bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4539: Anti-leukemic activity of mitochondria-targeted asymmetrical 2,2′-binaphthoquinones
Authors: Ricklis Rebecca M. , Vuica-Ross Milena , Brodsky Robert A. , Sausville Edward , Dang Chi V. , Karp Judith E. , Emadi Ashkan
Published: February 2011

Introduction: Binaphthoquinones are unique molecules consisting of two linked naphthoquinone units. By definition biquinones cause more effective oxidative stress than their monoquinone counterparts. Previously, we regiospecifically synthesized a series of asymmetrical binaphthoquinones, which possess HIV integrase inhibitory activity. Potent activity against HIV-uninfected CEM-T4 cells prompted us to investigate other cytotoxic mechanisms. A genome-wide yeast screen uncovered that mitochondria-related genes are required for sensitivity and resistance to these agents. Since mitochondria membrane permeabilization occurs early in the apoptotic program and is located downstream of most identified chemotherapy resistance mechanisms in hematological malignancies, we hypothesized that binaphthoquinones would be able to interfere with mitochondria function in leukemia cells and induce apoptosis by abrogating or bypassing other mechanisms of drug resistance.

Materials and Methods: Seventeen of our biquinones were incubated with four leukemia cell lines (MV411, MOLM14, HL-60, U937) grown under standard conditions. A standard MTT cell proliferation assay was used to identify IC50 values. Two of the most potent compounds, biquinones #7 and #10 (BiQ7 & BiQ10), were assayed against MV411 cells and analyzed by flow cytometry for loss of mitochondria membrane potential (via rhodamine 123 staining), as well as a variety of apoptosis markers, such as phosphatidylserine, activated caspases, and sub-2n DNA increases by using Annexin V-PE (with 7AAD), a fluorescent caspase inhibitor reagent, and propidium iodide (PI), respectively.

Results: The IC50 values for both compounds were less than 5 μM against all cell lines. A loss of mitochondria membrane potential was observed in 98-100% of the treated cells (control 5%). Loss of plasma membrane phosphatidylserine asymmetry was observed in 94-95% of the treated cells (control 18%). The cells showed increases in staining for activated caspases 3 & 7 (control 10%; BiQ7 98%; and BiQ10 96%). Finally, the cell cycle analyses showed increases in sub-2n DNA in the treated cells compared to the control (control 12%; BiQ7 34%; and BiQ10 54%). These assays were subsequently repeated to obtain triplicate results.

Conclusion: Our study shows that this new class of compounds possesses promising in vitro anti-leukemic effects by targeting mitochondria membrane potential. In vivo experiments in a xenograft model and ex vivo experiments on primary human cells are in progress. The cytotoxicities of binaphthoquinones could vary widely as a result of the array of structural configurations available.

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 4539.
Abstract 4543: Targeting the NADPH binding site of dihydrofolate reductase for cancer therapy
Authors: Hsieh Yi-Ching , Lawal Rialnat AdeBisi , Scotto Kathleen , Banerjee Debabrata , Abali Emine Ercikan , Bertino Joseph R.
Published: February 2011

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate into tetrahydrofolate using NADPH as the cofactor. Folate-dependent one carbon metabolism is essential for the de novo synthesis of purines, pyrimidines and certain amino acids. Thus, DHFR plays an essential role in production and maintaining reduced folates for cellular proliferation, and consequently it has been a critical target for chemotherapy. Methotrexate (MTX), an antifolate that targets DHFR, has been used for decades in the clinic. However, both intrinsic and acquired resistance occurs due to decreased uptake, gene amplification and protein mutation. We have initiated studies to find an inhibitor that targets the cofactor NADPH binding site that would bypass these resistance mechanisms to MTX in contrast to analogs that target the dihydrofolate binding site.

Fourteen NAD/NADP analogues were screened by the MTT assay in Chinese hamster ovary (CHO) cells which lack endogenous DHFR expression but were stably transfected with a DHFR-EGFP fusion construct driven by a CMV promoter (DG44 DHFR-EGFP). Two compounds, thionicotinamide adenine dinucleotide (NAD-S) and thionicotinamide adenine dinucleotide phosphate (NADP-S), had IC50 values of 1 μM. NAD-S and NADP-S were also found to be potent inhibitors of two other cancer cell lines, C85 (metastatic human colon cancer cell line) and CCRF-CEM (human T-cell lymphoblastic leukemia cell line).

Surprisingly, both NAD-S and NADP-S decreased the levels of DHFR in CEM-CCRF cells. Pulse-chase experiments showed that the half life of DHFR is significantly reduced upon treatment of cells with 10 μM NADP-S. Furthermore, this NADP analog caused an increase in cells at the G1 phase (11% at 10 μM and 44% at 40 μM NADP-S treatment) that was reversible by removing NADP-S from the cell culture. Because NADP-S decreased DHFR levels, the combination of NADP-S with MTX was tested and found to significantly reduce colony formation, and was synergetic in vitro (Chou-Talalay analysis). We conclude that NADPS has a novel mechanism of action, i.e., it causes degradation of DHFR, likely by competing with NADPH binding, known to stabilize DHFR.

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 4543.
Abstract 4548: Erythrose kill cancer cell in vitro and inhibit tumor growth in vivo
Authors: Wang Xingnong , Wei Youcheng
Published: February 2011

In general, mutations in oncogenes and tumor suppressor genes are believed to represent the fundamental cause of carcinogenesis. Mitochondrial deficiency (Warburg effect) may be a direct or indirect consequence of these mutations. Accordingly, tumor cells depend on glycolysis rather than mitochondrial oxidative metabolism as energy source for their survival and proliferation. Targeting this kind of metabolism could offer a possibility for cancer treatment. Erythrose was used as an alternative energy source to test its inhibitory effect on several tumor cell lines in vitro and in vivo.

Cancer cell lines (breast BT474 and MCF-7, brain U87mg, pancreas Panc-1, HEL, LL-2 Lewis lung, and colorectal CT26 and SW480 lines) were grown in DMEM + 10% FBS media with different concentrations of D-erythrose and cell growth was tested with the Trypan blue assay. In general, 70% of cancer cells did not survive after 24 hrs of culture with 500mg/L D-erythrose. Addition of ZnCl2 (40µM) doubled the cancer killing effect at 400mg/L D-erythrose. In addition, LL-2 Lewis lung cells were subcutaneously introduced into the immunocompetent C57BL/6 mice. After allowing tumor growth to 2.7±0.4mm wide diameters, a daily subcutaneous injection beside the tumor with D-erythrose (∼1g/kg bw) was administered for 25 days; controls received a PBS injection instead. Tumor growth in erythrose-treated mice was inhibited more than 90% by weight.

We hypothesize that the administration of erythrose leads to CO2 production from oxidation in cytosol or mitochondria. Carbonic acid is formed from CO2 and water, accelerated by carbonic anhydrase (a zinc enzyme), which can convert lactate to lactic acid, and cause intracellular acidosis and cancer death since cancer cells have increased lactate production.

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 4548.
Abstract 4561: Quantitative phosphoproteomics of an AKT inhibitor in a PTEN-LOF breast model by label free phospho-dMS demonstrates modulation of protein transcription, protein translation, and motility
Authors: Chi An , Keilhack Heike , Guo Hongbo , Nagashima Kumiko , Andersen Jannik , Paweletz Cloud , Hendrickson Ronald
Published: February 2011

Among the many types of molecular mechanisms that are currently understood, none is more important for the control of cellular faith than the network of protein kinases and phosphatases. The importance of understanding these signaling networks is furthermore highlighted by the fact that virtually all major pharmaceutical companies have significant projects focused on targeted interference against oncogenic kinases as potential anti-tumor therapies. Yet, a system's wide analysis of signaling phosphorylations by these inhibitors in in vivo (i.e. tissue or xenograft models) is cost prohibitive and rarely performed.

The use of mass spectrometry is a powerful approach to study complex mixtures, and some techniques have used either chemical or metabolical isotopic labeling strategies to quantify protein differences between cohorts. While these techniques have been shown to be useful and are routinely applied for cell culture systems, they do not extend readily to in vivo studies as these require either combining of samples or are dependent on the duty cycle of the mass spectrometer. Here we report a generally applicable proteomic approach to identify global protein phosphorylation changes in in-vivo systems. This label-free discovery platform, named phospho-differential mass spectrometry (phospho-dMS) does not require complex mixing or pooling strategies or isotope labeling, and instead identifies statistically significant changes in full scan mass spectrometry data. Hence, in-vivo samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis.

We demonstrate phospho-dMS on 27 breast tumor tissues samples (3 concentrations, 3 time points, 3 mice each) excised from a PTEN-LOF mouse model. We quantified in excess of 2000 high confidence phosphorylation events. The most prominent protein modules inhibited by compound treatment (P<0.0001) relate to cytoskeletal machinery such as cell polarity and cytoskeletal reorganization, transcription factors, co-activators, and protein translation with known relevance to cancer. For the first time, using quantitative MS, the topology and significance of phosphorylation networks may be investigated in xenograft, GEM models, and tumor tissue samples marking a new era of cancer signaling research

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 4561.
Abstract 4567: Urine biomarkers outperform serum biomarkers in the diagnosis of various human cancers
Authors: Nolen Brian M. , Lomakin Aleksey , Bast Robert , Lu Karen , Grizzle William E. , Jhala Nirag C. , Eloubeidi Mohamad A. , Christein John D. , Arnoletti Juan P. , Oelschlager Denise K. , Vickers Selwyn M. , Marrangoni Adele , Lokshin Anna
Published: February 2011

Background: The measurement of biomarkers present in the bodily fluids of cancer patients represents an important avenue for the development of minimally invasive tests to predict tumorigenesis, disease recurrence, or treatment response. A great deal of work along these lines has already been devoted to blood, given its systemic exposure and extensive availability through tissue banks. However, blood is a dynamic biofluid with a proteome under continuous metabolic and homeostatic regulation. Alternatively, urine represents a biofluid that is inherently quiescent in that all molecular and proteolytic activity is largely complete upon sampling. Additional characteristics of urine including its high level of temperature stability and low-complexity matrix resulting from renal filtration have led many to propose urine as an alternative or companion to blood for biomarker studies.

Methods: We performed an analysis of biomarkers present in the urine and serum of patients diagnosed with ovarian, pancreatic, and breast cancer utilizing multiplexed bead-based immunoassays developed for the Luminex® xMAP® platform. Ovarian and breast cancer patients were compared to matched healthy controls while the pancreatic cancer patients were compared to a group of patients diagnosed with benign pancreatic conditions. Each case/control group was tested for 10-15 biomarkers identified as informative for each cancer type through previous population-based analyses of serum biomarkers.

Results: In our analysis, nearly all of the tested biomarkers were detectable in urine and many of the biomarkers exhibited mean relative differences of greater magnitude in urine versus serum. Our multivariate analysis identified several urine multimarker panels capable of discriminating the cancer from the control groups with high sensitivity and specificity. The use of a 4-biomarker panel comprised of 3 urine biomarkers and one serum biomarker resulted in the discrimination of ovarian cancer patients from healthy controls with a sensitivity of 100% at 95% specificity. For the ovarian and pancreatic cancer comparisons, urine/serum multimarker panels outperformed the best serum biomarker panels identified. A urine 5-biomarker panel was identified in the breast cancer comparison that demonstrated a high discriminatory power in both training and validation sample sets.

Conclusions: Our results support the use of urine biomarkers as alternatives and/or companions to serum biomarkers for the diagnosis of selected human cancers.

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 4567.
Abstract 4570: Using laser capture microdissection functionally coupled with mass spectrometry for an enhanced study of solid tumor heterogeneity via tissue proteomics
Authors: Johann Donald J. , Mukherjee Sumana , Rodriquez-Canales Jaime , Hanson Jeffrey , Prieto DaRue , Veenstra Timothy D. , Emmert-Buck Michael , Blonder Josip
Published: February 2011

Introduction

Examining solid tumor heterogeneity using a tissue proteomics approach, which relies on a functional coupling between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS), is the purpose of this research study. A common characteristic of solid tumors is a heterogeneous architecture, which enables distinct cellular morphologies and molecular behaviors. This diverse composition of cells, specifically cancer cells proper along with both modified and unmodified stromal elements, collectively forms a microenvironment that serves to nurture the malignant process.

Methods

Using LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lesion of breast carcinoma metastasis. All slides were reviewed by a pathologist and the LCM session planned. Multiple specimens each containing ∼ 50,000 cells were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins.

Preliminary results

Prepared samples were analyzed by LC-MS/MS resulting in more than 500 unique protein identifications. High confidence measures were employed by including only proteins identified by ≥ 2 unique peptides. Decoy database searching revealed a false-positive rate between 5 and 10%. Machine learning methods assisted with molecular profiling activities to better discern the concurrent expression of multiple cellular phenotypes due to the heterogeneity of the solid tumor. Cross validation via Western Blot analysis confirmed specific linkage of 12 validated proteins to underlying pathology and their potential role in solid tumor heterogeneity.

Summary and conclusion

Solid tumors are composed of an assortment of cell types, which have diverse morphologies, as well as distinct molecular phenotypes. Tumor heterogeneity may be viewed as a memory of the malignant process and thus a novel proteomic study may yield insights regarding cancer development and more effective treatment. The biological background of such heterogeneity is not well understood. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery.

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 4570.
Abstract 458: A novel resveratrol analogue, HS-1793, down-regulates the expression levels of hypoxia-inducible factor-1α and VEGF, and inhibits hypoxia-induced cell migration of human prostate cancer cells
Authors: Kim Dong Hwan , Kim Min Young , Hossain Mohammad Akbar , Kim Jin-Ah , Kang Yong Jung , Suh Hongsuk , Choi Yung Hyun , Kim Gi Young , Kim Nam Deuk
Published: February 2011

Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, over- expressed in many human tumors and their metastases, and is closely associated with more aggressive tumor phenotype. An important objective of anti-cancer therapy is the development of new drugs that suppress hypoxic responses in solid tumors. In many studies, resveratrol has been shown to chemopreventive effect in various cancer cells. However, resveratrol's biological activity is limited by its photosensitivity and metabolic instability. This study was investigated the effects of a novel analogue of resveratrol, HS-1793, on the expression levels of HIF-1α and vascular endothelial growth factor (VEGF) in PC-3 human prostate cancer cells. Hypoxic condition induced a time-dependent increase in the level of HIF-1α protein in PC-3 cells, and treatment with HS-1793 markedly decreased HIF-1α expression level. HS-1793 also inhibited VEGF expression level. Mechanistically, HS-1793 inhibited HIF-1α and VEGF expression through multiple mechanisms. Firstly, HS-1793 inhibited both PI3K and Erk phosphorylations in PC-3 cells. Secondly, HS-1793 substantially induced HIF-1α protein degradation through the proteasome pathway. Finally, HS-1793 inhibited hypoxia-induced cell migration. These data suggested that HS-1793 may inhibit human prostate cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression. Moreover, HS-1793 showed more potent effect than resveratrol on the cytotoxic effects on PC-3 cells. Taken together, these results implied that HS-1793, a novel analogue of resveratrol, might be a new potent chemopreventive agent against human prostate cancer cells. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R01-2006-000-11117-0)].

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 458.
Abstract 4581: Implementation of a new cancer protein biomarker development pipeline - NCI's clinical proteomic technologies for cancer
Authors: Kinsinger Chris , Mesri Mehdi , Boja Emily , Hiltke Tara , Rahbar Amir , Rivers Robert , Rodriguez Henry
Published: February 2011

Proteomic technologies have revolutionized the area of protein biomarker discovery by providing powerful new tools such as protein arrays and mass spectrometry to characterize complex proteomes, multi-protein complexes and post-translational modifications. However, attempts to use proteomic approaches to discover cancer biomarkers in biofluids and tissues have been largely unsuccessful in the past. This indicates issues present in the biomarker candidate pipeline mainly due to variability within biomarker discovery and the lack of verification (or pre-validation) of biomarkers prior to costly clinical validation studies using immunosorbent assay (ELISA). Clinical Proteomic Technologies for Cancer (CPTC) initiative established by NCI in 2006 sought to develop a more refined, efficient, and reliable biomarker development pipeline anticipated to produce better-credentialed candidate leads, ultimately accelerating the translation of new cancer biomarkers into diagnostic tests. CPTC is a team science based five-year initiative composed of three components with an emphasis on the optimization of technology platforms, standards development, involvement with the U.S. Food and Drug Administration, and community reagents and resources development to address the challenges of clinical proteomics and to enhance technical abilities to identify and measure proteins accurately, quantitatively and reproducibly in biological systems. This presentation highlights the significant milestones of this initiative in building a more reliable and efficient protein biomarker development pipeline - one that consists of three development stages that we believe places the field of proteomics as a key component in initiatives for personalized medicine - Discovery, Verification, and Validation.

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 4581.
Abstract 4592: ER-beta inhibits cell proliferation in normal mammary epithelial cells
Authors: Pan Zhongzong , Zhong Yili , Song Xiaozheng
Published: February 2011

ER-beta is the second subtype of estrogen receptor and its role in mammary epithelial cell proliferation remains obscure. Studies using human breast cancer cell lines suggest that ER-beta may function as a tumor suppressor to inhibit cell proliferation. In the in vivo mouse studies, however, activation of ER-beta was shown to promote cell proliferation of normal mammary epithelial cells. It is not clear whether the difference is caused by the different experimental models, such as in vitro versus in vivo or breast cells versus normal cells. In our studies, we evaluated the effect of ER-beta on cell proliferation using immortalized normal mammary epithelial cell lines. We found that cell proliferation was significantly inhibited by ER-beta. FACS analysis on cell cycle showed that more cells were in the G2/M phase, suggesting that ER-beta may induce G2 arrest. The inhibitory function of ER-beta is independent of estrogen binding. In summary, our data support that ER-beta is a negative regulator of mammary epithelial cell proliferation.

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 4592.
Abstract 46: Interdependence of choline kinase and phospholipase D in human breast cancer cells
Authors: Krishnamachary Balaji , Gadiya Mayur , Mori Noriko , Mironchik Yelena , Glunde Kristine , Bhujwalla Zaver M.
Published: February 2011

One hallmark of cancer is an increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis. Enzymes in choline metabolism present attractive targets that can be exploited for treatment. Choline kinase (Chk) is a cytosolic enzyme that catalyzes the phosphorylation of choline (Cho) to phosphocholine (PC) by using ATP in the presence of magnesium. Over-expression of Chk-α has been observed in breast, prostate and lung cancers, making it an obvious choice for targeting. Down-regulation of Chk-α results in a significant reduction of cell proliferation and increased differentiation in highly invasive MDA-MB-231 human breast cancer cells after siRNA-Chk transfection. Another potential target is phosphatidylcholine-specific phospholipase D (PC-PLD). Two mammalian isoforms of PLD, PLD1 and PLD2 have currently been identified. PLD1 is activated by G proteins such as ARF, Rho and Rac. Elevated PLD1 has been reported in gastric, renal, colon and breast carcinoma.

We characterized the expression of PLD1 in metastatic MDA-MB-231 cells, poorly metastatic MCF-7 cells, and nonmalignant MCF-12A cells and used siRNA to transiently down-regulate PLD1 and Chk-α in MDA-MB-231 and MCF-7 cells. Forty-eight hours post-transfection cells were harvested for protein and RNA. To determine the effective knock-down of Chk-α or PLD1, about 30µg of protein was resolved on 7.5% acrylamide gel, transferred to nitrocellulose membrane and probed with antibody specific to either Chk-α or PLD1. The effects of Chk-α downregulation on PLD1 expression and the effects of PLD1 downregulation on Chk-α expression were determined by western blots, and the message was quantified with q-RT-PCR. Downregulation of Chk resulted in an increase of PLD1 in MDA-MB-231 cells stably expressing shRNA against Chk or transiently transfected with Chk siRNA. Similar data were obtained for MCF-7 cells. PLD1 levels were highest in the metastatic MDA-MB-231 cells. Effective downregulation of PLD1 was achieved with transient PLD1 siRNA transfection, which resulted in an increase of Chk-α in these cells. These data demonstrate the adaptability of cancer cells, the interdependence of Chk-α and PLD1, and the importance of multiple targeting to eliminate any adaptive compensatory effects that would allow cancer cells to survive. This work was supported by NIH P50 CA103175.

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 46.
Abstract 4630: Seminal AMACR protein: a non-invasive test in the detection of prostate cancer
Authors: Huang Wei , Jarrard David , Ritter Mark , Waknitz Michelle , Straus Jane , Wilding George
Published: February 2011

Introduction: Current prostate cancer screening relies on the PSA test and the digital rectum rectal examination (DRE), which have relatively low specificity. A more specific marker is in need for prostate cancer screening and risk stratification could reduce the burden of unnecessary biopsies. AMACR, has been proven to be an excellent tissue marker for prostate caner, could be an attractive candidate, but. Soso far, serum the detection of AMACR detection in serum has been unsuccessfuldifficult. This study explored the detection of AMACR protein in semen ejaculate and its potential as a non-invasive test in the diagnosis and management of prostate cancer.

Methods: Semen ejaculates were analyzed from 23 prostate cancer (biopsy proven) patients, 21 of which had prostatectomy, as well as 15 age-comparable controls (>50y, PSA<2 ng/mL, assumed prostate cancer free). An indirect sandwich ELISA chemiluminescence assay was used to test semen AMACR protein. Tissue AMACR protein was also quantified in 13 corresponding prostatectomy specimens using automated quantitative analysis (AQUATM).

Results: AMACR was detected in 19 of 23 (83%) semen ejaculates of prostate cancer patients. Among the other 4 patients with undetectable semen AMACR, one had no residual tumor in the prostatectomy specimen (#40), one had Gleason score of 9 cancer (#37) with no detectable tissue AMACR either, while the other two remaining patients (#31 and #33) had a very small volume of cancer on two separate sextant biopsies biopsy sets and are on watchful waiting list. AMACR was also detected in 7 of 15 (47%) control group. Using 100 ng/mL as a cutoff value, semen AMACR protein was positive in 16 of 23 (70%) cancer patients who had underwent prostatectomy and in 5 of 15 (33%) age-comparable controls.

Conclusions: This has been the first study first to demonstrated that AMACR protein is detectable in semen ejaculate. A high semen AMACR detection rate in cancer patients suggested suggests that semen AMACR may be useful, supplemental, as a non-invasive test in addition toto the PSA test for detecting prostate cancer, especially cancer of significant volume. Questions regarding specificity of AMACR are, however, raised. Given the lack of prostate biopsy confirmation, The the age-comparable controls in this study may not be an entirely representative sample of men free of prostate cancer without confirmation by prostate biopsy. This Our study's findings will form the basis for the design of a larger finding need to be further validated in a larger prospective screening trial of AMACR.

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 4630.
Abstract 4636: Increased expression of CSE1L/hCAS in colorectal cancer: Correlation with tumor progression
Authors: Shivers Steven C. , Coppola Domenico , Eschrich Steven A. , Chen Dung-Tsa , Shibata David , Yeatman Timothy J. , Malafa Mokenge P.
Published: February 2011

Introduction. The chromosome segregation and human cellular apoptosis susceptibility gene (CSE1L/hCAS) has been implicated in cancer and has been shown to be amplified in several human maligancies, including colorectal cancer. In this work, we sought to evaluate the gene expression of CSE1L/hCAS in colorectal cancer cell lines and tumor tissue and to examine CSE1L/hCAS expression in colorectal cancer progression.

Methods. In order to examine CSE1L/hCAS at the gene expression level, we surveyed three different datasets from studies in which Affymetrix U133+ gene chips had been performed on tissue samples from patients with colorectal cancer, as well as patients with adenomas and normal individuals. We examined relative gene expression levels for 3 probe sets on the U133+ gene chip that closely align to the RefSeq for the CSE1L/hCAS gene. In order to examine CSE1L/hCAS at the level of protein expression, we utilized a tissue microarray (TMA) that had been previously created at the Moffitt Cancer Center from primary colorectal tumors of various stages, adenomas and normal tissues and performed immunohistochemical (IHC) staining for the CSE1L/hCAS protein.

Results. CSE1L/hCAS showed increased gene expression in all of the adenoma and colorectal cancer patient samples tested by microarray, compared to tissue samples from normal individuals. However, there was not a significant difference in CSE1L/hCAS mRNA levels between patients with adenoma and those with invasive cancer, nor between patients compared by cancer stage, grade, vascular invasion, sex or age. IHC staining also demonstrated intense nuclear and/or cytoplasmic staining for the CSE1L/hCAS gene product in adenomas and invasive colorectal cancers. Furthermore, later stage specimens showed more intense nuclear staining, while early stage lesions, and even fibroblasts in nearby normal tissue, showed predominately cytoplasmic staining for CSE1L/hCAS.

Conclusions. These results demonstrate that increased expression of the CSE1L/hCAS gene, while not specific for invasive cancer cells, might be a good marker for early detection of colorectal cancer. This gene appears to be expressed early and across all stages of cancer development. Because of its early expression and potential involvement in a variety of important cellular processes, we believe that this gene product might represent a potential target for prevention and/or treatment of early colorectal cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4636.
Abstract 4645: CIP2A is a marker of reduced survival in serous ovarian cancer patients
Authors: Böckelman Camilla , Lassus Heini , Hemmes Annabrita , Leminen Arto , Westermarck Jukka , Haglund Caj , Bützow Ralf , Ristimäki Ari
Published: February 2011

Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein, but its role in ovarian cancer is not known. We analyzed CIP2A protein expression in 562 consecutive serous ovarian cancer patients, of which 524 were scored successfully and analyzed for presence of CIP2A immunopositivity using tissue microarrays. The association of CIP2A expression with survival was evaluated according to the Kaplan-Meier method. We found strong CIP2A positive immunostaining in 212 (40.4%) specimens, 222 (42.4%) were weakly positive, while the rest (n = 90, 17.2%) were immunonegative for CIP2A protein. Our results demonstrate that CIP2A immunopositivity is a marker of reduced overall survival in ovarian cancer patients (P < 0.0001). In addition, positive CIP2A expression was significantly more frequent in specimens with high grade (P < 0.0001), advanced stage (P = 0.0005), aberrant p53 immunoreactivity (P < 0.0001), high proliferation index (P < 0.0001), and aneuploidy (P = 0.001). Interestingly, in subgroups of patients with favorable clinical factors (i.e. low stage and optimal debulking surgery) CIP2A expression was strongly associated with reduced survival (P < 0.0001 for both parameters). This shows that CIP2A could be used to predict biological behavior in the group of patients with otherwise favorable prognosis. In conclusion, our results show that CIP2A protein expression is a novel marker of reduced survival in patients with serous ovarian carcinoma. Furthermore, these results suggest that CIP2A characterizes the aggressive type of this disease even within subgroups with initially favorable prognosis.

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 4645.
Abstract 4649: Comprehensive isoform expression analysis reveals that EGFR and sEGFR are prognostic determinants of cancer
Authors: Wilken Jason A. , Baron Andre T. , McCormick Daniel J. , Singh Meenakshi , Maihle Nita J.
Published: February 2011

In July, 2009 the United States’ Food and Drug Agency (FDA) approved the use of a new biologically-targeted drug called ‘cetuximab’ (Erbitux) for the treatment of patients with advanced colorectal cancer (CRC). Cetuximab is a chimeric antibody directed against the extracellular domain of the epidermal growth factor receptor (EGFR). Paradoxically, EGFR expression, as it is currently measured by commercially available assays, has not been useful for predicting who will respond to cetuximab. We propose that one problem with these assays is their inability to measure EGFR and soluble EGFR (sEGFR) isoforms independently. We previously have documented the expression of an sEGFR isoform in human tissues (1), and have generated antibodies directed against a 31-mer peptide (residues 604-634) located within the unique 78 amino acid carboxy-terminal sequence of sEGFR (2). Interestingly, this anti-sEGFR antibody recognizes a second protein of ∼140-kDa (p140), which we have identified as human α5-integrin. Biochemical and mass spectrometry analyses show that a short region in the calf domain of α5-integrin shares sequence identity with the 31-mer sEGFR peptide. We have used this 31-mer antibody with an antibody directed against the intracellular domain of full-length EGFR to develop a novel EGFR vs. sEGFR/α5-integrin expression assay. This assay demonstrates that sEGFR/α5-integrin is co-expressed with EGFR in normal as well as malignant tissues, including in a number of carcinoma-derived cell lines. We also observe that high expression of EGFR and sEGFR/α5-integrin in a large cohort of ovarian cancer patients defines a strong adverse prognostic determinant of survival. These results implicate EGFR, sEGFR, and integrins as cancer biomarkers and demonstrate the critical importance of independently assessing both full-length and soluble EGFR isoforms in prognostic EGFR expression assays.

*–JAW and ATB contributed equally to this study

(1). Reiter et al. (2001) Genomics. 71(1):1-20

(2). Christensen et al. (2002) Hybrid Hybridomics. 21(3):183-9

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 4649.
Abstract 465: Human enhancer of filamentation 1 is a mediator of hypoxia-inducible factor-1 alpha-mediated migration in colorectal carcinoma cells
Authors: Kim Sun-Hee , Xia Dianren , Kim Sang-Wook , Holla Vijaykumar , Menter David G. , DuBois Raymond N.
Published: February 2011

Human enhancer of filamentation 1 (HEF1, also known as NEDD9 or Cas-L) is a scaffolding protein that is implicated in regulating diverse cellular processes, including cellular attachment, motility, cell cycle progression, apoptosis and inflammation. Here, we identify HEF1 as a novel hypoxia-inducible factor-1α (HIF-1α) regulating gene and reveal that HEF1 mediates hypoxia-induced migration of colorectal cancer (CRC) cells. Moreover, we found that HIF-1α binds to a hypoxia-responsive element (HRE) of the HEF1 promoter to activate its transcription under hypoxic conditions. We further demonstrate that inhibition of HIF-1α expression abolished hypoxia-induced HEF1 expression and promoter activity in CRC cells. Importantly, silencing of HEF1 significantly inhibited hypoxia-stimulated HIF-1α transcriptional activity by preventing the interaction between HIF-1α and the transcriptional coactivator p300. HEF1 silencing also reduced the expression of hypoxia-inducible genes including those regulating cell motility and inhibited cell migration under hypoxic conditions. Thus, the positive feedback loop may contribute to adaptive responses by CRC cells that encounter hypoxia during CRC progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 465.
Abstract 4672: Tumor dihydropyrimide dehydrogenase (DPYD) expression is associated with survival in patients with metastatic colorectal cancer treated with gefitinib, 5-fluorouracil, leucovorin, and oxaliplatin (IFOX)
Authors: Francisco Edgar B. , Link James T. , Ramsey Meghan , Beck Andy H. , Schwartz Erich , Rouse Robert , Fisher George A. , Sikic Branimir I.
Published: February 2011

New drugs that target the EGFR signaling pathways suggest that EGFR inhibition exerts a chemosensitizing effect in colorectal cancer. We have previously reported that the combination of gefitinib, leucovorin, 5-flourouracil, and oxaliplatin (IFOX) resulted in higher response rates than those reported with FOLFOX-4 alone in patients with metastatic colorectal cancer. The aim of the present study was to examine genomic factors that might be associated with response or survival in the IFOX regimen.

Patients and Methods: Seventy-two patients had stage IV colorectal adenocarcinoma and were either untreated or previously treated prior to receiving the IFOX regimen. Formalin-fixed, paraffin-embedded tissues (FFPE; n=63) were obtained, and 4-um sections were cut, placed on slides, and deparaffinized in xylene, followed by laser capture microdissection (LCM; n=42) to enrich for tumor cells. Using real-time quantitative RT-PCR, thirty-five samples had good-quality actin RNA readings following LCM, and the expression levels of dihydropyrimidine dehydrogenase (DPYD), epidermal growth factor receptor (EGFR), excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1), thymidylate synthase (TYMS), and thymidine phosphorylase (TYMP) were separately quantified. To detect mutations in KRAS, DNA was extracted from fifty-eight patient samples and analyzed using the KRAS Mutector II kit (TrimGen, Sparks, MD).

Results: For the 33 patients available for RT-PCR analysis, expression levels of DPYD, EGFR, ERCC1, TYMP, or TYMS did not correlate significantly with response. However, in terms of survival, the overall survival rate was significantly worse in patients with a high DPYD expression level compared to patients with a low DPYD expression level (p<0.05), and in a multivariate analysis, patients with both a high DPYD expression and a low EGFR expression had the worst survival compared to all other patients with differing expression levels of these two genes (p<0.01). KRAS mutations were identified in 24 out of 58 patients (41%), but KRAS mutational status did not correlate with either response or survival.

Conclusion: Dihydropyrimidine dehydrogenase mRNA expression was a significant independent prognostic factor for overall survival in colorectal cancer patients treated with FOLFOX-4 along with EGFR inhibition by gefitinib.

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 4672.
Abstract 4684: Autophagy blockade sensitizes prostate cancer cells towards Src family kinase inhibitors
Authors: Wu Zhaoju , Chang Pei-Ching , Yang Joy C. , Chu Cheng-Ying , Wang Ling-Yu , Chen Nien-Tsu , Ma Ai-Hong , Desai Sonal J. , Lo Su Hao , Evans Christopher P. , Lam Kit S. , Kung Hsing-Jien
Published: February 2011

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. Our lab has been interested in the role of the Src complex in prostate cancer progression including the induction of androgen independence and metastasis. Previously, we reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells, but rarely apoptosis. Here, we have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, we showed a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth, compared with the control group. Taken together, these data suggest that 1) autophagy serves a protective role in SFK inhibitor-mediated cell killing; 2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors.

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 4684.
Abstract 4730: Exploring the nature of prostate cancer SNP associations: Results from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3)
Authors: Lindstrom Sara , Kraft Peter ,   BPC3
Published: February 2011

Genome-wide association studies have identified more than 35 single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. In order to characterize the associations by disease aggressiveness (stage and tumor grade) and to investigate gene-environment and gene-gene interactions that might increase our understanding of prostate cancer etiology, we genotyped 29 SNPs in 9,067 prostate cancer cases and 9,188 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We confirmed prostate cancer associations for all SNPs (P-values ranging from 0.03 to 10−26) and found no evidence that the SNPs are differentially associated with aggressive and non-aggressive disease.

We further tested each SNP for effect modification by age and family history. One SNP, rs6983267, showed a significant non-multiplicative interaction with age (P=7.3[[Unable to Display Character: ∙]]10−5) where a stronger risk association was observed in younger men. We observed no interaction between SNPs and family history. The prospective nature of cohorts allowed us to explore possible gene-environment interactions with environmental factors such as smoking, alcohol and BMI that have been inconsistently related to prostate cancer, and we hypothesized that hidden gene-environment interactions might explain previous weak associations. We observed no interaction between the SNPs and these factors. In addition, we found no evidence of SNP-SNP interactions (epistasis).

Our findings for established prostate cancer SNPs did not differ across prostate cancer clinical subtypes, and we observed no significant gene-environment interactions.

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 4730.