bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2327: Placenta-derived mesenchymal stem cells and their secreted exosomes inhibit the self-renewal and stemness of glioma stem cells in vitro and in vivo
Authors: Brodie Chaya , Buchris Edrat , Finniss Susan , Cazacu Simona , Xiang Cunli , Lee Hae Kyung , Poisson Laila
Published: August 2015

Mesenchymal stromal cells (MSCs) are multipotent stem cells that can be obtained from bone marrow and adipose tissues or from other sources such as placenta and umbilical cord. The latter allow the potential use of universal, allogeneic cell therapy because to reduced antigenicity due to low expression of MHC class II molecules. MSCs can be easily expanded in vitro for therapeutic applications and their safety and therapeutic impact have been demonstrated in various pre-clinical and clinical studies. MSCs have been shown to cross the blood brain barrier and migrate to sites of experimental GBM and can deliver cytotoxic compounds that exert anti-tumor effects. In this study we examined the effects of placenta-derived MSCs and their secreted exosomes on GSCs in vitro and in vivo. Conditioned medium of placenta MSCs or their derived exosomes decreased the self-renewal, stemness markers, Sox2 and Oct4 and the migration of these cells. Similarly, intracranial administration of the MSCs decreased the tumor volume of GSC-derived xenografts and prolonged animal survival. miRNA sequencing analysis of placenta MSC-derived exosomes revealed a set of specific miRNAs that were downregulated in GSCs and that acted as tumor suppressor in these cells. We demonstrated delivery of some of these miRNAs to GSCs following treatments with MSC-derived exosomes. We further demonstrated that MSCs or exosomes that were loaded with exogenous miR-124 delivered high levels of this miRNA into glioma cells as detected by a novel quantitative miRNA reporter. Moreover, administration of placenta MSCs loaded with exogenous miR-124 exerted a strong inhibitory effect on GSC-derived xenograft growth. These results demonstrate that placenta-derived MSCs may have important clinical applications in stem cell-based glioma therapeutics. Moreover, these studies provide a novel approach for the targeted delivery of endogenous and exogenous anti-tumor miRNAs to glioma cells as a miRNA replacement therapy for GBM.

Citation Format: Chaya Brodie, Edrat Buchris, Susan Finniss, Simona Cazacu, Cunli Xiang, Hae Kyung Lee, Laila Poisson. Placenta-derived mesenchymal stem cells and their secreted exosomes inhibit the self-renewal and stemness of glioma stem cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2015-2327
Abstract 2331: CD271 defines a cancer-initiating cell population in hypopharyngeal cancer
Authors: Tanaka Nobuyuki , Imai Takayuki , Mochizuki Mai , Tamai Keiichi , Yamaguchi Kazunori , Satoh Kennichi , Matsuura Kazuto , Sato Ikuro , Saijo Shigeru , Sugamura Kazuo
Published: August 2015

Cancer stem cells (CSCs) or Cancer initiating cells (CICs) contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. We found that the CD271+ but not CD271- cell population sorted from xenotransplanted HPCs possesses tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271+ cells contained both CD271+ and CD271- cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271+ cells localized to a perivascular niche near CD34+ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metaloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271+ compared to the CD271- cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Furthermore, RNA interference of CD271 in CD271+ cells decreased efficiency of tumor initiation and migration phenotypes, suggesting that CD271 is indeed functional. Taken together, our findings indicate that CD271 is a novel marker for cancer initiating cells and for HPC prognosis.

Citation Format: Nobuyuki Tanaka, Takayuki Imai, Mai Mochizuki, Keiichi Tamai, Kazunori Yamaguchi, Kennichi Satoh, Kazuto Matsuura, Ikuro Sato, Shigeru Saijo, Kazuo Sugamura. CD271 defines a cancer-initiating cell population in hypopharyngeal 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 2331. doi:10.1158/1538-7445.AM2015-2331
Abstract 2333: The tumor suppressive role of PRMT6 in regulating the pathogenesis of hepatocellular carcinoma
Authors: Chan Lok Hei , Ng Kai Yu , Ma Stephanie
Published: August 2015

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. The overall 5-year survival rate of HCC patients remains at a dismal 15% due to late diagnosis, metastasis and frequent tumor relapse. The presence of cancer stem cells (CSCs) has been proposed to account partly for the challenge of treating HCC. Our group has previously identified CD133 as a functional CSC marker of HCC. By adopting a PCR array encompassing diverse human chromatin modifiers, protein arginine methyltransferase 6 (PRMT6) was found to be differentially down-regulated in CD133+ human HCC cells. 55.8% (43/77) of clinical HCC tumor tissues showed down-regulated PRMT6 expression and patients with reduced PRMT6 expression were at significant risk of disrupted tumor encapsulation and manifested vascular invasion. An antagonistic relationship between PRMT6 and CD133 was observed in HCC cells, while modulation of PRMT6 led to an inversely altered CD133 expression. PRMT6 knockdown in liver cancer cells induced morphological alterations, where extensive cytoplasmic stretching and filopodia-like projections were observed. Moreover, PRMT6 regulated cancer and stem cell-like properties in vitro and in vivo including proliferation, colony formation, invasion, migration, angiogenesis, self-renewal, chemo-sensitivity and tumor initiation. In addition, stable knockdown of CD133 in HCC cells did not alter PRMT6 expression levels, which suggested that PRMT6 is potentially functioning upstream of CD133. Ongoing studies are in progress to delineate the underlying mechanisms by which PRMT6 suppress HCC.

Citation Format: Lok Hei Chan, Kai Yu Ng, Stephanie Ma. The tumor suppressive role of PRMT6 in regulating the pathogenesis of hepatocellular 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 2333. doi:10.1158/1538-7445.AM2015-2333
Abstract 235: SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma
Authors: chen Luyue , Han Lei , zhang kailiang , wei jianwei , pu peiyu , zhang jianning , kang chunsheng
Published: August 2015

Background

Gliomblastoma (GBM) is associated with disproportionately high morbidity and mortality, reflecting the need to develop new diagnostic and therapeutic targets for this disease. Recently, accumulating evidence has suggested that small nucleolar RNAs (snoRNAs) are gaining prominence and are more actively involved in tumorigenesis than previously thought. However, no report concerning the implication of snoRNAs in glioma has been published to date.

Methods

The expression of SNORD76 and its host gene, GAS5, in glioma patients were evaluated by quantitative PCR. Flow cytometry was used to analyze to cell cycle distribution after SNORD76 over-expression. After SNORD76 over-expression in GBM cell lines, cell growth and anti-tumor effect were investigated in vitro and in vivo.

Results

SNORD76 expression was selectively decreased in glioblastoma (WHO grade IV) compared with low grades glioma (WHO grade II and III). Forcibly expressed SNORD76 inhibited proliferation and anchorage-independent growth of glioma cells. Flow cytometry demonstrated that enforced SNORD76 expression arrested the cells at S phase of the cell cycle. Western blot Cell cycle-associated proteins cyclin A1, cyclin B1 and p107 were down-regulated after SNORD76 over-expression, while the expression of Rb and pRb increased. The growth of orthotopic tumors was significantly inhibited by forced expression of SNORD76.

Conclusion

In summary, engineered restoration of SNORD76 caused marked repression of glioma growth in vitro and in vivo. The tumor suppression could be achieved at least partially through inducing S phase arrest in an Rb-associated manner. Thus, SNORD76 may act as a potential tumor suppressive gene in the development and progression of glioblastoma and may be a potential specific glioblastoma biomarker.

Citation Format: Luyue chen, Lei Han, kailiang zhang, jianwei wei, peiyu pu, jianning zhang, chunsheng kang. SNORD76, a box C/D snoRNA, acts as a tumor suppressor in 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 235. doi:10.1158/1538-7445.AM2015-235
Abstract 2388: Interaction between connective tissue growth factor and a disintegrin and metalloproteinase with thrombospondin type I repeats 7 during cancer development
Authors: Pi Liya , Petersen Bryon E.
Published: August 2015

Introduction: Connective tissue growth factor (CTGF) is a secreted pro-angiogenic protein within the CCN (Cyr61/CTGF/Nov) family and modulates multiple angiogenic pathways through its broad binding ability to cysteine knot motifs presents in key angiogenic factors including vascular endothelial growth factor (VEGF)-A. Overexpression of CTGF has been found in many cancers such as Lewis lung carcinoma (LLC), cholangiocarcinoma (CC), and hepatocellular carcinoma (HCC). In an effort to understand the molecular action of CTGF, we identified a disintegrin and metalloproteinase with thrombospondin type I repeat 7 (ADAMTS7) as a CTGF binding protein in yeast two-hybrid analysis. This enzyme belongs to the ADAMTS family capable of cleaving extracellular matrix (ECM) components and extracellular regulatory molecules. Here, we show that ADAMTS7 binds to and degrades CTGF during cancer development.

Methods: Adamts7 knockout mice were used to investigate the importance of ADAMTS7 in CTGF interaction and cleavage during LLC development. Adamts7+/+ and Adamts7−/− mice were subcutaneously implanted with LLC cells. Tumor growth was quantitatively measured within two weeks after implantation. Tumor angiogenesis was assessed utilizing a microvascular density assay. In addition, Adamts7 expression was tracked via X-gal staining of LLC tumors grown in Adamts7−/− mice that contained a LacZ trap-in cassette in the targeted Adamts7 allele. VEGF-A, CTGF, and ADAMTS7 were determined at both the mRNA and protein levels.

Results: Adamts7 induction as indicated by the β-galactosidase activity in X-gal staining was found in intra-tumor stromal cells of LLC tumors grown in Adamts7−/− mice. The association of CTGF and ADAMTS7 proteins in protein lysates of LLC tumors grown in Adamts7+/+ mice was confirmed in immunoprecipitation assays. Higher levels of VEGF-A, CTGF and ADAMTS7 mRNAs were found in LLC tumors from Adamts7−/− animals as compared to Adamts7+/+ controls. Slower rate of CTGF turnover, faster growth of LLC tumors, and higher microvascular density were also found in Adamts7−/− animals than controls.

Conclusion: These observations confirmed the binding and processing of CTGF by ADAMTS7 during LLC growth. The host-derived ADAMTS7 appears to regulate CTGF turnover and provides a protective effect towards aberrant LLC tumorigenesis and angiogenesis.

Citation Format: Liya Pi, Bryon E. Petersen. Interaction between connective tissue growth factor and a disintegrin and metalloproteinase with thrombospondin type I repeats 7 during cancer development. [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 2388. doi:10.1158/1538-7445.AM2015-2388
Abstract 2404: Quality assessment of circulating cell-free DNA using multiplexed droplet-digital PCR
Authors: Contente-Cuomo Tania , Murtaza Muhammed
Published: August 2015

Recent proof-of-principle studies have demonstrated potential utility of sequencing cell-free DNA in cancer diagnostics. However, little is understood about the effect of fragment size distributions in plasma DNA on the performance of sequencing-based assays. We developed a multiplexed assay to perform one-step analysis of DNA quantity and integrity from minute amounts of cell-free DNA using picoliter droplet digital PCR. Primers were designed to amplify 5 short PCR products (67-71 bp) and 4 long PCR products (439-522 bp) from independent quiescent regions of the human genome. Oligonucleotide probes targeting each of the short and long products were labeled with fluorescent dyes FAM and TET respectively. We evaluated the performance of our assay using control human genomic DNA sheared using sonication to an average of 150 bp, 300 bp, 500 bp and 1000 bp fragment sizes. We observed that relative quantities of short and long PCR products reflect integrity of sheared input DNA. We further observed that accurate quantification and assessment of integrity could be achieved using picogram amounts of cell-free DNA (as few as 200 pg in current assessments). Finally, our results were predictive of diversity and obtainable depth-of-coverage in next-generation sequencing libraries made from cell-free DNA samples.

Citation Format: Tania Contente-Cuomo, Muhammed Murtaza. Quality assessment of circulating cell-free DNA using multiplexed droplet-digital PCR. [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 2404. doi:10.1158/1538-7445.AM2015-2404
Abstract 241: A novel tumor suppressor miRNA co-regulating EMT and p53-independent cell survival in breast cancer
Authors: Raza Umar , Uhlmann Stefan , Yurdusev Emre , Wiemann Stefan , Sahin Ozgur
Published: August 2015

Breast cancer is the most common cancer and the second leading cause of cancer-related deaths among women. MicroRNAs (miRNAs) have been shown to be key regulators of cancer-related processes including cell proliferation, survival, migration and invasion. Here, we performed a small scale miRNA mimic screen to identify miRNAs leading to inhibition of cell survival and epithelial-mesenchymal transition (EMT), both of which are key parameters for tumor growth and metastasis. We identified a miRNA (miR-644) which leads to apoptosis in p53-mutated breast cancer cell lines, but not in p53-wild type ones. Furthermore, in vitro migration and invasion assays revealed that miR-644 inhibits both migration and invasion by interfering with the EMT process. Then, we combined whole genome mRNA profiling with target prediction algorithms to determine the potential targets of this miRNA, and identified transcriptional co-repressor CTBP1 as a novel direct target which is already known for limiting apoptosis by repressing Bax transcription. CTBP1 has also been reported to bind ZEB transcription factors and assists EMT by enhancing ZEB-mediated downregulation of E-Cadherin. We showed that silencing of CTBP1 phenocopies the effects of miR-644 in induction of p53-independent apoptosis and blocking EMT and cell invasion. Currently, we are testing the role of miR-644 and its target CTBP1 in tumor growth and metastasis in in vivo assays. Overall, our results suggest that miR-644 is a novel tumor suppressor miRNA co-regulating EMT and cell survival and it could be a potential target for breast cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Umar Raza, Stefan Uhlmann, Emre Yurdusev, Stefan Wiemann, Ozgur Sahin. A novel tumor suppressor miRNA co-regulating EMT and p53-independent cell survival 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 241. doi:10.1158/1538-7445.AM2015-241
Abstract 2410: Cell-free DNA derived from ascites: Detection of copy number and somatic mutations using OncoScan FFPE® Assay
Authors: Husain Hatim , Venkatapathy Sumathi , Gomez German , Woodward Brian , Lee Suzanna , Khambaty Lubena , Chen Lily , Duttagupta Radha , Fung Eric T. , Kurzrock Razelle
Published: August 2015

Background: There is increased interest in molecular analysis of cell-free DNA isolated from body fluids for the evaluation of tumor progression. These “liquid” biopsies are usually obtained from blood. Their advantages include the fact that they can be repeatedly accessed, are less invasive, and may be sensitive to changes in tumor profile from multiple metastatic sites.

Materials and Methods: Eleven ascites samples from patients with metastatic epithelial neoplasms (gastric, N = 3; pancreas, N = 3; ovarian, N = 2; breast, N = 2; and lung cancer, N = 1) were investigated. Cell-free DNA was isolated from supernatant of ascites fluid (50 ml) after centrifugation using commercially available DNA purification kits (Norgen Bioteck Corp and Qiagen), and analyzed using the OncoScan FFPE Assay kit.

Results: Cell-free DNA yields ranged from 1.7 ng to 230 ng per mL of ascites fluid, indicating wide variability in DNA content. Of the 11 patients, all had detectable aberrations, including copy number alterations affecting from <1%- 37% of the genome. A sample from a patient with metastatic breast cancer had apparent chromosome 12 chromothripsis (chromosome breakage with numerous clustered chromosomal rearrangements). Another sample (metastatic lung cancer) had high overall genomic stability (T); the lung tumor tissue did not show these alterations though the patient has never smoked. Other aberrations were seen in genes including: p53 (N = 4 patients), EGFR (N = 3), ERBB2 (N = 2), PI3KCA (n = 2).

Conclusions. Combined copy number and somatic analysis of ascites cell-free DNA revealed clinically relevant aberrations, including ones not found in primary tumor tissue. Comparison to circulating cells in ascites, cell-free DNA in blood, as well as tumor molecular profile is ongoing.

Citation Format: Hatim Husain, Sumathi Venkatapathy, German Gomez, Brian Woodward, Suzanna Lee, Lubena Khambaty, Lily Chen, Radha Duttagupta, Eric T. Fung, Razelle Kurzrock. Cell-free DNA derived from ascites: Detection of copy number and somatic mutations using OncoScan FFPE® Assay. [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 2410. doi:10.1158/1538-7445.AM2015-2410
Abstract 2430: Treatment sequence and molecular biomarker in EGFR mutant lung cancer cells
Authors: Motes Hannah , Reyes Emma Borrego-Diaz , Kevern Jared , Huang Chao H. , Van Veldhuizen Peter J.
Published: August 2015

Introduction

Lung cancer is the leading cause of cancer related death in the US. A subset of patients with lung cancer has a mutation in the Epidermal Growth Factor Receptor (EGFR) which makes them extremely responsive to EGFR tyrosine kinase inhibitor (TKI) erlotinib. The emergence of EGFR TKI resistance is new challenge in the management of these patients. We conducted a study to determine the best treatment sequence after resistance to EGFR TKI, testing the use of chemotherapy Pemetrexed (P), Afatinib (A), Rociletinib (R) and the combination of these agents in lung cancer cell lines with EGFR mutation that were pretreated with erlotinib.

Material and methods

Lung cancer cell lines with EGFR mutation CRL-2868 (E746 - A750 deletion) and CRL-2871 (L747 - E749 deletion, A750P) mutation were treated with 5 nM erlotinib for 24h and 72 h.

The erlotinib resistance cells were then treated with 10nM of A or 10nM of P, or 21.5 nM of R, or the combination AP or the combination of RP. As control, we used lung cancer HTB-177 which does not have EGFR mutation. MTT proliferation assay was performed after 24, 48, 72 and 144h after the treatment. microRNA profiling was done by real time PCR in all cells after erlotinib treatment to find potential biomarkers as hallmark of erlotinib resistance cells.

Results

Rociletinib induced the maximum inhibition of cell proliferation at 144h in both cell lines with EGFR mutation. The RP also induced decreased cell proliferation in both cell lines with EGFR mutation. RP combination was more effective then R alone in CRL 2871. The use of A alone, P alone or AP combination was not different in CRL2871 but P had antagonistic effect when combined with A or R in CRL 2868. microRNA 10b, 27a and 27b were upregulated in the erlotinib resistant cells.

Conclusion.

EGFR resistance arises after treatment with erlotinib in lung cancer cell lines with EGFR mutation. Treatment with R and the combination of RP induced greater inhibition of proliferation compared to A, P alone. The combination of AP was antagonistic. This may have implications in the management of patients resistant to EGFR TKI. microRNA analysis showed up-regulation of several microRNA that should be tested in clinical setting.

Citation Format: Hannah Motes, Emma Borrego-Diaz Reyes, Jared Kevern, Chao H. Huang, Peter J. Van Veldhuizen. Treatment sequence and molecular biomarker in EGFR mutant lung 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 2430. doi:10.1158/1538-7445.AM2015-2430
Abstract 2433: A novel MDM2 inhibitor suppresses breast cancer growth and metastasis
Authors: Qin Jiang-Jiang , Wang Wei , Voruganti Sukesh , Zhang Ruiwen
Published: August 2015

The MDM2 oncogene is amplified and/or overexpressed in many human cancers, including breast cancer, and it has been suggested as a molecular target for treating human cancers. MDM2 is a major negative regulator of tumor suppressor p53. Most MDM2 inhibitors under development are targeting the MDM2-p53 binding, and have little or no effects on cancers without functional p53, such as advanced breast cancer. Therefore, new strategies to target MDM2 are desirable. The present study was designed to develop a new class of MDM2 inhibitors that exert anti-breast cancer activity, regardless of p53 status in the cancers. The selective MDM2 inhibitors were discovered by a computational structure-based screening, yielding a lead compound, termed JapA. Fluorescence polarization (FP)-based binding assay and cellular thermal shift assay were performed to determine the binding between MDM2 and JapA. Its cytotoxicity was initially evaluated in breast cancer cell lines (MCF-7, MCF-7/p53−/−, MDA-MB-231, and MDA-MB-468) and normal human breast cells (MCF-10A). Its anti-breast cancer activities were further assessed in in vitro and in vivo MCF-7 and MDA-MB-231 models. The molecular mechanisms for its targeting effects on MDM2 were elucidated in various cell models in vitro and in vivo. We further found that JapA selectively inhibited breast cancer cell growth, decreased cell proliferation, and induced G2/M phase arrest and apoptosis in breast cancer cells through an MDM2-dependent mechanism, regardless of p53 status. In MCF-7 and MDA-MB-231 xenograft models, JapA inhibited MDM2 and suppressed tumor growth and lung metastasis in a dose-dependent manner, without host toxicity. Furthermore, JapA directly bound to MDM2 protein and reduced MDM2 levels in cancer cells in vitro and in vivo by promoting MDM2 protein degradation and inhibiting MDM2 transcription, which is distinct from the existing MDM2 inhibitors. In conclusion, JapA represents a new class of MDM2 inhibitor that exerts its anticancer activity through directly down-regulating MDM2, and might be developed as a novel cancer therapeutic agent. (Supported by the NIH R01 CA112029, R01 CA121211, and R01 CA186662)

Citation Format: Jiang-Jiang Qin, Wei Wang, Sukesh Voruganti, Ruiwen Zhang. A novel MDM2 inhibitor suppresses breast cancer growth and 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 2433. doi:10.1158/1538-7445.AM2015-2433
Abstract 2605: The geranylgeranyltransferase I inhibitor GGTI-2418 suppresses multiple myeloma malignancy in the 5TMG1 mouse model
Authors: Yang Hua , Emmons Michael F. , Cubitt Christopher , Shain Ken , Coppola Domenico , Sullivan Daniel , Lynch Conor C. , Hazlehurst Lori , Sebti Said M.
Published: August 2015

Little is known about the contributions of geranylgeranylated proteins to multiple myeloma (MM) malignancy, and whether targeting geranylgeranyltransferase I (GGT-1) is a viable therapeutic approach in myeloma is yet to be explored. In this study, we investigated the effects of GGTI-2418, a GGT-1 inhibitor that has reached phase I clinical trials, on MM malignancy in the 5TMG1 mouse model, where GGTI-2418 effects were assessed in the confines of the bone microenvironment that recapitulates the clinical disease in humans. GGTI-2418 treatment significantly decreased the serum levels of the MM biomarker IgG2B (M-protein) as well as the percentage of MM tumors within the tibia as demonstrated by H&E staining. X-ray and micro-CT analyses also identified that GGTI-2418 prevented MM induced bone disease with significantly lower tumor induced osteolysis and trabecular bone volume loss noted. Furthermore, treatment of mice with GGTI-2418 resulted in a significant activation of caspase 3 (apoptosis induction), accumulation of nuclear p27Kip in MM tumors (a positive prognostic biomarker for MM patients), and reduction of α-SMA levels in myofibroblasts surrounding the sinusoidal vasculature, suggesting decreased vascularization of marrow blood vessels. Importantly, GGTI-2418 treatment resulted in a significant increase in mouse median survival and delay in the onset of limb paralysis. In cell culture studies, GGTI-2417, the methylester pro-drug of GGTI-2418 that is better taken up by cultured cells, inhibited MM proliferation, increased p27kip levels, induced apoptosis and sensitized MM cells to bortezomib. Taken together, our preclinical studies suggest targeting GGT-1 as a viable therapeutic approach in MM, and warrant the investigation of GGTI-2418 in the clinic.

Citation Format: Hua Yang, Michael F. Emmons, Christopher Cubitt, Ken Shain, Domenico Coppola, Daniel Sullivan, Conor C. Lynch, Lori Hazlehurst, Said M. Sebti. The geranylgeranyltransferase I inhibitor GGTI-2418 suppresses multiple myeloma malignancy in the 5TMG1 mouse model. [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 2605. doi:10.1158/1538-7445.AM2015-2605
Abstract 285: MEDI9447: enhancing anti-tumor immunity by targeting CD73 In the tumor microenvironment
Authors: Hay Carl , Sult Erin , Huang Qihui , Hammond Scott , Mulgrew Kathy , McGlinchey Kelly , Fuhrmann Stacy , Rothstein Raymond , Poon Edmund , Stewart Ross , Hollingsworth Robert , Sachsenmeier Kris
Published: August 2015

MEDI9447 is a monoclonal antibody specific for the ectoenzyme, CD73. Data is presented in support of the hypothesis that targeting the extracellular production of adenosine by CD73 reduces the immunosuppressive effects of adenosine. We report a range of activities for this antibody, including inhibition of both recombinant and cellular CD73 ectonucleotidase activity, relief from AMP-mediated lymphocyte suppression in vitro, and inhibition of syngeneic tumor growth. In contrast with many other cancer immunotherapy agents such as checkpoint inhibitors or T cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment. Changes include significant increases in CD8 effector cells and activated macrophages, as well as a reduction in the proportions of myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes. Furthermore, these changes correlate directly with responder and non-responder subpopulations within the arms of animal studies using syngeneic tumors. Data showing additive activity between MEDI9447 and other immune-mediated therapy antibodies demonstrates the importance of relieving adenosine-mediated immunosuppression within tumors.

Citation Format: Carl Hay, Erin Sult, Qihui Huang, Scott Hammond, Kathy Mulgrew, Kelly McGlinchey, Stacy Fuhrmann, Raymond Rothstein, Edmund Poon, Ross Stewart, Robert Hollingsworth, Kris Sachsenmeier. MEDI9447: enhancing anti-tumor immunity by targeting CD73 In the tumor microenvironment. [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 285. doi:10.1158/1538-7445.AM2015-285
Abstract 2942: TRAIL pathway inducer ONC201/TIC10 primes multiple myeloma cells (MM) for apoptosis by downregulating X-linked inhibitor of apoptosis
Authors: Kline Christina Leah , Lulla Amriti R. , Dicker David , Allen Joshua E. , El-Deiry Wafik
Published: August 2015

MM cells inherently have a high protein turnover rate that makes them heavily dependent on the proteasome and uniquely sensitive to ER stress induction. Proteasome inhibitors, such as bortezomib, are used in front-line therapy for MM to elicit the ER stress response that results in apoptosis of MM cells. Despite significantly improved clinical outcomes in MM patients treated with proteasome inhibitors, resistance to therapy invariably emerges and MM remains an incurable disease. The objective of this study is to assess the utility of the TRAIL pathway inducer ONC201/TIC10 in MM due to preliminary evidence that suggests ONC201/TIC10 activates the integrated stress response (ISR) in tumor cells. We found that MM cells are uniquely sensitive to ONC201/TIC10, with EC50 values in the nanomolar range. In addition, ONC201/TIC10 was effective as a single agent against bortezomib-resistant MM cells and exhibited an additive effect with bortezomib against these cells. Sub-G1 analyses of MM cells co-treated with ONC201/TIC10 and a pan-caspase inhibitor indicated that MM cells undergo ONC201/TIC10-induced apoptosis in a caspase-dependent manner. Given that ONC201/TIC10 can inactivate both Akt and ERK signaling pathways, we assessed Akt and ERK phosphorylation in ONC201/TIC10-treated KMS18 and MM1S MM cells. These MM cell lines showed no inhibition of Akt and ERK phosphorylation or decrease in the downstream phosphorylation of Fox3a in response to ONC201/TIC10 treatment. These results suggested that alternative mechanisms of cytotoxicity are operative in myeloma cells treated with ONC201/TIC10. Given that downregulation of X-linked inhibitor of apoptosis (XIAP) has been linked to the ISR, we assessed the effect of ONC201/TIC10 treatment on XIAP expression. In agreement with ISR activation, XIAP levels were significantly downregulated in MM cells in response to ONC201/TIC10. Ongoing experiments are unraveling the mechanistic basis for lowered XIAP expression in ONC201/TIC10-treated MM cells. In summary, our results indicate that ONC201/TIC10 is highly effective in preclinical MM models by engaging antitumor mechanisms that include the ISR and downstream attenuation of XIAP expression. ONC201/TIC10 appears to have significant therapeutic potential in MM as a monoagent and in combination with bortezomib.

Citation Format: Christina Leah Kline, Amriti R. Lulla, David Dicker, Joshua E. Allen, Wafik El-Deiry. TRAIL pathway inducer ONC201/TIC10 primes multiple myeloma cells (MM) for apoptosis by downregulating X-linked inhibitor of apoptosis. [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 2942. doi:10.1158/1538-7445.AM2015-2942
Abstract 2963: 5-hydroxymethylcytosine alterations at H3K9me3 marked genomic regions serve as potential biomarker for renal cell carcinoma patients
Authors: Meng Wei , Lautenschlaeger Tim , Frankhouser David , Ye Zhenqing , Huebner Alexander , Jin Victor , Yan Pearlly , Chakravarti Arnab
Published: August 2015

Background: Epigenetic alterations are frequently encountered in many malignancies. Control of DNA methylation is thought to play a critical role in the global epigenetic reprogramming observed during development and carcinogenesis. DNA demethylation is initiated by the transition from 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC patterns are relatively well characterized in many malignancies, not much is known about 5hmC patterns and the potential usefulness as biomarkers. We thus sought out to identify 5hmC alterations in renal cell carcinoma (RCC) and assess their potential usefulness as urine biomarkers.

Results: We performed genome-wide 5hmC MethylCap-seq on one pooled RCC tissue sample (n = 3) and the corresponding pooled matched normal kidney tissue (n = 3), as well as on one pooled urine sample obtained from RCC patients (n = 52) and one pooled urine sample obtained from control patients without malignancy (n = 65). Global 5hmC levels were dramatically reduced in RCC tissues compared to matched normal adjacent kidney tissues, and in urine samples compared to tissue samples. Assessing histone marked regions we found 5hmC levels to be highly enriched in H3K9me3 marked repressive genomic regions of RCC tissue compared to normal adjacent tissues. Given the low 5hmC signal in these regions in normal tissues, this difference was also clearly identified comparing urine samples from RCC patients to control patients without RCC.

Conclusions: We characterized the 5hmC distribution of RCC and normal human renal tissue as well as of urine samples from RCC patients and control patients without RCC. We identified dramatic genome-wide 5hmC changes to occur during carcinogenesis, which have potential for development as non-invasive urine biomarkers.

Citation Format: Wei Meng, Tim Lautenschlaeger, David Frankhouser, Zhenqing Ye, Alexander Huebner, Victor Jin, Pearlly Yan, Arnab Chakravarti. 5-hydroxymethylcytosine alterations at H3K9me3 marked genomic regions serve as potential biomarker for renal cell carcinoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2015-2963
Abstract 3433: Identification of potential salivary response biomarkers in subjects practicing yogic breathing
Authors: Balasubramanian Sundaravadivel , Janech Michael G. , Warren Graham W.
Published: August 2015

Yogic Breathing (YB; Pranayama) is a form of Yoga technique practiced since ancient times for healthy living. In the United States, the practice of Yoga has markedly increased in recent years. Recent data suggest that adjunctive Yoga in cancer patients may help patients with symptom management. Whereas there are data on Asana (Postures) that involve physical movements in cancer patients, there are limited data on YB. Considering the ease of practice when compared to Asana, YB might benefit patients who are already physically overwhelmed with symptom burden. Current data measure the outcomes of Yoga using subjective measures, but biomarkers in an easily accessible biospecimen might provide key information as to the effects of YB among practitioners. We hypothesized that YB stimulates alterations in the salivary biomarker expression. We conducted an Institutional Review Board approved pilot randomized controlled trial in 20 subjects (n = 10 in each group) to compare the salivary expression of biomarkers between a 20 min YB group vs. Attention Control (AC) group. The YB group performed two exercises (Pranava Pranayama, and Thirumoolar Pranayama) each for 10 min, and the AC group read a scientific article for the same amount of time. Tandem Mass Spectrometric (LC-LC-MS-MS), Multiplex, and Western blot analysis showed the differential expression of 22 salivary proteins associated with immune response, stress and cancer. As compared with the AC group, YB increased Deleted in Malignant Brain Tumor 1 (DMBT1) as early as 5 min and maintained significantly high levels in all time points tested. In contrast, YB reduced IL-1beta (at 10, 15 and 20 min) and IL-8 levels (at 5, 10, 15 and 20 min) in participants when compared to AC controls. Further analysis of salivary lipidomic profiles demonstrated marginal reductions in sphingosine and sphingosine-1-phosphate levels in YB group. Collectively, our data provide the first evidence that YB could alter salivary biomarkers associated with cancer, inflammation, and stress. These markers may be useful to evaluate symptom management via stress reduction and immune regulation.

Citation Format: Sundaravadivel Balasubramanian, Michael G. Janech, Graham W. Warren. Identification of potential salivary response biomarkers in subjects practicing yogic breathing. [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 3433. doi:10.1158/1538-7445.AM2015-3433
Abstract 3860: USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168
Authors: Zhu Qianzheng , Sharma Nidhi , He Jingshan , Wani Gulzar , Wani Altaf A.
Published: August 2015

In DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs the monoubiquitination of H2A (uH2A) and ultraviolet radiation (UVR)-induced monoubiquitination of γH2AX, and decreases the levels of pBmi1, Bmi1, RNF168, pChk1 and BRCA1. USP7 silencing by siRNA reduces uH2A and UVR-induced monoubiquititination of γH2AX, as well as the levels of Ring1B, Bmi1, RNF168 and BRCA1, recapitulating the chronic USP7 deficiency. USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of uH2A, poly-K63-ubiquititination and BRCA1, but does not affect UVRIF and IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytic-inactive or interaction-defective mutants, prevents UVR-induced RNF168 degradation. Both USP7 mutants are unable to cleave Ub-conjugates of RNF168 in vivo. Furthermore, ectopic expression of RNF168, or both RNF8 and RNF168 together, rescue the formation of UVRIF and IRIF of poly-K63-ubiquititination and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.

Note: This abstract was not presented at the meeting.

Citation Format: Qianzheng Zhu, Nidhi Sharma, Jingshan He, Gulzar Wani, Altaf A. Wani. USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168. [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 3860. doi:10.1158/1538-7445.AM2015-3860
Abstract 4018: The role of c-MET/HGF signaling as a critical mediator of an invasive and resistant phenotype in colorectal cancer
Authors: Bradley Conor A. , Dunne Philip , McArt Darragh , Arthur Ken , McQuaid Stephen , Salto-Tellez Manuel , Johnston Patrick , Van Schaeybroeck Sandra
Published: August 2015

The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (5-10%), and mutated (5%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-induced c-MET activation has been linked with migration, survival, invasion, and resistance to targeted therapy, and has been suggested as a possible prognostic biomarker for CRC. We are currently investigating the role of c-MET and microenvironment derived HGF in the acquisition of an invasive and migratory phenotype, and resistance to targeted therapy in CRC.

In order to model CRC tumour cell invasion and metastasis, we have generated invasive subpopulations from CRC cells using Boyden Invasion chambers. Invasive cell lines were characterised for protein expression/activity by Western blotting, and analysed for migratory and invasive potential using the xCELLigence System (Roche). To model the CRC microenvironment, we have utilised a range of co-culture techniques with CRC cell lines and colon fibroblasts. c-MET expression in FFPE tissues was measured using IHC in a tissue microarray (TMA) derived from early stage CRC patients.

HCT116 and LoVo invasive subpopulations showed an EMT-like, mesenchymal, migratory/invasive phenotype. In addition, increased expression and activation levels of c-MET were found in these sublines, which was determined to be ligand independent. Inhibition of c-MET using RNAi abrogated both basal and HGF-induced migration and invasion in CRC cell lines. Co-culture of CRC cells with HGF-expressing colon-derived fibroblasts leads to activation of c-MET, inducing CRC migration, invasion, and resistance to MEK inhibition, and this phenotype could be diminished using a HGF neutralising antibody. Significant increased expression levels of c-MET were also found in a CRC TMA compared to matched normal tissues, and also at the invasive edge of some CRC tumours, where it may be driving invasive biology.

The identification of key pathways driving metastasis has a huge potential to change treatment strategies in CRC. We intend to further investigate the role of c-MET in regulation of migration, invasion, and resistance to therapy in CRC. We hypothesise that patients with high tumoural c-MET at the invasive front may benefit in particular from anti- c-MET therapies.

Citation Format: Conor A. Bradley, Philip Dunne, Darragh McArt, Ken Arthur, Stephen McQuaid, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. The role of c-MET/HGF signaling as a critical mediator of an invasive and resistant phenotype in colorectal 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 4018. doi:10.1158/1538-7445.AM2015-4018
Abstract 451: The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints
Authors: Llosa Nicolas Jose , Cruise Michael , Tam Ada , Wick Elizabeth , Hechenbleikner Elizabeth , Taube Janis , Blosser Lee , Fan Hongni , Wang Hao , Zhang Ming , Luber Brandon , Papadopoulos Nickolas , Kinzler Kenneth , Vogelstein Bert , Sears Cynthia , Anders Robert , Pardoll Drew , Housseau Franck
Published: August 2015

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-γ production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC.

Significance. The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested.

Citation Format: Nicolas Jose Llosa, Michael Cruise, Ada Tam, Elizabeth Wick, Elizabeth Hechenbleikner, Janis Taube, Lee Blosser, Hongni Fan, Hao Wang, Ming Zhang, Brandon Luber, Nickolas Papadopoulos, Kenneth Kinzler, Bert Vogelstein, Cynthia Sears, Robert Anders, Drew Pardoll, Franck Housseau. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. [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 451. doi:10.1158/1538-7445.AM2015-451
Abstract 5315: Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma cells
Authors: Lee Hyung-Ok , Mustafa Aladdin , Hudes Gary R. , Kruger Warren D.
Published: August 2015

mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway which helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases glycolysis rates. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K, was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of the p70S6K. Treatment with the proteasome inhibitor bortezomib, restored phospho-S6 levels, suggesting that the reduction of phospho S6 is caused by increased degradation of phospho, but not total S6. Surprisingly, depletion of the key autophagy regulator Atg7 did not mimic the effects of HCQ, indicating that HCQ regulates S6 phosphorylation by ATG7-independent mechanism. Our finding suggests that HCQ may be useful in the treatment of renal cell carcinoma.

Citation Format: Hyung-Ok Lee, Aladdin Mustafa, Gary R. Hudes, Warren D. Kruger. Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma 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 5315. doi:10.1158/1538-7445.AM2015-5315