bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4989: FLIP transcriptional deregulation: A potential mechanism for development of androgen independent prostate cancer
Authors: Xie Jianping , Ghosh Rita , Kumar Addanki P.
Published: February 2011

The cellular Fas/FasL-associated death domain protein-like inhibitory protein (c-FLIP) plays a critical role in the regulation of apoptosis. Studies from our laboratory identified transcription factor Sp1 as an activator and Sp3 as an inhibitor of Sp1 transactivation of FLIP in prostate cancer cells. We have also shown that inhibition of FLIP by 2-methoxyestradiol (2-ME2) (i) induces apoptosis in prostate cancer cells and (ii) is associated with regression of prostate tumors in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. In addition FLIP promoter contains putative binding sites for various transcription factors including AP-1, c-Myc, NFκB, ARE (androgen response element) and p53, it is possible that these elements alone or through cooperative interactions may be important for the regulation of FLIP transcription and also in mediating 2-ME2 induced apoptosis. The present study was carried out to investigate the role of androgens in the transcriptional regulation of the FLIP using deletion analysis coupled with chromatin immunoprecipitation assay (ChIP), electrophoretic mobility shift assay (EMSA) and transient expression assays in prostate cancer cells. Our results show that treatment of androgen responsive LNCaP cells with DHT stimulates (i) cell proliferation; (ii) FLIP transcriptional activity; (iii) Sp1 and ARE DNA binding activity; (iv) PSA transcriptional activity and treatment with 2-ME2 inhibited the observed DHT-stimulated activities. Our findings suggest that although the FLIP promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of FLIP transcription, possibly through cooperative interactions between Sp1 and additional transcription factors including AR. Supported by ACS RSG-04-169 and NIH CA 135451 (APK).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4989.
Abstract 4990: Nuclear Factor-Y (NF-Y) regulates the transcription of the human aldo-keto reductase 1C1 (AKR1C1) gene*
Authors: Rajash Pallai , Simpkins Henry , Chen Jianli , Parekh Hemant K.
Published: February 2011

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Here, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene. The 5′ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5′-flanking, ∼3.0 kb region of the human AKR1C1 gene, identified the region between −128 to −88 as the minimal proximal promoter that is essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Based on computational analysis, a set of potential putative transcription factor binding sites for Oct1, Sp1, NF-Y, C/EBP, p40X, USF, NF1 and AP-2 were identified in this region. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were essential for controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at −109ATTGG−105 of the AKR1C1 gene. These results indicate that the NF-Y transcription factor regulates the basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4990.
Abstract 4998: Down-regulation of Hsulf-1 promotes tumorigenicity of ovarian cancer cells
Authors: He Xiaoping , Ohta Tsuyoshi , Shridhar Viji
Published: February 2011

Human Sulfatase1 enzyme (HSulf-1) is down-regulated in a majority of ovarian cancer cell lines and primary tumors, but the functional consequence of this downregulation remains unclear. Here, we showed that down-regulation of HSulf-1 by RNA interference in OV202 ovarian cancer cells resulted in an elevated level of phosphorylated AKT. Conversely, over-expression of HSulf-1 in SKOV3 ovarian cancer cells led to decreased phosphorylation of AKT. When cells were treated with CDDP, OV202 cells with knockdown of HSulf-1 displayed more resistance to drug-induced apoptosis, with IC50 value 3-fold higher than cells with non-targeting shRNA, while SKOV3 cells with overexpression of HSulf-1 were more sensitive to drug-induced apoptosis compared to the control cells. Moreover, knockdown of HSulf-1 also endowed ovarian cancer cells with increased resistance to detachment-induced apoptosis (anoikis). Cell proliferation assays revealed that knockdown of HSulf-1 significantly accelerated cell proliferation of ovarian cancer cells in vitro. Importantly, when cells were injected subcutaneously, 7 of 8 (87.5%) nude mice developed tumors with HSulf-1 knockdown cells within 2 weeks. However, none of the mice injected with nontargeted control transduced cells (control group) developed tumors. Four weeks after injection, 8 of 8 (100%) nude mice developed tumors with HSulf-1 down-regulated cells, while only 2 of 5 (40%) mice had tumors in the control group. The average tumor weight was 4.2±2.7g per mice in HSulf-1 knockdown group, significantly higher than 0.6±0.3g per mice in the control group. Collectively, these results strongly suggest that down-regulation of HSulf-1 promotes tumorigenicity of ovarian cancer possibly due to elevated activation of AKT pathway.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4998.
Abstract 5004: Functional modulation of insulin-like growth factor binding protein-3 in melanoma
Authors: Dar Altaf A.
Published: February 2011

Insulin-like growth factor binding protein-3 (IGFBP3) is a member of the IGFBP family, which regulates the mitogenic and anti-apoptotic effects of insulin-like growth factors. Hypermethylation of the IGFBP3 promoter has been found to suppress its expression in different tumors. In the present study we evaluated the role of IGFBP3 in melanoma. Quantitative real time PCR (qRT-PCR) of 12 melanoma cell lines indicated a significant down-regulation of IGFBP3 expression as compared to a normal melanocyte cell line. qRT-PCR of human melanoma tissues showed significant downregulation of IGFBP3 as compared to nevus samples. Melanoma cell lines treated with the demethylating agent 5-AZA-2′deoxycytidine (5AZA) re-expressed IGFBP3 at the mRNA level, indicating a possible role for methylation in the suppression of its expression. We examined the methylation status of the IGFBP3 promoter in cell lines and in human tissue samples by bisulfite DNA sequencing. The promoter region of IGFBP3 in C8161.9 and DO4 melanoma cell lines was hypermethylated, which was completely reversed by treatment of these cells with 5AZA. The IGFBP3 promoter region was also highly methylated in human melanoma samples as compared to normal nevus samples. Overexpressing IGFBP3 in melanoma cells in vitro suppressed cell survival and colony formation and increased rate of apoptosis. Stable overexpression of IGFBP3 suppressed melanoma cell growth in vivo. Our results indicate that silencing of IGFBP3 in melanoma is due to the methylation of its promoter, and overexpression of IGFBP3 induces apoptosis and suppresses cell survival and growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5004.
Abstract 5020: Role of LIN28 in aggressive breast cancers lacking REST
Authors: Gunsalus Kearney T.W. , Wagoner Matthew P. , Potter Wyatt B. , Schoenike Barry , Roopra Avtar
Published: February 2011

Our lab has discovered a highly aggressive subset of breast cancers characterized by a lack of function of the tumor suppressor RE1 Silencing Transcription Factor (REST). REST is a transcriptional repressor controlling over 2000 neuronal genes, and has been identified as a tumor suppressor in lung and colon cancers. Our lab has discovered that REST function is lost in 20% of breast tumors and that its loss is associated with a significantly decreased disease-free survival and poor prognosis (Wagoner et al. poster, this meeting, CB4).

In addressing the mechanisms underlying the aggressiveness of these RESTless tumors, we found that expression of the tumor promoter LIN28 is upregulated. We describe the direct regulation of LIN28 by REST in a range of mammary cell lines and show a correlation between REST loss and LIN28 overexpression in breast tumors. LIN28 inhibits maturation of the tumor suppressor microRNA let-7, which in turn suppresses expression of the oncogenes Myc and Ras. LIN28 overexpression has been found to promote metastasis in a mouse xenograft model of breast cancer.

To examine the effects of REST loss and LIN28 expression on cellular transformation and motility, we have knocked down REST and LIN28 in a variety of breast cell lines (including T47D, MCF7, MDA-MB-231, 3T3 and NMuMG cells). Here, we describe the results of our colony formation, soft agar growth and motility assays. We recapitulate in vitro aspects of tumor aggression seen in the patient population, and we find that these processes are promoted by REST loss in a LIN28-dependent manner. We also describe xenograft models looking at the growth and metastasis of tumors that express or lack REST and LIN28. We conclude that REST loss promotes tumor aggressiveness at least in part via upregulation of its target gene LIN28.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5020.
Abstract 5030: Mechanistic insight of differential sensitivity to MEK inhibition
Authors: Ji Zhenyu , Njauw Ching-Ni , Tsao Hensin
Published: February 2011

BRAF is mutated in melanomas and plays a role in tumorigenesis. Although depletion of oncogenic BRAF resulted in tumor regression in mouse melanoma model, clinical development of anti-BRAF signaling therapy has not been successful up to date. We performed a search of genomic alterations that are correlated to sensitivity to MEK inhibitors using human melanoma cell lines. Multiple genetic changes are identified to be of predictive value. Based on our genetic analysis, p53 is identified to be a major factor that controls sensitivity to MEK inhibitors. p53 is a major tumor suppressor and is mutated in a small set of human melanomas. Here, we show that p53 is inducible in most human melanoma cell lines in response to DNA damage. MDM2 inhibitor Nutlin-3a increases p53 protein expression as well as its activities in melanoma cells. Addition of nutlin-3a significantly increases sensitivity of melanoma cells to MEK inhibitor U0126 induced cell death or cell cycle arrest. In addition, knockdown p53 by specific shRNAs effectively rescues apoptosis induced by MEK inhibitor and/or p53 activation. This data suggests that p53 activity is required for cell death induced by MEK inhibitors and/or p53 activators. In conclusion, p53 is identified from a genetic screen for MEK inhibitor sensitivity. p53 reactivation potentiates cell death induced by BRAF/MEK signaling inhibitory agents in a group of melanomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5030.
Abstract 5042: Trop-2 is a universal cancer growth stimulator through a ubiquitous signaling platform
Authors: Alberti Saverio , Trerotola Marco , Guerra Emanuela , Havas Kristina , Lattanzio Rossano , Lasorda Rossana , Bonasera Veronica , Vacca Giovanna , Aloisi Anna Laura , Piantelli Mauro
Published: February 2011

Trop-2 is a transmembrane calcium signal transducer. Our findings show that Trop-2 is overexpressed by the majority of human cancers, suggesting strong selective pressure for a conserved function. Trop-2 was then demonstrated to be necessary and sufficient to stimulate cancer growth, with a linear relationship between growth rates and Trop-2 expression levels. Cell growth stimulation was shown to be conserved across cell-types and species. These findings indicated impingement on a ubiquitous downstream signal-transduction module. The Trop-2 signaling pathway was investigated by yeast 2 hybrid screening, coimmunoprecipitation / mass-spectrometry and proteomic chip analysis. Trop-2 was demonstrated to bind CD44 and tetraspanins, triggering their growth-promoting ability via a feed-forward activation loop of CD9-recruited PKCα and phosphorylation of the Trop-2 cytoplasmic tail. We demonstrate that PKCα stimulates growth in a Trop-2-restricted manner and that PKCα and Trop-2 are coordinately transported in recurrent waves to membrane ruffles and podosomes. Trop-2 induction was shown to activate the ERK pathway, to up-regulate NF-κB, and to modulate apoptotic factors, including p53 and Rb. Members of the Trop-2 pathway (CD9, CD44 and p53) were found coordinately upregulated in retrospective studies of human cancer case series. These findings reveal the existence of a novel, strikingly widespread mechanism of stimulation of cancer growth. This is quantitatively driven by a wild-type Trop-2 and acts upon ready-to-signal, ubiquitous signal-transduction platforms.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5042.
Abstract 5049: Functional significance of L1CAM in non-small cell lung cancer
Authors: Hai Josephine , Radulovich Nikolina , Zhu Chang-Qi , Der Sandy D. , Jurisica Igor , Tsao Ming-Sound
Published: February 2011

Background: Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers. Early stage NSCLC patients are treated by complete surgical resection, but the 5-year survival rate remains relatively poor at 50%. Deaths are mainly due to metastatic recurrence. Post-surgery (adjuvant) chemotherapy may significantly improve survival, but improved selection methods for patients who are most likely to benefit from the adjuvant treatment are required. Our laboratory has recently identified a 15-gene expression signature capable of classifying patients based on risk of death and at the same time predicting benefits of adjuvant chemotherapy in high-risk patients (J Clin Oncol 26: 2008, May 20 suppl; abstr 7510). We hypothesize that genes whose expression are de-regulated significantly in patients with a poor prognosis may play important roles in tumorigenesis and metastasis. Our goal is to functionally characterize the roles of these prognostic genes in the growth and metastatic potential of NSCLC. One of these signature genes is L1CAM, a cell adhesion molecule. Although extensively characterized in the nervous system, the role of L1CAM in NSCLC has not been defined.

Methods: We stably modified the expression of L1CAM at both the RNA and protein levels in NSCLC cell lines using the lentiviral cDNA/shRNA system. In vitro matrigel assays were used to assess invasion and cell migration by wound-healing assays.

Results: Downregulation of L1CAM expression resulted in decreased invasiveness and motility of metastatic NSCLC cell lines. In contrast, loss of L1CAM did not alter the growth rate on plastic. Studies on anchorage-independent growth and in vivo tumorigenicity and metastatic potential are ongoing and the results will be presented.

Conclusion: Considering the importance of migratory phenotype in the first steps of metastasis, the results suggest that L1CAM contributes to malignancy in NSCLC and may be a novel therapeutic gene for this disease. (Supported by Canadian Cancer Society Research Institute grant #020527)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5049.
Abstract 5050: Functional characterization of a newly identified growth regulatory gene SKCG-1 in human kidney cancer
Authors: Singh Kamaleshwar P. , Sarkar Shubhashish , Roy Deodutta
Published: February 2011

The mechanism of human kidney cancer development is not completely understood. Recently, we have identified a new growth regulatory gene, SKCG-1 and its expression was found to be decreased or lossed in human Wilms tumor. The objective of this study was to further characterize this gene and determine whether it has growth inhibitory function in human kidney cancer. The role of DNA hypermethylation in the inhibition of SKCG-1 expression in tumorigenic kidney epithelial cells was investigated by 5′-aza-2′-deoxycytidine (demethylating agent) treatment. The role of SKCG-1 in cell growth was tested by both, silencing its expression in normal cells, and overexpressing it in tumorigenic kidney epithelial cells. Reactivation of SKCG-1 transcripts in 5′ Aza-2′-deoxycytidine -treated HEK293 cells as compared to the untreated HEK293 cells suggest the hypermethylation as one of the causes for loss of SKCG-1expression in human kidney cancer cells. Colony formation assay revealed that ectopic expression of SKCG-1 suppresses the growth of tumorigenic HEK293 cells. Small interfering RNA (siRNA)-mediated silencing of SKCG-1 in HEK293 resulted in increased cell growth and this was further confirmed by increased DNA synthesis as revealed by Brdu assay. Silencing of SKCG-1 also resulted in increased expression of cell cycle gene CyclinD1 and decreased expression of P53. Immunocytochemical staining of SKCG-1 confirmed its localization in the nuclear membrane. Immunohistochemical staining further confirmed the loss of SKCG-1 expression in human kidney tumors (RCC) and its presence in surrounding normal human kidney tissue. These results suggest that the presence of SKCG-1 inhibits the growth of cancer cells and its absence allows the growth of cancer cells. The findings of this study further support the tumor suppressor role of SKCG-1 in kidney cancer. Therefore, the restoration of SKCG-1 function and its signaling pathway(s) represents a possible therapeutic target in kidney cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5050.
Abstract 5058: Phosphatase Nuclear Targeting Subunit (PNUTS) is phosphorylated by AKT kinase
Authors: Krucher Nancy A. , Perry Brittany , Lentine Brandon , Dzaferovic Violeta , Cardoso Filipa
Published: February 2011

Phosphorylation of the tumor suppressor protein, Retinoblastoma (Rb) is associated with a highly proliferative state of the cell and cancer. Rb phosphorylation state is partly controlled by the activity of Protein Phosphatase 1 (PP1) which is regulated by Phosphatase Nuclear Targeting Subunit (PNUTS). It is likely that PNUTS plays a role in cell survival and/or apoptosis because siRNA mediated knockdown of PNUTS causes apoptosis in breast and colon cancer cells. This occurs through activation of PP1 phosphatase activity toward Rb which leads to Rb dephosphorylation which can trigger apoptosis. Here we show that PNUTS may be regulated by the PI3K-AKT pathway which has been shown to be involved in many cellular functions including proliferation, growth and survival. In addition, signaling in this pathway has been shown to be disrupted in several cancers. In response to a sub-lethal dose of UV radiation of HCT116 colon cancer cells, activation of the AKT kinase occurs, with phosphorylation of known AKT substrates GSK-3 and mdm2 observed. Phosphorylation of Ser473 of AKT further verifies AKT activation. Under these conditions PNUTS is phosphorylated by the AKT kinase. Inhibition of PNUTS phosphorylation occurs when cells are treated with the LY294002 PI3K-AKT pathway inhibitor. Thus PNUTS is a putative substrate of AKT, and may play an important role in the survival of cells in response to stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5058.
Abstract 5061: Induction of B cell acute lymphoblastic leukemia by BCR/ABL requires c-Myb and its activation of Bmi-1
Authors: Waldron Todd J. , Soliera Angele R. , Zhang Ying , Martinez Robert , Hyslop Terry , Bender Timothy , Calabretta Bruno
Published: February 2011

Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL), carrying the e1a2 fusion BCR/ABL oncogene, is typically an aggressive B cell leukemia, often resistant to current treatment options. The transcription factor c-Myb plays a critical role in B cell development, but its role in B cell leukemia is unknown. To study the role of c-Myb in BCR/ABL-induced lymphoblastic leukemia, we compared disease induction, latency and survival in mice expressing the BCR/ABL transgene in a c-Myb knockout versus a wild-type background. BCR/ABL+Myb+/− mice show a distinct leukemia-resistant phenotype, whereas, BCR/ABL+Myb+/+ mice exhibit rapid induction of disease with shortened survival. Using microarray analysis to examine differential gene expression, we have identified putative downstream targets of c-Myb which play a role in leukemogenesis. Subsequent use of gene overexpression, RNAi for target gene knockdown and chIP have identified the polycomb gene, Bmi-1, as a target of c-Myb transactivation. Furthermore, inhibition of the p19ARF tumor suppressor, a Bmi-1 target, may be essential to the induction of ALL by BCR/ABL. In this report, we describe the essential role that c-Myb and its activation of Bmi-1, play in BCR/ABL-induced leukemogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5061.
Abstract 5062: Rac1 signaling is up-regulated in normal tissue adjacent to recurrent respiratory papillomas
Authors: Lucs Alexandra V. , Wu Rong , Symons Marc H. , Abramson Allan L. , Steinberg Bettie M.
Published: February 2011

Recurrent respiratory papillomas, caused by human papillomavirus infection, are benign tumors that block the airway and have significant potential for malignant conversion. Previously we have shown that the epidermal growth factor receptor (EGFR) is overexpressed in papillomas. This overexpression leads to the activation of a novel signaling pathway involving increased expression of the Rho-family member GTPase Rac1, activation of the p38 mitogen activated kinase and increased expression of cyclooxygenase 2 (COX-2). This pathway is not activated in normal laryngeal epithelial tissue samples from individuals without the disease, but is constitutively active in papilloma tissue. Here we have investigated the extent to which the EGFR → Rac1 → p38 → COX-2 pathway is activated in clinically normal airway tissue from patients with respiratory papillomas. Surprisingly, we found that the Rac1 and COX-2 levels are comparable in “normal” and papilloma tissues, suggesting widespread activation of this pathway in the laryngeal mucosa, without marked elevation of the EGFR in the “normal” adjacent tissue. Moreover, preliminary data suggests that overexpression of Rac1 in these patients may be systemic, as it is also found at higher levels in peripheral blood mononuclear cells. We hypothesize that Rac1 overexpression may be a rare genetic trait that predisposes these patients to develop recurrent respiratory papillomatosis. Furthermore, investigation into Rac1 levels revealed the presence of a splice variant known as Rac1b. This constitutively active form of Rac1 is found at higher levels in papilloma tissue when compared to “normal” adjacent tissue from the same patient, possibly contributing to the growth of the tumors. Our current research is focused on determining the mechanism of Rac1 overexpression and elucidating the role of the Rac1 isoforms in papilloma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5062.
Abstract 5066: Transformation of pancreatic epithelial cells by BrafV600E
Authors: Appleman Vicky , Klimstra David , Lewis Brian C.
Published: February 2011

Pancreatic cancer is the 4th leading cause of cancer-related deaths in the United States, with an estimated 35,000 deaths expected in 2009. The vast majority of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC), and activating mutations in the KRAS oncogene are present in over 90% of these tumors. Interestingly, one third of all KRAS wild-type PDAC harbor activating mutations in the gene encoding the downstream Kras effector Braf. Yet, the roles of specific Kras-stimulated effector pathways in pancreatic tumor initiation and progression remain unclear. We therefore sought to determine the consequences of expressing an activated Braf molecule in primary pancreatic duct epithelial cells (PDECs). Expression of an activated BrafV600E mutant results in the activation of the Mek-Erk and PI3K-Akt signaling cascades as determined by phosphorylation of Erk on Thr202 and Akt on Ser473. Measurement of cell proliferation demonstrated that BrafV600E stimulates the proliferation of PDECs, albeit to a lesser extent than an activated KrasG12D mutant. Activated Braf also promotes the survival of PDECs after exposure to apoptotic stimuli such as cycloheximide treatment and ultraviolet irradiation. This survival is dependent on activation of both the Mek-Erk and PI3K-Akt signaling cascades, as inhibition of these pathways abrogated Braf-induced survival. Consistent with its ability to stimulate the proliferation and survival of PDECs, orthotopic transplantation of PDECs expressing BrafV600E, and deleted at the Ink4a/Arf and Trp53 tumor suppressor loci, resulted in tumor formation, albeit with delayed kinetics relative to KrasG12D expressing PDECs. Interestingly, cell lines derived from orthotopic tumors required the activation of the PI3K-Akt signaling axis, but not the Mek-Erk signaling pathway, for survival after exposure to apoptotic stimuli, suggesting differential roles for these pathways during pancreatic tumor initiation and progression. Thus, our studies provide evidence that activating Braf mutations lead to the transformation of pancreatic epithelial cells, and additionally suggest that downstream Ras effectors may play different roles during pancreatic tumor initiation and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5066.
Abstract 5067: Neurofilament heavy polypeptide regulates the Akt-β-catenin pathway in human esophageal squamous cell carcinoma
Authors: Kim Myoung Sook , Chang Xiaofei , LeBron Cynthia , Nagpal Jatin K. , Lee Juna , Huang Yiping , Yamashita Keishi , Trink Barry , Ratovitski Edward A. , Sidransky David
Published: February 2011

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5067.
Abstract 5087: Mirk/Dyrk1B kinase mediates survival of quiescent tumor cells and is downstream of the cellular energy sensor AMPK
Authors: Deng Xiaobing , Friedman Eileen
Published: February 2011

The Mirk kinase gene has been localized to the 19q13 amplicon and is amplified in a subset of pancreatic cancers and ovarian cancers, and less frequently in colon cancers. Mirk mediates survival of these cancers at least in part by reducing reactive oxygen species (ROS). Mirk is expressed at very low levels in most normal tissues. However, Mirk is highly expressed in normal skeletal muscles and in C2C12 myoblasts where it mediates differentiation and survival. Using immunofluorescence techniques we now show that Mirk is localized in fast twitch skeletal muscles. Such muscle endogenously produces ROS in response to repeated contractions. Depletion of Mirk from C2C12 myoblasts led to an increase in ROS, consistent with ROS control being a major role of Mirk in muscle development and function. This protective ROS-decreasing role is likely to have provided a selective pressure to upregulate Mirk expression in tumor cells. Mirk is a co-activator for several transcription factors and increases expression of a series of antioxidant proteins in cancer cells including superoxide dismutase 2 (SOD2), the ferroxidase ceruloplasmin, glutathione peroxidase 2, sulfiredoxin, peroxiredoxin 2, COX2 and archidonate 12-lipoxygenase (12-LOX), as assayed by either Northern blotting or quantitative RT-PCR or both measurements. Mirk is most abundant and active as a kinase when cells are in a quiescent G0 state. Low intracellular energy levels due to nutrient deprivation lead to transient cellular arrest in G0 through the function of AMPK (AMP-activated protein kinase). Mirk expression is controlled through AMPK which serves as a metabolic checkpoint kinase, arresting cell growth in response to low nutrient levels which depress intracellular ATP levels. Thus the serine/threonine kinase Mirk mediates tumor cell survival through maintenance of its original function in skeletal muscle cells, depletion of ROS, when cells are in a transient quiescent state, with both Mirk expression and cell arrest controlled by AMPK.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5087.
Abstract 5090: The role of p32 in brain metastasis
Authors: Staflin Karin , Fogal Valentina , Fernandez-Santidrian Antonio , Ruoslahti Erkki , Felding-Habermann Brunhilde
Published: February 2011

Brain metastases are among the most feared complication in cancer. To develop effective and targeted therapies for treatment of brain metastases, we need a better understanding of the mechanisms that control cancer cell growth within the brain microenvironment. We previously found evidence that brain metastatic cells have increased rates of oxidative phosphorylation and redox potential compared to tumor cells from extracranial lesions. Here we sought to identify mechanisms involved, and to identify potential targets for the inhibition of metastatic growth in the brain. Many of the genes involved in transformation such as Myc, AKT, and p53 have extensive effects on the metabolism, suggesting that tumors might be susceptible to antimetabolic drugs. One of the proteins that has been shown to be upregulated in many solid tumors is the protein p32. P32 is a multi-compartmental and multi-functional protein which has a regulatory function in oxidative phosphorylation, in addition to being involved in the regulation of apoptosis and autophagy. We found p32 expressed in human cancer cells capable of seeding brain metastases in immune deficient mice, and we hypothesized that p32 contributes to cell survival and proliferation within the brain microenvironment. We knocked down p32 in brain homing variants of the MDA-MB-231 and MDA-MB-435 metastasis cell models. Following luciferase tagged tumor cells by non-invasive bioluminescence imaging revealed that stable p32 knockdown greatly reduced metastatic activity in both MDA-MB-231 brain specific variant and the MDA-MB 435 cell line. Furthermore, our genomic profiling of p32 knock-down and control cells identified candidate genes in the respiration chain involved in p32-dependent tumor cell survival and growth that may impact progression to brain metastasis. Thus, the expression and functionality of p32 in tumor cells capable of colonizing the brain appears associated with changes in crucial pathways that enable the tumor cells to proliferate and survive within the brain microenvironment.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5090.
Abstract 5094: Mutant specific mitochondrial localization of BRAFV600E: Clinically used BRAF kinase inhibitors are not able to block mitochondrial actions of BRAFV600E
Authors: Lee Min Hee , Kim Koon Soon , Lee Seong Eun , Ryu Min Jeong , Kim Soung Joong , Kim Yong Kyoung , Kweon Gi Ryang , Jo Young Suk , Shong Minho
Published: February 2011

Oncogenic mutation in BRAF kinase (BRAFV600E) results active structural conformation characterized with greatly elevated ERK activities. However, other cellular effects which are specifically influenced by mutation in BRAF kinase remains to be identified. We investigated differences in subcellular localizations of wild type and mutant BRAFV600E and observed the effects of the BRAF inhibitors on subcellular compartment-specific effects of BRAFV600E. We found that significant portion of endogenous and exogenous BRAFV600E, but not wild type BRAF, was detected in mitochondrial fraction. The other BRAF mutants, BRAFV600D, BRAFV600K and BRAFV600R which shows higher kinase activities were able to localize in mitochondria. The localization of BRAFV600E onto mitochondria provides anti-apoptotic activities against staurosporine and TNFα/cycloheximide. In addition, inducible expression of BRAFV600E increases glucose uptake rate, decreased O2 consumption, suggesting reduced mitochondrial oxidative phosphorylation which are signature features found in cancer cells. Interestingly, suppression of MEK activities using U0126 did not affect both mitochondrial localization and anti-apoptotic activities of BRAFV600E. The BRAF inhibitor such as Sorafenib was effective to inhibit MEK/ERK activation, however, they did not block the mitochondrial localization of BRAFV600E. Therefore, these inhibitors did not block the antiapoptotic activities and not reduce high glucose uptake rate and glycolytic activities induced by BRAFV600E. We found oncogenic BRAF mutants are able to localize in mitochondria and these actions might be related to altered responses to apoptotic stimuli and to characteristic metabolic phenotypes. In addition, currently BRAFV600E inhibitors were not sufficient to block mitochondrial localization of BRAFV600E and it may explain the limited efficacy of these drugs in clinically advanced BRAFV600E-positive tumors. These observations provide new mutation- specific roles of BRAFV600E which is potentially important for the development of therapeutics.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5094.
Abstract 5103: Analysis of the role of “ATM” through “omics” analysis
Authors: Cheema Amrita K. , Jung Mira , Timofeeva Olga , Varghese Rency , Ressom Habtom , Dritschilo Anatoly
Published: February 2011

The human genetic disease, ataxia-telangiectasia (AT) is characterized by exquisite radiation sensitivity, neurological degeneration, immune deficiencies, genetic instability and predisposition to cancer. Furthermore, cells from patients with AT show disruption of the DNA damage response (DDR), a global signaling network that senses damage and coordinates cellular responses that include cell cycle regulation, transcription, DNA repair processes and apoptosis. Previously, AT5BIVA cells have been was established by SV40 immortalization of fibroblasts derived from an AT group D patient. We derived ATCL8 cells line was then derived by transfecting AT5BIVA with a vector carrying expressing the full- length wild type ATM. ATCL8 cells are and was found to be less sensitive to radiation resistant (D0=1.7 Gy) as compared tothan are the parental cell line AT5BIVA cells (D0=0.7 Gy).

We have used a systems approach to understand compare the baseline changes that occur at the genomics, proteomics and metabolomic levels in these two near isogenic cell lines and integrate the information to understand the correlation. The. mMicroarray analysies wereas carried performed out to compare investigate basal level changes in gene expression. between AT5BIVA and ATCL8. The p Proteomics analysies for the same system was donewere performed using a 2-D gels and mass spectrometry approach. Finally, metabolomic profilesing was performed were obtained using UPLC-ESI-TOF mass spectrometry followed by multivariate data analysis using (SIMCA-P and random forest) software. The mMetabolites which were found observed to be significantly different in ATCL8 as compared to AT5BIVA were then subjected to functional pathway analysies using the ingenuity software. The proteomics and the microarray data were then scored for the presence of proteins and genes which were a part of the predominant signaling pathways in the two cell lines as determined by metabolomic analysis.

The model system used in this study involves studyingof basal level changes in expression resulting from perturbations in a single gene and our results demonstrate the feasibility of such an the approach to correlate the changes found at all three levels of expression. The findings will be discussed in the context of the “AT” phenotype will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5103.
Abstract 5133: Microtentacles and invadopodia: Functionally distinct plasma membrane protrusions of metastatic breast tumor cells separated mechanistically by c-Src
Authors: Balzer Eric M. , Whipple-Bettes Rebecca , Cho Edward H. , Matrone Michael A. , Mueller Susette C. , Martin Stuart S.
Published: February 2011

During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Our recent discovery of a filamentous actin (F-actin) component within thin tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells prompted an investigation of whether these extensions are structural or functional analogs of invadopodia. We show here that MDA-MB-231s are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by expression of a constitutively-active c-Src kinase, and repressed by expression of dominant negative, catalytically inactive form of c-Src. In contrast expression of inactive c-Src significantly increased the McTN formation. Additionally, direct inhibition of c-Src with the SU6656 inhibitor compound significantly enhanced McTN formation, suppressed the appearance of F-actin cores and phospho-cortactin foci, and completely blocked focal degradation of extracellular matrix. These results identify McTNs and invadopodia as separate structures that provide unique and sequential contributions to metastatic progression, namely endothelial attachment and invasion, respectively.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5133.
Abstract 5138: The regulation of CD44 expression in malignant brain tumor cells
Authors: Raines Timothy A. , Debinski Waldermar , Martin Patrick M.
Published: February 2011

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor that demonstrates a high growth rate and the capacity to invade surrounding brain tissue. These characteristics result in a dismal prognosis for GBM patients. Current treatment options for GBM tumors involve surgical resection followed by radiation and/or chemotherapy. Disappointingly, GBM tumors are or become resistant to radiation and chemotherapy, and the invasiveness of GBM precludes curative removal of the tumor. GBM cells infiltration has been associated with the transmembrane glycoprotein, CD44, which is the principle cell surface receptor for hyaluronic acid (HA). The molecular mechanisms that regulate CD44 in GBM are not fully understood. However, CD44 protein expression has been associated with an AP-1 transcription factor, fos related antigen 1 (Fra-1), in another aggressive cancer, mesothelioma. Therefore, we investigated the regulation of CD44 expression in human glioma cells by Fra-1. Human GBM cell lines, U-251 MG, U-1242 MG, and A-172, were exposed to either epidermal growth factor (EGF, 20 ng/ml) or hepatocyte growth factor (HGF, 50 ng/ml) at varying time points and Western blot analysis was used to determine the protein expression of CD44 and Fra-1. CD44 and Fra-1 expression levels increased with longer exposures (4 to 24 hr) to EGF and HGF. To determine whether Fra-1 takes part in the regulation of CD44 expression, we utilized transient gene knockdown using siRNA directed against fra-1. fra-1-siRNA decreased Fra-1 expression in EGF- and HGF-treated GBM cells. Following EGF stimulation and fra-1-siRNA treatments, CD44 expression also decreased. However, HGF stimulation of fra-1-siRNA treated GBM cells showed no change in CD44 expression. These data suggest that Fra-1 regulation of CD44 expression is growth factor-specific. To investigate whether CD44-mediated cell adhesion is promoted by the expression of Fra-1, we analyzed GBM cell adhesion to HA. U-251 MG and U-1242 MG cell lines demonstrated an increase adherence to HA after growth factor simulation, 4hr and 24 hr respectively. This data is comparable to the initial western blot analysis of the GBM cells after growth factor simulation. However, A-172 cells did not adhere to HA regardless of growth factor stimulation. These data suggest that CD44 binding to HA is cell line specific. Taken together, our results suggest that CD44-mediated HA binding of GBM cells can be regulated by Fra-1 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5138.