bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3086: From profiling to function: A role for microRNA miR-888 in promoting prostate cancer progression
Authors: Lewis Holly , Hasegawa Tsuyoshi , Glavich Garrison , Lance Raymond , Semmes O. John , Beydoun Hind , Drake Richard , Esquela-Kerscher Aurora
Published: August 2015

Prostate cancer remains the second leading cause of cancer-related deaths among men in the United States despite the widespread use of prostate specific antigen (PSA) as a diagnostic marker for prostate cancer and as a predictor of treatment outcome. MicroRNAs (MiRNAs) belong to a growing class of small non-coding RNAs that are often abnormally expressed in fluid and tissue specimens from prostate cancer patients compared to non-cancer individuals. These miRNAs are hypothesized to play a direct role in promoting disease progression and metastasis in the prostate and are intensely studied for both their diagnostic and therapeutic potential for human cancer. Our laboratory profiled candidate prostate-cancer associated miRNAs in an innovative prostatic fluid biomarker source called expressed prostatic secretions in urine (EPS urine) to determine their utility as discriminating biomarkers for advanced forms of prostate cancer. 10 out of 50 miRNA tested showed statistically significant differences in expression between cancer and non-cancer patient groups within the supernatant but not pellet fractions of whole EPS urine. We are investigating if differential miRNA expression in EPS supernatant is due to exosomal populations and how miRNA-loaded exosomes modulate prostate tumorigenesis in mice. Our profiling studies identified miR-888 as a novel factor that correlates with advanced prostate cancer in human prostate cancer cell lines, EPS urine, and primary tumors. Functional assays revealed that miR-888 promotes proliferation, migration, and colony formation of hormone-refractory and androgen-sensitive human prostate cancer cells. Our working model is that miR-888 overexpression in the diseased prostate suppresses SMAD4, RBL1, and TIMP2 targets leading to tumor progression and metastasis. Human miR-888 belongs to a genomic cluster of seven miRNAs on human chromosome Xq27.3. We are currently testing how this miR-888 cluster interacts within a growing network of coding and non-coding genes to regulate prostate tumor progression. This research will provide a better understanding how prostate tumor cells progress to a more metastatic, aggressive state and facilitate the development of more effective diagnostic and therapeutic strategies for this prevalent and deadly disease.

Citation Format: Holly Lewis, Tsuyoshi Hasegawa, Garrison Glavich, Raymond Lance, O. John Semmes, Hind Beydoun, Richard Drake, Aurora Esquela-Kerscher. From profiling to function: A role for microRNA miR-888 in promoting prostate cancer progression. [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 3086. doi:10.1158/1538-7445.AM2015-3086
Abstract 3104: Molecular features that determine the sensitivity of cancer cells to abemaciclib, an inhibitor of CDK4 and CDK6
Authors: Gong Xueqian , Chio Li-Chun , Lallena MaryJo , Merzoug Farhana , Chu Shaoyou , Webster Yue , Dempsey Jack , Ma Xiwen , De Dios Alfonso , Beckman Richard , Buchanan Sean G.
Published: August 2015

It is well established that phosphorylation of Rb-family pocket proteins by CDK4 and CDK6 is important for the commitment of cancer cells to a new cell cycle and the initiation of the G1-S phase transition. Abemaciclib is a potent inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing clinical testing. To better understand the molecular determinants of response to abemaciclib, we tested its anti-proliferative activity across a panel of over 500 well characterized cancer cell lines. Statistical approaches were employed to uncover genomic features associated with the response. Candidate markers of sensitivity and resistance were further tested by genetic manipulations in vitro. In vivo models representing the candidate molecular marker of sensitivity were identified and drug efficacy examined.

Three broad classes of response were identified. The class of tumors cells most resistant to abemaciclib showed enrichment for RB1 mutations. Conversely, cell lines with amplification of CCND2 and CCND3 were among the very most sensitive tumor cells and tumor cells with these markers showed evidence of senescence and apoptosis after either depletion of the cognate D-cyclin or treatment with abemaciclib. In vivo models of tumors harboring CCND2 and CCND3 gene amplification were very sensitive to abemaciclib treatment and showed evidence of tumor regression.

Citation Format: Xueqian Gong, Li-Chun Chio, MaryJo Lallena, Farhana Merzoug, Shaoyou Chu, Yue Webster, Jack Dempsey, Xiwen Ma, Alfonso De Dios, Richard Beckman, Sean G. Buchanan. Molecular features that determine the sensitivity of cancer cells to abemaciclib, an inhibitor of CDK4 and CDK6. [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 3104. doi:10.1158/1538-7445.AM2015-3104
Abstract 3110: Urinary miR-1825 and miR-484: An oncogene and a tumor- suppressor gene among prostate cancer patients
Authors: Abdalla Moemen , Haj-Ahmad Taha A. , Haj-Ahmad Yousef
Published: August 2015

MicroRNAs (miRNAs) are short, single-stranded RNA molecules with the ability of acting as post-transcriptional regulators of gene expression. MiRNAs regulate a number of important pathways, such as: cellular differentiation, proliferation, and apoptosis. MiRNA expression profiling patterns were demonstrated to have more potential than the common mRNA expression profiling for the characterization of poorly differentiated tumours. Studying miRNA expression profiles “signatures” will help in understanding the role of these miRNAs in the process of cancer development. The aim of this study was to profile the urinary miRNA expression signatures among prostate cancer patients in comparison to healthy male individuals. To identify the miRNA signatures specific for PCa, total RNA was isolated from 2.5mL of urine from all prostate cancer patients and from healthy males. RNA isolated from each group were pooled together and the differential expression analysis of miRNAs between the pooled samples was performed using MiRNA Microarray technology. Differential expression of two individual miRNAs, miR-1825 and miR-484, was observed between healthy males and prostate cancer patients. The relative expression of these two miRNAs were assessed among among the prostate cancer group and the healthy male group using RT-qPCR. MiR-1825 was up-regulated in seven out of eight PCa samples (88%), whereas miR-484 was down-regulated in six out of the eight PCa samples (75%). It has been found that the putative target for miR-1825 is member-1 of the Discoidin Domain family of Receptors (DDR1). The over-expression of DDR1 was reported to be a prognostic marker for the poor survival in several cancer types and therefore is proposed that miR-1825 might function as a tumor-suppressor by normally or in response to cancer, inhibiting DDR1's translation. As for miR-484, E3 ubiquitin-protein ligase (UBR5), which is thought to be involved with DNA repair, has been found to be the putative target for miR-484. It has be reported that the over-expression of a mutated version of UBR5 was found in breast and ovarian cancers where its deregulation was cited as a possible cause of malignant progression. It is therefore being proposed that miR-484 might function as a tumor- suppressor whereby its expression controls progression via the regulation of genes, such as UBR5, that have already been potentially associated with cancer progression.

Citation Format: Moemen Abdalla, Taha A. Haj-Ahmad, Yousef Haj-Ahmad. Urinary miR-1825 and miR-484: An oncogene and a tumor- suppressor gene among prostate cancer 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 3110. doi:10.1158/1538-7445.AM2015-3110
Abstract 3111: JNK/c-Jun- and NF-κB-mediated microRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver cancer
Authors: Kim Hyung Seok , Shen Qingyu , Eun Jung Woo , Shin Woo Chan , Yang Hee Doo , Park Won Sang , Lee Joung Young , Nam Suk Woo
Published: August 2015

Many clinical studies of cancer patients have established that the most common reason behind changes in HDAC function is its overexpression. However, among HDACs in liver cancer, HDAC6, a cytoplasmic tubulin deacetylase, is uniquely endowed with a tumor suppressor, but the mechanism underlying HDAC6 inactivation has yet to be uncovered. In this study we show that genetic and epigenetic dysregulation was not part of the HDAC6 inactivation mechanism. Instead, microRNA-221-3p (miR-221) directly suppressed HDAC6 in hepatocellular carcinoma (HCC) cells. A comprehensive miRNA profiling analysis identified seven putative endogenous miRNAs that are significantly upregulated in HCC. Ectopic expression of miRNA mimics in Dicer knockdown cells evidenced miR-221 to suppress HDAC6 in HCC cells. Notably, targeted-disruption of miR-221 repressed cancer cell growth and proliferation, and it recapitulated HDAC6 overexpression effects in HCC cells. Our results also demonstrate that c-Met-mediated c-Jun NH2-terminal kinase (JNK)/c-Jun signaling induced miR-221 to suppress HDAC6 and was sustained in liver cancer cells but not in normal hepatic cells. In addition, cytokine-induced NF-κBp65 independently regulated miR-221, thereby suppressing HDAC6 expression in liver cancer cells. HCC tissues derived from chemical-induced rat liver cancer models validated that JNK/c-Jun activation and NF-kBp65 nuclear translocation are essential for the transcriptional activation of oncogenic miR-221 to repress tumor suppressor HDAC6 in HCC. In conclusion, our finding suggest that the functional loss or suppression of the tumor suppressor HDAC6 is caused by miR-221 through coordinated JNK/c-Jun- and NF-κB-signaling pathways during liver tumorigenesis, providing a novel target for the molecular treatment of liver malignancies.

Citation Format: Hyung Seok Kim, Qingyu Shen, Jung Woo Eun, Woo Chan Shin, Hee Doo Yang, Won Sang Park, Joung Young Lee, Suk Woo Nam. JNK/c-Jun- and NF-κB-mediated microRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver 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 3111. doi:10.1158/1538-7445.AM2015-3111
Abstract 3114: MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis
Authors: Wu Meng-Ju , Chang Chun-Ju
Published: August 2015

Dysregulation of epigenetic controls is associated with tumorigenesis in response to microenvironmental stimuli; however, the regulatory pathways involved in epigenetic dysfunction are largely unclear. We have determined that a critical epigenetic regulator, microRNA-205 (miR-205), is repressed by the ligand jagged1, which is secreted from the tumor stroma to promote a cancer-associated stem cell phenotype. Knockdown of miR-205 in mammary epithelial cells promoted epithelial-mesenchymal transition (EMT), disrupted epithelial cell polarity, and enhanced symmetric division to expand the stem cell population. Furthermore, miR-205-deficient mice spontaneously developed mammary lesions, while activation of miR-205 markedly diminished breast cancer stemness. These data provide evidence that links tumor microenvironment and microRNA-dependent regulation to disruption of epithelial polarity and aberrant mammary stem cell division, which in turn leads to an expansion of stem cell population and tumorigenesis. This study elucidates an important role for miR-205 in the regulation of mammary stem cell fate, suggesting a potential therapeutic target for limiting breast cancer genesis.

Citation Format: Meng-Ju Wu, Chun-Ju Chang. MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis. [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 3114. doi:10.1158/1538-7445.AM2015-3114
Abstract 3119: miR-296-5p and miR-874-3p control cell proliferation and apoptosis in HCC via regulation of PIN1 expression
Authors: Leong Ka Wai , Cheng Chi Wai , Wong Chun Ming , Ng Oi Lin Irene , Kwong Yok Lam , Tse Eric Wai Choi
Published: August 2015

Introduction: PIN1 is a peptidyl-prolyl cis/trans isomerase (PPlase) that regulates multiple signaling pathways to control cell fate. It is found to be up-regulated in hepatocellular carcinomas (HCC), leading to increased cell proliferation and inhibition of apoptosis. However, the regulation of PIN1 remains poorly defined. MicroRNA (miRNA) plays a pivotal role in carcinogenesis by regulating tumor suppressor genes and oncogenes via targeting the 3′UTR of their encoded mRNAs. Therefore, we aimed at identifying miRNAs regulating PIN1 expression, and determining the contributions of these miRNAs to tumorigenesis through PIN1.

Methods: Potential miRNAs regulating PIN1 were identified through sequence prediction using the online database TargetScan Release 6.2. Western immunoblotting and qRT-PCR were used to assess PIN1 expression in HCC cells with over-expression of identified miRNAs. Dual-Luciferase reporter assay was conducted to examine the direct interaction between the identified miRNAs and PIN1 3′UTR. The tumor suppressive role of the identified miRNAs in HCC cells was investigated using MTT cell proliferation assay, colony formation assay and Annexin V apoptosis detection assay. To investigate the correlation between the levels of PIN1 and the identified miRNAs, qRT-PCR was used to determine the mRNA levels of PIN1 and the identified miRNAs in 20 pairs of normal liver tissue and HCC.

Results: We identified miR-296-5p and miR-874-3p as important negative regulators of PIN1. Consequently, over-expression of miR-296-5p and miR-874-3p enhanced cellular apoptosis, and inhibited cell growth and colony formation in HCC cells. Furthermore, the cellular functions of miR-296-5p and miR-874-3p were abrogated by over-expression of PIN1. Importantly, the expression of PIN1 was negatively correlated with that of miR-296-5p and miR-874-3p in HCC tissues, consistent with the regulatory role of miR-295-5p and miR-874-3p on PIN1 expression.

Conclusion: Taken together, our data showed that miR-296-5p and miR-874-3p controlled cell proliferation and apoptosis in HCC by regulating PIN1 expression.

Citation Format: Ka Wai Leong, Chi Wai Cheng, Chun Ming Wong, Oi Lin Irene Ng, Yok Lam Kwong, Eric Wai Choi Tse. miR-296-5p and miR-874-3p control cell proliferation and apoptosis in HCC via regulation of PIN1 expression. [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 3119. doi:10.1158/1538-7445.AM2015-3119
Abstract 3120: miR-489 function as tumor suppressor miRNA by targeting HER2 in HER2-positive breast cancer
Authors: Patel Yogin , Shah Nirav , Botbyl Racheal , Lee Jishin , Chen Hexin
Published: August 2015

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in 20-30% of all breast cancer and associated with poor prognosis and outcome. Previous studies have shown that dysregulation of several micro RNAs (miRs), play a key role in breast cancer progression and metastasis. In this study, we evaluated ∼330 miRs, to find specific miRs dysregualted by HER2 signaling. After further screening we focused our study on miR-489, whose expression was ∼6-7 fold downregulated specifically by HER2 signaling. Restoration of miR-489 expression in several HER2-positive breast cancer cell lines resulted in increased cell death and cell cycle arrest. To investigate the molecular mechanism of action of miR-489, we studied effect of miR-489 on HER2 signaling pathway and found that higher expression of miR-489 led to the decreased levels of HER2 (both mRNA and protein) and decreased Akt activation. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2 and miR-489 signaling pathways form a mutual regulatory loop. To further demonstrate clinical relevance of miR-489, we analyzed both normal and tumor human breast tissue samples using Fluorescent in situ hybridization technique (FISH). Our imaging data indicated that miR-489 is expressed at much lower level in tumor tissues compared to that of the normal breast tissue. Overall, our data indicates that miR-489 acts as a tumor suppressor miR at least partially by directly targeting HER2 in HER2 positive breast cancer cells.

Citation Format: Yogin Patel, Nirav Shah, Racheal Botbyl, Jishin Lee, Hexin Chen. miR-489 function as tumor suppressor miRNA by targeting HER2 in HER2-positive 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 3120. doi:10.1158/1538-7445.AM2015-3120
Abstract 3129: miR-340 is a modulator of oncogenic signaling in melanoma
Authors: Poenitzsch Strong Ashley M. , Adochio Emily G. , Setaluri Vijayasaradhi , Spiegelman Vladimir S.
Published: August 2015

Melanoma is responsible for only 4% of skin cancer diagnoses; nonetheless 80% of skin cancer deaths are attributed to this aggressive skin cancer each year. Growing evidence points to an important role miRNAs may play in the aberrant cellular signaling of cancers, including melanoma. microRNAs (miRNAs) are highly conserved 22-25nt non-coding RNAs involved in regulating RNA stability and translation and are predicted to regulate ∼60% of the human transcriptome. We are investigating the tumor suppressor role miR-340 plays in the development and progression of melanoma. A limited number of miR-340 targets were previously characterized, however, it is unlikely the effects of miR-340 on the melanoma phenotype are the result of directly targeting a few mRNAs, as miRNAs are likely to target upwards of 100 mRNAs each. Using an in silico target identification and pathway mapping system, we identified a multitude of potential miR-340 mRNA targets that encode proteins distributed amongst a variety of cellular signaling pathways. In silico analysis revealed a high proportion of miR-340 predicted mRNA targets that encode for members of the MAPK signaling pathway. Our results show that miR-340 modulated most of the assayed mRNAs that encode MAPK components. We also demonstrate that miR-340 inhibits the tumorigenic phenotype of melanoma cells. We recently developed a novel method to identify direct targets of miRNAs. Using this method, we demonstrate miR-340 directly targets mRNAs that encode protein kinases of the MAPK pathway. Further direct target identification is crucial toward understanding the role miR-340-directed regulation plays in triggering pleiotropic effects during melanoma development and progression. Elucidating the roles aberrant gene regulatory networks play in melanoma is critical for understanding the disease and developing treatment options to improve the melanoma prognosis.

Supported by NIH grants ES007015 (to A.M.P.S.), AR063361 (to V.S.S.) and CA191550 (to V.S.S.).

Citation Format: Ashley M. Poenitzsch Strong, Emily G. Adochio, Vijayasaradhi Setaluri, Vladimir S. Spiegelman. miR-340 is a modulator of oncogenic signaling in melanoma. [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 3129. doi:10.1158/1538-7445.AM2015-3129
Abstract 3141: Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified T cells
Authors: Smith Jillian P. , Long Adrienne H. , Mackall Crystal L.
Published: August 2015

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has proven effective at treating hematologic malignancies, however similar success has not been observed using CARs targeting other antigens. We have observed that several CARs, including a GD2-targeted CAR, are limited in efficacy due to T cell exhaustion induced by tonic, antigen-independent signaling. This phenomenon is triggered by oligomerization of scFvs on the surface of transduced T cells, leading to chronic CAR zeta signaling and early exhaustion characterized by a diminished proliferative potential, poor cytokine production, and overexpression of inhibitory receptors including PD1, TIM3 and LAG3. Early CAR T cell exhaustion is also associated with an altered cellular metabolism that is more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced, activated control cells. Using a Seahorse Extracellular Flux Analyzer, we found that the rate of glycolysis (ECAR) in GD2 CAR T cells was twice that of CD19 CAR T cells or untransduced controls, and no deficiencies in oxidative phosphorylation in GD2 CAR T cells were observed (normal OCR and spare respiratory capacity). To evaluate whether increased glycolytic metabolism contributes to the development of CAR T cell exhaustion, we sought to restrict glycolysis by culturing GD2 CAR T cells with the hexokinase inhibitor 2-deoxyglucose (2DG) or in media supplemented with galactose instead of glucose. Restricting CAR T cell glycolysis was associated with decreased TIM3 and LAG3 expression and enhanced cytokine production of GD2 CAR T cells but a modest decrease in cytokine production of CD19 CAR T cells. Given the previously reported role of mTOR on promoting glycolytic metabolism, we used rapamycin (sirolimus) in the cell culture during transduction and expansion. Culturing GD2 CAR T cells with rapamycin similarly resulted in slower acidification of cell culture medium and diminished expression of TIM3 and LAG3. Rapamycin treated GD2 CAR T cells retain specific lytic ability of GD2-positive tumor cells compared to non-treated GD2 CARs as measured by chromium release, and these cells persist longer than non-treated cells in a murine xenograft model of GD2-positive 143b osteosarcoma. These results suggest that modulation of T cell metabolism may provide functional benefit and enhance antitumor activity of adoptively transferred tumor antigen specific T cells by diminishing the susceptibility of highly activated T cells to exhaustion.

Citation Format: Jillian P. Smith, Adrienne H. Long, Crystal L. Mackall. Restricting aerobic glycolysis provides functional improvement of chimeric antigen receptor (CAR)-modified T 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 3141. doi:10.1158/1538-7445.AM2015-3141
Abstract 3161: Predictive Immunotherapy Models: Overcoming the challenge of T cells gene transfer
Authors: Bouillé Pascale , Duthoit Christine , Gayon Régis , Feuillet Vincent , Auffray Cédric
Published: August 2015

The initiation of an appropriate T cell response is a key event in the establishment of efficient anti-tumoral response. In a context of tumor development, different T cell populations are mobilized and understanding the pathways involved in their functions and in their interactions is a major field of research in cancer immunotherapy. Thus, while the activation of antigen-specific naive CD4+ T cells that can differentiate into appropriate effector T cells and home to the tumor might be highly beneficial, regulatory CD4+ T cells are considered as deleterious. Many efforts are thus made to understand the behavior of both CD4+ T cells subsets and to decipher their functions within the tumor micro-environment. A key step to reach this goal is the development of relevant predictive cancer models including genetic modifications of T cell populations in vitro and in vivo, either for gene target studies, or to create new tools for sensitive readouts.

While human and activated mouse T cells are easily transduced with lentiviral vectors, transduction of naive and regulatory murine T cells is a technical challenge since these target cells are poorly permissive to transfection or viral transduction. Here, extensive in vitro transduction data show that the use of highly concentrated (1E9 TU/mL) and pure (1E8 TU/mg of proteins) lentiviral vectors increases significantly the transduction level of naive and regulatory CD4+ T cells. Furthermore, transduced T cells exhibit an original cell phenotype without any changes of T cells specific markers expression (CD44, CD69, CD25, Ly-6C) attesting that the composition of this pure batch of lentiviral vector does not have any impact on T cells phenotype. Transduced T cells were then assessed for their properties once adoptively transferred into recipient mice. Analysis of these mice show that not only the genetically modified T cells/regulatory cells can be easily followed thanks to the fluorescent reporter but also their phenotype is not modified by the transduction, as shown in vitro.

Our data bring out that the composition of lentiviral vectors in terms of titer, specific activities and purity are the success keys to ensure transduction of murine T cells. This major technological leap will allow to tightly control expression of various modifiers of these cells and thus paves the way to design original tools and develop cell-based cancer.

Citation Format: Pascale Bouillé, Christine Duthoit, Régis Gayon, Vincent Feuillet, Cédric Auffray. Predictive Immunotherapy Models: Overcoming the challenge of T cells gene transfer. [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 3161. doi:10.1158/1538-7445.AM2015-3161
Abstract 3183: Role of danger signals in tumor elicited inflammation
Authors: Francescone Ralph , Vendramini-Costa Debora , Dmitrieva Oxana , Hou Vivi , Posocco David , Grivennikov Sergei
Published: August 2015

Spontaneous colon cancer (CRC) is traditionally thought to arise from a series of genetic mutations to key tumor suppressors and oncogenes that drive its progression. Recently, however, the importance of the immune system and inflammation in exacerbating CRC development has been demonstrated, as seemingly non-inflammatory tumors have massive infiltration of immune cells. This phenomenon has been deemed “Tumor elicited inflammation” (TEI), whereby the tumor cells recruit immune cells to the tumor, promoting tumor development. Danger Associated Molecular Patterns (DAMPs), proteins that are released during cellular stress or oncogenic transformation, promote a robust immune response and are often up-regulated in cancer. Thus, DAMPs may serve as an initial cue to induce TEI. The precise mechanisms of these so-called “danger signals” in CRC development and TEI have not been fully elucidated.

Using a number of in vitro and in vivo models, we sought to uncover the major cellular producers of danger signals in the tumor microenvironment, how these danger signals recruit various immune cells to the tumor, and finally their effects on tumor progression. We found an up-regulation of three danger signals, S100A8, S100A9, and HMGB-1, in tumor tissue from mouse models of spontaneous CRC, at the RNA and protein levels. In addition, using FACS, the myeloid compartment of sorted immune cells demonstrated the highest expression levels of S100A8 and S100A9, as measured by qPCR. In order to further explore role of danger signals, lentiviral overexpression of S100A8, S100A9, and HMGB-1 was performed in MC38 colon cancer cells. Subsequently, these new cell lines were injected subcutaneously into C57BL/6 mice and tumor development was followed. S100A8 and S100A9 overexpressing tumors showed accelerated development and a greater influx of myeloid cells into the tumor when compared to GFP overexpressing control cells. Future work will focus on the crosstalk between cancer cells and immune cells, the key cellular pathways affected in cancer cells and immune cells, and finally if blockade of these signals has any therapeutic benefit in the treatment of CRC.

Citation Format: Ralph Francescone, Debora Vendramini-Costa, Oxana Dmitrieva, Vivi Hou, David Posocco, Sergei Grivennikov. Role of danger signals in tumor elicited inflammation. [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 3183. doi:10.1158/1538-7445.AM2015-3183
Abstract 3189: Tunneling nanotube formation is significantly upregulated in invasive cancer cells
Authors: Desir Snider , Subramanian Subbaya , Steer Clifford , Lou Emil
Published: August 2015

Introduction: Intercellular communication is critical for cancer growth and progression. Research to date has focused on gap junctions, micro-vesicles/exosomes, and diffusible factors. There is limited understanding of how efficient cell-to-cell communication occurs between distant cells in the complex tumor microenvironment. Tunneling nanotubes (TNTs) are thin, non-adherent actin filament-based, open conduits that facilitate direct sharing of various cellular cargos between cells. TNTs can be 50-800 nanometers in diameter and up to several hundred micrometers in length in vitro. Our lab was the first to provide in vivo evidence of TNTs in human solid tumors. We hypothesized that TNT formation is significantly upregulated in human cancers compared to their non-malignant counterparts. In this study, we examined the rate of TNT formation in cell lines from multiple human cancers.

Methods: Cell lines from four cancers (colon, mesothelioma, pancreatic, and ovarian cancer) were plated at similar density in 6-well tissue culture plates. The number of TNTs and cells in 10 randomly chosen fields of each well were quantified every 24 hrs for up to 96 hrs and representative images were taken. Experiments were performed in duplicate for each cell line.

Results: The number of TNTs per cell (TNTs/cell), which we have titled “TNT index,” was calculated in order to control for cell proliferation. The ratio of TNTs formed between malignant colorectal cells compared benign adenoma ranged from 20:1 to 100:1 by 96 hrs.

For mesothelioma, malignant:mesothelial TNTs/cell ratio ranged from 8:1 to 78:1 over 72 hrs. The mesothelioma cells were too confluent at 96 hrs to quantify TNTs. The pancreatic malignant:stellate human pancreatic ductal epithelial TNTs/cell ratio ranged between 2:1 to 63:1. Interestingly, we found that non-malignant ovarian epithelial (IOSE) cells had a significantly higher rate of TNT formation than malignant ovarian cells.

Conclusions: We have demonstrated that malignant cells have significantly higher rates of TNT formation compared to stromal or premalignant cells of their corresponding tumor. TNTs potentially provide malignant cells a direct route for transmitting influential messages across long distances in the complex tumor microenvironment. We believe that upregulation of TNTs in cancer may amplify cellular cross-talk and thus increase rates of tumor recurrence and invasion.

Citation Format: Snider Desir, Subbaya Subramanian, Clifford Steer, Emil Lou. Tunneling nanotube formation is significantly upregulated in invasive 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 3189. doi:10.1158/1538-7445.AM2015-3189
Abstract 3193: Expression analysis reveals candidate genes involved in highly invasive high-hyaluronan binding subpopulations of prostate cancer cell lines
Authors: Leith Sean J. , Chambers Ann F. , McCarthy James B. , Chin Joseph L. , Turley Eva A.
Published: August 2015

Tumour heterogeneity is one of the hallmarks of aggressive cancers, which can be detected by differential gene expression, DNA mutation patterns, and ligand binding profiles. Hyaluronan (HA) is an extracellular matrix polysaccharide whose elevated accumulation is associated with tumour recurrence in colon and breast carcinomas. Previously, we reported that triple-negative breast cancer cell lines exhibit heterogeneous binding of HA, with high and low binding subpopulations showing differences in proliferation and invasion. Here we assessed whether or not a similar binding heterogeneity detects prostate cancer cell subsets with differential invasive capability. Flow cytometry HA-binding profiles reveal heterogeneous binding of a fluorescent HA probe to the PC3M-LN4 prostate cancer cell line. Fluorescence-activated cell sorting (FACS) was used to isolate low and high hyaluronan binding subpopulations. Sorted cells were cultured and, after one week of growth, were analyzed for cell proliferation and invasion using alamar blue, transwell invasion and gelatin degradation assays. Results confirmed that HAhigh tumor cells displayed reduced growth but increased invasion and degradation relative to HAlow subpopulations. We then employed human gene 2.0 expression arrays to assess transcriptome changes between these two subpopulations. The expression of 7 genes was significantly elevated or lowered in HAhigh vs. HAlow cells, and these were linked to inflammation, angiogenesis, MMP9 regulation and metastasis. These results identify a novel form of heterogeneity common to both breast and prostate cancer cell lines and suggest that subpopulations binding high levels of HA can be used as indicators of aggressive tumors.

Citation Format: Sean J. Leith, Ann F. Chambers, James B. McCarthy, Joseph L. Chin, Eva A. Turley. Expression analysis reveals candidate genes involved in highly invasive high-hyaluronan binding subpopulations of prostate cancer cell lines. [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 3193. doi:10.1158/1538-7445.AM2015-3193
Abstract 3208: Stromal up-regulation of gene expression in prostate cancer cells: Role of microenvironment
Authors: Ashida Shingo , Kawada Chiaki , Inoue Keiji , Shuin Taro
Published: August 2015

Recently, tumor microenvironment has been reported to be important in carcinogenesis and tumor progression. Especially in prostate cancer (PC), it has been suggested that tumor microenvironment plays an important role in progression, acquisition of androgen independence, and distant metastasis. However, little is known about the genetic basis of human PC microenvironment. To clarify the mechanism of PC progression and metastasis, we previously reported the analysis of genome-wide gene expression profiles using human PC cells (LNCaP), normal human prostate epithelial cells (PrEC), and normal human prostate stromal cells (PrSC). In that study, we identified genes that were up-regulated in LNCaP when co-cultured with PrSC, which included alpha-methylacyl-CoA racemase (AMACR), homeobox B13 (HOXB13), and UDP glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17). We often confront the discrepancy in the difference of gene expression between cancer cells and tissues. Our data reveals that stromal cells could affect on PC cells to increase their gene expression, which might contribute to aggressiveness of PC cells. We then focused on secreted proteins that can mutually affect in this experimental design and identified genes that were overexpressed only in stromal cells. They included chemokine (C-C motif) ligand 2 (CCL2), hepatocyte growth factor (HGF), insulin-like growth factor binding protein 5 (IGFBP5), wingless-type MMTV integration site family, member 5A (WNT5A), and collagens (COL1A1, COL1A2, COL3A1, COL6A3, COL15A1). We further investigated the influence of these genes on the expression of AMACR, HOXB13, and UGT2B17 in PC cells to identify crucial signaling pathways in this process. This study provides potential candidate genes the products of which might serve as microenvironmental targets for the treatment of PC.

Citation Format: Shingo Ashida, Chiaki Kawada, Keiji Inoue, Taro Shuin. Stromal up-regulation of gene expression in prostate cancer cells: Role of 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 3208. doi:10.1158/1538-7445.AM2015-3208
Abstract 3209: The expression of SLC7A11 transporter in lung and pancreatic cancer tissues at different stages of development
Authors: Moreno Jonathan A. , Lambros Maria P.
Published: August 2015

Purpose: The purpose is to investigate the expression of the SLC7A11 transporter in human biopsy samples from patients with lung or pancreatic cancer at different stages of development. The SLC7A11 is a membrane antiporter highly specific for cystine. It is a major transporter for the uptake of cystine in exchange of intracellular glutamate. It plays a role in the growth and progression of cancers because they need extracellular cystine for growth. The expression of SLC7A11 was compared between normal and malignant tissues.

Methods: Lung and pancreatic tissue array slides were purchased from US Biomax, Inc. Pancreatic samples were categorized into adenocarcinoma, normal and adjacent to cancer tissues. Lung cancer samples were categorized into: adenocarcinoma or squamous cell carcinoma, and normal tissue. The slides were hydrated with a series of washes using xylene and graded alcohols. The tissue arrays underwent blocking of endogenous peroxidase and albumin. The tissue arrays were processed with a primary antibody (1:500) dilution of a rabbit polyclonal antibody against SLC7A11, and a secondary antibody (1:3000) dilution of goat anti-rabbit conjugated to horseradish peroxidase (HRP). The slides were counterstained with hematoxylin and images were taken with the EVOS® FL Cell Imaging System. The software ImageJ was used to determine proportion of stained cells. The staining intensity was determined by comparing the samples to their negative control supplied on the tissue arrays. The immune-reactive score (IRS) was calculated as SI x PP and it ranged from 0 to 12. An IRS within 0-3 indicated no, or low expression, 4-7 medium expression and 8-12 high expression. The distribution of the IRS score frequency in the different stages was compared to that of the normal tissues and compared using the chi square test for trend.

Results: The SLC7A11 is significantly overexpressed on all cancer tissues compared to normal tissues or adjacent to cancer tissues. Pancreatic and lung adenocarcinomas as well as lung squamous cells carcinomas have significantly different distributions of IRS score staining for SLC7A11 compared to normal tissues. However, there is no significant difference between the different cancer stages and the distributions of the IRS staining for SLC7A11.

Conclusion: The results indicate that expression of the SLC7A11 transporter is significantly overexpressed in cancer compared to the normal tissues, but there is no significant difference between the expression levels of SLC7A11 at different stages of lung or pancreatic cancer.

Citation Format: Jonathan A. Moreno, Maria P. Lambros. The expression of SLC7A11 transporter in lung and pancreatic cancer tissues at different stages of 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 3209. doi:10.1158/1538-7445.AM2015-3209
Abstract 3211: Heterogeneity of PPARβ/δ expression in colorectal tumor stroma
Authors: Tan Eddie Han Pin , Sng Ming Keat , Chan Jeremy Soon Kiat , Tan Nguan Soon
Published: August 2015

Background: The role of PPARβ/δ in colorectal cancer has been a hot topic for debate, with both tumor-promoting and anti-tumor roles been reported. Current studies on colorectal cancer have focused on either the whole intestine or on the major tissue type, the intestinal epithelium. In recent years, various studies have emphasized the pro-inflammatory and tumor promoting effects of tumor stroma in various cancers. However, the involvement of tumor stroma in adenocarcinoma development remains a largely unexplored realm of colorectal research. Hence, we hypothesized that PPARB/D expression in colorectal tumor stroma may play an important role in colorectal carcinogenesis.

Methods: In this study, we aimed to investigate the role of PPARβ/δ in the colorectal tumor stroma. To study the role of the tumor stroma in vivo, we have adopted a model organism approach involving adult APCmin mice and AOM/DSS-treated wild-type mice models. We collected intestinal adenocarcinomas from these mice and extracted tumor stroma using microscopy-directed laser capture micro-dissection. The collected stroma were recovered for mRNA and analyzed for gene expression levels using real-time PCR.

Results and conclusion: We observed contrasting trends in the PPARβ/δ mRNA expression, with elevated levels in stroma of APCmin adenocarcinoma but decreased in AOM/DSS-induced tumors. Our results to date demonstrate the heterogeneity of colorectal tumor stroma and provide an insight to the distinct differences between the two often co-studied murine models of colorectal carcinogenesis. We therefore plan to study if this trend is also observed in human colorectal tumor samples and in murine colorectal cancer models carrying stromal-specific deletion of PPARβ/δ expression.

Citation Format: Eddie Han Pin Tan, Ming Keat Sng, Jeremy Soon Kiat Chan, Nguan Soon Tan. Heterogeneity of PPARβ/δ expression in colorectal tumor stroma. [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 3211. doi:10.1158/1538-7445.AM2015-3211
Abstract 3212: Metastatic breast cancer cell communication within a pro-dormancy bone marrow niche
Authors: Price Trevor T. , Lee Clara H. , Cheng Qing , Lyerly H. Kim , Fogler William E. , Magnani John L. , Sipkins Dorothy A.
Published: August 2015

Distant metastases are the major cause of death from breast cancer. In estrogen receptor positive (ER+) breast cancer (BC) in particular, metastatic relapse can occur years after initial treatment, suggesting that disseminated tumor cells have a prolonged dormant phase before becoming proliferative. A major site of early disease dissemination and metastatic relapse of ER+ BC is the bone, although the critical signals that allow circulating breast cancer cells (BCCs) to identify bone marrow (BM) vasculature, enter the tissue, and tether to the BM microenvironment (BMM) are little understood. Investigating these interactions of BCCs with the BMM, however, could enable us to target the key biologic determinants allowing safe harbor for metastases within bone. To address the features of breast tumors associated with late recurrence, but not confounded by variations in systemic treatment, we compiled breast tumor gene expression data from 4,767 patients and established a discovery cohort consisting of 743 lymph node-negative patients who did not receive systemic neoadjuvant or adjuvant therapy. Using this data set, we found that expression of E-selectin ligands and CXCR4 were significantly elevated in ER+ tumors that had late recurrences. Based on these observations, we used intravital fluorescence microscopy to study the precise functions of E-selectin ligands and CXCR4 (SDF-1 receptor) in tumor metastasis in in vivo xenograft models and found that these play distinct roles in ER+ BCC migration in the BMM. Using highly specific E-selectin (GMI-1271) and CXCR4 (AMD3100) inhibitors, we determined that E-selectin interactions were critical for BCC entry into bone, while CXCR4/SDF-1 interactions anchored BCCs to the BMM. CXCR4 inhibition led to significant mobilization of BC micrometastases from the BMM into the peripheral circulation. Moreover, we found that dormant and proliferating BCCs occupy distinct regions of the BMM, with dormant BCCs predominantly found in SDF-1 and E-selectin rich regions and thus highly susceptible to AMD3100 mobilization. These findings shed light on how dormant and proliferating BCCs interact with the BMM and suggest new interventions to break the foothold of dormant BC micrometastases in bone.

Citation Format: Trevor T. Price, Clara H. Lee, Qing Cheng, H. Kim Lyerly, William E. Fogler, John L. Magnani, Dorothy A. Sipkins. Metastatic breast cancer cell communication within a pro-dormancy bone marrow niche. [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 3212. doi:10.1158/1538-7445.AM2015-3212
Abstract 3223: A true orthotopic ovarian cancer patient-derived xenograft (PDX) model
Authors: Cui Hongmei , He Yingyan , Krepler Clemens , Tanyi Janos , Morgan Mark A. , Burger Robert A. , Kim Sarah , Ko Emily , Ince Tan , Herlyn Meenhard , Simpkins Fiona
Published: August 2015

Introduction: Patient derived xenografts (PDXs) show promise as models to study cancer as they recapitulate the principle characteristics of the patients’ original tumor such as histology, mutational status, DNA copy number, gene expression patterns, and clinical behavior while remaining biologically stable through passaged in mice. We used an orthotopic model transplanting tumor directly to the fallopian tube/ovary in order to accurately study the native environment and the progression of the ovarian cancer (OVCA).

Experimental Design: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week old mice. Tumor growth was followed over time. Tumors were evaluated by IHC. Alu II probe staining was performed to evaluate human stroma content. RPPA and targeted DNA sequencing are in-process. Drug studies are in progress. Primary tumor lines are being generated.

Results: To date, we have transplanted tumor from 18 primary, 2 interval, and 5 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 85% success rate in generating tumors in mouse passage 1 (MP1). The median time for tumors to reach ∼2 cm is 3 months when tumors are then passaged again (MP2/MP3). We have generated 8 BRCA 1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrates human cells in the stroma. We have generated 8 primary tumor cell lines from original tumors/PDXs.

Conclusions: We conclude that although technically more challenging, the orthotopic transplantation technique is feasible in generating ovarian cancer PDXs that most closely resemble the natural environment for ovarian cancer progression.

Citation Format: Hongmei Cui, Yingyan He, Clemens Krepler, Janos Tanyi, Mark A. Morgan, Robert A. Burger, Sarah Kim, Emily Ko, Tan Ince, Meenhard Herlyn, Fiona Simpkins. A true orthotopic ovarian cancer patient-derived xenograft (PDX) 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 3223. doi:10.1158/1538-7445.AM2015-3223