bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4015: Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer.
Authors: Yung Man Kuen , Yip Chi Wai , Cheung Phyllis Fung Yi , Cheung Siu Tim
Published: August 2014

Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer

Hermann M.K. Yung, Wallace C.W. Yip, Phyllis F.Y. Cheung and Siu Tim Cheung

Department of Surgery, The University of Hong Kong.

Introduction:

Hepatocellular carcinoma (HCC) is the 3rd most lethal cancer worldwide. Curative treatments, such as surgical resection, are limited to only a small group of patients. Granulin-epithelin precursor (GEP), which is a secretory growth factor, has been shown to enhance the growth, invasion, metastasis and chemo-resistance of HCC. GEP was found to overexpress in tumors compared to the non-tumor counterpart, and the overexpression correlates to poor prognosis in HCC patients. Antibody targeting GEP inhibited tumor growth in vivo. However, the overexpression mechanisms in HCC are not clear.

Hypothesis:

Gene amplification may be one of the mechanisms for GEP overexpression as its locus, 17q21, is frequently amplified.

Method:

Quantitative Microsatellite Analysis (QuMA) was used to determine the copy number alteration of the GEP gene locus. In this method, quantitative PCR was used to quantify the GEP and microsatellite loci. The genomic stable microsatellite loci were used as reference. Copy number variations were determined by normalization of HCC data to the copy numbers of blood samples from healthy donors, which were defined as diploid 2N. GEP mRNA level was quantified by real-time RT-PCR. Correlations between GEP copy number and its mRNA expression level were analyzed.

Results:

GEP DNA copy numbers were quantified in a panel of clinical HCC (n=60). Amplification of GEP locus was observed in 30% (20/60) HCC, and the amplification frequency was comparable to published reports on chromosome 17q. Overall, GEP copy number correlated to the mRNA overexpression levels (n=60, r=0.267, P=0.039). For HCC with GEP gene locus amplification (n=20), tight correlation with GEP expression levels were observed (r=0.646, P=0.002).

Summary:

We showed that GEP gene was frequently amplified in HCC, and this amplification correlates to the expression level of GEP. Fluorescent in situ hybridization (FISH) will be performed to further investigate the amplification status.

Citation Format: Man Kuen Yung, Chi Wai Yip, Phyllis Fung Yi Cheung, Siu Tim Cheung. Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2013-4015
Abstract 4018: Whole exome sequencing analysis of familial Medulloblastoma.
Authors: Sasaki Mark M. , Skol Andrew D. , Pugh Trevor J. , Meyerson Matthew , Onel Kenan
Published: August 2014

Medulloblastoma (MB) is the most common malignant brain cancer in children. Although MB can be categorized into four distinct subtypes, the underlying genetic changes required to promote MB remain unclear. We performed whole exome sequencing was performed on germ-line DNA from two affected cousins who were diagnosed at similar ages with classic WNT subtype MB, and their unaffected mothers, who are full sisters. We found cousins to be homozygous for a variant in a gene regulating WNT-signaling (variant 1), while their mothers were heterozygous. In a mouse model of WNT-type MB, incomplete penetrance and the requirement for inactivation of TP53 were observed. We, therefore, hypothesized that a deleterious mutation in the TP53 pathway may be present. Indeed, we found a single copy of a novel mutation in a regulator of TP53 shared between the affected cousins (variant 2). This allele segregated through the cousins’ maternal line as evidenced by both mothers being heterozygous for this allele. Finally, we identified a common homozygous variant in a gene involved in promoting RAS-mediated cell proliferation (variant 3). We found that mutations in the genes of variants 1 and 3 have been previously reported to be involved in sporadic MB, suggesting that what we observe in this family translates to sporadic disease. Thus, our model for MB development is that of a threshold effect in which combinations of both familial and common variants interact to increase susceptibility. We hypothesize that this model with incomplete penetrance may underlie cancer susceptibility in many families with apparent cancer predisposition syndromes but in whom no causative mutation has been identified. We also hypothesize that this multi-gene model with incomplete penetrance may also explain a proportion of apparently sporadic cases of cancer. This work underscores the advantages of analyzing sequence data from multiple well-chosen affected and unaffected individuals in well-chosen families for gene discovery in cancer and other complex diseases. It also puts forth a new model for analysis, incorporating both agnostic and hypothesis driven models using data from the literature and complementary datasets.

Citation Format: Mark M. Sasaki, Andrew D. Skol, Trevor J. Pugh, Matthew Meyerson, Kenan Onel. Whole exome sequencing analysis of familial Medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2013-4018
Abstract 4031: Distinct gene expression profile of recurrent stage II colorectal cancers in African American and Caucasian American patients.
Authors: Jadhav Trafina , Chen Dongquan , Putcha Balananda-Dhurjati Kumar , Samuel Temesgen , Posey James , Heslin Martin , Manne Upender
Published: August 2014

Objective: To determine gene expression profile of Stage II colorectal cancers (CRCs) based on disease recurrence and patient race/ethnicity.

Background: About 25-35% of Stage II CRCs recur after potentially curative surgery. Furthermore, the incidence, recurrence, and mortality rates for CRC are higher in African Americans (AAs) relative to Caucasian Americans (CAs), even when they are diagnosed with early stage disease. Differences in tumor biology may contribute to this race-based disparity. Thus, identification of these ‘high-risk’ patients is an unmet medical need.

Methods: Using GeneChip microarray technology, we obtained gene expression profiles for Stage II CRCs from 16 AA and 30 CA patients. Samples from recurrent disease, within 5 years post-surgery (n=5 in AA, n= 9 in CA) were compared against non-recurrent, within 10 years post-surgery, (n=11 in AA, and n=21 in CA) samples. The data were analyzed for expression differences at the gene level comparing AA with CA patients and further analyzed for functions and pathways.

Results: In the samples of recurrent CRCs, a significant number of genes (n=561) were differentially expressed, with a false-discovery rate of ≤5%. Further analyses showed 253 genes differentially expressed within the AA recurrent subgroup and 317 genes in CA recurrent subgroup. In recurrent CRCs, there were 9 genes common to AA and CA patients, suggesting a common gene signature specific for Stage II disease. Canonical pathway analyses and interaction network analyses for these differentially expressed genes revealed that ERK/MAPK, protein kinase A, and CDK5 signaling were the top three affected signaling pathways in recurrent samples from AAs. In contrast, RhoA, integrin, and cell cycle control signaling were the top three affected pathways in Stage II CRCs of CAs. This suggests, for samples from CAs and AAs, a distinct race-based gene expression and downstream effects on pathways.

Conclusion: A common gene expression signature, specific for stage II, together with a race-specific expression profile may have therapeutic implications for AA and CA patients.

Citation Format: Trafina Jadhav, Dongquan Chen, Balananda-Dhurjati Kumar Putcha, Temesgen Samuel, James Posey, Martin Heslin, Upender Manne. Distinct gene expression profile of recurrent stage II colorectal cancers in African American and Caucasian American patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2013-4031
Abstract 4036: Curcumin induces ER stress-mediated apoptosis through cell type-specific interruption of redox balance in cervical cancer cells.
Authors: Kim Boyun , Jung Eun-Ji , Song Yong Sang
Published: August 2014

Prolonged accumulation of misfolded or unfolded proteins prompts a cellular stress condition known as ER stress which activates unfolded protein response (UPR). The ER stress is usually sustained at higher level in cancers than in normal cells due to altered metabolism of the cancer. Recent studies have shown that curcumin, a major yellow pigment of tumeric derived from Curcuma longa plant, triggers apoptosis through induction of ER stress in several cancer cells. Here, we found that ER stress was induced by interruption of redox balance when cervical cancer cells were exposed to curcumin. We used four cervical cancer cell lines, C33A, CaSki, HeLa and ME180. Curcumin inhibited proliferation of cervical cancer cells in a dose- and time-dependent manner and induced apoptotic cell death (increase of AnnexinV-positive and SubG1 fraction). Additionally, proteolytic activation of caspase-9 and -3 and PARP cleavage were shown in cervical cancer cells. We next evaluated activation of unfolded protein response (UPR) in response to curcumin. Three ER-resident sensors (PERK, IRE1α and ATF6) and their down-signaling proteins (XBP1, ATF4 and eIF2α) were activated in all cervical cancer cells (C33A, CaSki, HeLa and ME180), but not in normal cervical epithelial cell. Also, one of UPR down-signaling proteins CHOP/GADD153, which is a transcription factor inducing ER stress-mediated apoptosis, was up-regulated by curcumin. CHOP/GADD153 decreased anti-apoptotic protein Bcl2 and subsequently promoted apoptosis of cervical cancer cells exposed to curcumin. Furthermore, curcumin led to elevation of intracellular ROS in CaSki and ME180 cells, but not in C33A, HeLa and normal cervical epithelial cell. Pretreatment of N-acetyl-cysteine (ROS scavenger) resulted in inhibition of ER stress and cell death at least in part. Taken together, these results show that curcumin promotes ER stress-mediated apoptosis through cell type-specific ROS generation in cervical cancer cells.

Citation Format: Boyun Kim, Eun-Ji Jung, Yong Sang Song. Curcumin induces ER stress-mediated apoptosis through cell type-specific interruption of redox balance in cervical cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4036. doi:10.1158/1538-7445.AM2013-4036
Abstract 4057: Functional domain of Annexin A4: which domain is indispensable for chemoresistance to platinum drugs.
Authors: Morimoto Akiko , Enomoto Takayuki , Matsuzaki Shinya , Hiramatsu Kousuke , Ueda Yutaka , Yoshino Kiyoshi , Fujita Masami , Yokoyama Takuhei , Serada Satoshi , Naka Tetsuji , Kimura Tadashi
Published: August 2014

Background: Platinum drugs are often used for the postoperative adjuvant therapy or treatment in advanced and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtype of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin.

Purpose: To identify the domain of Anx A4 relevant to chemoresistance.

Materials and methods: Anx A4 possesses the NH2-terminal domain and four annexin repeat domains with one Ca2+-binding site in each domain. Deleting annexin repeat domains one by one from C-terminal site, we generated four deletion mutants of Anx A4 named R1, 2, 3, and 4 (arabic figure shows the number of annexin repeat domains). Stable cell lines overexpressing each deletion mutants were established in NUGC3 cells. Chemosensitivity to platinum drugs was evaluated using the MTT assay.

Results. Compared with mock transfected cells, all the four deletion mutants induced chemoresistance to both cisplatin and carboplatin.

Conclusions. The deletion derivative covering only one annexin repeat domain can induce chemoresistance to platinum drugs. Further study is underway to determine the specific site for chemoresistance of Anx A4.

Citation Format: Akiko Morimoto, Takayuki Enomoto, Shinya Matsuzaki, Kousuke Hiramatsu, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takuhei Yokoyama, Satoshi Serada, Tetsuji Naka, Tadashi Kimura. Functional domain of Annexin A4: which domain is indispensable for chemoresistance to platinum drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2013-4057
Abstract 4059: Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A.
Authors: Matsuzaki Shinya , Morimoto Akiko , Serada Satoshi , Yokoyama Takuhei , Kimura Toshihiro , Kobayashi Eiji , Ueda Yutaka , Yoshino Kiyoshi , Fujita Masami , Enomoto Takayuki , Naka Tetsuji , Kimura Tadashi
Published: August 2014

Because platinum drugs are often used for the chemotherapy of human cancers, when platinum resistance occurs it is a major issue. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to Carboplatin via increased extracellular efflux of the drug. However, the precise mechanisms of that chemoresistance, and the relationship of Anx A4 to platinum resistance in vivo, remain unclear. In this report we investigate in vitro the mechanism of platinum resistance induced by Anx A4 in endometrial carcinoma cells (HEC1 cells) which normally have a low level of expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, we compared HEC1 control cells with Anx A4-overexpressing HEC1 cells when both were xenografted to mice, Anx A4-overexpressing xenografted mice presented with significantly greater chemoresistance to Cisplatin in vivo. By immunofluorescence analysis we found that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became co-localized with ATP7A, a copper transporter also well known to be a platinum effluxer. When the expression of ATP7A was suppressed by small interfering RNA in HEC1 control cells, they showed no change of chemosensitivity to platinum drugs. However, suppressed ATP7A in Anx A4 overexpressing platinum-resistant cells, they showed improved chemosensitivity to platinum drugs, to a level comparable of that of control cells. Our results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A.

Citation Format: Shinya Matsuzaki, Akiko Morimoto, Satoshi Serada, Takuhei Yokoyama, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takayuki Enomoto, Tetsuji Naka, Tadashi Kimura. Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2013-4059
Abstract 4067: Detection of S-nitrosylated protein in renal cell cancer.
Authors: Hongo Fumiya , Ueda Takashi , Ito-Ueda Saya , Oishi Masakatsu , Nakamura Terukazu , Naya Yoshio , Miki Tsuneharu
Published: August 2014

Purpose: Nitric oxide (NO) is a versatile signaling molecule. Its roles in a variety of physiological functions in mammals are beginning to be understood. It has been demonstrated that NO up-regulates the expression of the Fas receptor on human tumor cells via specific inactivation of transcription repressor YY1 DNA binding activity to the silencer region of the Fas promoter (Hermes G., et al. 2001). We also showed that inactivation of YY1 via protein S-nitrosylated by NO in prostatic cancer could be identified (Hongo F., et al. 2005). This post-translational modification, known as S-nitrosylation, has emerged as a highly conserved and spatiotemporally specific signaling mechanism. The objective of our study was to examine the effect of NO donor treatment on renal cell cancer.

Methods: We examined the effect of the NO donor DETA-NONOate (100-500uM) on the following renal cell cancer cell lines; CAKI-1, NC65, and ACHN. Anti-S-nitroso-Cysteine antibody in the rabbit (SIGMA-ALDRICH) was applied as a primary antibody for immunohistochemistry. The biotin-switch technique was applied to detect specific S-nitrosylated proteins. Whole cell lysates were extracted from ACHN cells employed with DETA-NONOate. Using an S-nitrosylated detection kit (Cayman Chemical Company), S-NO was replaced by Biotin. Cell lysates were incubated at 4 oC with 30 μL of Dynabeads M-280 Streptavidin (Invitrogen) for 3 hrs. After five washes with wash buffer (S-Nitrosylation Wash buffer), the protein complex was eluted from anti-biotin dynabeads by incubation with 1x volume of elution buffer (0.1M glycine (pH2.0)) twice for 15 min. Each sample was added to an SDS-polyacrylamide gel for mass spectrometry.

Results: Significant up-regulation of S-nitrosylated proteins employed with DETA-NONOate was observed by immunohistochemistry. Specific S-nitrosylated proteins in ACHN cells were detected by the biotin-switch technique and included HSP90 (heat shock protein 90), GRP78 (HSP70 family), HSP70, and pyruvate kinase M2.

Conclusions: Our data showed that NO treatment significantly increased S-nitrosylation of several proteins. S-nitrosylation should be considered as one of the mechanisms by which NO acts on cancer cells.

Citation Format: Fumiya Hongo, Takashi Ueda, Saya Ito-Ueda, Masakatsu Oishi, Terukazu Nakamura, Yoshio Naya, Tsuneharu Miki. Detection of S-nitrosylated protein in renal cell cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2013-4067
Abstract 407: Urinary C-reactive protein: A biomarker of metastatic kidney cancer.
Authors: Morrissey Jeremiah , Kharasch Evan D.
Published: August 2014

Background: Approximately one-third of patients with kidney cancer who do not have detectable metastatic disease at the time of diagnosis and/or nephrectomy will nonetheless go on to develop metastasis. Presently, there is no method to predict which patients with kidney cancer and no evidence of metastases; will later develop metastatic disease. This investigation evaluated pre-nephrectomy urine C-reactive protein as an early diagnostic predictor of subsequent metastatic kidney cancer, and its use for tumor staging by molecular pathology to supplement conventional observational pathology.

Methods: C-reactive protein was measured by a sensitive ELISA in the pre- and post-nephrectomy urine of 31 patients with a pathologic diagnosis of clear cell or papillary carcinoma (tumor size 1-11 cm) and in 23 surgical control patients matched for age and sex. C-reactive protein in the tumors was determined by immunohistochemistry.

Results: By immunohistochemistry, kidney tumors synthesize C-reactive protein, thus making it available for potential excretion into the urine. The urine C-reactive protein of all control patients was 0.6 (0.2, 1.0) ng/ml (median, (1st and 3rd quintile)) while in the pre-nephrectomy urine of 31 patients with clear cell or papillary carcinoma was 3.9 (2, 11) ng/ml (P<0.0001, Wilcoxon rank sum test). The c-statistic for all controls and patients with kidney cancer was 0.89 (95% CI, 0.80 to 0.97) while the Spearman regression coefficient to tumor size was 0.72 (95% CI, 0.57-0.83). There was a significant 82% decrease in the urinary C-reactive protein concentration in the post-nephrectomy urine (P=0.0156, Wilcoxon test for paired samples). All stage 3 tumors and almost half of the stage 1 tumors excreted high amounts of biomarker. Four patients with stage 1 tumors and with concentrations of urine C-reactive protein significantly above the control range and no noted metastases at the time of partial nephrectomy subsequently developed clinical metastases over 32-34 months. Four patients with stage 1 tumors and urinary C-reactive protein concentrations equivalent to the controls had not developed metastatic disease in the same time frame.

Conclusion: The measurement of C-reactive protein in the urine may be a non-invasive biopsy of the renal tumor indicating sub-clinical metastatic potential and may pre-surgically stage the tumor by molecular pathology.

Citation Format: Jeremiah Morrissey, Evan D. Kharasch. Urinary C-reactive protein: A biomarker of metastatic kidney cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 407. doi:10.1158/1538-7445.AM2013-407
Abstract 4072: Wnt1 gene expression alters heterogeneous population of prostate cancer cells; decreased expression pattern observed in CD133+/CD44+prostate cancer stem cells.
Authors: Goksel Gamze , Bilir Ayhan , Uslu Ruchan , Akbulut Hakan , Guven Ummu , Oktem Gulperi
Published: August 2014

Tumor mass contains a complex and heterogeneous phenotypic population including a rare group of cancer cells that are capable of serving cancer- initiating cells or cancer stem cells (CSCs). Established cancer cell lines contain CSCs, which can be propagated, to form in vitro 3D tumor spheroids. Spheroids reflect differentiation properties of CSCs in serum contained medium and more accurately reflect the complex in vitro microenvironment than simple two-dimensional monolayer. Aberrant activation of Wnt signaling is strongly implicated in the progression of cancer and controls CSCs properties. In this study we hypothesize that when cells maintained as spheroids the structure of CSCs could be show differentiation between CSCs and non- CSCs counterpart. It is possible to determine target molecules for CSCs eradication and specify a clue in therapeutic strategies of cancer. With this aim, CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with remained heterogeneous cancer cells bulk population. Wnt1, Fzd1, Adar, Apc, Axin, Btrc, Frat1 gene expression analysis was applied and protein expression levels of Wnt1, Fzd and Axin were shown by immunohistochemistry. Results show that Wnt1 expression significantly higher in non cancer stem cells compared to CSCs. Nevertheless Fzd1, Adar, Apc, Axin, Btrc, Frat1 gene expressions were higher in CSCs group than the other population. Increased Wnt1 immunoreactivity was demonstrated in the non-cancer stem cells. It is possible to assume that intracellular signaling of Wnt pathway and related molecules in the nucleus and/or cytoplasm might play an important role independent of Wnt ligand. This unexpected expression could be important for CSCs behavior and targeting of this pathway could have therapeutic value in cancer.

Citation Format: Gamze Goksel, Ayhan Bilir, Ruchan Uslu, Hakan Akbulut, Ummu Guven, Gulperi Oktem. Wnt1 gene expression alters heterogeneous population of prostate cancer cells; decreased expression pattern observed in CD133+/CD44+ prostate cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2013-4072
Abstract 4075: Regulation of NKX3.1 by RMND5 proteins.
Authors: Louw Alison , Thomas Marc A. , Harvey Jennet , Bentel Jacqueline M.
Published: August 2014

Expression of the prostatic tumour suppressor, NKX3.1 is reduced or undetectable in a large proportion of advanced human prostate tumours. Although loss of heterozygosity (LOH) is common at the 8p21.2 locus which encompasses the NKX3.1 gene, mutations of the remaining NKX3.1 allele that would account for loss of NKX3.1 expression have not been identified. Indeed, the documented discordance between NKX3.1 mRNA and protein expression suggests that translational or posttranslational mechanisms contribute to the loss of NKX3.1 expression in prostate tumour tissues. We have identified two novel NKX3.1 binding proteins in LNCaP cells, RMND5A and RMND5B. The RMND5A gene locus at 2p11.1 is deleted or amplified in a variety of cancers, while the RMND5B locus at 5q35.3 undergoes frequent LOH in breast tumours from BRCA1 and BRCA2 mutation carriers and is located within an uncharacterised prostate cancer heritability locus. RMND5 proteins are widely expressed, multidomain proteins that each contain a putative RING domain, suggesting that they are able to function as E3 ubiquitin ligases. In in vitro ubiquitin assays, the RING domain of RMND5A was able to associate with UbcH2, UbcH5b and UbcH5c, while the RING domain of RMND5B interacted with UbcH5b and UbcH5c to mediate ubiquitin transfer. Both RMND5A and RMND5B were associated with ubiquitinated proteins in vivo and the increased expression of RMND5A or RMND5B augmented cellular levels of ubiquitinated proteins, providing further evidence of their E3 ubiquitin ligase activity. Overexpression of either RMND5A or RMND5B in LNCaP cells resulted in dose-dependent declines in endogenous NKX3.1 levels, which were attenuated following proteasome inhibition. In addition, RMND5A or RMND5B overexpression enhanced cellular levels of ubiquitinated NKX3.1. In LNCaP cells, RMND5 proteins were diffusely distributed in the cytoplasm and nucleus, with RMND5B occasionally exhibiting a punctate cytoplasmic distribution. When NKX3.1 and RMND5A or RMND5B were overexpressed, RMND5 proteins became predominantly nuclear, while the nuclear levels of NKX3.1 were markedly reduced. Together, these findings indicate that RMND5 proteins promote NKX3.1 ubiquitination and proteasome-dependent degradation and additionally modify the intracellular localisation of NKX3.1, potentially regulating its function as a transcription factor.

Citation Format: Alison Louw, Marc A. Thomas, Jennet Harvey, Jacqueline M. Bentel. Regulation of NKX3.1 by RMND5 proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2013-4075
Abstract 4078: Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer.
Authors: Fang Xiaolan , Sivanandane Sittadjody , Gyabaah Kenneth , Balaji K.C.
Published: August 2014

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in both normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial stromal interactions including paracrine function. Yet an ideal 3-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model, using double layered alginate hydrogel microspheres to incorporate prostate cancer epithelial and stromal cells. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also proved that the amount of sE-cad is influenced by epithelial stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction.

Citation Format: Xiaolan Fang, Sittadjody Sivanandane, Kenneth Gyabaah, K.C. Balaji. Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2013-4078
Abstract 4083: Inhibition of constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen indepenent signaling and growth in (C4-2) prostate cancer cells.
Authors: Tran Cindy , Richmond Oliver , Aaron LaTayia , Powell Joann B.
Published: August 2014

The aryl hydrocarbon receptor is a member of the basic-helix-loop-helix family of transcription factors. AhR mediates the biochemical and toxic effects of a number of polyaromatic hydrocarbons such as 2,3,7,8,-tetrachloro-dibenzo-dioxin (TCDD). AhR is widely known for regulating the transcription of drug metabolizing enzymes involved in the xenobiotic metabolism of carcinogens and therapeutic agents, such as cytochrome P450-1B1 (CYP1B1). Additionally, AhR has also been reported to interact with multiple signaling pathways during prostate development. Here we investigate the effect of sustained AhR signaling on androgen receptor function in prostate cancer cells. Immunoblot analysis shows that AhR expression is increased in androgen independent (C4-2) prostate cancer cells when compared to androgen sensitive (LNCaP) cells. RT-PCR studies revealed constitutive AhR signaling in C4-2 cells without the ligand induced activation required in LNCaP cells. A reduction of AhR activity by pharmacologic inhibitors or short RNA mediated silencing in C4-2 cells reduced expression of both AhR and androgen responsive genes. The decrease in androgen responsive genes correlates to a decrease in phosphorylated androgen receptor and androgen receptor expression in the nucleus. Furthermore, the forced decrease in AhR expression resulted in a 50% decline in the growth rate of C4-2 cells. These data indicates that AhR is required to maintain hormone independent signaling and growth by the androgen receptor in C4-2 cells. Collectively, these data provide evidence of a direct role for AhR in androgen independent signaling and provides insight into the molecular mechanisms responsible for sustained androgen receptor signaling in hormone refractory prostate cancer.

Citation Format: Cindy Tran, Oliver Richmond, LaTayia Aaron, Joann B. Powell. Inhibition of constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen indepenent signaling and growth in (C4-2) prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4083. doi:10.1158/1538-7445.AM2013-4083
Abstract 4090: Akt2-specific signaling in prostate cancer maintenance.
Authors: Chin Yuet Ming Rebecca , Balk Steve , Toker Alex
Published: August 2014

Mutations and amplification in genes regulating the PI 3-K/Akt pathway are common in prostate cancer, with deletion or mutations of PTEN being the most frequent. This has led to major efforts in developing anti-cancer drugs targeting the Akt pathway. Although the role of Akt in tumorigenesis has been studied extensively, little is known regarding isoform-specificity, and it is largely assumed that Akt isoforms play redundant roles in regulating cancer cell survival.

In this study, we have developed a tet-on inducible system for expressing Akt1/2/3 shRNAs to determine the role of Akt isoforms in prostate cancer progression. Three-dimensional (3D) matrigel cultures are employed to better recapitulate tumors growing in vivo. Administration of doxycycline (dox) in Akt1 or Akt2 shRNA PTEN-deficient LNCaP cells at the start of culture resulted in the inhibition of spheroid formation, suggesting an important role of both Akt1 and Akt2 in the initial growth of spheroids. Conversely, when spheroids were allowed to form for 7 days, followed by dox for further 10 days, no difference was observed between Akt1-depleted and control spheroids. In contrast, silencing of Akt2 resulted in complete disintegration of spheroids. Confocal microscopy studies showed a significant induction of active caspase-3 and fragmented nuclei in Akt2-silenced cells. Moreover, whereas Akt1 is uniformly expressed, Akt2 is localized in the cytoplasm and/or on the plasma membrane. Interestingly, the differences of effect of Akt isoforms on cell survival in conventional 2D culture are considerably more modest. The exclusive requirement of Akt2 for survival is also observed in PC3 prostate cancer cells, which are also PTEN-deficient. Importantly, our preliminary data indicated that Akt2, but not Akt1 knockdown, led to tumor regression in the LnCaP xenograft model.

These results indicate that Akt2, but not Akt1, plays a critical role in prostate cancer maintenance. Current efforts are aimed at identifying novel Akt isoform-specific substrates that contribute to prostate cancer progression using a genome-wide proteomic screen. The results of these studies could point to new targets for anti-cancer therapeutics.

This work was supported by grants from NIH (A.T., CA122099; R.C., 1K99CA157945-01, T32 CA081156-09)

Citation Format: Yuet Ming Rebecca Chin, Steve Balk, Alex Toker. Akt2-specific signaling in prostate cancer maintenance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2013-4090
Abstract 4097: Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell.
Authors: Ahmed Maqbool , Hussain Azhar R. , Al Dayel Fouad , Uddin Shahab , Al-Kuraya Khawla S.
Published: August 2014

The mammalian target rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of mRNA in mammalian cells. Torin2 is a potent, selective and orally available mTOR inhibitor for treatment of cancer. Dysregulation of mTOR signaling has been found in many cancers, however its role Papillary Thyroid Cancer (PTC) has not been explained. Therefore in this study, we investigated the role of mTOR and its associated molecules in PTC cell lines using MTT, flow cytometry and DNA fragmentation assays and western blotting. Our data showed that Torin2 caused a dose dependent growth inhibition in all cell lines studied. Cell cycle assay showed that the sub-G1 population in TPC-1 cell increased from 12.44% in untreated control sample to 31.50% after 200nM of Torin2 treatment and 44.08% after 400nM of Torin2 treatment for 48 hours. Similarly BCPAP cells increased from 8.47% in untreated control sample to 33.54% after 200nM of Torin2 treatment and 41.16% after 400nM of Torin2 treatment for 48 hours. Further confirmation using FITC-conjugated Annexin V and PI assay showed that apoptotic cell increased from 8.89% in untreated cells to 58.54% and 73.54% after 200nM and 400nM treatment in TPC-1 cells, and from 13.24% in control to 46.74% and 70.64% in BCPAP cells, indicating a dose dependent apoptosis induced in both cell lines. Treatment of PTC cell lines with Torin2 inactivated downstream targets of mTOR; p70S6 and 4E-BP1. Torin2 treatment of PTC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspase-9, and 3, and cleavage of PARP. Furthermore Torin2 positively regulated autophagy by disrupting the Beclin-1 complex, releasing Beclin-1 to induce autophagy. Our results suggest mTOR and its associated signaling pathway play a critical role in PTC tumorigenesis, therefore targeting of mTOR expression may be a viable therapeutic intervention for treatment of these cancers.

Citation Format: Maqbool Ahmed, Azhar R. Hussain, Fouad Al Dayel, Shahab Uddin, Khawla S. Al-Kuraya. Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2013-4097
Abstract 4098: CXCR4 regulation of neuroblastoma migration and differentiation.
Authors: Shan Yufei , Van Golen Cynthia M.
Published: August 2014

CXCR4 is a chemokine involved in the migration of numerous cell types, including hematopoietic cells, neuronal cells during differentiation, and metastatic tumor cells. CXCR4 has been implicated in the invasion of metastasis of multiple tumor types that metastasize to bone, including prostate carcinoma and breast carcinoma. However, in neuroblastoma, a pediatric tumor that frequently metastasizes to bone and causes osteolytic lesions, the role of CXCR4 has not been elucidated. Several studies have suggested multiple potential roles of CXCR4 activation and signaling in neuroblastoma progression, including proliferation, migration, and invasion. Our previous studies have shown that blocking the CXCR4 receptor using a neutralizing antibody reduces migration toward the CXCR4 ligand CXCL12. However, when CXCL12 is added to neuroblastoma cells, activating the receptor, rather than an increase in migration, neurite extension and differentiation occurs. Through subsequent studies, we have demonstrated that upon stimulation, CXCR4 interacts with the actin cytoskeleton, potentially through the adaptor protein drebrin. Furthermore, this interaction appears to be dependent upon Akt. Our current hypothesis is that at low levels of CXCL12, migration is induced; upon increased activation of the receptor, however, CXCR4 is internalized, reducing the amount of receptor on the cell surface, and the internalized receptor then interacts with actin to increase process extension. This enables the neuroblastoma cells to migrate toward favorable conditions, but once the cells have reached those conditions (such as high CXCL12 levels in bone), the cells to remain in that environment. Experiments are currently being performed to test this working hypothesis.

Citation Format: Yufei Shan, Cynthia M. Van Golen. CXCR4 regulation of neuroblastoma migration and differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4098. doi:10.1158/1538-7445.AM2013-4098
Abstract 4103: Ptprz gene encoding a transmembrane-type protein phosphatase is up-regulated by Sox-2 in glioma.
Authors: Yamashita Yoji , Tanuma Nobuhiro , Kumabe Toshihiro , Tominaga Teiji , Shima Hiroshi , Katakura Ryuichi
Published: August 2014

Ptprz is a receptor-like protein-tyrosine phosphatase (PTP) abundantly expressed in neuronal and glial cells in the central nervous system. To date, several potential substrates of Ptprz such as p190RhoGAP, ErbB4, and Git-1, have been identified. In terms of its biological functions, for example, it was shown that Ptprz regulates differentiation of glial cells, but its role(s) in gliomagenesis remains obscue.

In this study, expression of Ptprz mRNA in human glioma samples were analyzed. Ptprz mRNA was up-regulated in glioma when compared to normal brain. We found that the levels of Ptprz mRNA in specimens are correlated with those of Sox-2, a transcription factor associating with glioma stemness. Knock-down of Sox-2 by siRNA decreased Ptprz mRNA of several glioma cell-lines. ChIP analysis showed the physical association of Sox2 to the putative promoter region of Ptprz gene, suggesting that Ptprz is a direct transcriptional target of Sox-2. Up-regulation of Ptprz expression may be a part of transcriptional program elicited by Sox-2, which may contribute to glioma stemness

Citation Format: Yoji Yamashita, Nobuhiro Tanuma, Toshihiro Kumabe, Teiji Tominaga, Hiroshi Shima, Ryuichi Katakura. Ptprz gene encoding a transmembrane-type protein phosphatase is up-regulated by Sox-2 in glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4103. doi:10.1158/1538-7445.AM2013-4103
Abstract 4105: Signaling network-based analyses of sonic hedgehog pathway components: predictions and possibilities.
Authors: Malhotra Anshu , Potts Chad R. , Fernandez-Lopez Africa , Rotenberry Rachel , Kenney Anna M.
Published: August 2014

A subclass of Medulloblastoma shows considerable dependence on the Sonic Hedgehog (Shh) pathway. Since cellular functions are a consequence of coordinated networks involving protein-protein interactions, it is imperative to explore different events cooperating with Shh entities in order to explore novel targets for medulloblastoma therapy. The Yes Associated Protein (YAP), a component of the Hippo pathway, has been previously established as a downstream target of the Shh pathway (Fernandez et al 2011). Several interesting gene targets featured in the networks developed by us using YAP overexpression in CGNPs. A concurrent upregulation of IGF2 and H19, both imprinted genes, was observed. Possible links between the Shh/Hippo pathways and IGF2 signaling also featured in the networks developed in the study. Important links to lipid metabolism, a process highly active in medulloblastomas and essential for the proliferation of CGNPs, were also predicted. The study includes results of signaling interactions between the Shh/Hippo pathways and entities from other relevant pathways that together play a role in medulloblastoma development and proliferation.

Citation Format: Anshu Malhotra, Chad R. Potts, Africa Fernandez-Lopez, Rachel Rotenberry, Anna M. Kenney. Signaling network-based analyses of sonic hedgehog pathway components: predictions and possibilities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2013-4105
Abstract 4111: SDF1/CXCR4 regulates stiffness of bone marrow stromal cell through RhoA and Rac1 GTPases pathway to support myeloma cell proliferation in multiple myeloma.
Authors: Wu Dan , Su Jing , Song Qianqian , Zhou Xiaobo
Published: August 2014

Abstract

As the second most prevalent hematologic malignancy, multiple myeloma (MM) remains high incurable and relapse owing to intrinsic or acquired drug resistance. Therefore, new therapeutic strategies that are active in drug resistance and target molecular processes of MM progress are attractive. MM is characterized by bone marrow infiltration of malignant plasma cells. The bone marrow microenvironment is a complex structure of various extracellular components and many cell types including haematopoietic stem cells, bone marrow stromal cells (BMSCs), immune cells, bone marrow endothelial cells, as well as osteoclasts and osteoblasts. Recently study showed the physical interaction of multiple myeloma cells with BMSCs from patients of multiple myeloma in the bone marrow milieu, provokes a positive feedback loop and results in multiple myeloma pathogenesis.

We observed the myeloma BMSCs are much stiffer and more contractile than normal BMSCs. Stiffer hydrogels support colony formation and adhension of MM better than softer hydrogels. These suggested that myeloma BMSCs provide myeloma cell-friendly microenvironments for myeloma cells via exerting biomechanical forces. In order to elucidate the molecular mechanisms leading to the change of myeloma BMSCs biochemical phenotype, we analyzed myeloma BMSCs and normal BMSCs gene expression profiles. Microarray data analysis demonstrated stromal cell derived factor-1(SDF1) was over expressed in MM

BMSCs. In vitro, 100ng/ml SDF1 significantly induced actin polymerization and polarization in MM BMSCs, but not in normal BMSCs. In order to understand how SDF1 regulate BMSCs’ contractile, we conducted reverse phase protein array (RPPA) experiment. Based on RPPA data, we determined RhoA and Rac1 GTPases played important roles in SDF1 induced stiffer of BMSCs. Western blot results also approved SDF1 increased phosphate RhoA and phosphate Rac1 expression level.

In conclusion, we characterized the pivotal role of RhoA and Rac1 GTPases in regulating BMSCs stiffness and BMSCs-MM interaction. Targeting biomechanics of BMSCs in myeloma patients, restoring to their normal state would be essential for the long term cure of myeloma, provoking RhoA and Rac1 pathway as novel MM therapy.

Citation Format: Dan Wu, Jing Su, Qianqian Song, Xiaobo Zhou. SDF1/CXCR4 regulates stiffness of bone marrow stromal cell through RhoA and Rac1 GTPases pathway to support myeloma cell proliferation in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4111. doi:10.1158/1538-7445.AM2013-4111
Abstract 4113: Discovery and optimization of potent SDF1α antagonist peptides.
Authors: Coronella Julia A. , Fu Yanwen , Johnson Kimberly , Zhong Jingping , Campbell Lioudmila , Chen Gang , Willhite Dorian , Rosario Joselyn Del , Bhat Abhijit , Woodnutt Gary , Levin Nancy
Published: August 2014

The SDF1α / CXCR4 axis contributes to cancer metastasis, proliferation, angiogenesis and resistance to standard of care drugs. Here we describe novel phage display derived peptide SDF1α antagonists. Phage displayed peptides were optimized through a multistep process that resulted in a 400-fold improvement in activity, from 6.4 uM IC50 inhibition of SDF1α -driven Calcium flux to 16 nM IC50. First, peptides were affinity matured via panning of focused phage libraries. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold IgG (CVX-2000), generating bivalent CovX-Bodies with 5-fold improved potency in SDF1α driven Calcium flux assays. N and C-terminal extension of the peptide and introduction of unnatural amino acids produced an additional 11-fold improvement in activity to 16 nM. Constructs with optimal pharmacokinetic stability were identified, and demonstrated single agent tumor inhibition in a Ramos B cell lymphoma xenograft model.

Citation Format: Julia A. Coronella, Yanwen Fu, Kimberly Johnson, Jingping Zhong, Lioudmila Campbell, Gang Chen, Dorian Willhite, Joselyn Del Rosario, Abhijit Bhat, Gary Woodnutt, Nancy Levin. Discovery and optimization of potent SDF1α antagonist peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2013-4113