bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C200: Second-generation PIM inhibitors exhibit improved activity in solid tumor models in vitro.
Authors: Foulks Jason M. , Xu Yong , Saunders Michael , Liu Xiao-hui , Brenning Benjamin , Clifford Adrianne , Wilkes Marcus , Luo Bai , Lai Shuping , Merx Shannon , Chan Ashley , Huang Liwen , Vollmer David , Senina Anna , Liu Jihua , Ho Kan , McCullar Michael V. , Kanner Steven B.
Published: June 2012

The proto-oncogene PIM kinase family (PIM-1, -2 and -3) comprises constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, bladder, gastric and head & neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, promoting tumor cell survival and proliferation. PIM-1 and PIM-2 overexpression models were obtained in the human prostate cancer cell lines PC-3M and 22RV-1 and the non-tumorigenic mouse NIH-3T3 background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In the PC-3M cell line, enhanced phosphorylation of the PIM kinase substrate BAD (pBAD) was observed following PIM overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, as well as next generation PIM inhibitors exhibiting 4–10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-μM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the PC-3M prostate cancer cell line, the UM-UC-3 bladder cancer cell line, and the HSC3 head & neck cancer cell line. The second generation PIM inhibitors possess favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using both PIM overexpression models and clinically relevant solid tumor models will facilitate identification of a clinical candidate.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C200.
Abstract 1008: Prevalence of FoxM1 expression in Middle Eastern epithelial ovarian carcinoma patients and its potential role as a therapeutic target
Authors: Ahmed Maqbool , Jehan Zeenath , Bavi Prashant , Hussain Azhar R. , Olayan Aisha Saad , Saravanan Thangavel , Dayel Fouad Al , Ghourab Samir , Uddin Shahab , Al Kuraya Khawla S.
Published: June 2012

FoxM1 is a member of Forkhead transcription factors and its expression has been found to be altered in a variety of cancers. Several studies have indicated the importance of FoxM1 in cell proliferation, as it is a key cell-cycle regulator of both the transition from G1 to S phase and the progression to mitosis. In this study, we investigated the functional consequences of FoxM1 signaling pathway in a large cohort of epithelial ovarian cancer (EOC) tumor (patient) samples, panel of cell lines, and in nude mouse model. Using immunohistochemistry, FoxM1 was detected in 47.4% (64/135) of the ovarian tumor samples. EOC with FOX-M1 expression showed a significantly better disease free survival(p=0.0148) of 21.9 months as compared to the EOC with low FOX-M1 expression(14.4 months). Overexpression of FoxM1 was also significantly associated with activated p-AKT(p=0.0072), SKP2(p=0.0121), p-Rb(p=0.0357), UBE2C(p=0.0106), FAS-N(p=0.0179), PIK3CA p110αIHC(p=0.0362), XIAP(p=0.0033) and tumor proliferative marker Ki167 (p<0.0001). Treatment of a panel of EOC cell lines with thiostrepton, an inhibitor of FoxM1, inhibited cell proliferation and induced apoptosis. Thiostrepton treatment caused apoptosis by involving the mitochondrial pathway, through activation of caspases and down regulation of XIAP and survivin. FoxM1 inhibition in EOC cells by thiostrepton resulted in the down regulation of Skp2 and accumulation of p21, possibly causing G2/M arrest. Furthermore, FoxM1 inhibition also down regulated cell migration and cell invasion in EOC cells by inducing decreased secretion of VEGF and MMP2. In addition, pharmacological inhibition of FoxM1 or its gene silencing with siRNA causes downregulation of FoxM1, AKT and its associated downstream signaling molecules. Altogether, these results suggest that FoxM1 and its role in the deregulated survival pathway make this a potential target for therapeutic intervention of EOC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1008. doi:10.1158/1538-7445.AM2011-1008
Abstract 1020: Investigating LKB1 regulation in osteosarcoma
Authors: Eskandarpour Malihe , Duhamel Laure , Tirabosco Roberto , Flanagan Adrienne
Published: June 2012

Osteosarcoma (OS) accounts for approximately 60% of primary malignant bone tumours diagnosed in the first two decades of life. The incidence of osteosarcoma is 200/year in the UK, but is higher in children and young adults. On introduction of multimodal chemotherapy for the treatment of osteosarcoma, patients’ average 5 year survival rate increased from 20% to 70% but there has been little improvement since. Also, despite this improvement in survival it is not possible to predict which patients are likely to respond to therapy. Knowledge of the molecular biological characteristics of osteosarcoma may resolve this problem and help in selecting patients for targeted therapy.

LKB1 is a serine/threonine kinase and a tumour suppressor gene that is frequently lost in some cancer types. However, its role in the pathogenesis of human OS pathogenesis is unknown although it has been reported that mice deficient in one LKB1 allele (LKB1+/-) develop osteoblastic tumours. We therefore investigated the role of this gene in the pathogenesis of osteosarcoma.

We showed that 85% (17/20) of osteosarcomas have a low or undetectable protein expression level as assessed by western blotting (WB), and 59% (153/259) by immunohistochemistry. However, no cases showed LKB1 copy number loss when analyzed by fluoresence in situ hybridisation, and none of the previously reported somatic mutations in this gene was detected in 21 tumours. Since no significant differences were detected in the mRNA level by q-RT-PCR in these cases it is postulated that the low level of LKB1 protein is regulated by a post-transcriptional mechanism.

Recently has been shown that SIRT1 protein antagonizes LKB1-dependent AMPK activation through promoting the acetylation, ubiquitination and proteasome-mediated degradation of LKB1. SIRT1 is a class III deacetylase and is implicated in a wide range of cellular functions. We hypothesised that SIRT1 regulates LKB1 degradation. We first demonstrated an inverse correlation between LKB1 and SIRT1 in OS cell lines by WB, and in OS tumours by double immunofluoresence. Using the proteosome inhibitor (PS-341), we showed protection of LKB1 from degradation. We also showed prevention of LKB1 degradation by applying the SIRT1 inhibitor (sirtinol) or SIRT1 suppressor (morpholinos) in OS cell lines, SaOS2, U2OS and MNNG/ HOS.

In conclusion, absence of LKB1 protein expression is a common event in human osteosarcomas; the mechanism appears to be a post-transcriptional event and our findings implicate a role for SIRT1 in LKB1 regulation through degradation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1020. doi:10.1158/1538-7445.AM2011-1020
Abstract 1029: Identification of novel MEK1 inhibitors by computer docking and virtual screening
Authors: Kim Dong Joon , Reddy Srinivasa , Li Yan , Bode Ann M. , Dong Zigang
Published: June 2012

The mitogen-activated protein kinase kinase 1 (MEK1) signaling pathway is a major component of the RAS/RAF/MEK/ERK signaling axis that regulates tumorigenesis and cancer cell proliferation. MEK1 is frequently activated in various cancers, especially in tumors that have mutations in the KRAS and BRAF oncogenes. Therefore MEK1 is an important cancer therapy target and the discovery of novel MEK1 inhibitors is of great interest. We used the MEK1 protein crystal structure and supercomputer screening of chemical libraries combined with simulation modeling and in vitro, ex vivo and in vivo assays to identify novel inhibitors of MEK1. Herein, we report the discovery of threee novel MEK1 inhibitors, herein referred to as Compound #7, #9 and #12. All three were highly effective to suppress MEK1 activity in HCT116 and HCT15 colorectal cancer cells. Based on in vitro and ex vivo data, these compounds inhibited MEK1 activity and phosphorylation of ERK1/2 in a dose-dependent manner and were twice as effective than the known MEK1 inhibitor, U0126. The compounds completely inhibited anchorage-independent cell growth and proliferation of colon cancer cells. Overall these results suggest that these novel MEK1 inhibitors might be used for chemotherapy; and computer modeling and simulation could be very useful for finding additional MEK1 or other inhibitors for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1029. doi:10.1158/1538-7445.AM2011-1029
Abstract 1045: Src-mediated Tyr20 phosphorylation of transferrin receptor 1 (TfR1) promotes its anti-apoptotic effect: A novel function of TfR1 in cancer cells
Authors: Huang Xi , Jian Jinlong , Yang Qing
Published: June 2012

Transferrin receptor 1 (TfR1) is highly expressed in cancer cells and its overexpression is widely thought to be a result of the increased requirement of iron uptake for cell growth. In the present study, we have found that two ligands of TfR1, gambogic acid (GA) but not holo-transferrin (Tf) induced apoptosis in breast cancer MDA-MB-231 cells and activated the JNK and p38 signal pathways. Furthermore, PP2, an inhibitor of steroid receptor coactivator (Src), a tyrosine kinase, greatly promoted GA-mediated apoptosis. These results indicate that Src is involved in GA-mediated TfR1-dependent apoptotic pathway. Following immunoprecipitation of TfR1 and immunoblotting of Src or verse versa, TfR1 was found to be associated with Src. This association was further illustrated by their co-localization with confocal fluorescence microscope and shown resulting in constitutive tyrosine phosphorylations of the TfR1. A type II membrane receptor, TfR1 contains a short intracellular N-terminal domain with 61 amino acids. 20YTRF23, the only Tyr(Y) site within the cytoplasmic domain, has been shown as the motif essential for TfR1-Tf complex internalization. To elucidate which Tyr is phosphorylated, we used a Chinese hamster ovary (CHO)-TRVb cell line that does not express TfR1. Point mutation Y20L showed that Tyr20 of TfR1 was the phosphorylation site by Src. Further treatments of wild type and point mutant revealed that it is the Tyr20 phosphorylation that enhanced the anti-apoptotic effects of TfR1 against GA. Our results indicate that in addition to the known function of iron transport, TfR1 has a novel function of regulating cell survival and antiapoptosis through its Tyr20 phosphorylation by Src. This new function is particularly significant and relevant to cancer cells since high expression of TfR1 not only fulfills the requirement of more iron as a co-factor in DNA synthesis but also enhances their survival. This work is supported by R21 CA132684.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2011-1045
Abstract 1046: Differential interactions between c-Src and c-Met mediate resistance to c-Src inhibitors
Authors: Sen Banibrata , Peng Shaohua , Saigal Babita , Williams Michelle , Johnson Faye M.
Published: June 2012

Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can also significantly alter facial appearance. One promising molecular target for which new agents have been developed is the Src family kinases (SFKs). SFK inhibition in cancer cells leads to an universal abrogation of invasion but a variable and weak effects on apoptosis and proliferation. The pathways downstream of c-Src promoting survival are not well-characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal for head and neck cancers, we sought to characterize the mechanisms of resistance to SFK inhibition in Head and Neck squamous cell carcinoma (HNSCC). SFKs were inhibited in a panel of oral cancer cell lines and the effects on subsequent survival and signaling were measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and their intrinsic kinase activity by in vitro kinase assay. Cytotoxicity was measured using an MTT assay and the Chou-Talalay combination index calculated. The in vivo effects of c-Met and SFK inhibitors were assessed using an orthotopic model of oral cancer. SFK inhibition resulted in c-Met inhibition in cell lines that were sensitive to SFK inhibitors, but not in resistant cell lines. Isolated c-Met act as a c-Src substrate in both sensitive and resistant cells, whereas distinct difference exists in c-Src and c-Met interaction in intact sensitive and resistant cells. The epidermal growth factor receptor contributed to c-Met activation in resistant cells whereas in sensitive cells c-Met acts as a direct downstream target for c-Src. We demonstrated the biological consequences of this mechanism in vitro with synergistic cytotoxicity and enhanced apoptosis when SFK and c-Met inhibitors were combined. Likewise, the combination resulted in decreased tumor size in vivo. In conclusion, we demonstrate that sustained c-Met activation can mediate resistance to SFK inhibition. The differences between c-Src and c-Met signaling in sensitive and resistant cells are not due to intrinsic structural changes in c-Src or c-Met, but rather to distinct interactions within the intact cells. The synergistic cytotoxic effects of SFK and c-Met inhibition may be important for the treatment of head and neck cancers and should be tested in future clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2011-1046
Abstract 105: An epigenetic vicious cycle of DNMT, protein Tyrosine phosphatase receptor type R, and the MAPK pathway in cancer metastasis
Authors: Su Po-Hsuan , Lin Ya-Wen , Liao Yu-Ping , Wang Yu-Chi , Lee Hsin-Yi , Wang Hui-Chen , Chao Tai-Kuang , Chen Chi-Kuan , Chan Michael W.Y. , Chu Tang-Yuan , Yu Mu-Hsien , Lai Hung-Cheng
Published: June 2012

Introduction: Epigenetic modifications are a driving force in carcinogenesis. In addition to its role in initiation and promotion, DNA methylation is thought to participate in cancer metastasis. The present study is to interrogate the role of de novo DNA methylation in cervical cancer invasions.

Methods and Results: Here, we report evidence of the overexpression of DNA methyltransferases 3B (DNMT3B) in invasive cervical cancer and that DNMT3B interference inhibits metastasis. Using methyl-DNA immunoprecipitation coupled with microarray analysis (mDIP-on-chip), we found that Protein Tyrosine Phosphatase Receptor Type R (PTPRR) is targeted by DNMT3B. PTPRR inhibited metastasis by inhibiting p44/42 MAPK signaling, epithelial-mesenchymal transition (EMT), and matrix metalloproteinase 9 (MMP9) expression. PTPRR also inhibited the expression of transcription factor AP1, HPV oncogenes, and DNMTs, and reciprocally demethylated the PTPRR promoter. The methylation status of PTPRR was increased in cervical scrapings in accordance with disease severities, and immunohistochemical staining confirmed that PTPRR protein was expressed in precancerous lesions but was silenced in invasive cancers. The clinical correlation results suggest that methylation of the PTPRR promoter is a potential biomarker for cervical cancer detection.

Conclusion: This study demonstrates for the first time that DNMT3B plays an important role in the cervical cancer invasion and metastasis. The DNMT3B-mediated silencing of PTPRR activates p44/42 MAPK signaling and its multiple downstream effectors such as EMT, enabling the in situ tumors becoming invasive. MAPK signaling also augments the expression of DNMTs, which further consolidates the methylation silencing of PTPRR. A vicious cycle between epigenetic machinery and oncogenic signaling promotes cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2011-105
Abstract 1058: LKB1 negatively regulates IGF-1 induced ERK phosphorylation
Authors: Zhong Diansheng , Liu Xiuju , Fu Haian , Sun Shi-Yong , Khuri Fadlo R. , Zhou Wei
Published: June 2012

The LKB1/AMPK metabolic checkpoint can be activated by various energetic stresses, and a common reagent to induced LKB1/AMPK signaling is 2-deoxyglucose (2-DG), a glycolytic inhibitor. We recently discovered a novel function of 2-DG because it is capable of disrupting the interaction between IGF-1 and IGFBP3 to release free IGF-1 and activate IGF1R signaling. Therefore, 2-DG activates LKB1/AMPK and IGF1R signaling pathways through two distinct mechanisms. Interestingly, while 2-DG induced IGF1R signaling activates PI3K/Akt function in most cell lines; the induction of MEK/ERK signaling was only observed in some cancer cells. Here, we demonstrated that 2-DG induced ERK phosphorylation in LKB1 mutant cells but suppressed ERK phosphorylation in cells with wild-type LKB1. The phenomenon is LKB1-dependent because restoration of LKB1 expression in LKB1-null cells attenuated IGF-1 induced ERK phosphorylation, and the depletion of LKB1 by RNAi in LKB1-wild type cells promoted ERK phosphorylation. Mechanistically, LKB1 mediates ERK suppression through its down-stream target, AMPK, as the inhibition of AMPK function either by RNAi or small molecular inhibitors promotes IGF-1 induced ERK phosphorylation. In summary, this study has demonstrated a novel function of LKB1/AMPK in the negative regulation of IGF-1 induced ERK signaling. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH/NCI PO1 CA116676-030002. SY Sun, FR Khuri and W Zhou are Georgia Cancer Coalition Distinguished Cancer Scholars. W Zhou is an American Cancer Society Research Scholar.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2011-1058
Abstract 1072: Activation of the Ras signaling pathway promotes the over-expression of anti-apoptotic heme oxygenase-1 in human renal cancer
Authors: Banerjee Pallavi , Basu Aninda , Pal Soumitro
Published: June 2012

Activation of the proto-oncogene ras may play a critical role in the development and rapid progression of different types of human cancers, including renal cancer. It has been shown that the Ras pathway can mediate pro-survival signals in cancer cells by inhibiting cellular apoptosis. It is established that the cytoprotective enzyme heme oxygenase-1 (HO-1) is over-expressed in several cancers, and may play a significant role in the growth and metastasis of tumors by inducing cell survival pathways. However, it is not known if there is an association between Ras activation and HO-1 over-expression in cancer. We have recently demonstrated that the treatment of human renal cancer cells with the immunosuppressive agent cyclosporine may activate the H-Ras pathway, and promote a rapid progression of the disease. We have also shown that HO-1 is over-expressed in human renal cancer, and plays a key role in the survival of renal cancer cells by inhibiting cellular apoptosis. Here, we wished to analyze if the activation of H-Ras pathway can regulate the expression of HO-1 in two different human renal cancer cell lines 786-0 and Caki-1. In co-transfection assays, using a full-length HO-1 promoter-luciferase construct, we found that the activated form of H-Ras (H-Ras12V) promoted HO-1 transcriptional activation as observed by increased luciferase activity. We also found that the activation of H-Ras increased the mRNA expression of HO-1 as observed by real-time PCR. By using a dominant-inhibitory mutant construct H-Ras17N, we confirmed that the inhibition of H-Ras expression significantly decreased HO-1 promoter activity. We next wanted to determine which effector molecule(s) (Raf, Rho and PI-3K) is functional for H-Ras-mediated HO-1 over-expression. We made use of three different effector domain mutant constructs of H-Ras; Ras(12V,35S) retains full-length Raf-1 binding activity, Ras(12V,37G) retains Rho binding activity, and Ras(12V,40C) retains PI-3K binding activity. In co-transfection assays using these effector domain mutant constructs, we observed that the Ras-induced HO-1 transcriptional activation is mediated primarily through the Raf-1 signaling pathway. Together, our findings suggest that the Ras-Raf signaling pathway might play an important role in the survival of human renal cancer cells through the induction of HO-1 over-expression. Thus, targeting this H-Ras pathway for HO-1 over-expression might act as novel therapeutics to inhibit human renal tumors through the induction of cellular apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2011-1072
Abstract 1073: A systematic approach to identify the Ras-dependent C-RAF(Ser338)-phosphorylating kinase
Authors: Johannessen Cory M. , Boehm Jesse , Hamm Luke , Yang Xiaoping , Garraway Levi
Published: June 2012

Phosphorylation of c-RAF at Ser338 is an absolute requirement for c-RAF activation and Ras-mediated signaling; however, definitive identification of the c-RAF(Ser338)-phosphorylating kinase has remained elusive. As a first step towards this end, we screened a collection of ∼600 expressible kinase open reading frames to determine which are sufficient to induce phosphorylation of c-RAF on Ser338. In total, 12 kinases induced c-RAF(Ser338) phosphorylation, including 9 Ser/Thr kinases, 1 kinase with unknown site specificity, 1 adapter factor and 1 scaffolding protein. Notably, two proteins previously shown to regulate c-RAF(Ser338) phosphorylation (PAK3 and KSR) also scored in this assay, highlighting the utility of this approach to uncover plausible effectors. We have initiated follow-up studies, including recombinant protein-based kinase assays and loss of function experiments, to characterize the kinase(s) responsible for Ras-mediated c-RAF(Ser338) phosphorylation in vivo. Given the inherent difficulty of targeting Ras-dependent cancers, a more detailed understanding of c-RAF phosphorylation and Ras-dependent activation may inform new therapeutic inroads to targeting these lesions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1073. doi:10.1158/1538-7445.AM2011-1073
Abstract 1074: Dishevelled 3 (Dvl3) is upregulated and enhances cell migration and invasion in hepatocellular carcinoma
Authors: Tsui Yu Man , Tung Edmund Kwok-Kwan , Mak Carmen Ka-Man , Ng Irene Oi-Lin
Published: June 2012

Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies in the world and is particularly prevalent in China and Southeast Asia. Although the risk factors are well known, the molecular mechanisms underlying hepatocarcinogenesis is unclear. Alteration of Wnt signaling pathway is one of the key events in HCC development and so we performed a low-density array (LDA) analysis on 38 pairs of HCC samples and their corresponding non-tumorous livers to examine the expression profiles of different Wnt signaling components. We found that, among the genes studied, Dishevelled 3 (Dvl3) was significantly overexpressed in HCCs as compared with the corresponding non-tumorous liver tissues. In addition, Dvl3 overexpression in HCC was significantly associated with presence of venous invasion (P = .021). The overexpression result was confirmed by expansion of the sample size using quantitative real-time PCR (q-PCR). Next, we investigated the effects of Dvl3 on the migratory and invasive abilities of HCC cells. We established stable shRNA Dvl3-knockdown clones in PLC/PRF/5 HCC cells and found that knockdown of Dvl3 suppressed the migratory and invasive abilities of HCC cells using transwell migration and invasion assays, respectively, suggesting an enhancing role of Dvl3 in HCC cell movement and cell invasion. Although cell proliferation assay in vitro showed no significant difference between the stable Dvl3 knockdown and non-target control PLC/PRF/5 cells, in vivo study in SCID mice showed that the stable Dvl3-knockdown cells resulted in a lower incidence of tumors formed and a smaller tumor size. Furthermore, since Dvl3 is an upstream regulator of the Wnt/β-catenin pathway, using the TOPFOP luciferase reporter assay, we found that transient transfection of Dvl3 in PLC/PRF/5 and BEL-7402 HCC cell lines enhanced the β-catenin-dependent transcriptional activity. In conclusion, upregulation of Dvl3 was frequent in human HCCs. Dvl3 enhanced cell migration and invasion in vitro and tumorigenicity in vivo in SCID mice, and activated canonical β-catenin pathway. It may play an oncogenic role in HCC development.

(This study was funded by Hong Kong Research Grants Council CRF grants (RGC CRF HKU1/06C and HKU 7/CRG/09).)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2011-1074
Abstract 1078: PPM1A acts as a novel RelA phosphatase to negatively regulate NF-κB signaling
Authors: Lu Xinyuan , An Hanbing , Yarbrough Wendell G.
Published: June 2012

Phosphorylation of RelA is required to fully activate NF-κB transcription activity and dephosphorylation of RelA can inhibit its activity. To date, Wip1 (PPM1D) and PP2A have been described as RelA phosphatases. The purpose of this study was to identify novel RelA phosphatase(s) and determine if they play a role in regulation of NF-κB activity related to tumorigenesis. PPM1A belongs to PP2C protein phosphatase family, the same family of Wip1. To begin exploring PPM1A activity toward RelA, serine 536 (S536) phosphorylation of RelA was determined with and without expression or knockdown of PPM1A. Expression of PPM1A in U2OS cells decreased RelA phosphorylation, whereas siRNA-mediated loss of PPM1A increased S536RelA phosphorylation. Luciferase assay using a κB responsive reporter with expression of either PPM1A or phosphatase-dead PPM1A (PPM1A-PD) revealed that wild-type, but not PPM1A-PD, inhibited RelA transcription. Furthermore, in vitro phosphatase assays using bacterially expressed PPM1A with substrate of either full length RelA or phosphorylated RelA peptides, suggest that PPM1A can function as a direct RelA phosphatase at residuesS536 and S276. We could not identify reports of additional phosphatases with activity toward the S276 residue of RelA. Using liquid chromatography and tandem mass spectroscopy (LC-MS/MS) LZAP-associated proteins (LAPs) were sought with PPM1A being identified. LZAP (C53, CDK5RAP3) has tumor suppressor-like activity that is mediated at least partially through inhibition of RelA S536 phosphorylation and activity. PPM1A's ability to decrease RelA phosphorylation at S536 site was largely inhibited in cells with siRNA-mediated loss of LZAP. To determine if PPM1A has tumor suppressor activity, HeLa cells were infected with retrovirus directing shRNA-mediated knockdown of PPM1A and tumorigenic properties of these cells were determined. Preliminary xenograft tumor data suggest PPM1A has a negative effect on tumor initiation and proliferation and studies are underway to determine if PPM1A activity toward NF-κB plays a role. In summary, our data suggest that PPM1A is a novel RelA phosphatase with activity to dephosphorylate RelA at serines 536 and 276. PPM1A inhibits NF-κB transcriptional activity and that PPM1A activity toward RelA is at least partially dependent on LZAP. Our preliminary data also suggest PPM1A might have tumor suppressor-like activity that could be dependent on LZAP or RelA.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2011-1078
Abstract 1079: Function and modulation of Pdcd4 in ovarian cancer
Authors: Liu Stephanie S. , Wei Tina N. , Chan Karen KL , Ngan Hextan YS
Published: June 2012

Programmed cell death 4 (Pdcd4), a novel gene and originally identified as the neoplastic transformation inhibitor, is attenuated in various cancer types. It has been shown to have tumor suppressor properties such as inhibiting cell proliferation and inducing apoptosis. Our previous study demonstrated a continuous down-regulation of Pdcd4 expression in the sequence of normal-borderline-malignant ovarian tissue samples, and a significant correlation of Pdcd4 expression with disease-free survival. The objective of the current study is to further investigate the function and modulation of Pdcd4 in ovarian cancer cells.

Our in vitro studies demonstrated that ectopic Pdcd4 expression significantly inhibited ovarian cancer cell proliferation by up-regulation of cyclin dependent kinase inhibitors of p27 and p21 and subsequently leading to cell cycle arrest at G1 stage. Unlike other cancers, Pdcd4 neither induce ovarian cancer cell apoptosis, nor promote the cellular sensitivity in response to cisplatin treatment. Pdcd4 over-expressing ovarian cancer cells exhibited elevated phosphatase and tensin homolog (PTEN) and decreased p-Akt expressions. In addition, Pdcd4 expression was up-regulated and translocated from cell nucleus to cytoplasm upon serum withdrawal treatment, but was rapidly depleted via proteasomal degradation upon serum re-administration. Treatment of a phosphoinositide 3-kinase (PI3K) inhibitor prevented the degradation of Pdcd4.

In conclusion, loss of Pdcd4 may result in diminished cell ability to arrest at key checkpoint, leading to tumor development. The PI3K-Akt pathway may be involved in the regulation of Pdcd4 degradation in ovarian cancer cells. In response to the stress condition, Pdcd4 expression was altered and it was translocated to cytoplasm, suggesting that Pdcd4 was able to shuttle between cell compartments to perform its diverted functions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2011-1079
Abstract 1083: Caveolin-1 in cutaneous squamous cell carcinoma development
Authors: Trimmer Casey , Katiyar Sanjay , Pestell Richard G. , Lisanti Michael P. , Capozza Franco
Published: June 2012

Caveolin-1 (Cav1) is the main protein component of a specialized form of lipid raft termed caveolae and serves to compartmentalize and negatively regulate the activity of signaling molecules involved in cell proliferation and survival. Cav1 has been shown to act as a tumor suppressor in several types of cancer, including breast cancer and melanoma, but its role in the pathogenesis of non-melanoma skin cancer remains largely unexplored. Previous work has indicated that Cav1 is expressed in the basal cell layer of both murine and human epidermis; additionally, loss of Cav1 protein has been shown by our lab to render murine skin more susceptible to papilloma development following treatment with a carcinogenic compound. The purpose of the current study was to further examine the role of Cav1 in non-melanoma skin cancer development using various experimental approaches.

Given the previous research, our hypothesis is that Cav1 acts as a suppressor of tumor growth and development in the skin. In order to explore this hypothesis, we have used several experimental systems, including human tissue arrays and loss of function and gain of function studies in animal models of cutaneous squamous cell carcinoma. Manipulation of Cav1 expression in (1) a keratinocyte cell line followed by xenotransplantation and (2) an induced model of skin cancer allowed us to assess the effect of this protein on tumor development, growth, and progression. Using these approaches, we were able to show that Cav1 ablation dramatically increases not only in vitro cell growth and invasion, but also in vivo tumorigenesis, tumor growth, and invasion. In conclusion, these studies provide further evidence in support of the hypothesis that Cav1 acts as a tumor suppressor protein in the skin and serve to identify new potential therapeutic targets in the treatment of cutaneous malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2011-1083
Abstract 1090: Development of predictive biomarkers for anti-TGF-beta therapy
Authors: Yang Yu-an , Weng Jia , Welsh Michael , Ford Heide L. , Lonning Scott M. , Wakefield Lalage
Published: June 2012

TGF-betas are pleiotropic growth factors with complex roles in tumorigenesis. The TGF-betas have many activities that could promote metastasis, including the enhancement of invasion and migration, the promotion of angiogenesis, and the suppression of immune surveillance. Overexpression of TGF-beta in many advanced human tumors correlates with metastasis and poor prognosis, and there is now considerable interest in developing TGF-beta antagonists for cancer therapy. Indeed, a Phase I oncology trial using a fully humanized anti-TGF-beta monoclonal antibody was recently successfully completed. There is now an urgent need for good predictive biomarkers for patient stratification in future clinical trials. Using different syngeneic mouse transplant models of metastatic breast cancer, we have found that anti-TGF-beta antibodies suppress metastasis in several models, including the widely-used 4T1 model. However, TGF-beta antibody treatment has no effect or even stimulates metastasis in other models. This panel of mouse models with differing responses to anti-TGF-beta therapy represents a powerful experimental platform for the identification of predictive biomarkers. Based on existing knowledge, we have assessed a number of plausible candidates. Global gene expression analyses have defined several distinct molecular subtypes of breast cancer with different clinical characteristics. The “Claudin-low” subtype is a particularly poor prognosis subset of the triple negative ER-PR-HER2- breast cancers. By definition these tumors have low claudin and E-cadherin expression and a mesenchymal phenotype. Since TGF-betas are strong inducers of the mesenchymal phenotype, the claudin-low subtype may represent a particularly good target for anti-TGF-beta therapy. Similarly, overexpression of the homeobox protein Six-1 has recently been shown to cause TGF-beta to switch from tumor suppressor to pro-metastatic factor in breast cancer and might also be a predictor of a positive therapeutic response to TGF-beta antagonism. Here we have assessed the claudin-low status of a number of mouse mammary tumor models, by RTQ-PCR for key markers, using cells cultured in vitro. In addition, we determined nuclear Six1 levels by Western blot. Neither claudin-low status nor Six1 expression were predictive of response to anti-TGF-beta therapy. We are assessing additional candidate biomarkers and moving forward with discovery approaches.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1090. doi:10.1158/1538-7445.AM2011-1090
Abstract 1098: Interactions between P53 and IGF2 in human esophageal adenocarcinoma tissues and cell lines
Authors: Zhao Ronghua , Casson Alan G.
Published: June 2012

To define the role of the insulin-like growth factor (IGF) axis in esophageal adenocarcinoma (EADC), the objective of this study was to evaluate interactions between IGF2 and P53 in a well characterized series of surgically-resected esophageal tissues and the following cell lines: Het1A (derived from immortalized normal esophageal epithelium), OE33 and JH-EsoAd1 (each derived from a primary EADC). Nucleic acids were extracted from 68 primary EADC (and matched histologically normal) tissues. PCR was performed on gDNA and RT-PCR on RNA followed by ApaI digestion to identify informative (heterozygous) cases and imprinting status, respectively. Expression of IGF2 mRNA was determined by quantitative PCR, and of IGF2 and P53 protein by Western analysis and immunohistochemistry. Molecular findings in tissues were correlated with pathologic and clinical characteristics including patient survival. Functional studies utilizing cell lines evaluated IGF2/P53 interactions. Of 44 informative cases, P53 protein overexpression was significantly higher in normally imprinted tumors (12/30) vs. tumors with loss of IGF2 imprinting (1/14; p<0.05). Multivariable logistic regression confirmed IGF2 imprinting status to be independently associated with P53 expression (OR 0.12, 95% CI 0.01-1.00; p=0.05). Tumors overexpressing both IGF2 and P53 were found to be of advanced stage with poor survival. In patients below age 65 years, IGF2 mRNA expression was significantly higher in tumors with wild type P53, and multivariate analysis showed IGF2 mRNA expression to be independently associated with p53 mutation (OR 0.74, 95% CI 0.58-0.94; p<0.05) and P53 protein expression (OR 0.83, 95% CI 0.69-1.00; p<0.05). Whereas treatment of Het1A cells with IGF2 significantly increased P53 expression (1.34+/-0.06 vs. 1.00+/-0.00 untreated cells; p=0.003), IGF2 did not modulate P53 expression in either of the two tumor cell lines (OE33 and JH-EsoAd1). IGF1 Receptor (IGF1R) inhibition with AG1024 reduced P53 expression only in OE33 (0.48+/-0.22 vs. 1.00+/-0.00 untreated cells; p=0.05) and JH-EsoAd1 (0.71 +/- 0.23 vs. 1.00+/- 0.00 untreated; p = 0.15) cells, an effect not reversed by IGF2 treatment. In conclusion: 1) these studies identify novel molecular regulatory interactions between P53 and IGF2 in esophageal malignancy, which are dependent on IGF2 imprinting status and tissue/cell type. 2) Overexpression of both IGF2 and P53 in esophageal tumors is associated with aggressive disease in younger patients (below age 65 years), and may represent clinically useful biomarkers to define a biologically distinct subset of EADC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2011-1098
Abstract 1109: Characterization of hepatoma-derived growth factor (HDGF) as a novel survival related protein in prostate cancer cells
Authors: Munirathinam Gnanasekar , Shetty Aditya , Samykutty Abhilash , Dakshinamoorthy Gajalakshmi , Zheng Guoxing , Chen Aoshuang , Bosland Maarten C. , Kajdacsy-Balla André , Kalyanasundaram Ramaswamy
Published: June 2012

Prostate cancer (PCa) is a leading cause of death by cancer in men, resulting in approximately 32,000 deaths each year. While the cause of PCa is not clearly known at this time, some risk factors include age, diet, race, and activation of certain oncogenes. Hepatoma-derived growth factor (HDGF) is a recently discovered oncogene and a key mitogenic growth factor which is activated in lung, liver, and skin cancers, however, its role in the development of PCa is unknown. Therefore, the goal of this study is to characterize the role of HDGF in prostate carcinogenesis. A normal prostate cell line, RWPE-1, and four different PCa cell lines, LNCaP, 22RV1, DU145, and PC-3, were analyzed for their expression level of HDGF by western blot analysis. Results of this evaluation revealed that HDGF is over-expressed in multiple PCa cell lines while minimally expressed in RWPE-1 cells. When the HDGF gene is ectopically over-expressed in normal RWPE-1 cells, proliferative rate was increased by 1.5 fold compared to the normal cells, suggesting that HDGF has growth stimulating activity. On the contrary, down regulation of HDGF expression by siRNA significantly inhibited the survival of both androgen dependent (LNCaP) and androgen independent (PC-3) PCa cells. DNA content analysis of RWPE-1 cells stably expressing HDGF by flow cytometry showed that the majority of the cells were in the G2/M phase (55%) of the cell cycle while only 28% of the control RWPE-1 cells were at G2/M phase implying HDGF may have a mitotic function in prostate cells. Furthermore, AKT (phosphorylated form) and NFκB (p65 domain) signaling molecules were found to be activated in RWPE-1 cells expressing HDGF. This activation resulted in the increase of anti-apoptotic molecules Cyclin-E and BCL-2 and the simultaneous decrease in the pro-apoptotic molecule Bax. Activation of AKT-NFκB pathway plays a vital role in the carcinogenesis of several cancers, including PCa. Thus, HDGF over-expression may play a role in regulating this survival pathway in prostate carcinogenesis. Collectively, the results of this study suggest that HDGF is a key player in the development of PCa and may serve as a target for chemopreventive or chemotherapeutic strategies in PCa.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2011-1109
Abstract 1117: Macrophage migration inhibitory factor (MIF) plays a critical role in clear cell renal carcinoma
Authors: Wright Bradley M. , Du Weinan , Li Xiaofeng , Finke James , Rini Brian I. , Zhou Ming , He Huiying , Lal Priti , Welford Scott M.
Published: June 2012

Clear cell renal carcinoma (CCRC) is the most common form of renal cancer, resulting in 50,000 new cases per year in the US, and greater than 10,000 deaths. CCRC is frequently characterized by alterations in the von Hippel-Lindau protein (VHL), an E3 ligase that mediates oxygen dependent degradation of the alpha subunit of the hypoxia inducible factor (HIF). Activation of HIF and many of its transcriptional target genes are critical for renal tumorigenesis. The macrophage migration inhibitor factor (MIF) is a pleiotropic cytokine that has roles in inflammation, cell signaling, and cell cycle regulation. MIF is regulated by HIF, and is expressed in a variety of tumor types wherein MIF expression correlates with poor outcome. Functionally, MIF can negatively regulate p53, and thus may be particularly relevant in cancers like clear cell carcinoma in which p53 is not commonly mutated. The purpose if this study is to determine the role of MIF in CCRC.

In order to determine if MIF is expressed in CCRC patient samples, we stained tumor microarrays with over 95 primary CCRC tumors for MIF expression. In keeping with its reported regulation by the VHL/HIF pathway, MIF demonstrated high expression in greater than 90% of the tumors compared to normal kidney samples. Likewise, in CCRC tumor cell lines MIF expression was highly expressed compared to cell lines in which VHL expression has been reconstituted. To determine the significance of MIF expression in CCRC tumor cell lines, MIF was depleted using two independent shRNAs and we observed significant decreases cell proliferation and clonogenic survival in a variety of CCRC cell lines. These observations were replicated by silencing the expression of the MIF receptor CD74, suggesting the MIF signaling pathway is intact and functional CCRC.

At the mechanistic level, MIF is known to inhibit the function of p53 through promotion of MDM2 mediated degradation. To assess whether the role of MIF in CCRC is to regulate p53, we determined the effects on p53 signaling when MIF expression was inhibited. We found that inhibition of MIF lead to an increase in p53 stability and subsequent expression of the canonical p53 target gene p21. Together these observations suggest that MIF expression may play a critical role as a target of the HIF/VHL pathway that leads to the development of clear cell carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1117. doi:10.1158/1538-7445.AM2011-1117