bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3230: Human stem cell-based model of MYCN-driven neuroblastoma
Authors: Huang Miller , McHenry Lauren K. , Miller Matthew L. , Kim Grace E. , Moriarity Branden S. , Miyaoka Yuichiro , Bronner Marianne E. , Largaespada David A. , Conklin Bruce R. , Ji Hanlee P. , Maris John M. , Matthay Katherine K. , Weiss William A.
Published: August 2015

Neuroblastoma (NB), a disease of neural crest (NC) origin, is the most common extracranial solid tumor in childhood. High-risk NB patients represent the subgroup with the worst prognosis and frequently harbor amplification of MYCN. Due to its biochemical structure as a transcription factor, MYCN has been difficult to target directly with small molecules. Alternatively, identification of cooperating partners of MYCN-induced tumorigenesis can reveal a more therapeutically viable target, such as anaplastic lymphoma kinase (ALK). While genetically engineered mouse models (GEMMs) of NB driven by MYCN and ALK exist, recent studies have found significant differences between mouse models of disease and the human tumors they are intended to represent. In support of this, mouse and human NC cells differ in development and marker expression, and the genetic requirement for transformation of human cells has been shown to be more complex than mouse cells, suggesting that a human cell-based NB model would be more relevant. To develop a human cell-based model of NB, we started with a normal human induced pluripotent stem (iPS) cell line derived integration-free from a healthy adult and transduced empty vector, ALK F1174L (active mutant), doxycycline-inducible MYCN (DOX-MYCN), or ALK F1174L/DOX-MYCN. These iPS cells were differentiated towards NC cells and subsequently implanted orthotopically into renal capsules of mice fed on dox chow. Within 3 months, 60% of mice developed tumors with ALK F1174L/DOX-MYCN, 10% with DOX-MYCN, and 0% with both empty vector and ALK F1174L alone. Tumors were transplantable and demonstrated histologic characteristics consistent with NB, including morphology and expression markers. Thus, we are demonstrating the first, to our knowledge, human stem cell-based model of NB. We are using this system to further investigate similarities and differences with GEMMs of NB, test and establish novel candidate drivers of NB, and evaluate potential therapeutic options.

Citation Format: Miller Huang, Lauren K. McHenry, Matthew L. Miller, Grace E. Kim, Branden S. Moriarity, Yuichiro Miyaoka, Marianne E. Bronner, David A. Largaespada, Bruce R. Conklin, Hanlee P. Ji, John M. Maris, Katherine K. Matthay, William A. Weiss. Human stem cell-based model of MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2015-3230
Abstract 3252: LGR5 suppresses colon cancer metastasis
Authors: Zhou Xiaolin , Wang Jing Jenny
Published: August 2015

Metastasis is the major cause of death for patients with solid tumors, mainly due to the lack of effective therapies once metastases begin to form. Further research into the mechanism of metastasis is therefore essential in order to have a better understanding of this process and eventually develop more efficient cancer therapies to prevent or treat metastasis. LGR5 is a leucin-rich G protein coupled receptor and a Wnt target gene. It marks the proliferating adult stem cells in the small intestine and has been shown to play an important role in the maintenance of intestine stem cell proliferation by enhancing the canonical Wnt signaling. It is overexpressed in human colorectal, ovary and liver tumors due to the inappropriate activation of Wnt signaling at the onset of those diseases. Recent studies are conflicting regarding the functional role of LGR5 in colon cancer initiation. However, its role in colon cancer progression and metastasis has not been studied. Analysis of copy number variation (CNV) of LGR5 from the Cancer Genome Atlas (TCGA) database revealed that CNV loss of LGR5 is enriched in late stage colon cancer (stage III and IV) with high lymph node spread and/or distant metastasis and in patients with recurrence whereas CNV gain is enriched in early stage disease with no regional or distant metastasis. These results suggest that LGR5 expression may increase during the onset of colon tumorigenesis and decrease thereafter during tumor progression. Functional studies showed that knockdown of LGR5 expression in colon cancer cells increased their clonogenecity, survival capacity and motility in vitro and metastatic dissemination to the liver and lungs in an orthotopic model in vivo. Our results demonstrate that LGR5 functions as a suppressor of colon cancer metastasis and that reduction of LGR5 expression contributes to colon cancer progression and metastasis.

Citation Format: Xiaolin Zhou, Jing Jenny Wang. LGR5 suppresses colon cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3252. doi:10.1158/1538-7445.AM2015-3252
Abstract 3260: Identification of a novel signaling axis that leads to co-induction of two major actin-regulatory proteins with anti-migratory properties in breast cancer cells
Authors: Joy Marion , Roy Partha
Published: August 2015

Dysregulation of actin-regulatory proteins is a hallmark feature of tumor malignancy. Along this line, recent studies have established causal relationships between downregulation of expression of actin-binding proteins profilin-1 and -2 (Pfn1, Pfn2 - genes located on chromosomes 17 and 3, respectively) and increasing invasive ability of breast cancer cells. Conversely, genetically elevating expression of these Pfn isoforms suppresses aggressiveness of breast cancer cells. Therefore, identification of a common signaling pathway linking to regulation of these two Pfn isoforms may lead to a novel conceptual strategy to suppress metastatic potential of breast cancer cells in Pfn-dependent manner. We found that hyperactivating RhoA, a member of Rho-family GTPases, causes a marked depletion of cellular Pfn1 and Pfn2 expression, suggesting that a pathway downstream of activated RhoA is one of the major signaling axes for regulating Pfn1 and Pfn2 expression. Through overexpression and knockdown experiments, we further identified MKL1 (megakaryoblastic leukemia-1), a transcriptional co-factor that activates serum-response factor (SRF)-mediated gene transcription downstream of activated Rho, to be a major regulator of expression of Pfn isoforms. However, SRF does not appear to play a role in Rho-dependent Pfn regulation and furthermore, Rho-dependent Pfn regulation occurs post-transcriptionally suggesting that a non-canonical MKL1-linked pathway is involved in co-regulation of Pfn isoforms. Finally, MKL1-dependent induction of Pfn isoforms involves STAT1 and this pathway can be activated pharmacologically resulting in inhibition of breast cancer cell motility. In conclusion, we have identified a novel MKL1-STAT1 signaling axis for co-induction of the two major cellular isoforms of Pfn. This study could open up promising pharmacological points of attack in the context of metastatic breast cancer.

Citation Format: Marion Joy, Partha Roy. Identification of a novel signaling axis that leads to co-induction of two major actin-regulatory proteins with anti-migratory properties in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3260. doi:10.1158/1538-7445.AM2015-3260
Abstract 3281: Oklahoma Nitrone-007: A novel compound for treating pediatric glioblastoma
Authors: Mallory Samantha , McNall-Knapp Rene , Coutinho deSouza Patricia , Fung Kar-Ming , Saunders Debra , Smith Nataliya , Smartwood Shanna , Towner Rheal
Published: August 2015

Brain tumors are the most common solid tumor in children, and despite current treatment options including surgical resection, chemotherapy, and radiation for pediatric high-grade gliomas, survival in these patients remains very poor. This research represents promising pre-clinical data of a novel compound, OKN-007 (OKlahoma Nitrone 007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)), as a new clinical therapy for pediatric high-grade gliomas (pHGGs). The objective is to establish if pHGGs, which are often hard to surgically resect, can be treated with OKN-007, either as a single agent or in combination with currently used chemotherapeutic agents. OKN-007 may be an ideal candidate that can have an anti-cancer effect on pHGG cells, due to its multiple anti-cancer effects, including induction of apoptosis, and inhibition of cell proliferation and angiogenesis, and may play an important role in the anti-cancer activity against pHGGs. Morphological magnetic resonance imaging (MRI) of xenograft nude athymic and SCID mice injected with pHGG cells TCCC3752 and KNS42 is used in this study to assess brain tumor volumes for response to therapy. Preliminary data regarding the regression of pHGGs in mice following treatment with OKN-007 is presented. Mean animal survival was found to be significantly increased with OKN-007 treatment in responsive animals compared to an untreated group, p<0.05. Immunohistochemistry and microarray will be used to assess levels of angiogenic (VEGF (vascular endothelial growth factor), HIF-1α (hypoxia inducible factor-1α), MVD (microvessel density)), cell differentiation (CAIX (carbonic anhydrase IX)), cell proliferation (Glut-1 (glucose transporter 1), MIB-1), apoptosis (cleaved caspase 3) markers and assess inhibition of TGFB and PDGFR complexes and downstream pathways, which are overexpressed in tumors and thought to play a role in oncogenesis and progression. OKN-007 is currently an investigational drug undergoing clinical trial evaluation for recurrent gliomas in adult patients but will still require FDA approval for use in pediatric glioma patients. An outcome of the proposed research is to incorporate the pre-clinical data in an IND application to the FDA in order to translate this research to the clinic and benefit children with pHGGs.

Citation Format: Samantha Mallory, Rene McNall-Knapp, Patricia Coutinho deSouza, Kar-Ming Fung, Debra Saunders, Nataliya Smith, Shanna Smartwood, Rheal Towner. Oklahoma Nitrone-007: A novel compound for treating pediatric glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3281. doi:10.1158/1538-7445.AM2015-3281
Abstract 3300: Concerted epigenetic and signaling mechanisms regulate normal stem cell radiosensitivity
Authors: Jacobs Keith M. , Misri Sandeep , Meyer Barbara , Raj Suyash , Zobel Cheri L. , Sleckman Barry P. , Hallahan Dennis E. , Sharma Girdhar G.
Published: August 2015

Side effects resulting from ionizing radiation (IR) during cancer radiotherapy often involve normal tissue injury including depletion of tissue regenerative capacity. Utilizing multiple in vivo tissue niches and cell culture models, we demonstrate that normal stem cells are highly radiosensitive while differentiated progeny are radioresistant, and IR-induced apoptosis of stem cells is observed broadly throughout the cell cycle independent of proliferation status.

Our study demonstrates that normal stem cells fail to activate the DDR, do not display IR-induced foci in vivo as well as in culture, and exhibit severely attenuated DNA repair. Despite normal sensing of DNA breaks, recruitment/ retention of repair factors at DNA break sites is repressed in stem cells. Stem cells are unable to eliminate constitutive phosphorylation of H2AX-Y142 around break sites, abetting stem cells toward an apoptotic instead of repair pathway. The abrogated DDR in normal stem cells is associated with a constitutively enhanced histone-3 lysine-56 acetylation, which epigenetically contributes to muted retention of repair factors. Reinforced PP2A phosphatase expression was also identified as substantial regulators of stem cell radiosensitivity and transient inhibition of phosphatase activity restores stem cell survival.

We thus identify pluralistic interacting molecular and epigenetic mechanisms that collectively control and impart IR hypersensitive phenotype to the normal stem cells. These insights will be crucial in development of prevention and intervention therapeutic strategies to minimize IR-induced stem cell dropout and increase the efficacy of radiotherapy.

Citation Format: Keith M. Jacobs, Sandeep Misri, Barbara Meyer, Suyash Raj, Cheri L. Zobel, Barry P. Sleckman, Dennis E. Hallahan, Girdhar G. Sharma. Concerted epigenetic and signaling mechanisms regulate normal stem cell radiosensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2015-3300
Abstract 3304: Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer
Authors: Haberlau Steffi , Hönscheid Pia , Jimenez Rafael E. , Baretton Gustavo B. , Tindall Donald J. , Krause Mechthild , Datta Kaustubh , Muders Michael H.
Published: August 2015

Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples.

The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed.

VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity.

We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation.

Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2015-3304
Abstract 3310: Targeting MUC1-mediated metabolism sensitizes pancreatic cancer cells to radiation therapy
Authors: Gunda Venugopal , Abrego Jaime , Shukla Surendra K. , Chaika Nina V. , Yu Fang , Bessho Tadayoshi , Lin Chi , Singh Pankaj K.
Published: August 2015

Overexpression of MUC1 promotes growth promoting metabolic alterations in pancreatic cancer cells. We investigated the role played by MUC1-mediated metabolic alterations in radiation response of pancreatic cancer cells. Our findings indicate that cell survival and clonogenicity of pancreatic cancer cells upon radiation treatment correlate with MUC1 expression. MUC1 expression reduced radiation-induced reactive oxygen species (ROS) production and DNA damage, whereas, increased glucose uptake and lactate secretion upon irradiation. Metabolomic analysis through liquid chromatography-coupled tandem mass spectrometry approach revealed that pancreatic cancer cells exhibit spontaneous metabolic alterations upon irradiation. MUC1 expression further altered radiation-induced carbon flux especially in glycolysis and pentose phosphate pathway. We further investigated radiation-sensitization of MUC1-expressing cells by targeting glycolysis or pentose phosphate pathway using metabolic inhibitors, and observed that pre-treatment with 3-bromopyruvate (BrPA) effectively abrogates MUC1-mediated radiation-resistance by elevating ROS and DNA damage. BrPA treatment also reduced MUC1-induced carbon flux into glycolysis and pentose phosphate pathway, upon irradiation. Hence, our findings demonstrate that MUC1-mediated radiation-resistance in pancreatic cancer cells can be effectively targeted through inhibition of glycolysis.

Citation Format: Venugopal Gunda, Jaime Abrego, Surendra K. Shukla, Nina V. Chaika, Fang Yu, Tadayoshi Bessho, Chi Lin, Pankaj K. Singh. Targeting MUC1-mediated metabolism sensitizes pancreatic cancer cells to radiation therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2015-3310
Abstract 3312: Growth inhibition of cancer cells by 5-aminolevulinic acid
Authors: Horiuchi Toshikatsu , Serizawa Yusuke , Tateishi Syoichiro , Iwaya Keiichi , Watanabe Junichi , Kobayashi Shinichi , Sato Ken , Kimura Fumihiko , Shinomiya Nariyoshi , Morimoto Yuji
Published: August 2015

Introduction: 5-aminolevulinic acid (ALA) is intracellularly metabolized to protoporphyrin IX (PpIX) in heme synthetic pathway. As PpIX is a fluorescent photosensitizer and has been shown to accumulate preferentially in cancer cells, exogenously administered ALA is used for photodynamic diagnosis (PDD) and photodynamic therapy (PDT). However, the influence of exogenously administered ALA on cellular proliferation is not elucidated. Overproduction of heme owing to exogenously administered ALA may induce an upregulation of heme oxygenase (HO-1), which is a potential inhibitor of cellular proliferation via a generation of carbon monoxide (CO) and an induction of P21. We therefore examined cytotoxicity when I was administered.

Methods and Materials: Human cancer cell lines (10 types) and human normal cell lines (2 types) were incubated with ALA (1.0 mM) for 24 hrs. A fluorometric measurement for PpIX (ex. 405 nm. em. 630 nm) and a proliferation assay using WST-8 were carried out. The expression of HO-1 was evaluated using Western blotting.

Results: In 8 out of 10 cancer cell lines, an amount of intracellularly synthesized PpIX after the administration of ALA was highly correlated with a decrease in number of cells (cell death) (R = 0.73), and two out of the eight cell lines (HepG2 and Caki-1) showed a significant reduction in number of cells (> 15%) accompanied with a significant increase in PpIX generation (> 90 a.u.). On the other hand, normal cell lines (HUVEC, SMC) showed less changes in cell count (within 1.5%) accompanied with less PpIX generation (within 35 a.u.). The HO-1 production rate, defined by HO-1 expression level in ALA-treated cells / HO-1 expression level in non-treated cells was highly correlated with the cell death (R = 0.67).

Conclusion: In some types of cancer cell lines, an exogenously administered ALA leads to cell death, probably due to the upregulation of HO-1 that is produced by the accumulation of PpIX. Meanwhile, in normal cell lines, the exogenously administered ALA is less likely to induce cell death.

Citation Format: Toshikatsu Horiuchi, Yusuke Serizawa, Syoichiro Tateishi, Keiichi Iwaya, Junichi Watanabe, Shinichi Kobayashi, Ken Sato, Fumihiko Kimura, Nariyoshi Shinomiya, Yuji Morimoto. Growth inhibition of cancer cells by 5-aminolevulinic acid. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3312. doi:10.1158/1538-7445.AM2015-3312
Abstract 3346: The ketone body β-hydroxybutyrate increases radiosensitivity in glioma cell lines in vitro
Authors: Rossi Alex P. , Woolf Eric C. , Brooks Kenneth S. , Fairres Marshall J. , Scheck Adrienne C.
Published: August 2015

Glioblastoma (GB) is the most common and aggressive primary malignant brain tumor. Despite aggressive treatment, including maximum surgical resection followed by both chemotherapy and radiotherapy, median survival for patients remains at approximately 1.5 years after diagnosis. Recent preclinical studies examining the potential of the ketogenic diet (KD) as an adjuvant therapy for the treatment of malignant gliomas demonstrated that animals fed a KD and treated with chemotherapy or radiation survived significantly longer than those treated with chemotherapy or radiation and fed a standard diet. One of the key results of the KD is an increase in the blood level of the ketone body β-hydroxybutyrate (BHB). To more fully examine the mechanism through which the KD potentiates radiation, we examined the potential of BHB to increase radiosensitivity in mouse glioma cells in vitro. Plating cells in 10mM BHB followed by treatment for 2 weeks following radiation prevented colony formation in both the control and the radiation groups. We therefore assessed the effect of pretreatment with 5mM or 10mM BHB alone prior to radiation with 4 Gy. A statistically significant change in survival was observed when cultures were pre-treated with 10mM BHB for 24 hours prior to irradiation but not with 5mM BHB. However, significant changes in clonogenic survival were observed with a dose of 5mM BHB when cells were treated for 7 days post irradiation, starting 24 hours after irradiation. The effect of BHB on cell growth was then assessed using live cell counts. Both 5mM and 10mM BHB were effective in significantly reducing the cell number by 192 hours post plating. Growth was similarly affected by 10mM BHB or 2 Gy of radiation alone; however, when 10mM BHB was added to 2 Gy of radiation there was a very strong potentiating effect. These data suggest that BHB is an effective radiosensitizer for mouse glioma cells when used alone. In addition, they provide evidence that the extended survival seen in our in vivo mouse model of glioma when radiation is used in addition to the ketogenic diet may be modulated, to a large extent, by blood ketone levels. Whether this is due to differences in radiation-induced DNA damage, DNA repair or changes in the growth rate of the tumor cells is currently under investigation.

Citation Format: Alex P. Rossi, Eric C. Woolf, Kenneth S. Brooks, Marshall J. Fairres, Adrienne C. Scheck. The ketone body β-hydroxybutyrate increases radiosensitivity in glioma cell lines in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2015-3346
Abstract 3356: Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker
Authors: Kline Justin Paul , Liu Xiao , Venkataraman Girish , Lin Jiaying , Kiyotani Kazuma , Smith Sonali M. , Montoya Magdeline , Nakamura Yusuke
Published: August 2015

Gene expression profiling experiments have demonstrated that the immune microenvironment in follicular lymphoma (FL) plays an important role in controlling the behavior of the disease. FL patients with tumors enriched for T cell-related transcripts have a more favorable prognosis. As T cells have been shown to mediate anti-tumor immunity in other cancers, they may also be critical in controlling the progression of FL. T cells are often identified in and around malignant follicles in FL by immunohistochemistry, but it is not known whether their presence relates to their specificity for antigens expressed by FL cells. Hypothesizing that at least a fraction of T cells in FL are truly lymphoma antigen-specific, we have begun to characterize the T cell receptor (TCR) repertoire of FL tissues using a next generation sequencing platform, and have deeply sequenced T cell receptor (TCR) cDNAs of T cell subset populations present in pretreatment FL biopsy specimens. Regulatory T cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion, compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who has received no anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both HLA class I and class II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease.

Citation Format: Justin Paul Kline, Xiao Liu, Girish Venkataraman, Jiaying Lin, Kazuma Kiyotani, Sonali M. Smith, Magdeline Montoya, Yusuke Nakamura. Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3356. doi:10.1158/1538-7445.AM2015-3356
Abstract 3367: BMP-6 regulates expression of androgen receptor via NF-κB signaling pathway and activated macrophages
Authors: Wilcox Emma , Kwon Seok Joo , Lee Geun Taek , Kim Isaac Yi
Published: August 2015

Prostate cancer (CaP) is the most common non-cutaneous cancer among men in the United States (US). Recent report estimated that around 233,000 new CaP cases will be occurred in 2014 and about 29,480 men will be death because of CaP in the US. Despite recent progress in CaP treatment, metastatic CaP patients still have a poor prognosis due to the inevitable emergence of castration resistant prostate cancer (CRPC). It has been reported that androgen hypersensitivity and tumor microenvironment were critical factors for developing CRPC. In the present study, we investigate the role of BMP-6 on androgen hypersensitivity in the context of macrophages. Initially, we co-cultured androgen responsive murine CaP cell lines TRAMP-C1 and PTEN-CaP8 with RAW 264.7 murine macrophage cell lines and treated with BMP-6. We found that androgen receptor (AR) expression was increased in both cell lines by treatment of BMP-6 and BAY 11-7082 NF-κB inhibitor neutralized this augmentation. The result indicates that NF-κB signaling pathway is involved in up-regulation of AR in CaP cells. To find which NF-κB is activated, we analyzed NF-κB signaling pathway with immunoblot and found that canonical NF-κB pathway was activated in CaP cells. Cytokines such as IL-6 and TNF-α are potential inducer of NF-κB signaling pathway and these cytokines were up-regulated in RAW cells in our co-culture system. Based on these observations, we suggest that BMP-6 potentially regulates expression of AR in CaP cells via activated macrophages and canonical NF-κB pathway.

Citation Format: Emma Wilcox, Seok Joo Kwon, Geun Taek Lee, Isaac Yi Kim. BMP-6 regulates expression of androgen receptor via NF-κB signaling pathway and activated macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3367. doi:10.1158/1538-7445.AM2015-3367
Abstract 3369: Nuclear hormone receptor profiling of skin cancer-associated fibroblasts for targeted pharmacological modulation of skin squamous cell carcinoma
Authors: Chan Jeremy Soon Kiat , Sng Ming Keat , Teo Zi Qiang , Tan Nguan Soon
Published: August 2015

Background: Cutaneous squamous cell carcinoma (SCC) is the second-most frequently diagnosed non-melanoma skin cancer that commonly manifests in sun-exposed areas of the skin, resulting in permanent disfigurement and potentially death when untreated. The development and malignant progression of skin SCC is dependent not only on the properties of the tumor epithelia but also the effects of the tumor stroma. Cancer associated fibroblasts (CAFs) are enriched in the stroma of skin SCC tumors and supply paracrine factors that encourage tumor growth. However, the transcription regulatory networks enabling this pro-tumor secretory phenotype of CAFs remain largely unknown. Nuclear receptors, being ligand-inducible transcription factors, are relevant therapeutic candidates for modulation of CAF-dependent SCC tumor advancement.

Materials and methods: Laser capture microdissected CAFs from archived patient SCC biopsies were profiled for their expression of 48 known nuclear receptors. siRNA knockdown and overexpression of specific nuclear receptors in CAFs was performed, and their impact on the SCC transcriptome and phenotype was evaluated in a co-culture system. Co-administration of nuclear receptor ligands and conventional anticancer chemotherapeutics was also carried out in a 3D-organotypic skin tumor model to investigate synergistic and sensitizing effects on SCC.

Findings and conclusions: Our study is the first to define a nuclear receptor profile for SCC CAFs, wherein the expression of 5 nuclear receptors was unchanged, 18 nuclear receptors were highly expressed and 3 nuclear receptors were lowly expressed in CAFs relative to normal fibroblasts. We showed that SCC tumor epithelia are transcriptionally and functionally sensitive to this nuclear receptor profile of CAFs. Simultaneous and differential attenuation of multiple aggressive SCC phenotypes was possible through the manipulation of specific nuclear receptor expression and activity in CAFs. Our results highlight the utility of CAF nuclear receptor profiling and a role for nuclear receptor-directed drugs in the multimodal treatment of skin SCC.

Citation Format: Jeremy Soon Kiat Chan, Ming Keat Sng, Zi Qiang Teo, Nguan Soon Tan. Nuclear hormone receptor profiling of skin cancer-associated fibroblasts for targeted pharmacological modulation of skin squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2015-3369
Abstract 3372: Molecular characteristics of cancer-associated adipocytes in breast cancer
Authors: Moon Hyeong-Gon , Lee Jiwoo , Lee Han-Byoel , Ryu Han Suk , Han Wonshik , Noh Dong-Young
Published: August 2015

Adipose tissue in the tumor microenvironment is now regarded to be an active player in cancer development and progression. Recent studies have suggested critical roles of the cancer-associated adipocytes in breast cancer proliferation and invasion. However, unlike the well-known cancer-associated fibroblasts, the molecular profiles of cancer-associated adipocytes are largely unknown.

We studied the histo-morphologic changes in adipose tissue adjacent to the breast cancer cells in human tissues. Adipocytes had smaller fat droplets and often associated with immune cell infiltration. We investigated the changes of mRNA expression profiles of differentiated adipocytes when they were cultured with breast cancer cells (MCF-7 and MDA-MB-231) by using the NIH-3T3-L1 cell line. Indirect coculture with cancer cells resulted in significantly altered expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. IL6 and its downstream genes, transcription factors such as RUNX1, as well as PTX3 and TIMP1 were most unregulated genes. Increased secretion of IL6 was further validated in the culture media by ELISA. Also, the gene expression data showed that adipocytes cocultured with cancer cells showed decreased adipocyte-differentiation genes suggesting the cells underwent de-differentiation process. Microscopic examination of co-cultured adipocytes showed smaller fat droplets similar to human specimens. The co-cultured adipocytes showed different effect on cancer cells according to the molecular subtypes of the breast cancer cells. The conditioned media from co-cultured adipocytes resulted in increased proliferation of ER+ breast cancer cells, suggesting a subtype-dependent effect of co-cultured adipocytes in breast cancer.

Our results suggest that cancer-associated adipocytes show increased expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. Futhermore, these cancer-associated adipocytes show features toward the preadipocyte state suggesting the possibility of de-differentiation of adipocyte under the influence of adjacent cancer cells. The functional impact of the cancer-associated adipocyte seem to vary according to the molecular subtype of epithelial cancer cells.

Citation Format: Hyeong-Gon Moon, Jiwoo Lee, Han-Byoel Lee, Han Suk Ryu, Wonshik Han, Dong-Young Noh. Molecular characteristics of cancer-associated adipocytes in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2015-3372
Abstract 3392: Ex vivo analysis of RAD51 foci induction in fresh tumor tissue: a promising new tool for the identification of homologous recombination-deficient tumors
Authors: Vreeswijk Maaike , Meijers M. , Naipal K.A.T. , Verkaik N.S. , Kanaar R. , Hoeijmakers J.H.J. , Jager A. , van Gent D.C. , Gaarenstroom K.N. , Smit V.T.H.B.M. , Vrieling H.
Published: August 2015

Purpose. BRCA1 and BRCA2 related breast and ovarian tumors respond very well to treatment with PARP inhibitors (PARPi) because of their deficiency in homologous recombination (HR). We have indications that a substantial number of ovarian tumors might have deficiencies in HR unrelated to germline mutations in BRCA1 or BRCA2. A functional assay to assess HR efficacy might therefore allow identification of additional ovarian cancer patients that could benefit from PARPi treatment.

Experimental procedure. Unselected fresh ovarian tumor samples were collected from patients undergoing surgery or ascites/pleural fluid drainage. Samples were irradiated with ionizing radiation ex vivo, fixed after 2 hours and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined by pathologist. The presence of RAD51 foci in replicating cells as visualized by immunofluorescence was used as a functional read out for HR proficiency.

Results. We determined the ability to induce RAD51 foci following radiation in ex vivo cultured ovarian tumor samples that contained sufficient numbers of replicating cells (n = 13). RAD51 foci formation was absent (<20% replicating cells showed RAD51 foci) in 46% of the samples. In one tumor sample that did not form RAD51 foci in replicating cells upon ionizing radiation, a pathogenic mutation in BRCA1 was identified (c.5277+1G>A). Genetic and epigenetic analyses of the other samples are ongoing.

Conclusion. The ex vivo analysis of RAD51 foci induction in fresh tumor tissue is a promising new tool to identify HR deficient tumors. Not only is it an important time saving strategy compared to BRCA1/BRCA2 mutation analysis, it may also identify a much broader patient population that will be eligible for the treatment with PARP inhibitors.

Citation Format: Maaike Vreeswijk, M. Meijers, K.A.T. Naipal, N.S. Verkaik, R. Kanaar, J.H.J. Hoeijmakers, A. Jager, D.C. van Gent, K.N. Gaarenstroom, V.T.H.B.M. Smit, H. Vrieling. Ex vivo analysis of RAD51 foci induction in fresh tumor tissue: a promising new tool for the identification of homologous recombination-deficient tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3392. doi:10.1158/1538-7445.AM2015-3392
Abstract 3393: Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology
Authors: Chen Caiwei , Tang Yanlin , Wei Haitao , Yuan Kehu , Wu Guiyin , Chen Jian , Chu Boyang , Wang Guangli , Shu Youmin , He Wei-Wu , Ma Donghui
Published: August 2015

Programmed death-1 (PD-1) expresses in many tumors in response to inflammation. It is up-regulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands PD-L1 and PDL2 and serves as a key checkpoint of the immune system. Pembrolizumab is the first anti-PD-1 therapy approved in the United States and received FDA's Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin-fixed paraffin-embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed.

To develop the best monoclonal antibody for PD-1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono-specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross-reactivity tests were also explored.

In summary, two newly generated monoclonal antibodies demonstrated ultra-specificity against PD-1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before pembrolizumab prescription.

Citation Format: Caiwei Chen, Yanlin Tang, Haitao Wei, Kehu Yuan, Guiyin Wu, Jian Chen, Boyang Chu, Guangli Wang, Youmin Shu, Wei-Wu He, Donghui Ma. Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2015-3393
Abstract 3402: Activin signal promotes cancer progression and is involved in cachexia in a subset of pancreatic cancer
Authors: Togashi Yosuke , Kogita Akihiro , Sakamoto Hiroki , Hayashi Hidetoshi , Terashima Masato , de Velasco Marco A. , Sakai Kazuko , Fujita Yoshihiko , Tomida Shuta , Kitano Masayuki , Kudo Masatoshi , Nishio Kazuto
Published: August 2015

Purpose: We previously reported that activin produces a signal with a tumor suppressive role in pancreatic cancer (PC). Here, the association between plasma activin A and survival in patients with advanced PC was investigated.

Experimental Design: The plasma activin A concentrations of patients with advanced PC were determined using an ELISA. The influence of ligands on PC cell lines was evaluated using an MTT assay and western blot analyses. The INHBA gene (beta subunit of inhibin) was overexpressed in PC cell lines, and this phenotype was investigated.

Results: Contrary to our expectations, patients with high plasma activin A levels had a significantly shorter survival period than those with low levels (median survival, 314 days vs. 482 days, P = 0.034). The cellular growth of the MIA PaCa-2 cell line was greatly enhanced by activin A via non-SMAD pathways. The cellular growth and colony formation of an INHBA-overexpressed cell line were also enhanced. In a xenograft study, INHBA-overexpressed cells tended to result in a larger tumor volume, compared with a control. The bodyweights of mice inoculated with INHBA-overexpressed cells decreased dramatically, and these mice all died at an early stage, suggesting the occurrence of activin-induced cachexia.

Conclusions: Our findings indicated that the activin signal can promote cancer progression in a subset of PC and might be involved in cachexia and that plasma activin A can be a novel prognostic factor in patients with advanced PC. The activin signal might be a novel target for the treatment of PC.

Citation Format: Yosuke Togashi, Akihiro Kogita, Hiroki Sakamoto, Hidetoshi Hayashi, Masato Terashima, Marco A. de Velasco, Kazuko Sakai, Yoshihiko Fujita, Shuta Tomida, Masayuki Kitano, Masatoshi Kudo, Kazuto Nishio. Activin signal promotes cancer progression and is involved in cachexia in a subset of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3402. doi:10.1158/1538-7445.AM2015-3402
Abstract 3438: Whole pelvis versus prostate-only radiotherapy with or without short course androgen deprivation therapy and mortality risk
Authors: Braunstein Lior Z. , Chen Ming-Hui , Dosoretz Daniel E. , Salenius Sharon , Katin Michael , Nanda Akash , D'Amico Anthony V.
Published: August 2015

Purpose: To determine whether extent of prostate radiotherapy (i.e. whole-pelvic radiotherapy (WPRT) versus prostate/seminal-vesicle radiotherapy [PSVRT]) is associated with all-cause mortality (ACM) in men treated with or without androgen deprivation therapy (ADT).

Methods: A multi-institutional cohort of 3,709 prostate cancer patients was prospectively assembled from 1991-2006. Median age was 72 years and all patients had T1c-T3N0M0 adenocarcinoma of the prostate. Patients were treated with WPRT or PSVRT followed by a brachytherapy boost, with or without neoadjuvant ADT (median duration 4.2months). 70% of patients had unfavorable-risk disease (Gleason ≥7; PSA ≥10ng/mL; or stage ≥T2b). Cox regression was applied to determine whether the radiation treatment volume affected the risk of ACM. A test for interaction between radiation volume and ADT use was conducted.

Results: After a median follow-up of 3.3 years, 561 deaths were observed. A decreased risk of ACM was noted with the use of WPRT versus PSVRT (adjusted hazard ratio [AHR] 0.58; 95% confidence interval [CI] 0.38-0.89; p = 0.01), or with ADT (AHR 0.71; 95%CI 0.58-0.90; p = 0.004). However, combining WPRT and ADT did not further improve ACM in comparison to either WPRT alone or PSVRT with ADT. Moreover, there was a significant interaction between the RT treatment volume and ADT (AHR 1.61; 95%CI 1.004-2.58; p = 0.048).

Conclusions: Treatment with WPRT or short-course ADT is associated with a decreased risk of ACM, though combining the two does not yield greater benefit. This observation suggests a shared mechanism for this risk-reduction which we hypothesize to be sterilization of micrometastatic disease within the pelvic lymph nodes.

Citation Format: Lior Z. Braunstein, Ming-Hui Chen, Daniel E. Dosoretz, Sharon Salenius, Michael Katin, Akash Nanda, Anthony V. D'Amico. Whole pelvis versus prostate-only radiotherapy with or without short course androgen deprivation therapy and mortality risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3438. doi:10.1158/1538-7445.AM2015-3438
Abstract 3497: Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations
Authors: Koh Siang-Boon , Richards Frances M. , Rodriguez Esther , Lyons Scott K. , Jodrell Duncan I.
Published: August 2015

Sensitization of cancer cells to gemcitabine has been shown with checkpoint kinase CHK1 and WEE1 inhibitors. To rationalize and optimize the concomitant use of these three agents, we first performed growth inhibition assays on MIA PaCa-2 pancreatic cancer cells using gemcitabine in combination with either CHIR-124 (CHK1 inhibitor) or MK-1775 (WEE1 inhibitor). In silico analysis with three mathematical models (Bliss Independence, Loewe and Highest Single Agent) identified synergistic growth inhibition at submaximal (i.e. <GI50) concentrations of the single agents (10nM gemcitabine, 20nM CHIR-124, 300nM MK-1775) in MIA PaCa-2 cells. At these concentrations of gemcitabine+CHIR-124, quantitative image-based cytometry demonstrated S-phase redistribution, with 31% of >8000 individual cells undergoing extensive fork collapse (compared to 0-1% with single agents) as evidenced by co-staining of yH2AX and hyper-phosphorylated RPA32. Conversely, submaximal gemcitabine+MK-1775 induced clear reduction of inhibitory CDK1 Y15 level compared to single agents. Consistent with this G2/M abrogation, in a Fucci-based CDT1/geminin-expressing MIA PaCa-2 stable cell line, gemcitabine+MK-1775 partially reversed the accumulation of the geminin-expressing (S/G2) population induced by gemcitabine alone. This was accompanied by an increase in the CDT1-expressing (G1) population, alongside aberrant mitotic cells with features of perturbed kinetochore-microtubule integrity. Based on these findings, we performed a series of long-term, kinetic live-cell imaging assays to determine the optimal scheduling for gemcitabine/CHK1i/WEE1i as a triplet at synergistic concentrations. In contrast to the widely proposed scheduling regimen, we found that delayed administration of CHK1i (at 24 hours), relative to gemcitabine, did not lead to synergy. However, concurrent administration yielded durable growth inhibition, even when the two agents were removed after 24 hours. This inhibition was further enhanced with the subsequent addition of WEE1i at 24 hours, but not continuation of a CHK1i. Together, our results illustrate differential mechanistic effects of CHK1 and WEE1 inhibitors with gemcitabine, when combined at sub-GI50 concentrations, and propose a novel triple agent schedule that will be evaluated in vivo.

Citation Format: Siang-Boon Koh, Frances M. Richards, Esther Rodriguez, Scott K. Lyons, Duncan I. Jodrell. Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2015-3497