bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4517: HDAC inhibitors suppress p53 expression through transcriptional repression: Implication for therapy against metastatic breast cancer
Authors: Zhao Lisa Y. , Li Qiang , Seto Edward , Liao Daiqing
Published: February 2011

Cancer specific mutations of the p53 tumor suppressor gene are the most prevalent genetic lesions that underpin breast cancer pathogenesis and progression. In most cases, mutations result in a single amino acid substitution in the DNA-binding domain of p53. Such missense mutant forms of p53 accumulate at high levels in tumor cells. Recent advances demonstrated that missense p53 mutants have a critical role in driving cancer metastasis. Thus elimination of mutant p53 can potentially prevent metastatic progression of breast cancer. Inhibitors of histone deacetylases (HDACi) have proven anticancer effects and vorinostat, an FDA approved HDACi, has been in clinical use for treating certain malignancies. HDACi shapes epigenetic landscapes by restoring histone acetylation that is often suppressed in breast and other cancer types. One consequence of the restoration of histone acetylation is reactivation of tumor suppressors and other genes. Other effects of HDACi include modulation of posttranslational acetylation of non-histone proteins, which can influence their biochemical functions, resulting in a net effect of enhanced cell cycle arrest and apoptosis. Interestingly, HDACi profoundly suppresses the expression levels of p53. We found that this suppression occurs irrespectively of the mutational status of p53, but surprisingly only in certain types of cancer cells such as breast cancer. We further showed that the expression of exogenous p53 under a heterologous promoter was not affected by HDACi. These results suggest that HDACi might downregulate p53 through gene repression. To further test this idea, we assessed the effects of HDACi treatment and ectopic expression of classical HDACs (Classes I, II and IV) on luciferase reporter gene under the control of the p53 promoter. Among the 11 classical HDACs, ectopic expression of HDAC7 and HDAC10 seem to upregulate the reporter gene. We are currently investigating how these HDACs interact with the p53 promoter and what transcription factors and corepressors are involved in regulating p53 expression. Additionally, we are studying roles of depleting mutant forms of p53 by HDACi in suppressing migration, invasion and metastasis of breast cancer cells and assessing the efficacy of HDACi in conjunction with p53 gene therapy for treating breast cancer in a mouse model. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health, Stop! Children's Cancer, Inc. and the NIH.)

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 4517.
Abstract 452: The hypoxic response is differentially regulated in small cell lung carcinoma (SCLC) cells as compared to non-SCLC cells
Authors: Persson Matilda , Johansson Martin , Monsef Nastaran , Planck-Sturegård Maria , Påhlman Sven , Pettersson Helen M.
Published: February 2011

Here we study the adaptive capacity of small cell lung carcinoma (SCLC) cells to hypoxia and the importance of activation of hypoxia-inducible factor (HIF) transcription factors in these cells. SCLC is an extremely aggressive tumor form and the 5-year survival rate for these patients is only approximately 9-15%. Regions of hypoxia (∼1% oxygen) often appear in solid tumors and are associated with aggressive tumor behaviour and poor outcome in several tumor forms. Tumor cells adapt to a hypoxic environment by changing the transcription of genes involved in a variety of processes, like angiogenesis, cell survival, and metabolism. This adaptive response is primarily regulated by the transcription factors HIF-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others neuroblastoma, breast carcinoma and lung adenocarcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia.

We investigated cell survival in a panel of SCLC cell lines cultured at different oxygen pressures. In these cells we also studied expression levels of well known hypoxia-driven genes using quantitative real-time PCR. Quantitative real-time PCR, Western blot and immunohistochemistry were used to characterise the expression of HIF transcription factors in SCLC cells as well as in SCLC specimens. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison.

Since tumor hypoxia correlates with poor prognosis it is important to elucidate how tumor cells adapt in response to hypoxia. Our results show that SCLC cells survive well at hypoxia, although the proliferation is somewhat reduced with lower oxygen pressure. Even though the SCLC cells have a high adaptive capacity, only a moderate up-regulation was observed for the investigated hypoxia-regulated genes at 1% oxygen. In addition, the expression patterns of HIF-1α and HIF-2α differ in SCLC as compared to non-SCLC, indicating that a “typical” adaptive response to hypoxia does not occur in SCLC cells. Based on these results a gene expression profile of hypoxic SCLC cells using microarray analyses are performed to further characterise the hypoxic adaptive response.

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 452.
Abstract 4526: Suppression of Alox5 gene is involved in DZNep induced apoptosis in multiple myeloma cells
Authors: Xie Zhigang , Bi Chonglei , Cheong Lip Lee , Chng Wee Joo
Published: February 2011

The 3-Deazaneplanocin A (DZNep), one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. It was found that DZNep effectively depletes EZH2 levels and inhibits trimethylation of lysine 27 on histone H3. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines(8226, H929, KMS11, KMS12BM, KMS18, MM1S, OPM-2 and U266). We then attempted to identify molecular mechanisms of sensitivity to DZNep. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by qRT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Furthermore, down-regulation of ALOX5 by RNA interference can also induce apoptosis in H929. Since it was reported ALOX5 can regulate IL-6 mRNA level, we also determined IL-6 and IL-6R expression by qRT-PCR. As expected, IL-6 and IL-6R mRNA levels were decreased (>2-fold) in H929 upon DZNep treatment. May-Grunwald-Giemsa staining demonstrated that treatment with DZNep also dose dependently induced nuclear and cytoplasmic morphologic features of apoptosis in H929. These findings in vitro warrants further investigation of DZNep for the treatment of MM in vivo.

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 4526.
Abstract 4533: In vivo activation of the p53 pathway leading to tumor regression by a novel and potent HDM2 antagonist SCH 1450206
Authors: Wang Yaolin , Wang Xiaoying , Lipari Philip , Ponery Abdul-Samad , Liu Suxing , Mirza Asra , Liu Yinghui , Bogen Stephane , Ma Yao , Doll Ronald , Shipps Gerald , Liu Ming , Seidel-Dugan Cynthia , Hicklin Daniel J. , Bishop Walter Robert
Published: February 2011

The HDM2-p53 protein-protein interaction is well characterized through X-ray crystallography. Disrupting this HDM2-p53 protein-protein interaction by a small molecule would therefore release the p53 from the negative inhibition of HDM2 and restore its anti-tumor activities. Peptides, antisense oligonucleotides and small molecules have been identified which disrupt this interaction and result in the stabilization of p53 protein and activation of its downstream targets. We have recently discovered a novel, potent HDM2 antagonist (SCH 1450206) with mechanism-based activity both in vitro and in vivo. Oral administration of SCH 1450206 as a single agent resulted in tumor regression in the SJSA-1 osteosarcoma xenograft model without any observable toxicity. Analysis of the pharmacodynamic markers demonstrated that the anti-tumor activity of SCH 1450206 correlated with the robust activation of p53 pathway in vivo. In addition to its single agent anti-tumor activity, combination of HDM2 antagonist SCH 1450206 with various cytotoxics resulted in further tumor growth inhibition in various human cancer xenograft models. The activation of p53 pathway in vivo targets preferentially to tumor tissues compared to high proliferating and radio sensitive organs of the mouse at the efficacious dose. The lack of single agent toxicity at the efficacious dose and schedule would potentially allow the combination of this type of HDM2 antagonist with other anti-cancer agents at full dose in the clinic.

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 4533.
Abstract 4535: MDM2 inhibitor MI-319 in combination with cisplatin/oxaliplatin is an effective treatment for pancreatic cancer independent of p53 function
Authors: Azmi Asfar S. , Philip Philip A. , Aboukameel Amro , Banerjee Sanjeev , Wang Zhiwei , Wang Shaomeng , Yang Dajun , Sarkar Fazlul H. , Mohammad Ramzi M.
Published: February 2011

Pancreatic cancer (PC) is considered to be among the most intractable malignancies as it is refractory to chemotherapy and radiation. Among the various genetic alterations in PC, mutation in the tumor suppressor p53 (mut-p53) gene has been reported to be about 50%. In the remaining 50%, the p53 is normal, but its function is inhibited by MDM2 protein which plays a key role in promoting ubiquitination and proteasomal-dependent degradation of p53. An attractive approach to p53 activation is the use of MDM2 inhibitors that disrupt the MDM2-p53 interaction and restore the p53 function. However, these small molecule inhibitors (SMIs) are not effective against tumors with mut-p53. Our group has designed a new class of SMI of MDM2 that is different from Nutlin-3. Here we are reporting, for the first time, that MI-319 can induce cell growth inhibition and apoptosis in PC irrespective of p53 mutational status when combined with cisplatin/oxaliplatin. MI-319-cisplatin or oxaliplatin combination synergistically suppressed cell growth (MTT CI<1), colony formation (clonogenic assay) and induced apoptosis in both mut-p53 Colo-357, BxPC-3 and Panc-28 or wt-p53 Capan-2 cells. Western blot analysis and siRNA silencing studies in mutant as well as p53 null cells highlighted a mechanism involving p73 which is under the regulation of MDM2, and unlike p53, it is rarely mutated in PC. In order to substantiate its in vivo relevance we tested this potent combination in mice xenograft models carrying Capan-2 and BxPC-3 tumors. 4 groups, each containing 8 animals were treated either with; Control (Vehicle only), MI-319 treated 200mg/Kg orally twice a day for three weeks, Cisplatin treated 4 mg/kg (i.v.) twice a week for two weeks and combination treated (MI-319 200 mg/Kg orally + Cisplatin 4 mg/kg). The combination effectively reduced tumor growth in both wt-p53 and mut-p53 tumor xenograft models (50% Capan-2 animals were tumor free). Consistent with our in vitro results, remnant tumor tissue analysis showed up-regulation of p73 and the cell cycle regulator p21. In conclusion, this study highlights a new role of MDM2 inhibitors in combination with cisplatin or oxaliplatin, and thus warrants further clinical investigation in human pancreatic tumors containing both wt-p53 and mut-p53.

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 4535.
Abstract 4537: SHLP6: A novel, naturally occurring, mitochondrial peptide has therapeutic and prognostic potential in prostate cancer
Authors: Cobb Laura , Nakamura Hiromi , Cohen Pinchas
Published: February 2011

Mitochondrial DNA (mtDNA) mutations have been demonstrated in prostate cancer, particularly at the 16S rRNA region; and abnormal mRNA transcripts from that locus have been described. We and others showed that a novel open reading frame (ORF) within the mitochondrial 16S rRNA region, encoding for a 24 AA peptide called humanin, is a potent cell-survival and metabolic factor (PNAS 2003, PLoS-ONE 2009).

Hypothesizing that additional bioactive peptides may be encoded from this region we have identified an additional six ORFs that encode peptides transcribed from within the 16s rRNA gene in the mtDNA, which we have named small humanin-like peptides, or SHLPs. SHLP1-5 are functionally analogous to humanin, and act as potent survival factors.

Intriguingly an additional peptide, SHLP6, functions in a completely opposing manner. We demonstrate that endogenous SHLP6 is expressed in serum and in multiple tissues, including the prostate, and that its expression declines with age. In addition, the addition of exogenous SHLP6 promotes apoptosis in prostate cancer cell lines including 22RV1, LNCaP and DU145. Analysis of gene and protein expression of SHLP6 treated cells reveals modulation of cell cycle and apoptosis genes and a dramatic inhibition of VEGF expression.

To assess the in vivo potency of SHLP6, we treated 22RV1 xenografts in SCID mice with SHLP6 for 1 week, and observed a potent inhibition of CaP xenograft growth and angiogenesis in vivo. In addition, we show that SHLP6 expression is decreased in preliminary staining of human prostate cancer, compared with normal prostate tissue.

Thus, our findings not only introduce the revolutionary concept that a previously unrecognized family of peptides is produced from the mitochondria, challenging existing paradigms about mitochondrial function, but also demonstrate that SHLP6 is altered in prostate cancer, and may serve as a novel therapeutic and diagnostic agent in this disease.

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 4537.
Abstract 455: Hypoxia induced carbonic anhydrase IX is essential for the growth and metastasis of breast tumors
Authors: Lou Yuanmei , McDonald Paul C. , Oloumi Arusha , Chia Stephen , Kyle Alastair , dem Keller Ulrich auf , Fotovati Abbas , Gray Virginia , Leung Samuel , Huntsman David , Clarke Blaise , Waterhouse Dawn , Bally Marcel , Roskelley Calvin , Overall Christopher , Minchinton Andrew , Supuran Claudiu T. , Dedhar Shoukat
Published: February 2011

Metastasis of primary malignancies is a multi-step process and remains the principal cause of cancer deaths. Carbonic anhydrase IX (CAIX) is a hypoxia inducible protein and a poor prognostic marker for several types of cancer, including breast cancer. However, the functional role of CAIX in the metastatic progression of breast cancer is unclear. Here, we have investigated its role in the growth and metastasis of breast tumors. Orthotopic mouse mammary tumors derived from metastatic 4T1 and 66cl4 cells or non-metastatic 67NR cells were examined for levels of proliferation (BrdU), hypoxia, (pimonidazole), perfusion (DiOC7), vasculature (CD31), apoptosis (TUNEL) and lymphangiogenesis (LYVE-1). Metastatic 4T1 and 66cl4 tumors expressed a hypoxia gene signature and were characterized as being poorly vascularized, with high levels of hypoxia. Large numbers of apoptotic cells and well developed intratumoral lymphatic vessels were also evident. Inhibition of expression of CAIX in the metastatic 4T1 cells by stable expression of short hairpin RNA (shRNA) resulted in cell death and reversal of extracellular acidosis in hypoxia in vitro, dramatic regression of tumors in vivo, and inhibition of metastasis. These properties were rescued by constitutive expression of human CAIX. Treatment of mice harboring 4T1 tumors with a novel CAIX-specific inhibitor resulted in significant inhibition of tumor growth. Interrogation by immunohistochemistry of a large (3992 patient samples) primary breast tumor tissue microarray showed that CAIX expression was significantly associated with worse distant relapse free survival (p<10−16) and was most prominent in the basal breast cancers (51%). Our data show that CAIX-mediated function is required for the survival and metastasis of hypoxic breast tumors, and suggest that CAIX is a promising therapeutic target for metastatic breast cancer.

This work was supported by the Canadian Breast Cancer Research Alliance, with special funding from the Canadian Breast Cancer Foundation.

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 455.
Abstract 4551: 111In-NLS-Nimotuzumab: A potent auger electron-emitting radiotherapeutic agent for EGFR-overexpressing breast cancer
Authors: Fasih Aisha , Reilly Ray , Tikhomirov Ilia
Published: February 2011

Objective: To evaluate the cytotoxicity of nimotuzumab (Nmab), a monoclonal antibody specific for EGFR labeled with 111In and modified with nuclear localization sequence (NLS) peptides for importation into the nucleus of human breast cancer (BC) cells, where the emitted Auger electrons are most damaging to DNA and lethal.

Methods: NMab was derivatized with sulfo-SMCC to introduce maleimide groups for reaction with NLS-peptides (CGYGPKKKRKVGG) and benzyl-DTPA (bn-DTPA) for complexing 111In. NLS substitution was quantified by a band shift on SDS-PAGE and bnDTPA substitution by an 111In-binding assay. The immunoreactivity (Kd) of NLS-Nmab was determined by the displacement of binding of 111In-Nmab to MDA-MB-468 BC cells (106 EGFR/cell). Internalization and nuclear importation were determined in MDA-MB-468 and MCF-7 (104 EGFR/cell) BC cells by subcellular fractionation at 37 oC and 4 oC up to 3 h. Clonogenic survival (CS) was measured in MDA-MB-468 and MCF-7 cells exposed to 2 to 40 nM (450-900 KBq/mL) of 111In-NLS-Nmab for 3 days.

Results: Nmab was modified with 8.5±0.2 NLS and 2.1±2.1 bn-DTPA groups. The Kd for binding of 111In-NLS-Mab to MDA-MB-468 cells was 9.8±0.9 nM. NLS-Nmab was labeled with 111In to a radiochemical purity of 99.2±0.1% (specific activity 350±9 MBq/mg). The proportion of cell-bound 111In internalized into MDA-MB-468 cells at 3 h was 27.8±0.8% at 37 oC and 4.4±0.2 at 4 oC. Nuclear uptake was 31.2±2.6% for 111In-NLS-Nmab and 16.0±0.6% for 111In-Nmab (P<0.001). Binding and nuclear uptake were 20-fold and 6-fold lower, respectively for MCF-7 cells. The CS of MDA-MB-468 cells exposed to 111In-NLS-Nmab (20 nM) was 2.8±0.2% compared to 21.0±5.3% for 111In-Nmab (P=0.004) and 60.0±7.0% for Nmab (P=0.001). The CS of MCF-7 cells exposed to 111In-NLS-Nmab (40 nM) was 82.6±6.4% vs. 80.0±1.6% for 111In-Nmab (p>0.05) and 95±19.4% for Nmab (p>0.05).

Conclusion: 111In-NLS-Nmab is a promising targeted radiotherapeutic agent for EGFR-overexpressing BC as evidenced by a 20-fold increased toxicity towards MDA-MB-468 cells compared to Nmab but lack of toxicity towards MCF-7 cells with 10-fold lower EGFR expression similar to that on most normal epithelial cells. Supported by the Canadian Breast Cancer Research Alliance (Grant 019513).

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 4551.
Abstract 4553: Novel bispecific 111In-labeled immunoconjugates (bsICs) for imaging HER2/HER3 heterodimerization in breast cancer
Authors: Eva Razumienko J. , Reilly Raymond M.
Published: February 2011

Objective: Our objective was to construct novel bsICs capable of binding HER2 and HER3 labeled with 111In for imaging HER2-HER3 heterodimerization in breast cancer (BC) possibly through higher avidity for dimeric vs. monomeric complexes.

Methods: bsICs were composed of trastuzumab Fab recognizing HER2 linked to a 7.5 kDa HER3-binding peptide of heregulin-β1 (pHRG). Fab were produced by digestion of trastuzumab IgG with papain. Fab were modified with Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or related analogues with polyethyleneglycol (PEG) spacers (SM-PEG12-NHS and SM-PEG24-NHS) to introduce maleimide groups for cross-linking to pHRG thiolated with Traut's reagent. bsICs were derivatized with diethylenetriaminepentaacetic acid (DTPA) for labeling with 111In. The ability to independently bind HER2 or HER3 was determined in competition assays using 111In-bsICs on cells expressing HER2, HER3 or both receptors competed with a 25-fold excess of Fab, pHRG or combined ligands.

Results: Conjugation of Fab to pHRG was confirmed by SDS-PAGE, Western blot and size-exclusion HPLC. bsICs were labeled with 111In to a radiochemical purity of 98.9 ± 0.2% (specific activity 375 ± 14 MBq/mg). Insertion of a PEG spacer between Fab and pHRG was required for displacement of binding of 111In-bsICs to HER3 but not HER2. For 111In-bsICs with the longest spacer (PEG24), the binding to SKBR-3 cells (HER2+/HER3+) was reduced to 32.7 ± 0.7%, 85.9 ± 1.0% or 30.4 ± 1.6%, respectively by Fab, pHRG or both ligands. The binding to SKOV-3 cells (HER2+/HER3-) was reduced to 16.7 ± 0.8%, 88.8 ± 5.3% or 13.4 ± 0.1% by Fab, pHRG or both ligands. The binding to MDA-MB-468 cells (HER2-/HER3+) was reduced to 98.5 ± 1.5%, 68.8 ± 0.3% or 64.8 ± 4.5% by Fab, pHRG or both ligands. Less efficient competition for HER3 was observed for the PEG12 spacer and no competition for 111In-bsICs without a spacer.

Conclusion: 111In-bsICs composed of trastuzumab Fab and pHRG bound specifically to HER2 and HER3 when an appropriate length PEG spacer was inserted between these two binding moieies. These 111In-bsICs may be useful for imaging HER2-HER3 heterodimerization in BC. Supported by the Ontario Institute of Cancer Research (1 mm Challenge) and the Canadian Breast Cancer Foundation (Ontario Branch).

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 4553.
Abstract 456: Adaptation of cancer cells to hypoxic environments for tumor progression
Authors: Mizushima Tomoko , Kawai Kenji , Nishime Chiyoko , Ohmura Mitsuyo , Hayakawa Eri , Matsuyama Masahiro , Asakura Keisuke , Izumi Yotaro , Suemizu Hiroshi , Ohnishi Yasuyuki , Nakamura Masato , Suematsu Makoto , Wakui Masatoshi
Published: February 2011

There have been a number of reports mentioning the significance of adaptation of cancer cells to hypoxic environments in tumor growth, invasion, and metastases. However, understanding of molecular aspects based on in vivo evidence still remains insufficient. We established an in vivo tumor progression model by transfer of human colorectal cancer HCT116 cells cultured under hypoxia to NOD/Shi-scid, IL-2Rγnull (NOG) mouse recipients. The hypoxic culture resulted in invasive proliferation of subcutaneous tumors. Immnohistochemical staining for molecular markers, such as E-cadherin, vimentin and BCRP1, demonstrated that adaptation of HCT116 cells to hypoxia was associated with increased frequencies of epithelial-mesenchymal transition and cancer stem cell-like appearance. To investigate the molecular basis of such effects of hypoxia on tumor progression, biological and molecular profiles of HCT116 cultured under hypoxia were analyzed. The hypoxic culture resulted in slowing cell cycling and increase in the side population cell frequency, suggesting induction of stemness. Capillary electrophoresis-mass spectrometric analysis revealed altered metabolism in hypoxic HCT116 cells, which appeared to be associated with adaptation to hypoxia. Gene expression profiling of the cultured cells under pharmacological inhibition of p53, which is not mutated in HCT116 cells, presented evidence for dependence of the adaptation to chronic mild but not acute severe hypoxia on p53 function. Our results provided insights into molecular basis of tumor progression critically directed by hypoxia.

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 456.
Abstract 4577: Comparative proteomic biomarker screening in laser microdissected tissues from colon adenoma and cancer
Authors: Meyer Markus , Spörl Monika , Harasym Agnieszka , Spangenberg Jörg , Unger Florian , Juhl Hartmut , David Kerstin A.
Published: February 2011

Colorectal (CRC) malignancies rank world wide at the third place for tumor diseases and account for an annual mortality rate of 492 000 cases. Although several molecular events are known to be involved in the transition from normal tissue to adenoma and finally to undifferentiated carcinoma it remains a challenge to discover new and more reliable biomarkers for diagnosis, prognosis and prediction of outcome. Towards this end a study was designed to identify potential biomarkers which are associated with the molecular events leading from epithelial adenoma to the early stages of carcinoma.

A new biomarker discovery strategy was developed to combine the cell specificity and selectivity of laser capture microdissection (LCM) with the resolution power and sensitivity of liquid-chromatography (LC)-matrix-assisted-laser-desorption/ionization mass spectrometry (LC-MALDI-MS). We carefully selected a group of closely matched patients (n = 10 for each group) afflicted with epithelial adenoma (high dysplasia) or early stages of carcinoma (stage I) and used the derived normal as well as the matched tumor tissue samples to reveal protein expression differences. According to this LC-MALDI-MS strategy microdissected cells (around 10 000 cells) were lysed and extracted proteins were digested with Trypsin. Obtained peptides were separated by capillary reversed phase HPLC (Agilent). The resulting LC-fractions (300) were spotted on prespotted AnchorChip targets (PAC, Bruker) and tryptic fragments subsequently detected by reflector MALDI-MS (ultraflex III, Bruker) measurements. This generated between 5000-7000 ion signals ranging from m/z 800 to 4000.

Differential peptide analysis was then performed with the goal to discover robust and significant expression differences between patient groups. Therefore, only m/z ions displaying a minimum twofold difference and a p-value of 0.01 between the tissue samples were considered for further analysis. The selected peptides were subsequently fragmented by MS/MS experiments to reveal their primary sequence and protein identity. Our targeted biomarker discovery approach resulted in the identification of more than 30 biomarker candidates which act in diverse cellular functions and can now be linked to early events (e.g. adenoma vs. normal tissue) or later events (e.g. carcinoma vs. normal) of tumor progression. Currently these biomarkers are validated using antibody based assays to further analyze their potential as markers in a clinical setting.

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 4577.
Abstract 4578: Comparative subcellular proteomics study of hepatocellular carcinoma cell lines differentially expressed alpha fetoprotein: Annexin A2 could serve as a potential histological and serological biomarker
Authors: Zhao Xiaohang , Sun Yulin , Cai Jianqiang , Qiao Yuanyuan , Zhou Lanping , Bi Xinyu , Liu Fang , Bai Xiaofeng
Published: February 2011

Hepatocellular carcinoma (HCC) is a common tumor worldwide with an extremely poor prognosis. Alpha-fetoprotein (AFP) is the only available HCC serological biomarker and has limited effect at early stages. Previous work has proven that AFP-producing and -nonproducing HCC cells have different gene/protein expression profiles. To identify additional candidates that might aid in HCC diagnosis, in addition to giving a better understanding of the mechanisms underlying hepatocarcinogenesis, subcellular fraction proteins from multiple liver cancer (HepG2, Hep3B and SK-HEP-1) and normal cells (HL-7702) were separated and identified using a 2-DE/MALDI-TOF-TOF approach. In total, 50 differentially-expressed proteins between liver cancer and normal liver cells were identified from different fractions including the cytosol, membrane/organelles and nuclear proteins. Among them, the tumor-related protein Annexin A2, was verified to be significantly upregulated in HCC tissues (63.5%, 80/126) especially in AFP-normal cases based on large-scale clinical specimens. Serum levels of Annexin A2 measured by an established ELISA assay were found to be significantly elevated in HCC patients compared with healthy individuals. The serum concentration was also correlated with histological grade. Combined use of AFP and Annexin A2 could increase the diagnostic performance of HCC. These data suggest that Annexin A2 may serve as a novel serological/histological candidate involved in HCC, especially among individuals with normal levels of serum AFP.

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 4578.
Abstract 4579: Effect of pre-adipocyte and differentiated adipocyte conditioned media on galectin-3 expression in mouse intestinal epithelial cells
Authors: Kim Jong-Sang , Lim Ji-Sun , Garcia Annalene , Seo Ji-Yeon , Park Jia , Nam Dae Hwan , Sung Mi Kyung , Seo Young-Rok
Published: February 2011

We hypothesized that obesity cause inflammation which, in turn, promotes colon carcinogenesis. To test the hypothesis mouse intestinal IEC-6 cells were incubated in the presence of pre-conditioned media collected from culturing LPS-stimulated macrophage, followed by comparing protein expression profile with control cells. Our proteomic study revealed 45 differentially regulated proteins in mouse intestinal IEC-6 cells under state of inflammation. Among others, galectin-3, a beta-galactoside-binding protein, was up-regulated. Galectin-3 has been reported to play anti-apoptotic role in tumor progression and metastasis, and its over-expression has been observed in several types of cancer such as colon cancer. In order to determine relationship between obesity-induced inflammation and colon carcinogenesis, conditioned media (CM) from LPS-stimulated 3T3-L1 pre-adipocytes or differentiated adipocytes were utilized to culture IEC-6 cells. The CM was analyzed for levels of nitric oxide (NO), TNF-α and IL-6, and IEC lysates were analyzed for galectin-3 expression by western blot. Results showed that enhanced generation of NO, TNF-α and IL-6 resulted in upregulation of galectin-3 with respect to incubation time. Also, cytokine concentration was correlated to level of galectin-3, as verified by incubating IEC with increasing concentration of TNF-α or IL-6. These results indicate involvement of pro-inflammatory mediators from pre-adipocytes and adipocytes in colon carcinogenesis, in which galectin-3 may play a crucial role.

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 4579.
Abstract 4580: Detection of early-stage pancreatic cancer by mass spectrometry: a multi-institutional validation study
Authors: Honda Kazufumi , Ono Masaya , Okusaka Takuji , Kosuge Tomoo , Felix Klaus , Nakamori Shoji , Sata Naohiro , Nagai Hideo , Ioka Tatsuya , Tsuchida Akihiko , Shimahara Masashi , Yasunami Yoichi , Hirohashi Setsuo , Büchler Markus W. , Yamada Tesshi
Published: February 2011

Background Invasive ductal carcinoma of the pancreas is one of the worst prognoses among common human malignancies. The poor outcome of pancreatic cancer patients seems to be attributable to difficulty in early detection.

Methods Mass spectra of peptide were obtained from a total of 1314 plasma/serum samples, consisting 4 cohorts (namely Cohorts 1-4) obtained from 7 medical institutions participating in the “Third-Term Comprehensive Control Research for Cancer” conducted by the Ministry of Health, Labor and Welfare of Japan and one institute in Germany using orthogonal matrix assisted laser/desorption ionization time of flight (oMALDI-qQTOF-MS). The amino acid sequences and posttranslational modifications of marker proteins were identified by tandem mass spectrometry (MS/MS).

Results Plasma apolipoprotein AII heterodimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summated value differed between 112 pancreatic cancer patients and 103 healthy controls (Cohort 1) with high statistical significance [P = 1.36 × 10−21, P = 4.35 × 10−14, and P = 1.83 × 10−24 (Mann-Whitney U-test)] and area-under-curve (AUC) values of 0.877, 0.798, and 0.903, respectively. The significance was further verified in 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a hospital-based prospective cohort [Cohort 4 (n = 833)]. Patients with invasive ductal adenocarcinoma of the pancreas, including those with stage-I disease, could be discriminated from healthy controls with the sensitivity of 97.6% (243/249) and specificity of 95.3% (122/128) by the combination of DUPAN-2, CA19-9, ApoAII-2, and ApoCIII-0.

Interpretation Although the reduction of the 2 modified forms of plasma/serum apolipoproteins was not pancreatic cancer-specific, the accurate measurement of plasma/serum proteins had a high capability in detecting pancreatic cancer. This test is essentially safe and might be applicable to the primary mass screening of pancreatic cancer in asymptomatic populations.

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 4580.
Abstract 4583: Multiplex mass spectrometric immunoassays
Authors: Nedelkov Dobrin
Published: February 2011

Targeted proteomics approaches can facilitate the study of the human protein variations and their association with cancer. In Mass Spectrometric Immunoassays, affinity interactions are utilized to selectively retrieve specific proteins from complex biological fluids, and are followed by mass spectrometric analysis of the targeted proteins. These assays are similar to traditional enzyme immunoassays in that they use antibodies as reagents for affinity retrieval of specific proteins, but instead of enzymatic reaction for indirect protein detection, mass spectrometric analysis is used for direct identification of the proteins and their modifications. This rather straightforward concept is realized through affinity pipette devices that enable high-throughput integration and assaying of thousands of samples per day.

Described here is the development of several multiplex mass spectrometric immunoassays toward cancer-related proteins. The project is part of the NCI's Clinical Proteomic Technologies for Cancer initiative to assess and apply proteomic technologies and data resources to solve critical problems in cancer research. Antibodies to several proteins (including one that serves as an internal reference standard - IRS) were immobilized in various ratios in the affinity pipettes, and following analysis of human plasma spiked with constant amount of the IRS, the ratio of the peaks representing the proteins were monitored to determine the optimal ratio of antibodies in the affinity pipettes. Detailed assaying protocols were developed and optimized, including pre-and post-sample rinses, and elution matrix composition. Increasingly-diluted plasma samples were analyzed and a decrease of the protein signals in reference to the IRS signal was observed, indicating non-saturating conditions for the assays. The assays were tested with numerous human plasma samples, and the intra- and inter-assay CVs were determined to be <10%. The results obtained with the MSIA assay were also compared to commercially available ELISA assays.

The introduction of mass spectrometric-based assays and platforms into clinical laboratories will facilitate the ongoing biomarker discovery, evaluation, and validation efforts. The multiplex mass spectrometric immunoassays are idealy suited for analysis of protein biomarkers exhibiting post-translational modifications because this protein microheterogeneity can only be detected via mass spectrometry. It is important to note that the assays target intact/native proteins (in contrast to MRM-based peptide assays), and in such are capable of detecting any and all PTMs, without a priori knowledge of their existence.

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 4583.
Abstract 4587: Quantitative analysis of IGF-1R signaling pathway activation in FFPE tissue
Authors: Krizman David , Hembrough Todd , Hiedbrink-Thompson Jenny , Thyparambil Sheeno , Burrows Jon , Darfler Marlene , Prakash Amol , Krastins Bryan , Sarracino David , Rezai Taha , Athanas Michael , Lopez Mary
Published: February 2011

Multiple Ewing's sarcoma clinical trials targeting insulin-like growth factor receptor (IGF-1R) with IGF-1R small molecules and antibody inhibitors are currently underway. The IGF-1R signal pathway is commonly activated in many cancer types so this targeted therapeutic approach holds great promise for not only treating Ewing's sarcoma but other types of cancers as well. There is currently no good method to quantitatively measure the IGF-1R protein, its phosphorylation status, or the immediate downstream signal pathway target IRS-1. Being able to quantify these proteins and determine their phosphorylation status directly in patient tissue biopsies could help identify and select patients most likely to benefit from anti-IGF-1R therapies. We have developed an approach to determine both absolute IGF-1R and IRS-1 levels and the phosphorylation status of both proteins directly in formalin-fixed paraffin-embedded (FFPE) patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which turns dissected tissue to a complete solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. This approach was used to measure the IGF-1R protein and its phosphorylation sites pY1131, pY1135, and pY1136 as well as the IRS-1 protein and its phosphorylation sites pY612 and pY896 in formalin fixed tissue culture cells and xenograft tumors. Results indicate the potential to detect and quantify the IGF-1R and IRS-1 proteins and to monitor their phosphorylation status directly in FFPE tumor tissue which could be used to identify and stratify patients most likely to benefit from anti-IGF-1R therapies.

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 4587.
Abstract 459: Caveolin-1 mediated signaling and proliferation is regulated through the oxygen sensing pathway
Authors: Xu Chao Ying , Wang Yi , Roche Olga , Ohh Michael
Published: February 2011

Caveolin-1 (Cav-1) is a multifunctional scaffolding protein with multiple binding partners that is associated with cell surface caveolae and the regulation of lipid raft domains. Cav-1 regulates multiple cancer-associated processes including cellular transformation, tumour growth, cell death and survival, and multidrug resistance. A high expression of Cav-1 is found in various cancer , including clear cell renal cell carcinoma (CCRCC), correlating with large, high grade tumors and poor prognosis. However, the role of Cav-1 in the oncogenesis of CCRCC remains unclear, and its role as either an oncogene or tumour suppressor is still under debate. Here, we investigated the mechanism of Cav-1 in controlling cell proliferation using in vitro and in vivo assays. We report that Cav-1 is highly expressed in tumour compared to normal tissue in patients with CCRCC, thus Cav-1 likely functions as an oncogene. Upon knock-down of Cav-1, tumour cells (786-VHL-/-) showed a decrease in proliferation. We showed that Cav-1 is associating with EGFR, a receptor tyrosine kinase, to activate the Ras-RAF-MEK-ERK kinase pathway and induce cell proliferation. We found that Cav-1 binds to EGFR in immunoprecipitation. The Ras pathway is upregulated in cells overexpressing Cav-1 after EGF stimulation. Upon knockdown of Cav-1, the level of p-ERK, p-MEK, and p-c-Raf decreased, as well as the rate of cell proliferation. Upstream of the Ras pathway, we report that Cav-1 appears to be under the control of hypoxia-inducible factor (HIF). Under hypoxia, VHL (the principal negative regulator of HIF) is downregulated, thus leading to upregulation of HIF. We showed that under hypoxia, Cav-1 increased in 786-VHL, Hela and A431 cell lines. Similarly, cells with VHL loss (786-VHL-/-), thus high HIF level, had high levels of Cav-1. We are currently examining the role of Cav-1 in tumour growth by injecting SCID mice with either 786-VHL-/-, or Cav-1 knock-down 786-VHL-/- tumour cells. Since Cav-1 level seems to be under the control of HIF in renal, cervical and squamous carcinoma cell lines, it suggests that the oxygen sensing pathway plays a general role in controlling cell proliferation through upregulating Cav-1. Cav-1 should be considered as a target for drug design as the knock down of Cav-1 decreased cell proliferation and could potentially decrease tumour 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 459.
Abstract 4591: Estrogen receptor beta controls a wide variety of functions in breast cancer cells
Authors: Cekanova Maria , Woraratphoka Jirayus , Sukhthankar Mugdha , Dharmawardhana S , Siriwardana Nalin , Moustaid-Moussa Naima , Strom Anders , Baek Seung J. , Wong Q , Zou M , Donnell Robert , Wimalasena Jay
Published: February 2011

The role of ER alpha as the mediator of estrogen action in breast cancer (BC) cells is well known, however, the role of ER beta is controversial. The majority of BC expressing ER alpha also express ER beta. Some clinical studies suggest that ER beta expression is a good prognosticator; however, in others, especially where expression levels of ER beta > ER alpha, ER beta may be associated with more advanced BC. Previously, we showed that ER beta may regulate a number of signaling pathways (AACR, 2008). Our recent work demonstrates that ER beta has even wider actions in MCF7 and T47D cells stably overexpressing ER beta. Thus, ER beta decreased phospho (p)/ total Stat1, p Stat3, p Stat5/total Stat5. Expression of the oncogenic miR cluster 17-92 was highly reduced (p<0.01) when ER beta overexpressing vs. control cells were compared. ER beta also decreased PPAR gamma, superoxide dismutase, as well as the concentration of the mRNA processing factor, RBM5, and the activated form of AMPK. All these changes were significant (p<0.05) in triplicate measurements. While ER beta overexpression increased migration through Matrigel, it significantly decreased CXCR4 expression (p<0.05, n=4). All of these effects of ER beta were estrogen-independent. On the other hand, estrogen increased CXCR4 expression in MCF7 cells significantly. In our study, we analyzed the expression of ER beta and BAD using tissue microarrays with normal and neoplastic breast tissue. Our immunohistochemical (IHC) data revealed a significant increase of ER beta cytoplasmic staining in neoplastic cells compared to normal cells (p<0.001). In addition, the nuclear expression of ER beta was significantly decreased in neoplastic cells compared to normal cells (p<0.01). From our correlation analysis of IHC data obtained from tissue microarray, we identified that cytoplasmic expression of BAD was negatively correlated with cytoplasmic expression of ER beta. In addition, the nuclear expression of BAD was significantly positively correlated with nuclear ER beta.

These results suggest that ER beta has a wide variety of actions in BC cells, as predicted by the regulation of 62 miRs (p<0.01) as determined by Exiqon Inc. Taken together our results suggest that ER beta may have a multitude of actions in breast cancer and that the ratio of ER alpha/beta may determine the prognostic outcome.

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 4591.