chart review of physician, dermatology, and pathology notes confirmed SJS/TEN in 47 patients (in-patient + historical) and possible SJS/TEN in an additional 17 patients, corresponding to an overall prevalence of 0.06%. Confirmed in-patient cases of SJS/TEN were more common in hematologic malignancies compared to solid tumors (n = 12 vs. n = 7, respectively). Notably, 5 of the 19 (26.3%) confirmed cases were observed in patients with acute myeloid leukemia. Physician-reported culprits for SJS/TEN diagnoses included antibiotics, immunomodulators, anticonvulsants, and anti-cancer agents. The observed prevalence of SJS/TEN was higher in cancer patients than previous reports from the general USA population. There were 19 confirmed in-patient diagnoses of SJS/TEN and an additional 45 historical and possible cases of SJS/TEN in 104,917 cancer patients, corresponding to an overall prevalence of 0.018% to 0.06%, an approximately 90-fold increase when compared to the general population. Possible explanations for increased risk in cancer patients include increased exposure to culprit medications, cancer disease process, immunocompromised state, or synergy between risk factors. A thorough understanding of the factors that increase risk to SJS/TEN in cancer patients is critical to facilitate culprit withdrawal and maximize patient outcomes and survival.

Citation Format: Nancy K. Gillis, Gillian C. Bell, Howard L. McLeod, Amy J. Brandt. Prevalence and triggers of drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in a cancer patient cohort. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4304.