bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A42: Small molecule activators of p53 for the treatment of inflammatory breast cancer
Authors: Chatterjee Namita , Ensslin Courtney J. , Tenniswood Martin P.
Published: May 2012

Although rare, inflammatory breast cancer (IBC) is the most aggressive form of breast cancer with a 5 year survival of 40% compared to 85% for ductal carcinoma in situ. The standard therapy for this disease is chemo- and radiation therapy; however there are currently no targeted therapies for IBC. Histopathology data suggests that 40–70% of IBC tumors express wild type p53, while approximately 40% are estrogen receptor positive (ER+). The disease is characterized by early and diffuse intravasation of the breast lymphatic vessels by the tumor cells which form emboli that block the drainage of the ducts leading to swelling of the affected breast and erythema. Efforts to increase survival from this often lethal disease have been hindered by the paucity of model systems for studying the underlying biology of IBC. We have used the SUM 149PT and SUM 190PT cell lines, which were originally derived from IBC to investigate the utility of two histone deacetylase (HDAC) inhibitors (CG-1521 and TSA), alone and in combination with anti-estrogens as a novel therapy for the disease. In prostate cancer cell lines CG-1521 has been shown to acetylate p53 at Lys 373 and induce cell death, while TSA induces specific acetylation of Lys382 and induces cell cycle arrest. Both SUM 149PT and SUM 190PT express ERα and ERβ, although neither cell line requires estradiol for cell growth. SUM149PT cells express wild type p53, while the SUM 190PT express a truncated isoform of the protein. CG-1521 induces DNA fragmentation and cell death in both cell lines. While TSA also induces cell death in the SUM 149PT cells, it only induces cell cycle arrest in the SUM 190PT cells, which we attribute to the truncation of the p53 protein. Treatment of SUM149PT cells with low doses of either tamoxifen or CG-1521 individually does not induce apoptosis but in combination, causes a substantial decrease in cell number and indicates that the two compounds act synergistically in this context. These data suggest that HDAC inhibitors such as CG-1521 and TSA, which target specific Lys residues in p53 may be useful for the treatment of IBC.

Citation Information: Cancer Res 2009;69(23 Suppl):A42.
Abstract A50: Interleukin-1β induced HIF-1α transcriptional activity in glioma cells is regulated by Ras via NFκB
Authors: Sharma Vivek , Koul Nitin , Mehta Veer Singh , Sen Ellora
Published: May 2012

Purpose: We observed elevated levels of the pro-inflammatory cytokine IL-1β in glioblastoma multiforme (GBM) tumor samples. Since Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in linking inflammatory and oncogenic pathways, we investigated the effect of IL-1β on HIF-1α expression in glioma cells under normoxia.

Experimental Procedures: Cytokine bead array was performed to determine the status of pro-inflammatory cytokine IL-1β in control and GBM human tissues. The effect of IL-1β on HIF-1α expression and transcriptional activity in glioma cell lines was determined by immunoblotting and reporter assay. The pathways involved in IL-1β mediated HIF-induction was determined by examining NFκB transcriptional activity and Ras activity, siRNA and immunoblotting blotting. To dissect the existence of autocrine loop between IL-1β and HIF-1α Chromatin immunoprecipitation (ChIP) assay was performed.

Results and Conclusions: IL-1β levels were elevated in GBM tissue compared to adjacent normal tissue as assessed by cytokine bead array. IL-1β induced HIF-1α expression and transcriptional activity in glioma cells under normoxia. Antagonizing of IL-1β signaling by blocking MyD88 activation downstream of the IL-1R or direct NFκB inhibition abolished HIF-1α activity, indicating a role of IL-1β induced NFκB in HIF-1α regulation. IL-1β mediated increased Ras activity is responsible for increased HIF-1α activity as down regulation of Ras resulted in a significant reduction in both NFκB and HIF-1α of IL-1β activity. These results implicate Ras as an upstream mediator of IL-1β induced NFκB and HIF-1α activation. Increased Ras expression was accompanied by increased phosphorylation of Ras effectors AKT, ERK, JNK and p38MAPK. However, inhibition of these effectors failed to block IL-1β mediated increase in HIF-1α induction. Interestingly, IL-1β elevated Wnt-1 expression in a Ras dependent manner and siRNA mediated knock-down of Wnt-1 decreased HIF-1α activity. Although Wnt-1 mediated HIF-1α was independent of the canonical Wnt/β-catenin signaling pathway, it regulated HIF-1α through NF-κB. IL-1β induced HIF-1α expression resulted in its increased interaction with HIF responsive element (HRE) on the IL-1β promoter, as demonstrated by chromatin immunoprecipitation (ChIP) assay.

Conclusions: Our studies suggest that under normoxia, IL-1β induced IL-1β HIF-1α autocrine loop establishes a vicious cycle of IL-1β secretion and HIF-1α stabilization.

Citation Information: Cancer Res 2009;69(23 Suppl):A50.
Abstract A51: Development and initial characterization of isogenic cell lines stably overexpressing prostate cancer candidate genes
Authors: Kacprzyk Lukasz , Blaich Stephanie , Wittig Rainer , Hudler Melanie , Sültmann Holger , Mollenhauer Jan
Published: May 2012

Microarray analyses of global changes in gene expression patterns have recovered a large number of genes, which are deregulated in prostate cancer, when compared to normal prostate tissue. In order to successfully translate these data into clinical intervention strategies, it is crucial to determine genes that truly contribute to tumor development (which may be referred to as ‘driver genes’) and to separate them from the majority of secondary anomalies in expression levels (‘passenger genes’). To obtain the missing functional information, we construct isogenic cell line libraries derived from commonly used prostate (RWPE-1) and prostate cancer (LNCaP, PC-3) cell lines and subject them to a range of functional assays.

Using a system for rapid generation of stably-transfected isogenic cell lines, that was developed in our group, we create a prostate cancer library in a two-step procedure. At first, acceptor clones are generated from cancer and normal prostate cell lines by stable integration of a plasmid carrying a recombinase target sequence. Incorporation of a second expression plasmid with the gene of interest is then mediated by site-specific recombination. This procedure allows a facilitated transgene integration into a predefined, transcriptionally active locus. It also minimizes the influence of genetic background, as clones generated within such a library differ among each other only by the presence of the gene of interest in the same chromosomal location. Therefore, in contrast to traditional knock-in strategies, our system ensures that readily interpretable phenotypes are obtained and thereby provides a highly standardized resource for functional gene analysis. The technology is well suited for medium throughput and was designed to link data from high-throughput genomic analyses with functional gene analysis in animal models.

LNCaP and PC-3 acceptor cell lines have been generated and characterized. Recombination of PC-3 with several well-described, cancer-relevant genes under the control of an inducible promoter indicated the feasibility of the method. The next step will include initial screening of the library of roughly 100 recombinants for changes in the cell viability and proliferation. The most promising effectors from the primary screen will be followed with an expanded range of functional assays, addressing their impact on cell cycle, apoptosis and cell migration. This can then serve as a starting point for an individual characterization of the novel prostate cancer genes.

Citation Information: Cancer Res 2009;69(23 Suppl):A51.
Abstract A56: Role of p63 in cell cycle progression in epidermis and in squamous cell carcinoma
Authors: Antonini Dario , Russo Monia T. , Missero Caterina
Published: May 2012

Genomic amplification and/or overexpression of the p63 gene are one of the most frequently observed abnormalities in human cutaneous and noncutaneous squamous cell carcinoma (SCC), however, in spite of several studies in genetically engineered mouse models and in human tumors, the contribution of p63 in SCC development is still controversial. As a member of the p53 family, p63 can bind to the canonical p53-binding site, transactivating or repressing a subset of p53 or p73 target genes in vivo, while it also affects a subset of specific target genes. In contrast to p53, p63, and more specifically its DeltaNp63 alpha isoform, has a tissue-restricted pattern of expression in stratified epithelia and its impact on tumorigenesis is likely to be tumor-specific as well.

Here we present data indicating that p63 is required for proliferation and G1 progression in a p53 independent manner in mouse primary keratinocyte, as well as in mouse embryonic epidermis. In addition we identified a number of p63 early target genes that are involved in cell cycle progression and in DNA replication. Among direct p63 targets we identified members of a specific microRNA (miRNA) family that are highly expressed in the epidermis. p63 binds to their genomic regulatory regions thereby directly repressing their expression. Overexpression of these specific miRNA arrests cell proliferation, whereas specific anti-miRNA expression substantially rescues the cell proliferation defect in p63 ablated keratinocytes. We propose that control of this specific miRNA family by p63 may play a role in sustaining cell proliferation in SCC development.

Citation Information: Cancer Res 2009;69(23 Suppl):A56.
Abstract A60: Role of telomere dysfunction in the premature cellular senescence of Hutchinson-Gilford progeria syndrome
Authors: Benson Erica K. , Aaronson Stuart A.
Published: May 2012

Introduction: Telomere dysfunction promotes genomic instability and is often associated with loss of tumor suppressor gene function and tumorigenesis. Conversely, in aging, telomere dysfunction is hypothesized to mediate cellular senescence by activating intact tumor suppressor pathways. Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by a dominant mutation in the nuclear lamina intermediate filament protein lamin A that leads to a truncated and permanently farnesylated protein termed progerin. Progerin expression is associated with early cellular senescence involving shortened telomeres and increased DNA damage. Herein, we investigated whether telomere dysfunction contributes to the DNA damage accumulation and premature cellular senescence phenotypes observed with cells from HGPS patients.

Methods: Using HGPS cell lines from patients or normal fibroblasts ectopically expressing progerin, we investigated the effects of telomerase on cell growth, senescence, p53 and Rb tumor suppressor pathway activation, and DNA damage. We also determined whether progerin-induced DNA damage localized to telomeres.

Results: We demonstrate that addition of telomerase, which has been reported to immortalize HGPS fibroblasts, abrogated premature senescence, reduced p53 and Rb tumor suppressor pathway activation, and decreased the DNA damage phenotype. These effects of telomerase required both its catalytic and DNA binding activities. Furthermore, progerin-DNA damage signaling localized to telomeres indicating that progerin causes damage at telomeres.

Conclusions: These results establish that progerin causes a defect in telomere maintenance linked to a chronic DNA damage response that activates tumor suppressor genes and induces the HGPS premature senescence phenotype.

Citation Information: Cancer Res 2009;69(23 Suppl):A60.
Abstract A63: Inhibition of ROCK signaling prevents breast cancer metastasis to human bone
Authors: Liu Sijin , Goldstein Robert H. , Scepansky Ellen M. , Rosenblatt Michael
Published: May 2012

ROCK signaling plays a fundamental role in regulating cell morphology, adhesion and motility. Aberrant expression of ROCK is related to tumor metastases and correlates with poor clinical outcome. However, the molecular mechanisms responsible for ROCK-mediated effects and the potential utility of ROCK inhibition as a therapeutic target have not been fully investigated. Here, we show that ROCK expression is increased in metastatic human breast cancer (BrCa) cell lines and clinical mammary tumors compared to non-metastatic cell lines and tumors. Inhibition of ROCK activity, either by a specific ROCK inhibitor (Y27632) or ROCK-targeted siRNAs, reduces BrCa cell migration and proliferation in vitro. We evaluated the effect of ROCK inhibition on metastasis to bone in vivo using a novel “human BrCa metastasis to human bone” mouse model. Metastases to human bone cores (hBCs) were inhibited in Y27632-treated mice (5 out of 14, 36%) compared to control mice (8 out of 12, 67%), based on luminescent imaging. The overall frequency of metastasis to mouse skeleton in Y27632-treated mice was reduced by 36% compared to controls. Moreover, the mass of metastatic tumors in the mouse skeleton was diminished by 77% (P<0.05) in Y27632-treated mice. ROCK1 siRNAs also decrease the frequency of BrCa metastasis to hBCs and mouse skeleton, but to a lesser extent than Y27632. We demonstrate that ROCK's effects on tumor cell invasion/motility and growth may derive from regulating the actin-myosin cytoskeleton, modulating the c-Myc pathway by increasing the stabilization of c-Myc protein via phosphorylation, and targeting c-miRNAs. Coupled to increased c-Myc, expression of the c-Myc-regulated miR-17-92 cluster is shown here to be elevated in metastatic BrCa cells compared to non-metastatic cells, and diminished upon Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease BrCa cell invasion/migration in vitro and metastasis in vivo. Collectively, these findings suggest that augmented ROCK signaling contributes to BrCa metastasis in at least 3 ways, by 1) regulating the actin cytoskeleton and actin-myosin-dependent contractility; 2) targeting mRNAs controlled by the transcription factor c-Myc to potentiate cell growth and viability; and 3) modulating the expression of certain miRNAs, such as the c-Myc-regulated oncogenic miR-17-92 cluster. Inhibition of ROCK-mediated signaling appears to be a promising and potentially specific approach to suppress the occurrence of bone metastases in BrCa.

Citation Information: Cancer Res 2009;69(23 Suppl):A63.
Abstract A64: Calprotectin suppresses PIWIL1 and tumor growth in oral squamous cell carcinoma
Authors: Khammanivong Ali , Sorenson Brent , Guenther Brian , Ross Karen F. , Herzberg Mark C.
Published: May 2012

Calprotectin (a heterodimer of S100A8 and S100A9), normally expressed in oral epithelial cells, is down-regulated in oral squamous cell carcinoma (OSCC). Carcinoma cells over-expressing calprotectin show reduced tumor growth in vivo and in vitro and are morphologically more differentiated than carcinoma cells negative for calprotectin expression. Phosphorylation of histone H3 at serine 10 (Ser10) is a marker for increased cell division, CD24 is expressed on differentiated cells and PIWIL1 plays a role in cellular self-renewal.

Objective: To determine the molecular pathways involved in calprotectin-associated suppression of tumor growth and self-renewal.

Methods: To study the regulatory function of calprotectin in tumor growth, a non-calprotectin expressing human epithelial carcinoma cell line (KB) was transfected with either an empty vector (KB-EGFP) or vector containing S100A8 and S100A9 genes (KB-S100A8/A9). To study the loss of function, S100A8 and S100A9 expression was silenced in calprotectin-positive OSCC cell line (TR146) by small interfering RNA. Genome wide analysis of gene regulation was compared between calprotectin-negative and calprotectin-positive KB cells using Affymetrix HG-U133A microarrays and validated by quantitative real-time RT-PCR. Changes in protein expression were examined by immunofluorescence flow cytometry.

Results: Calprotectin expression in carcinoma cells increases phosphosphorylation of histone H3 (Ser10) correlating with a larger population at the cell cycle checkpoint G2/M and significant reduction in growths in vitro (KB-S100A8/A9 and TR146) and in vivo (KB-S100A8/A9). Among the cellular differentiation-associated molecules, expression of CD24 is significantly up-regulated, whereas, the expression of self-renewal factor PIWIL1 is reduced at both mRNA and protein levels when calprotectin is over-expressed.

Conclusion: Calprotectin may play essential roles in suppressing tumor growth by decreasing self-renewal capability and maintaining cellular differentiation. Loss of calprotectin expression in OSCC may lead to unregulated growth and de-differentiation. This study was supported by the NIH/NIDCR grant R01DE11831.

Citation Information: Cancer Res 2009;69(23 Suppl):A64.
Abstract A66: K12/SECTM1 promotes melanoma tumor progression via upregulation of integrin β3 expression
Authors: Wang Tao , Lopez-Coral Alfonso , Roesch Alexander , Huang Catherine , Cha In-Chul , Hwang Wei-Ting , Meyer Stefanie , Vogt Thomas , Herlyn Meenhard , Kaufman Russel E.
Published: May 2012

Metastatic melanoma is a leading cause of mortality in skin cancers with few therapeutic options. However, molecular mechanisms underlying melanoma tumorigenesis and metastasis remain poorly understood. Here, for the first time, we have determined a specific role of K12/SECTM1(SECTM1) in melanoma pathogenesis. Microarray analysis of melanoma revealed that SECTM1 is higher expressed in melanoma cell lines compared to melanocytes. We confirmed that SECTM1 was upregulated in most melanoma cell lines both at the transcriptional and post-transcriptional level. In addition, SECTM1 is highly expressed in patient melanoma lesions both in primary and metastasis samples. Furthermore, overexpression of SECTM1 in melanoma cell line WM3899 dramatically increased cell invasion in vitro, and promoted tumor growth and metastasis in vivo. Consistently, reduction of SECTM1 expression in melanoma cell line WM9 inhibited tumor growth and metastasis in vivo. Mechanistically, SECTM1 upregulated expression of integrin β3 (ITGβ3), thus contributing to the progressive phenotype of SECTM1 in melanoma. Hence, this study identified SECTM1 as a novel melanoma metastasis gene. We anticipate that our findings will provide new avenues in the pathogenesis of melanoma metastasis and that targeting of SECTM1 could be a novel approach to inhibit melanoma progression.

Citation Information: Cancer Res 2009;69(23 Suppl):A66.
Abstract A73: Understanding specificity in Notch receptor signaling
Authors: Bell Alisa , Okamura Heidi , Keane David , Perino Samantha , Lorusso Jeanine , Gyuris Jeno , O'Hagan Ronan
Published: May 2012

Notch signaling plays an integral role in cell growth and differentiation. Alterations in Notch signaling caused by mutations in receptors, amplification of pathway components, and increased expression of receptors and ligands have been shown to be important in tumor growth and maintenance. There are multiple Notch receptors and multiple ligands that can potentially interact to elicit a signal. Furthermore, many tumor cells express more than one Notch receptor. The complexity of these interactions and the potential specificity of resultant signals necessitate further understanding of the context of signaling to better determine how to treat Notch driven tumors. To this end, we have developed cell based reporter assays to investigate total Notch signal from cells stimulated by cell surface ligands in a co-culture system or by plate-bound ligands. In addition, we have developed assays to determine signal from individual Notch receptors. Human cancer cell lines that express multiple Notch receptors were engineered to express a Notch-dependent luciferase reporter. These cells were stimulated with plate-bound or cell surface ligands and assayed for activation of the Notch receptors. Subsequently, specific blocking of individual Notch receptors with antibodies was used to determine which specific receptors acted as effectors of Notch signal. Understanding both the array of Notch receptors on a particular cell type and more specifically the ability of each receptor to produce active signal will be important in determining how to effectively treat Notch driven tumors.

Citation Information: Cancer Res 2009;69(23 Suppl):A73.
Abstract A76: A novel in vivo model for pancreatic restructuring and suppression of transformation
Authors: DiRenzo Daniel , Shi Guanglu , Konieczny Stephen F.
Published: May 2012

Our studies are focused on the identification and prevention of the initial transformation events associated with pancreatic ductal adenocarcinoma (PDA) development, an extremely lethal malignancy with poor prognosis and ineffective treatments. PDA initiates through activating mutations in the KRAS proto-oncogene (KrasG12D) and recent studies from our laboratory have determined that targeted expression of an endogenous KrasG12D allele in pancreatic acinar cells is sufficient to produce PDA precursor lesions (PanINs). These results suggest that PDA arises via acinar cells undergoing a process of acinar-to-ductal metaplasia (ADM) which is characterized by a loss of acinar cell properties with the acquisition of duct-like characteristics. Several studies indicate that the process of ADM requires inactivation of the acinar-specific transcription factor Mist1, since Mist1 null (Mist1KO) mice display accelerated ADM and PanIN formation upon KrasG12D expression. Additionally, acinar cell polarity is disrupted in Mist1KO mice with diffuse zymogen granule organization and mis-localization of nuclei. These results suggest that Mist1 has a key role in suppressing PDA formation by maintaining the acinar cell differentiation program. To identify the downstream genes affected by the absence of Mist1, we have generated an inducible Mist1 transgenic mouse line (LSL-Mist1myc) in which constitutive Mist1 expression is activated through Cre-mediated recombination. When LSL-Mist1myc mice are crossed to Mist1Cre-ER mice, Mist1myc expression can be induced in acinar cells upon Tamoxifen (TM) addition. Analysis of Mist1Cre-ER/Cre-ER; LSL-Mist1myc mice confirmed that exogenous Mist1myc expression is detected as early as 6 hours post-TM. Time course studies in a Mist1KO background revealed that nuclear and zymogen granule organization can be restored within 24 hours of Cre activation. We conclude that Mist1 actively controls pancreatic acinar cell organization and that induction of Mist1 in Mist1KO mice leads to complete reorganization of pancreatic tissues. Our current efforts are focused on establishing how Mist1 negatively regulates ADM and PanIN formation and to identify Mist1 targets that negatively regulate Kras-induced pancreas metaplasia and transformation.

Citation Information: Cancer Res 2009;69(23 Suppl):A76.
Abstract A78: Sgt1 heterozygous mutation suppresses phenotypes of p53-null mice
Authors: Sakuraba Yoshiyuki , Ohta Satoshi , Kitagawa Katsumi
Published: May 2012

Sgt1 is an Hsp90 co-chaperone that is highly conserved among species, from yeast to humans. In vitro studies using siRNA and small-molecule inhibitors have indicated that Sgt1 is required for kinetochore assembly, proper localization of the spindle checkpoint proteins during mitosis, and genomic stability in humans. In vivo studies using knockout (KO) mice have revealed that kinetochore and spindle checkpoint genes are related to tumorigenesis. To examine the in vivo functions of Sgt1 gene in mammals, especially in tumorigenesis, we generated and analyzed Sgt1 KO mice by using a gene-trap line.

No Sgt1 homozygous KO mice were obtained from heterozygous intercrosses, even in E8.5 embryos, suggesting that Sgt1 homozygous mice are early embryonic lethal. In contrast, Sgt1 heterozygous mutants grew normally and did not exhibit any obvious phenotypes. We are now monitoring carcinogen-induced and spontaneous tumorigenesis in Sgt1 heterozygous mutants. We generated Sgt1 p53 double mutants in order to enhance the phenotypes associated with tumorigenesis. p53 is a major tumor suppressor gene, and p53 −/− KO mice usually die when they are approximately 20 weeks old, mainly because of lymphoma. Interestingly, Sgt1+/− p53−/− double mutants, especially females, survive significantly longer than p53−/− single mutants, suggesting that the Sgt1 heterozygous mutation suppresses p53−/− -induced tumorigenesis. In addition to the survival phenotype, the unequal gender ratio (more males than females) in p53−/− mutants was also overcome by the Sgt1 heterozygous mutation.

These results suggest that the Sgt1 heterozygous mutation suppresses the p53 KO phenotypes not only in tumorigenesis but also in embryonic development defects of females. We are currently examining whether the female infertility in p53−/− is also suppressed by the Sgt1 mutation. We will discuss how Sgt1 suppresses the p53 phenotypes at the meeting.

Citation Information: Cancer Res 2009;69(23 Suppl):A78.
Abstract B10: A novel mechanism of prostate cancer metastasis through the PDGF D/β-PDGFR axis
Authors: Najy Abdo J. , Saliganan Allen , Bonfil Daniel , Kim Hyeong-Reh C.
Published: May 2012

Cancer metastasis is a multi-step process permitting malignant cells to invade into a secondary niche. Prostate cancer has a high prevalence of bone metastasis where 90% of diagnosed cases exhibit metastatic bone lesions. Analysis of these metastatic tissues revealed that PDGFRβ (platelet-growth factor receptor β) is upregulated and activated in 80% of the cases. This receptor was thought to be stimulated by its classical ligand PDGF B. However, within bone metastatic prostate cancer, PDGF B is not detected, suggesting another ligand may exist for PDGFR β. Recently, a new PDGF family member, PDGF D, was identified and shown to bind to and activate PDGFR β. Using Oncomine data analysis and immunohistochemical analysis, we demonstrated upregulation of PDGF D in metastatic prostate cancer. To investigate the role of PDGF D in bone metastasis, we established several prostate cancer cell lines engineered to overexpress PDGF D. We observed an increase in prostate carcinoma cell migration as well as adhesion on osteoblast deposited matrix in response to PDGF D upregulation. Cell surface integrins are known to play a crucial role in cell migration and adhesion. Our studies revealed that PDGF D overexpression modulated α1 and α3 expression to mediate the cancer cell malignant phenotype. In the intratibial metastatic mouse model, PDGF D enhanced prostate cancer tumorigenesis and resulted in osteoblastic lesions. Consistent with our in vivo findings, we found that PDGF D supports osteoblast proliferation and differentiation in vitro. Taken together, we propose that PDGF D contributes to prostate cancer bone metastasis through tumor cell surface integrin modulation and stimulation of stromal components within the bone microenvironment.

Citation Information: Cancer Res 2009;69(23 Suppl):B10.
Abstract B11: Cyclin D1 overexpression antagonizes Notch inhibition in the esophageal squamous epithelium
Authors: Nakagawa Momo , Wong Gabrielle , Ohashi Shinya , Natsuizaka Mitsuteru , Vega Maria E. , Stairs Doug B. , Rustgi Anil K.
Published: May 2012

Cyclin D1 overexpression is common in esophageal squamous cell carcinoma (ESCC) and other squamous cell cancers. Notch signaling is implicated in cell proliferation and differentiation, and may act as a tumor suppressor. While squamous differentiation is maintained in ESCC and its precursor lesions, the role of Notch in esophageal carcinogenesis remains elusive. The role of genetic interactions between cyclin D1 and the Notch signaling were explored in this study. Immortalized human esophageal cells EPC2-hTERT (keratinocytes) were retrovirally transduced with tetracycline-regulatable (tet-cyclin D1. Notch was activated by the Notch1 intracellular domain and inhibited by dominant negative mastermind-like1 (DN-MAML1), a genetic pan-Notch inhibitor. Notch activity was assessed by a 8xCSL-luciferase reporter assay. Quantitative RT-PCR, Western blotting and immunohistochemistry were performed. The squamous epithelium was reconstituted in organotypic (3-D) culture, a form of tissue engineering. Notch activation led to inhibition of cell proliferation and terminal differentiation in EPC2-hTERT with a robust induction of involucrin (an early differentiation marker). By contrast, DN-MAML1 inhibited the ICN-induced CSL-dependent transcriptional activity and Notch target genes, including Hey1, Hes5 as well as involucrin. In organotypic culture, the parental EPC2-hTERT cells formed a well-organized stratified squamous epithelium. In organotypic culture, DN-MAML did not affect basal cell proliferation, but severely impaired squamous maturation and terminal differentiation with loss of filaggrin expression (a late differentiation marker) in the suprabasal layer. In 3-D culture, Cyclin D1 overexpression resulted in basal cell hyperplasia and antagonized the DN-MAML1 effect. Thus, cyclin D1 appears to stimulate cell proliferation while restraining the Notch-mediated squamous differentiation.

Citation Information: Cancer Res 2009;69(23 Suppl):B11.
Abstract B14: The novel function and trafficking mechanism of nuclear ErbB-2
Authors: Li Long-Yuan , Chen Hsiuyi , Hsieh Yi-Hsien , Wang Ying-Nai , Chu Hsiao-Ju , Hung Mien-Chie
Published: May 2012

We have demonstrated that ErbB-2 translocates into the nucleus through importin β1—mediated nuclear import by endosomal sorting, and transactivates transcription of cyclooxygenase-2 which has been known to be involved in tumor progression and metastasis, suggesting nuclear ErbB-2 may also contribute to the tumor malignancy. However, how the membranebound ErbB-2 can be released from the lipid bilayer and translocates into the nucleus; and the biological functions of the nuclear ErbB-2 are not yet completely understood. Here, we have identified novel ErbB-2-interacting nuclear proteins. The interplay between nuclear ErbB-2 and interacting protein was investigated, which may elucidate a novel function of nuclear ErbB-2 in contributing to tumorigenesis. In addition, ErbB-2 interacts with endoplasmic reticulum (ER) proteins, Sec61α, a subunit of Sec61 channel, and EDEM. Sec61α and EDEM are known to mediate retrotranslocation of misfolded proteins in the ER to the cytosol (called ER-associated degradation pathway). We have demonstrated that Sec61α and EDEM also play important roles in retrotranslocation of ErbB-2 to the cytosol and to the nucleus, suggesting part of ER retrotranslocation pathway is likely involved in nuclear trafficking of ErbB-2. [This work was supported by National Health Research Institutes NHRI-EX98-9603BC, National Science Council NSC952311-B-039-002 and NSC 97-3111-B-039-004 and Department of Health DOH97-TD-I-111-TM003 and DOH98-TD-I-111-TM002 (to LYL)].

Citation Information: Cancer Res 2009;69(23 Suppl):B14.
Abstract B28: Trisomy suppress tumorgenesis in the NP-cis tumor model
Authors: Yang Annan , Reeves Roger
Published: May 2012

Down syndrome results from trisomy for human chromosome 21 (chr21). People with DS have an increased risk of getting leukemia, but the likelihood of solid tumors might be reduced by contribution of several genes. We showed that trisomy in Ts65Dn “Down syndrome” mice reduces tumor number in the ApcMin model of intestinal cancer and that tumor number is inversely proportional to copy number of the Ets2 gene [Sussan TE, Nature 451, 73–75]. The current study provides evidence for additional dosage sensitive tumor repressor gene(s) in the cancer model, NP-cis. These mice carry a single copy of both the tightly linked Nf1 and p53 genes in cis. Tumors form by LOH. When NP-cis mice were crossed with Ts65Dn, trisomic offspring survived significantly longer than euploid. In contrast to the intestinal adenoma finding, introduction of a null allele of Ets2 in either trisomic or euploid mice (resulting in 2 or one copies of Ets2, respectively) had no effect on survival. This result demonstrates further dosage effects of genes trisomic in these mice and in Down syndrome. The lack of Ets2 effect in the NP-cis model is consistent with a protective mechanism that involves up-regulation of p53 by Ets2, thereby sensitizing trisomic cells to p53-mediated apoptosis. This mechanism would not work in NP-cis since tumors form due to loss of p53. In this regard, in vitro analysis of fibroblast cell lines from several genotypes showed that trisomic cells expressing more Ets2 than euploid were more sensitive to induction of apoptosis by camptothecin and showed a higher frequency of senescence. Other genes, like Endostatin, a cleavage product of Col18a1, and Rcan1 have been shown to inhibit angiogenesis in adosage dependent manner in xenograft models of tumor growth [Zorick TS, EJHG 9, 811-4, Baek KH, Nature 459, 1126-30]. In contrast to results from xenografted tumors, endogenous tumors formed in trisomic, NP-cis mice showed no reduction in angiogenesis. Our findings indicate that multiple mechanisms involving other genes trisomic in Ts65Dn mice likely contribute to reduced tumor incidence in Down syndrome.

Citation Information: Cancer Res 2009;69(23 Suppl):B28.
Abstract B3: Potential new mechanism whereby IGFBP-5 contributes to cancer progression: Binding of GADD34 and enhancement of eIF2α dephosphorylation
Authors: Ho Penny PY , Grkovic Steve , Firth Sue M.
Published: May 2012

Insulin-like Growth Factor Binding Protein (IGFBP-5) has a well-defined role in normal mammary gland physiology and has recently been shown to play an important role in the biology of breast cancer; however the IGF-independent mechanisms of action remain obscure. Over-expression of IGFBP-5, but not the addition of the exogenous protein, has been shown to inhibit the growth of breast cancer cells in vitro. This suggests novel intracellular mechanisms where IGFBP-5 interacts with unidentified binding partners and thereby modulates the proliferation and survival of breast cancer cells. To address this issue, we performed a yeast two-hybrid screen utilizing a normal human mammary gland cDNA library and full-length IGFBP-5 as the bait. The pre-eminent binding partner identified was Growth Arrest and DNA Damage-inducible protein 34 (GADD34), which assembles an eukaryotic initiation factor 2 alpha subunit (eIF2α) dephosphorylation complex at the endoplasmic reticulum (ER). We have confirmed that IGFBP-5 and GADD34 interact through coimmunoprecipitation experiments using ectopically expressed IGFBP-5 and GADD34 in mammalian cells. We have also shown that IGFBP-3, which belongs to the same protein family as IGFBP-5, interacts with GADD34 through coimmunoprecipitation studies. Furthermore, IGFBP-5 or IGFBP-3 expression was found to dramatically increase GADD34 protein levels as demonstrated through immunoblot analysis. However, IGFBP-2, another protein related to IGFBP-5, did not interact with GADD34 or increase its protein expression. Because GADD34-mediated eIF2α dephosphorylation is required to promote resumption of protein translation after periods of ER stress, we next examined eIF2α phosphorylation levels in the presence and absence of IGFBPs. Expression of IGFBP-5 or IGFBP-3 decreased the levels of phosphorylated eIF2α, suggesting that the interactions diminish the magnitude and/or duration of ER stress-induced inhibition of translation. Our results demonstrate not only a physical interaction between IGFBP-5 or IGFBP-3 and GADD34, but also show that these IGFBPs appear to increase the stability and activity of GADD34. This discovery suggests a novel role for IGFBP-5 and IGFBP-3 in the regulation of a protective mechanism induced by ER stress. Since ER stress is one of the hallmarks of solid tumor progression, by modifying the tumor response to hostile microenvironments, it is likely that these IGFBPs can have significant impact on cancer growth, survival, and apoptosis.

Citation Information: Cancer Res 2009;69(23 Suppl):B3.
Abstract B31: B-raf deletion and pharmacologic inhibition enhance K-ras-driven tumorigenesis
Authors: Murriel Christopher L. , Kenski Denise , Yuan Wenlin , Chen Honglin , Hatzivassiliou Georgia , Hoeflich Klaus , Wallin Jeffrey , Belvin Marcia , Liang Yuxin , Seshagiri Somasekar , Davis David P. , Johnson Leisa
Published: May 2012

The Ras GTPase family controls numerous downstream signaling cascades in response to signals that regulate cellular processes including proliferation and survival. While Ras is one of the most prevalent targets for gain-of-function mutations in human tumors, questions remain regarding how the Ras effector pathway functions in mutant K-ras-driven tumorigenesis. Since an important function of K-ras involves B-Raf activation within the canonical MAPK signaling pathway, we initiated a study to determine B-raf's role in the context of mutant K-ras-driven tumor promotion and maintenance.

We began by delivering adenovirus expressing the Cre recombinase to the lungs of genetically engineered mice possessing a conditional K-rasG12D allele (K-rasLSL-G12D) and either 0, 1 or both copies of the B-raf gene flanked by LoxP sites (B-rafCKO). This procedure results in expression of mutant K-rasG12D in the presence or absence of one or both B-raf alleles deleted within the mouse lung. Surprisingly, we observe that B-raf deletion significantly enhances lung tumor number and burden and decreases overall survival. When we used a highly specific small-molecule inhibitor that targets B-raf in a murine non-small cell lung carcinoma line harboring the K-rasG12D mutation, we observed an increase in cell proliferation and soft agar colony formation. Further investigation revealed that treating K-rasG12D expressing cells with the B-raf inhibitor enhanced MEK and Erk phosphorylation. Therefore, our data suggests that while B-raf deletion does not inhibit K-ras-driven tumor initiation and disease progression, its presence may play a pivotal role in establishing negative feedback regulation of constitutive mutant K-ras activity.

Citation Information: Cancer Res 2009;69(23 Suppl):B31.
Abstract B35: Dynamics of MHC class II tumor antigen presentation is critical for effector antitumor CD4+ T-cell generation and tumor rejection
Authors: Bonorino Cristina , Maito Fabio , Souza Ana Paula
Published: May 2012

Absence of CD4+ T cell help leads to defects in CD8+ T cell memory responses.Although this phenomenon is well characterized in infectious immunity, little is known about the generation of anti-tumor CD4+ T cell responses in vivo, or even how relevant is T cell help for tumor elimination. CD4+ T cells appear differ from CD8+ T cells in their requirements for differentiation, the dynamics of encounter with antigen being critical in this process. We analyzed the relevance of MHC class II antigen presentation for tumor rejection, developing a B16EaRFP line to study class II restricted tumor antigen presentation and the cognate CD4+ T cell help in vivo. We found that, in a tumor heavily infiltrated by dendritic cells (DC), a progressive decline in different cell subpopulations is observed in draining lymph nodes (DLNs), suggesting an interruption of local cell traffic. Artificial increase in precursor frequency of tumor specific CD8+ T cells by adoptive transfer of naïve OT-I cells in C57Bl/6 mice injected with B16OVA tumor was able to delay tumor growth, but was not sufficient to block it completely. While tumor antigen presentation in class II molecules was detected in the DLN, and could be increased by delivery of necrotic or apoptotic tumor cells, tumor specific CD4+ T cells showed proliferation arrest in the DLN. Tumor growth was blocked only when dendritic cells (DCs) were activated by intra-tumoral LPS injection, reestablishing cell traffic to the LN, resulting in expansion of INF-γ producing anti-tumor CD4+ T cells. Our results suggest that CD4+ T cells are a major target of tumor mediated antigen presentation interruption, and that CD4+ T cell help is necessary for rejection of an established tumor. These observations have major implications for the design of cancer immunotherapy strategies.

Citation Information: Cancer Res 2009;69(23 Suppl):B35.
Abstract B38: Silencing of mortalin, the mitochondrial hsp70, as a novel anticancer treatment strategy
Authors: Masarwa Muhammad , Donin Natalie , Ziporen Lea , Fishelson Zvi
Published: May 2012

A handful of monoclonal antibodies have recently been introduced in treatment of hematopoietic malignant neoplasms and solid tumors. The complement system, an important arm of the immune system, is activated following anti-tumor antibody treatment. Complement proteins assemble to form channel-like pores called MAC (Membrane Attack Complexes) that inflict damage to target cells. Unfortunately, cancer cells have many defensive strategies against antibody-dependent complement-mediated cytotoxicity, including MAC shedding by exocytosis. Our recent results have implicated mortalin, the mitochondrial heat shock protein 70, in the removal of the MAC from the cell surface. Mortalin is known to have tumor promoting and anti-apoptotic effects. Our research hypothesis states that mortalin is a vital protector of tumor cells from immune damage. In order to examine this hypothesis, we set up two mouse models of cancer, of K562 erythroleukemia cells in nude mice and EL4 T-lymphoma cells in C57BL/6 mice. Mortalin siRNA was administered to tumor bearing mice with or without concomitant tumor directed antibodies. First, in vitro experiments showed that mortalin siRNA acts synergistically with anti-cancer antibodies in lysis of tumor cells. In vitro mortalin silencing in tumor cells by siRNA and then injection of the cells into mice demonstrated reduced tumor growth, relative to cells pretreated with scrambled siRNA. Mortalin silencing in vivo by intratumoral or intravenous injection of mortalin siRNA led to a significant decrease in tumor growth rate. Furthermore, mortalin siRNA also reduced tumor blood supply and caused tumor necrosis. Growth rate of EL4 and K562 tumors was also significantly inhibited by injection of the mortalin inhibitor MKT-077. Mortalin inhibition in tumors may thus be regarded as a novel adjuvant treatment to immunotherapy.

Citation Information: Cancer Res 2009;69(23 Suppl):B38.
Abstract B4: Specificity of ▵Np63α-repressed target genes is determined by changes in p63-interacting proteins
Authors: Ramsey Matthew R. , Ory Benjamin , He Lei , Ellisen Leif W.
Published: May 2012

The transcription factor p63 has been reported to have many functions, including regulation of proliferation, suppression of apoptosis, suppressing differentiation, and maintaining stem cell self-renewal. These various functions are carried out by activation or repression of a diverse set of target genes. However, the mechanisms that lead to selective regulation of target genes have remained elusive. In human Squamous Cell Carcinoma (SCC) ▵Np63α is expressed at high levels, and serves to block TAp73-mediated induction of pro-apoptotic target genes such as Puma. Our previous work has shown that ▵Np63α and TAp73 physically associate, and that p63 binds to the promoter of TAp73-activated target genes such as Puma. We have constructed a panel of p63 point and deletion mutants in order to examine the role of different functional domains in p63-mediated repression. Using Luciferase reporter assays and a physiologic reconstitution model with mutant p63 in SCC cells, we have identified mutants defective in suppression of apoptosis and TAp73 target genes. Examination of point mutants defective in DNA binding, but able to bind to TAp73 normally has revealed a requirement for DNA binding in suppression of TAp73 target gene activation and apoptosis, suggesting that ▵Np63α-TAp73 heteromeric complexes on DNA are repressive. We have also performed a si-RNA based screen for regulators of p73 activity, and identified Daxx as a p63-interacting protein involved in the repression of p73 activity in SCC cells. These data demonstrate that ▵Np63α functions as part of a repressive complex to regulate pro-apoptotic target genes in human SCC. Thus, ▵Np63α plays a crucial role in maintaining the survival of SCC cells and promoting tumorigenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):B4.
Abstract B45: Epigenetic signature of human breast cancer subtypes
Authors: Badola Sunita , Parker Alexander , Campbel Mara , Boedigheimer Michael , Grubinska Barbara , Turci Susan , Tsui Kim , Damore Michael A. , Wilson Cindy A.
Published: May 2012

Gene expression profiling has been used to extend classification of human breast cancers into five main sub-types having distinct clinical outcomes. The established prognostic markers ER, PR and HER-2 remain prominent components of this refined scheme that delineates luminal (luminal A, luminal B and HER-2) from non-luminal (basal/BRCA1 mutant and normal-like) breast cancers. Gene expression combined with immunohistochemical localization of various tumor markers in the normal mammary gland support the hypothesis that luminal and non-luminal breast tumors originate from different cell types. In the current study we explore epigenetic determinants of the different breast cancer sub-types using the genome-wide DNA methylation patterns (Illumina Infinium) of fifty human breast cancer cell lines. Principal component analysis of baseline gene expression profiles (Agilent) showed a clear separation between the luminal and non-luminal sub-types in agreement with previously published studies using a largely overlapping panel of breast cell lines. Comparative marker selection analysis using the GenePattern analysis package was then performed independently on the DNA methylation and gene expression datasets. When the results of these two analyses were compared, a statistically significant reciprocal overlap of the selected markers was observed. These results strongly suggest that a large fraction of the most robust luminal gene expression classifiers are silenced by promoter methylation in non-luminal cells, and conversely that many of the best non-luminal classifiers are silenced in luminal cells. The classifier genes indentified in the breast cancer cell line panel include the most highly referenced luminal (KRT8, ESR1, AR, TFF1, TFF3, TOB1 and XBP-1) and non-luminal markers (DSC3, VIM, SFRP1, KRT5, KRT16, KRT17, and RUNX3 that have been used to classify primary breast cancers. These striking differences in DNA methylation patterns strengthen the hypotheses that luminal and non-luminal sub-types arise from different precursor cells and that their transcriptional programs may be largely established during the process of cell specification in the mammary epithelial bi-layer. Since CpG island methylation is stable and robustly preserved in archival tissues, it is likely that a methylation signature comprised of a few key genes could be used to, for example, identify poor-prognosis, basal-type breast cancers. It is also possible that such a test would be more quantitative, accurate and reproducible than the “triple-negative” diagnosis currently based on the absence of immumohistochemical staining for ER, PR and HER-2.

Citation Information: Cancer Res 2009;69(23 Suppl):B45.