bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 193: Epigenetic regulation of Notch1 signaling-driven melanoma progression
Authors: Yin Ling , Huang Qinghua , Livingstone Alan S. , Velazquez Omaida C. , Liu Zhao-Jun
Published: August 2015

Objectives. Aberrant Notch signaling promotes melanoma progression, yet the underlying molecular mechanism remains largely unknown. We hypothesized a potential involvement of epigenetic mechanism in this process and sought to identify Notch-regulated miRNAs and their functional targets.

Methods. Notch-regulated miRNAs were identified by examining miRNA profiles in response to Notch activation/inhibition in human melanoma cells using RT2-miRNAArray. The role of specific miRNA in mediating the oncogenic effect of the Notch signaling was studied by testing whether alteration of miRNA expression could reverse the Notch's effects on melanoma cell growth, survival and colony-formation in vitro, and lung metastasis in vivo. The targets of the Notch-regulated miRNA were identified by searching miRNA target database and validated by immunoblot. The roles of miRNA targets in mediating the Notch's oncogenic effects were further assessed by in vitro colony-formation and in vivo lung metastasis assays.

Results. Notch activation down-regulated mi143 in melanoma cells. Reconstituted expression of mi143 antagonized Notch1's oncogenic effect. The anti-oncogenic effect of mi143 is mediated by inhibiting Notch1-induced expression of FGF-1, uPA and uPAR since expression of these targets was up-regulated by the Notch1 activation while down-regulated by reconstituted overexpression of mi143. Supplementation of exogenous FGF-1 or reconstituted expression of uPA/uPAR, but not uPA alone, reversed the suppressive effect of mir143 on Notch-enhanced melanoma cells colony formation in soft-agar or lung metastasis in SCID mice.

Conclusions. Notch activation up-regulates expression of FGF-1, uPA and uPAR through down-regulating mi143 expression in melanoma cells. mi143 is responsible for mediating aberrant Notch signaling-driven melanoma progression. The effect of down-regulated mi143 is transmitted through elevated FGF-1 and enhanced uPA-uPAR signaling activity, but not enzyme activity of uPA. This study unveils novel Notch1/mi143/FGF-1 and Notch1/mi143/uPA-uPAR cascades and uncovers an epigenetic mechanism underlying the oncogenic effect of Notch signaling, and provides potential novel targets for melanoma prognosis and therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Ling Yin, Qinghua Huang, Alan S. Livingstone, Omaida C. Velazquez, Zhao-Jun Liu. Epigenetic regulation of Notch1 signaling-driven melanoma 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 193. doi:10.1158/1538-7445.AM2015-193
Abstract 1934: Improved deconvolution of heterogeneous tumor data to reconstruct clonal evolution from bulk genomic samples
Authors: Roman Theodore , Schwartz Russell
Published: August 2015

Intratumor heterogeneity has long been a confounding factor in interpreting cancer genomic data, but has also been useful in reconstructing progression processes based on variation between clonal populations in single tumors. We previously developed a strategy of applying computational deconvolution algorithms to gene expression or DNA copy number data to reconstruct models of progression of cell populations from bulk tumor samples. Many tools have since been proposed for similar deconvolution analysis, but all are limited by the computational difficulty of unambiguously distinguishing small cell populations and variants from noise in genomic assays. We present a novel approach to infer tumor progression pathways from deconvolved genomic data designed to leverage the fact that tumors that partition into subtypes with similar evolutionary trajectories would be expected to lead to a mathematical substructure in the genomic data, known as a simplicial complex, which can be modeled computationally to better deconvolve cell populations across tumor types. We have developed a computational pipeline to perform tumor deconvolution while taking into consideration this kind of mathematical structure. The pipeline clusters tumors to identify genetically distinct subgroups, fits mixture models to these subgroups, and uses overlap between them to infer a refined deconvolution of major cellular populations and possible pathways of progression between them.

We apply our methods to a set of RNASeq data from the TCGA breast cancer data set and to synthetic data modeling distinct scenarios of tumor progression. We first compare our methods on the synthetic data to our earlier work and to a comparative Gaussian mixture model. All methods perform comparably on unstructured data but the new method substantially outperforms the others on data consistent with simple scenarios for tumor progression along multiple discrete subtypes. The novel method, however, shows lower tolerance for noisy data than a Gaussian mixture model. Application to the TCGA RNASeq data showed our method could partition tumors into discrete subcategories associated with HER2+, ER/PR+, and triple-negative status and could exploit the resulting substructure of the data to deconvolve tumor data and infer progression models reflecting partial sharing of progression states between subtypes. Gene enrichment analysis showed association of deconvolved cell populations with a variety of gene functional categories suggestive of distinct progression mechanisms of the subtypes.

Citation Format: Theodore Roman, Russell Schwartz. Improved deconvolution of heterogeneous tumor data to reconstruct clonal evolution from bulk genomic samples. [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 1934. doi:10.1158/1538-7445.AM2015-1934
Abstract 1941: Distinct roles of c-Jun N-terminal kinase (JNK) isoforms in skin cancer
Authors: Du Lili , Zhang Tinghu , Kaoud Tamer , Gray Nathanael , Dalby Kevin , Tsai Kenneth Y.
Published: August 2015

The c-Jun-N-terminal kinases (JNKs), are activated by stress, growth factors and cytokines, and are encoded by three separate genes (JNK 1, 2, and 3), with 10 total isoforms. Our published data has demonstrated that off-target effects on JNK signaling by BRAF inhibitors (BRAFi) are important for the induction of cutaneous squamous carcinoma (cuSCC). Overexpression of JNK2, has been found to promote transformation of keratinocytes, also suggesting an important role of JNK signaling pathway in promoting tumor development. However, it is still unclear how different JNK isoforms, especially ubiquitously expressed JNK1 and JNK2, really function in skin cancer.

To delineate the specific roles of JNK1 and JNK2, we expressed C116S mutants, which rendered them insensitive to the pan-JNK inhibitor JNK-IN-8, without affecting their activity. Wild-type and mutant JNK1 and JNK2 were transduced into cuSCC (SRB1) and keratinocyte (HaCaT) cell lines and application of JNK-IN-8 enabled chemical-genetic dissection of JNK1 and JNK2 activity. JNK-IN-8 significantly attenuated UV-induced apoptosis by 80-90% in SRB1 and HaCat cells overexpressing WT JNK1, WT JNK2 or C116S JNK2 mutant compared to vehicle control while having only a slight effect (0-10%) on cells overexpressing C116S JNK1 mutant, suggesting JNK1 is the key player in UV- induced apoptosis. This difference served as the basis for a gene expression profiling study in which we identified differentially expressed genes regulated by JNK1 vs. JNK2.

We also showed that the Leucine-zipper and sterile-α motif kinase (ZAK), which is a MAP3K, is an important upstream activator of JNK-mediated UV-induced apoptosis in skin cancer. To probe how ZAK activates JNK signaling, we knocked down ZAK in SRB1 and HaCaT cells with lentiviral shRNA. Following UV exposure, we found that both JNK1 and JNK2 activation occurred with JNK1 activity peaking within 6 hours and JNK2 activity sustained over 24 hours. In ZAK knockdown cells, JNK1 activation was selectively suppressed, suggesting that ZAK preferentially activates JNK1 in this context.

In conclusion, our results indicate that JNK signaling pathway, part of which is regulated by ZAK, plays a critical role in skin cancer. JNK1 and JNK2 exert opposing influences that center on the regulation of apoptosis and cell proliferation in skin cancer.

Citation Format: Lili Du, Tinghu Zhang, Tamer Kaoud, Nathanael Gray, Kevin Dalby, Kenneth Y. Tsai. Distinct roles of c-Jun N-terminal kinase (JNK) isoforms in skin 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 1941. doi:10.1158/1538-7445.AM2015-1941
Abstract 1943: Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF
Authors: Carpenter Brittany L. , Chen Min , Knifley Teresa , Davis Kelley A. , Harrison Susan M. , Stewart Rachel L. , O'Connor Kathleen L.
Published: August 2015

Integrin α6β4 is upregulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion, in part, through its coordinated modulation of the transcriptome. Here, we find that integrin α6β4 signaling dramatically upregulates the expression of several genes involved in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (Areg), epiregulin (Ereg), and ectodomain cleavage protease MMP1. Notably, these genes correlate well with integrin α6β4 expression in publicly available pancreatic cancer databases. We previously reported that integrin α6β4 promotes HGF-stimulated migration and invasion; therefore, we tested the role of autocrine EGFR signaling in these processes. We discovered that blocking EGFR signaling with EGFR-specific blocking antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Furthermore, we found that both Areg and Ereg are required for these autocrine effects, as knocking down either ligand prevented migration and invasion. Notably, AsPC1 cells, isolated from abdominal metastases, are not dependent on EGFR signaling for migration or invasion; thus suggesting a mechanism exists for circumventing EGFR dependency. We also demonstrated that HGF stimulates the secretion of Areg, which is dependent on integrin signaling pathways including MAPK, PI3K and PKC and that Areg was transported to the leading edge of cells treated with HGF. Moreover, MMP activity and integrin α6β4 signaling are required for Areg secretion. Finally, we found that EGFR is phosphorylated in response to HGF stimulation which was dependent on EGFR protein kinase activity; however, Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, we show that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through the upregulation of key genes and facilitating HGF-stimulated EGFR ligand secretion.

Citation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kelley A. Davis, Susan M. Harrison, Rachel L. Stewart, Kathleen L. O'Connor. Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF. [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 1943. doi:10.1158/1538-7445.AM2015-1943
Abstract 1946: Activating transcription factor 5 (ATF5) in highly expressed in Stage 4, MYCN-amplified neuroblastoma
Authors: Banerjee Debarshi , Zhang Shuobo , Lopez Gonzalo , Califano Andrea , Kadenhe-Chiweshe Angela , Yamashiro Darrell
Published: August 2015

Background: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been shown to regulate the differentiation of neuroprogenitor cells into mature neurons. ATF5 has also been implicated in progression of glioblastoma multiforme. We have recently identified ATF5 as a potential synthetic lethal gene to MYCN-amplified neuroblastoma (NB) through a whole genome shRNA screen. In the present study we investigate the role of ATF5 in NB tumorigenesis.

Methods: We analyzed the human NB patient data GEO (GSE45480, Kocak et al.) to investigate and correlate the expression of ATF5 with different stages of NB. Immunohistochemical staining for ATF5 was performed on primary human NB tumors. Quantitative RT-PCR and western blot for ATF5 were conducted on a panel of NB cell lines. Immunocytochemistry was performed to determine the intercellular localization of ATF5. ATF5 was silenced by a tetracycline inducible (tet-on) shRNA construct in BE(2)-C and SK-N-DZ cells. The cells were subjected to cell viability assay and soft agar colony formation assay to assess the function of ATF5.

Results: Analysis of NB patient data GEO (GSE45480) from Kocak et al., demonstrated that ATF5 is highly expressed in Stage 4 MYCN-amplified tumors compared to Stage 4, MYCN-non-amplified tumors (P = 2.56e-10) or Stage 1 tumors (P = 7.30e-18). ATF5 is also highly expressed in Stage 4, MYCN-non-amplified tumors vs. Stage 1 tumors (P = 2.8e-7). High expression of ATF5 strongly correlated with poor survival of NB patients (P = 2.88e-6, NCI Oncogenomics, Obertheur data set). Quantitative PCR detected ATF5 transcript in all eight NB cell lines tested. Western blot analyses demonstrated high expression of ATF5 in the MYCN-amplified neuroblastoma cell lines BE(2)-C, SK-N-DZ, and IMR32. Nuclear ATF5 staining was detected in all three cell lines as well as in Stage 4 NB tumors. Silencing of ATF5 reduced BE(2)-C cell viability to 30%, 48 hr after the addition of doxycycline. Silencing of ATF5 significantly reduced the colony formation of BE(2)-C and SK-N-DZ cells in soft agar (p<0.05)

Conclusions: Our study demonstrates that ATF5 is highly expressed in Stage 4 MYCN-amplified tumors, and that silencing of ATF5 inhibits proliferation. These results suggest that ATF5 has a role in NB tumorigenesis and it represents a potential therapeutic target in MYCN-amplified neuroblastoma.

Citation Format: Debarshi Banerjee, Shuobo Zhang, Gonzalo Lopez, Andrea Califano, Angela Kadenhe-Chiweshe, Darrell Yamashiro. Activating transcription factor 5 (ATF5) in highly expressed in Stage 4, MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2015-1946
Abstract 195: Insight into microRNA regulation of autophagy genes expression in crizotinib-treated Anaplastic Large Cell Lymphoma
Authors: Frentzel Julie , Mitou Geraldine , Brousset Pierre , Giuriato Sylvie
Published: August 2015

The anaplastic lymphoma kinase (ALK) is an oncogenic tyrosine kinase involved in 80% of Anaplastic Large Cell Lymphoma (ALCL), and 5% of non-small cell lung carcinoma (NSCLC), among other cancers. The ongoing therapies in ALK+ ALCL, based on aggressive chemotherapy, are not optimal. One compound so far, the crizotinib, is used in clinics for the treatment of ALK-dependent lung cancers. However, the first mutations conferring resistance to crizotinib, have been reported and hampered its use for the treatment of ALK+ ALCL. Thus, despite the therapeutic success of tyrosine kinase inhibitors in cancer treatments, acquired drug resistances universally develop. In this context, cytoprotective autophagy has been proposed as the next target for elimination of resistance to tyrosine kinase inhibitors. We recently demonstrated that cytoprotective autophagy is indeed activated shortly upon ALK inactivation in ALCL and that its inhibition enhances the anti-tumoral action of Crizotinib. A transcriptome analysis showed an increased expression of many genes from the autophagy machinery such as ULK1, MAP1LC3B, or ATG16L2 after crizotinib treatment in ALCL. Moreover, new preliminary results enabled us to identify a set of miRNAs differentially expressed in ALCL upon Crizotinib treatment. As miRNAs are known as fine regulators of gene expression, we anticipate that some of them could be efficient regulators of core autophagy genes expression. The aim of our study is to select a few miRNAs of interest and to confirm their regulatory role on the autophagic machinery. This could lead to the development of new therapeutical strategies which could ultimately help to overcome emerging resistances against crizotinib.

Citation Format: Julie Frentzel, Geraldine Mitou, Pierre Brousset, Sylvie Giuriato. Insight into microRNA regulation of autophagy genes expression in crizotinib-treated Anaplastic Large Cell Lymphoma. [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 195. doi:10.1158/1538-7445.AM2015-195
Abstract 1956: A subset of prostate cancer cells with loss of MST1 expression confers resistance to castration through YAP1-AR interactions
Authors: Cinar Bekir , Kuser Abali Gamze , Lewis Michael , Morrissey Colm , Garraway Isla
Published: August 2015

MST1 is a core kinase component of the Hippo tumor suppressor pathway. YAP1 is a transcriptional cofactor and a prominent nuclear effector of the Hippo signaling. YAP1 activation is linked to cancer promotion with poor prognosis. However, the role of MST1-YAP axis in prostate cancer (PC) progression remains unknown. Here we isolated MST1 high and MST1 low/- expressing cell subsets from a well-characterized LNCaP PC cell model by employing novel method that utilizes an epigenetically regulated-MST1 promoter-driven GFP reporter combined with FACS. We performed molecular and functional characterizations of these cells utilizing a wide variety of experimental approaches. We demonstrated that unlike MST1 high, MST1 low/- cells shared the features of cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotype, showed superior osteogenic abilities, and were resistant to androgen depletion and enzalutamide (ENZ), a potent inhibitor of androgen receptor (AR). Consistently, silencing of MST1 expression by siRNA recapitulated the above molecular and function behaviors of MST1 low/- cells. In addition, MST1 low/- cells showed superior invasive growth abilities in vitro and tumor-forming abilities with metastatic potentials in mice compared with those of MST1 high cells. Moreover, relative YAP1 expression and its nuclear localization were much higher in MST1 low/- cells than MST1 high cells. Likewise, YAP1 protein levels and its nuclear abundance were increased in PC clinical samples compared to non-cancerous counters. We further showed that YAP1 interacted with AR in an androgen-dependent manner in LNCaP cells, whereas the interactions between the two proteins were androgen-independent (AI) in C4-2 CRPC cells. Furthermore we found that unlike LNCaP, YAP1-AR interactions were resistant to ENZ exposure in C4-2 cells. Consistently, silencing of MST1 in LNCaP cells augmented AI interactions between YAP1 and AR proteins. Finally, the disruption of YAP1 signaling by RNAi and Verteporfin (VP) attenuated C4-2 cell growth and promoted apoptosis, accompanied with the attenuation of AR-dependent gene expression. In conclusion, our findings suggest that the Hippo-YAP1-AR signaling axis is one of the key mechanisms for metastatic PC progression and may represent as a promising therapeutic target for patients with the lethal disease.

Note: This abstract was not presented at the meeting.

Citation Format: Bekir Cinar, Gamze Kuser Abali, Michael Lewis, Colm Morrissey, Isla Garraway. A subset of prostate cancer cells with loss of MST1 expression confers resistance to castration through YAP1-AR interactions. [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 1956. doi:10.1158/1538-7445.AM2015-1956
Abstract 1960: Ginger consumption may delay the onset of prostate cancer
Authors: Lane Tonya S. , Richardson Ricardo M.
Published: August 2015

Ginger active components are widely known for being a potent antioxidant and recently as a potential anticancer agent. To date, the precise mechanism(s) for the antitumor effect of ginger is not well understood. An active chemical component of ginger, 6-gingerol, was recently shown to inhibit angiogenesis and tumorigenesis. In this study we investigated the role of ginger in prostate cancer growth and proliferation using PCa cell lines from African American (AA) and Caucasian American (CA) men. To that end, MDA-PCa 2b (AA), LnCap (CA) and PC-3 (CA) cells were treated with different concentrations of 6-gingerol or 6-shogaol for 24 hours and cell proliferations were measured by XTT assay. To determine the effect of 6-gingerol in cell signaling, cell lysates were assayed for β-arrestin 1 and 2 expression, MAP kinase and caspase activity by Western Blotting. The data demonstrated that 6-gingerol inhibits cellular proliferation and promotes apoptosis in both AA and CA PCa cell lines. These effects correlated with decrease in β -arrestin 2, not β -arrestin 1, expression and Akt phosphorylation but increased caspase activity. Taken together the data suggests that 6-gingerol inhibits PCa growth and promotes PCa apoptosis via a βarr-2/Akt-dependent mechanism. Since PC-3 cells do not express AR, the data likely indicates that 6-gingerol effect is independent of AR activation and PSA expression.

Citation Format: Tonya S. Lane, Ricardo M. Richardson. Ginger consumption may delay the onset of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2015-1960
Abstract 2016: Evaluation of prostate cancer biomarkers and therapeutic targets in FFPE specimens using the NanoString platform
Authors: Lee Jocelyn , Yan Wusheng , Young Denise , Song Yingjie , Chen Yongmei , Katta Shilpa , Mohamed Ahmed , Ravindranath Lakshmi , Srinivasan Alagarsamy , Cullen Jennifer , Kagan Jacob , Srivastava Sudhir , Dobi Albert , Rosner Inger , McLeod David G. , Sesterhenn Isabell A. , Srivastava Shiv , Petrovics Gyorgy
Published: August 2015

Introduction: Identification of prognostic indicators of disease progression is one of the highest priorities in prostate cancer (CaP) research. The majority of CaP tissue specimens available for biomarker discovery and validation are formalin fixed paraffin embedded (FFPE) specimens. The RNA quality of these tissues for traditional gene expression analysis approaches is not adequate, especially of cases with long term- follow up necessary for prognostic marker studies. We systematically evaluated and optimized the NanoString platform for CaP gene expression analysis in FFPE whole mounted prostate specimens with up to 15 years follow up.

Methods: NanoString readouts were compared between a series of input total RNA prepared through different tissue dissection methods. We then performed NanoString analysis of 151-CaP probe set on 63 archived CaP samples to evaluate this modified protocol. ERG gene expression was validated by immunohistochemistry (IHC) assay using CPDR ERG-MAb (9FY).

Results: We have performed NanoString analysis of CPDR designed 151- CaP gene panel on whole-mounted prostate tissue specimens using our optimized protocol. The prostate epithelial cell markers KLK3, MSMB and PAP had the highest signals in all samples reflecting the prostate epithelial origin of the specimens. Established CaP specific genes, such as AMACR, PCA3 and PSGR were most highly over-expressed in tumors compared to matched benign prostate epithelium. ERG positive tumors had strong expression of both TMPRSS2-ERG fusion probes and probes for several ERG splice forms (ERG 1,2,3 and 8), with no signals in the matched benign samples. The ERG gene expression status was consistent with blinded ERG IHC results.

Conclusions: The feasibility of NanoString profiling in archived whole mounted FFPE prostate specimens was demonstrated. This approach is well suited for a medium throughput evaluation and validation of prognostic biomarker and therapeutic target candidates in CaP.

Citation Format: Jocelyn Lee, Wusheng Yan, Denise Young, Yingjie Song, Yongmei Chen, Shilpa Katta, Ahmed Mohamed, Lakshmi Ravindranath, Alagarsamy Srinivasan, Jennifer Cullen, Jacob Kagan, Sudhir Srivastava, Albert Dobi, Inger Rosner, David G. McLeod, Isabell A. Sesterhenn, Shiv Srivastava, Gyorgy Petrovics. Evaluation of prostate cancer biomarkers and therapeutic targets in FFPE specimens using the NanoString platform. [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 2016. doi:10.1158/1538-7445.AM2015-2016
Abstract 2017: Development of ultrasensitive Singulex immunoassays for CCL3 and CCL4, important biomarkers for the BTK inhibitor studies
Authors: Wu Jenny Q. , Burton Luciana , Raja Rajiv , McCaffery Ian , Penuel Elicia , Darbonne Walter
Published: August 2015

Disregulation of B-Cell Receptor (BCR) leading to activation has been associated with a number of B-cell malignancies including Chronic Lymphocytic Leukemia (CLL) [1]. In response to BCR activation, B-cells secrete chemokines CCL3 and CCL4 that foster interactions between CLL cells and the leukemia microenvironment and to recruit T-cells. High levels of CCL3 have also been correlated with poor prognosis in CLL [2]. Bruton tyrosine kinase (BTK) has long been known to be a key component of B cell receptor (BCR) signaling that regulates B cell proliferation and survival. BTK inhibitors such as lbrutinib have been shown to disrupt secretion of BCR-dependent chemokines (CCL3/CCL4) and thereby reduce plasma concentrations in patients [3]. Consequently, these chemokines have been measured as pharmacodynamic biomarkers in a number of studies of molecules that inhibit BCR signaling [3]. However, baseline CCL3/CCL4 levels in CLL patients vary widely and are less than 10 pg/ml in a large proportion of patient samples, making detection of these cytokines in clinical samples challenging using existing immunoassays. In an effort to improve sensitivity of CCL3/CCL4 measurements in plasma samples, we developed ultrasensitive assays for CCL3 and CCL4 using the Singulex immunoassay platform. A direct comparison of multiple, commercially available immunoassays technologies and our optimized CCL3/CCL4 Singulex assay were performed using plasma samples from both healthy donors and CLL patients. Singulex proprietary Single Molecule Counting (SMCTM) technology combining digital counting enables the quantification of CCL3 and CCL4 with sensitivity of 0.37pg/ml and 0.15pg/ml respectively in plasma. This ultrasensitive assay has the potential to enable more robust and quantifiable measurements of CCL3 and CCL4 in the context of clinical studies testing BTK inhibitors.

Citation Format: Jenny Q. Wu, Luciana Burton, Rajiv Raja, Ian McCaffery, Elicia Penuel, Walter Darbonne. Development of ultrasensitive Singulex immunoassays for CCL3 and CCL4, important biomarkers for the BTK inhibitor studies. [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 2017. doi:10.1158/1538-7445.AM2015-2017
Abstract 2019: Photoimmunotherapy with an anti-EGFR antibody conjugated to an IRDye700 results in extensive and rapid cell death in vitro and in vivo in human pancreatic cancer cell lines
Authors: de Magalhães Nzola , Murakami Takashi , Heim Roger , Makings Lew , Garcia-Guzman Miguel , Kobayashi Hisataka , Hoffman Robert M. , Bouvet Michael
Published: August 2015

Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in a nude mouse model. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT for Panc-1, MiaPaCa2, and BxPC-3 human pancreatic cancer cells. For in vivo determination of PIT efficacy, nude mice were implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: treatment with anti-EGFR-IR700 + 690 nm laser and treatment with 690 nm laser only. Anti-EGFR-IR700 (100 μg) was administered to the treatment group via tail vein injection 24 hours prior to therapy. Tumors were then exposed and treated with phototherapy at an intensity of 150 mW/cm2 for 30 minutes. Whole body imaging was done using an OV-100 small animal imaging system. Anti-EGFR-IR700 antibody bound to the BxPC3 cells to a high degree based on image co-localization. Anti-EGFR-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the mouse experiments, the treatment group had significantly smaller tumors as measured by GFP than the control after just 96 hours. The present results indicate the high potential of PIT for pancreatic cancer therapy.

Citation Format: Nzola de Magalhães, Takashi Murakami, Roger Heim, Lew Makings, Miguel Garcia-Guzman, Hisataka Kobayashi, Robert M. Hoffman, Michael Bouvet. Photoimmunotherapy with an anti-EGFR antibody conjugated to an IRDye700 results in extensive and rapid cell death in vitro and in vivo in human pancreatic 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 2019. doi:10.1158/1538-7445.AM2015-2019
Abstract 2021: Integrin-alpha 8 as a potential therapeutic target in multiple myeloma early relapse
Authors: Ryu Jiyeon , Koh Youngil , Lee Hyun Jung , Chung Hyun Sub , Yoon Sung-Soo
Published: August 2015

Introduction: Multiple myeloma (MM) is an incurable neoplastic plasma cell disorder and many studies about MM biologic complexity have been reported. Despite the advances of treatment for MM, still many patients of MM exhibit early relapses and the mechanism of MM relapse is not well understood Patient with early relapse after autologous bone marrow transplantation need to be biologically characterized to better understand and reach complete remission.

Materials and Methods: To define altered gene expression and pathways, we performed microarray with bone marrow from 16 relapsed MM patients who underwent autologous bone marrow transplantation. Gene expression profile was compared (P < 0.01) in 16 MM patients divided into two groups on the basis of their progression-free survival (PFS) at the point of 12 months or only four patients with shortest and longest duration of PFS.

Results: 77 genes over two fold up-regulated were identified and inputted in pathway enrichment analysis. To ascertain of the result, only four patients with shortest and longest duration of PFS were examined with same way of analysis that 163 genes over two fold up-regulated were inputted in KEGG pathway database. Interestingly, both of the analysis showed common up-regulated Integrin-α8 (ITGA8) and its associated pathway, ECM receptor interaction and focal adhesion, was identified (P < 0.05). Several integrin family members are well documented to involve in MM disease progression but ITGA8 in MM is largely unknown. Functionally, we have investigated ITGA8 in MM cell lines that ITAG8 mediates MM cell adhesion, migration and drug resistance through bone marrow microenvironment which is regarded as leading events of MM relapse. Furthermore, its expression in MM is correlated with poor survival outcomes after autologous bone marrow transplantation.

Conclusion: Our study demonstrated that gene expression in MM patients showed distinctly different pattern between early relapse and late relapse. Additionally, our speculated specific gene up-regulated in early relapse in MM, ITGA8, is a potential prognostic marker and suggests attractive target for future research.

Citation Format: Jiyeon Ryu, Youngil Koh, Hyun Jung Lee, Hyun Sub Chung, Sung-Soo Yoon. Integrin-alpha 8 as a potential therapeutic target in multiple myeloma early relapse. [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 2021. doi:10.1158/1538-7445.AM2015-2021
Abstract 2026: G0S2 functions as a tumor suppressor gene through inhibition of c-Myc
Authors: Yim Christina Y. , Sekula David J. , Freemantle Sarah J. , Dmitrovsky Ethan , Spinella Michael J.
Published: August 2015

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target gene that is widely expressed in diverse organs and has been reported to have a role in adipose lipolysis by negatively regulating adipose triglyceride lipase (ATGL). G0S2 is also commonly silenced by DNA methylation in a variety of solid tumors including glioma, head and neck and lung cancers, and G0S2 induction is associated with retinoic acid-mediated clinical remissions in acute promyelocytic leukemia. This evidence suggests that G0S2 may possess tumor suppressor activity although definitive proof and mechanistic details are lacking. We now clearly demonstrate using G0S2 null immortalized mouse embryonic fibroblasts (MEFs) that G0S2 can function to oppose oncogene-induced transformation. G0S2 null MEFs were readily transformed with H-RAS or EGFR while wild-type MEFs were not. Importantly, re-introduction of G0S2 reversed H-RAS transformation of G0S2 null MEFs. Although G0S2 is a known regulator of fat metabolism through attenuating ATGL activity, we found that G0S2 repression of oncogene-induced transformation was independent of ATGL inhibition. Detailed microarray analysis demonstrated that G0S2 knockout alone upregulated gene signatures associated with transformation, proliferation and Myc transcriptional responses. G0S2 knockout also induced c-Myc expression at the transcript and protein level. Finally, transient and stable shRNA-mediated repression of c-Myc completely abrogated oncogene-induced transformation of G0S2 null MEFs. Taken together, these findings indicate that G0S2 can function as a tumor suppressor by opposing Myc. Studies to elucidate the mechanism underlying G0S2 regulation of Myc are ongoing. Understanding the functions and mechanisms of this novel tumor suppressor pathway may lead to new pharmacologic strategies for many cancers where G0S2 is silenced.

Citation Format: Christina Y. Yim, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky, Michael J. Spinella. G0S2 functions as a tumor suppressor gene through inhibition of c-Myc. [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 2026. doi:10.1158/1538-7445.AM2015-2026
Abstract 2028: RUNX3 knockdown inhibited oral cancer-induced bone destruction via reducing TGF-β-dependent responses
Authors: Park Junhee , Chung Won-Yoon , Park Kwang-Kyun
Published: August 2015

Oral cancer ranks among the top 10 commonly diagnosed cancers. It easily invades underlying jaw bones and causes severe psychological and physical problems. When oral cancer cells infiltrate into jaw bones, survival rate of the patient is significantly decreased. Therefore, attention has been focused on molecular diagnostics and targeted therapy for oral cancer-induced bone destruction. However, there remains a lack of reliable biomarkers that can distinguish between bone-invasive and non-invasive oral cancers despite the recent advances in the field. The Runt domain transcription factor, RUNX3, has been found to be a tumor suppressor in many cancers through promoting transforming growth factor-β (TGF-β)-induced growth inhibition. However, the role of RUNX3 in oral cancer remains controversial. Here, we investigated whether RUNX3 has a critical role in oral cancer-induced bone destruction. In mice inoculated with RUNX3 knockdown oral cancer cells, tumor growth and bone destruction were significantly reduced. In oral cancer cells, RUNX3 knockdown significantly reduced cell viability in TGF-β-stimulated cells compared to unstimulated cells. Cell cycle analysis revealed that RUNX3 knockdown induced G1-specific cell-cycle arrest in the cancer cells without TGF-β treatment but G2-specific cell-cycle arrest in response to TGF-β treatment. RUNX3 knockdown inhibited cell migration and invasion in the absence or presence of TGF-β by changing both basal and TGF-β-induced level of epithelial-to-mesenchymal transition-related proteins. Furthermore, RUNX3 knockdown reduced parathyroid hormone-like protein expression, a major osteolytic factor, in oral cancer cells with or without TGF-β. We confirmed that RUNX3 knockdown in cancer cells decreased receptor activator of nuclear factor-kappaB ligand/osteoprotegerin ratio in osteoblastic hFOB1.19 cells treated with cancer cell-derived conditioned media. In summary, RUNX3 promotes oral cancer-induced bone destruction by enhancing interactions between cancer cells and bone cells as well as promoting malignant behavior of cancer cells. Therefore, RUNX3 may be a useful biomarker and potential therapeutic target for bone invasion of oral cancer.

Citation Format: Junhee Park, Won-Yoon Chung, Kwang-Kyun Park. RUNX3 knockdown inhibited oral cancer-induced bone destruction via reducing TGF-β-dependent responses. [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 2028. doi:10.1158/1538-7445.AM2015-2028
Abstract 2035: Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer
Authors: Nkembo Augustine T. , Salako Olufisayo , Poku Rosemary , Mazu Tryphon , Aguilar Byron , Flores-Rozas Hernan , Lamango Nazarius S.
Published: August 2015

Pancreatic cancer (PC) incidence is a major healthcare problem as the five-year survival rate remains below 5%. Over 90% of PC cases harbor the hyperactive K-Ras mutant protein. Polyisoprenylation processes facilitate the proper localization and signaling of K-Ras whose gain-in-function mutations drive uncontrolled cell survival, differentiation and proliferation. Efforts to target and disrupt this pathway and signaling for cancer therapy have been unsuccessful. The purpose of this study was to determine the effects of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as a potential novel class of small molecules for PC therapy. The effects of the PCAIs on cell viability, proliferation, survival, apoptosis, migration, colony formation and cytoskeletal F-actin organization in four human PC cell lines representing wild type K-Ras (BxPC-3), Mutant K-Ras (MIAPaCa-2 and Panc10.05) and mucins (HPAF-II) were examined. PCAIs induced apoptosis with EC50 values ranging from 2.4 to 5.1, 2.0 to 7.3, 1.8 to 3.3 and 7.0 to 33 μM in MIAPaCa-2, BxPC-3, Panc10.05 and HPAF-II cells, respectively. At 0.2 μM, PCAIs arrested MIAPaCa-2 cells at the G0/G1 phase and significantly inhibited cell migration in the wound-healing assay. MIAPaCa-2 cells transfected with a plasmid expressing a protein that fluoresces red when interacting with F-actin were used to determine the PCAIs effects on F-actin organization. Treatment with PCAIs disrupted the F-actin structures causing the cells to shrink and become rounded upon treatment with PCAIs concentrations as low as 0.2 μM. The ability of PCAIs to disrupt these biological phenomena in pancreatic cancer cells shows that PCAIs may halt progression in tumors with the hyperactive K-Ras mutations.

Citation Format: Augustine T. Nkembo, Olufisayo Salako, Rosemary Poku, Tryphon Mazu, Byron Aguilar, Hernan Flores-Rozas, Nazarius S. Lamango. Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2015-2035
Abstract 2048: Evidence that MTAP acts as a genetic switch to inhibit tumor formation
Authors: Tang Baqing , Kruger Warren
Published: August 2015

Methylthioadenosine Phosphorylase (MTAP) deficiency is widely observed in a variety of human tumor and tumor derived cell lines, suggesting that MTAP may play a role in tumorigenesis. It is an enzyme that catalyzes phosphorylation of 5′deoxy-5′-methylthioadenosine (MTA), a byproduct of in vivo polyamine biosynthesis. To elucidate the mechanism by which MTAP inhibits tumor formation, we created a MCF-7 breast cancer derived cell line that expresses MTAP under a doxycycline (DOX) regulated promoter. In this cell line MTAP expression was undetectable in the absence of DOX and could be detected as early as 4 hours after DOX induction. MTAP expression caused a decrease in cell proliferation, soft-agar colony formation, and cell migration. Microarray studies revealed several genes that were regulated by MTAP. For two of the genes that were identified, AGPAT4 and IGFBP1, we examined the relationship between their mRNA levels and MTAP mRNA levels by qRT-PCR. Surprisingly, a brief pulse of MTAP expression was able to alter mRNA levels of these genes and this change in mRNA persisted even when MTAP RNA levels were no longer detectable. Our findings show that MTAP expression suppresses tumorigenesis and it may act as a genetic switch.

Citation Format: Baqing Tang, Warren Kruger. Evidence that MTAP acts as a genetic switch to inhibit tumor formation. [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 2048. doi:10.1158/1538-7445.AM2015-2048
Abstract 2056: SIRT1 silencing suppresses prostate cancer growth and metastasis in an orthotopic prostate cancer mouse model
Authors: Dai Yan , Zhu Lijia , Cho Elizabeth , Hagiwara Audrey , Faller Douglas , Kim Sun-Jin , Fidler Isaiah J. , Amaro Marina
Published: August 2015

Tumor environment is critical for tumor growth and progression, and orthotopic mouse model allows accurately study tumor development in an appropriate tumor environment. SIRT1, a class III histone deacetylase, has been shown overexpressed in prostate cancer and is a biomarker for prostate cancer recurrence. Although these data have suggested the importance of the role SIRT1 plays in prostate cancer progression, the role of SIRT1 in tumor growth and progression within, specifically, a prostate tumor environment has not been established.

In this study, we established an advanced prostate cancer PC3MM2 orthotopic mouse model to study the effects of SIRT1 silencing on prostate cancer growth and progression. Our data has shown that SIRT1 knockdown greatly suppresses tumor formation within a prostate tumor environment, along with an inhibition of metastasis to bone and liver. To further study the mechanism by which SIRT1 controls tumor growth and progression, we demonstrated that SIRT1 silencing reduces prostate cancer cell adhesion to cell matrix, and that this silencing also suppresses cancer cell growth and colony formation in a cell matrix dependent manner. Moreover, our data showed that SIRT1 regulates integrin expression and focal adhesion complex formation. Furthermore, we shows that SIRT1 silencing from prostate cancer cell significantly inhibited human umbilical vein endothelial cell (HUVEC) tube formation, suggests an important role of SIRT1 in prostate cancer angiogenesis. Thus, we have reported for the first time that SIRT1 plays a critical role in prostate cancer growth and progression using orthotopic mouse model, and demonstrated for the first time that SIRT1 regulates integrin expression, focal adhesion formation, tumor cell and matrix interaction, and angiogenesis. These results suggest SIRT1 regulates prostate cancer growth and progression through multiple mechanisms and that SIRT1 is a potential target in prostate cancer treatment.

Citation Format: Yan Dai, Lijia Zhu, Elizabeth Cho, Audrey Hagiwara, Douglas Faller, Sun-Jin Kim, Isaiah J. Fidler, Marina Amaro. SIRT1 silencing suppresses prostate cancer growth and metastasis in an orthotopic prostate cancer 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 2056. doi:10.1158/1538-7445.AM2015-2056
Abstract 206: Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate cancer cells
Authors: Jain Pallavi , Neveu Bertrand , Fradet Yves , Pouliot Frederic
Published: August 2015

Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate cancer cells

OBJECTIVE: Molecular imaging plays an important role in oncology for staging of tumors. Unfortunately, the specificity and sensitivity of current techniques remains low. This study aims to improve the existing imaging system based on transcriptional activation, named as “Two -Step- Transcriptional Amplification (TSTA) system”, with the goal to precisely image in vivo prostate cancer cells (PCa) by Positron Emission Tomography (PET). We have studied the potential of DD3/PCA3 promoter, a gene specifically expressed in PCa, to achieve this goal.

METHODS: Various adenovirus constructs incorporating the DD3 promoter, the TSTA system and the Firefly luciferase reporter gene were generated and their specificity for PCa cells was tested in transient infection. By molecular engineering, we have improved the TSTA system and generated the 3STA. The luciferase activities of DD3-TSTA, DD3-3STA and PSA-TSTA (Prostate Specific Antigen) were compared in vivo by bioluminescence in xenograft mouse models. Ex vivo prostate biopsy from RP (radical prostatectomy) specimen were exposed to DD3-3STA and luciferase expression was evaluated using bioluminescence and Immunohistochemistry.

RESULTS: The DD3 promoter activity is both specific to prostate and cancer cells. When DD3 promoter is incorporated in the amplification systems TSTA and 3STA, it is specific to PCa cells and its activity is amplified 32 and 95 times, respectively, when compared to the activity of the DD3 promoter alone. Moreover, activity of DD3-TSTA and DD3-3STA is androgen-independent. In vivo, DD3-3STA activity is comparable to that obtained with the PSA-TSTA whose activity can be imaged by PET. DD3-3STA could detect primary prostate cancer cells ex vivo in prostate biopsy obtained from RP specimen (Figure 1).

CONCLUSIONS: The new system DD3-3STA allows specific and sensitive imaging of PCa cells. The new amplification system 3STA allows the DD3 promoter to produce reporter signal high enough to be detected by PET, a technology available in the clinic.

Citation Format: Pallavi Jain, Bertrand Neveu, Yves Fradet, Frederic Pouliot. Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate 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 206. doi:10.1158/1538-7445.AM2015-206