bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5332: Snail transcription factor contributes to prostate cancer tumor progression via reactive oxygen species and Rac1 activation
Authors: Barnett Petrina K. , Henderson Veronica , Arnold Rebecca S. , Mezencev Roman , Kundu Kousik , Hudson Tamaro , Murthy Niren , Chung Leland W K , Zayzafoon Majd , Odero-Marah Valerie
Published: August 2012

Epithelial-mesenchymal transition (EMT) represses cell adhesion associated genes, making cells more migratory. Snail is the master gene that regulates the EMT process by down regulating tight junction proteins, such as E-cadherin, and up-regulating mesenchymal genes, such as vimentin. Cell migration can also be regulated by the Rho family of GTPases such as Rac1, which can induce reactive oxygen species (ROS), specifically superoxide, via the NADPH oxidase (Nox) pathways. ROS, specifically hydrogen peroxide has been shown to mediate EMT induced by MMP-3. The objective of this study is to determine whether Snail Transcription factor contributes to tumor progression via ROS and Rac1 and to study the effect(s) of antioxidants on Snail signaling. We utilized androgen-dependent LNCaP and androgen-independent 22Rv1 and ARCaP cell lines overexpressing Snail to examine Rac1 activity in vitro. ROS (superoxide and hydrogen peroxide) levels were examined in vitro and in vivo. Western blot analysis of EMT markers, cell migration on collagen and Real Time PCR Array to assay oxidative stress enzymes was also performed. We found that LNCaP, 22Rv1 and ARCaP cells overexpressing Snail displayed increased Rac1 activity while LNCaP and ARCaP cells with Snail overexpression expressed higher levels of superoxide and hydrogen peroxide in vitro. Furthermore, ARCaP cells overexpressing Snail injected into nude mice showed higher tumorigenicity as compared to ARCaP Neo control cells and excised tumors displayed increased levels of superoxide and hydrogen peroxide ex vivo. Real Time PCR Array revealed significant increase in key enzymes involved in oxidative stress such as aldehyde oxidase I and peroxidasin. Treatment with the hydrogen peroxide scavenger, N-acetyl cysteine (NAC), could partially revert Snail-mediated EMT in ARCaP cells as evidenced by re-expression of E-cadherin after 3 days. The muscadine grape skin extract (MSKE) antioxidant decreased ROS levels and Rac1 activity and reverted Snail-mediated EMT as seen by decreased vimentin, re-induction of E-cadherin, and decreased cell migration. This is the first report of Snail regulation of ROS by regulating oxidative stress enzymes, leading to EMT and tumor progression. We are also showing novel data that Snail can regulate cell motility via Rac1 activation. Therefore, targeting Snail-mediated EMT with antioxidants offers a promising therapy to reduce prostate cancer progression in future. Research supported by NIH Grants: 1P20MD002285 and G12RR03062.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5332. doi:1538-7445.AM2012-5332
Abstract 5333: EpCAM overexpression in metastasizing gastric carcinomas
Authors: Ensinger Christian , Reinold Susanne , Galler Daniela , Drach Mathias , Hofer Carina
Published: August 2012

EpCAM, a homophilic Ca2+-independent adhesion molecule, is expressed in various epithelial tissues. Especially proliferating tissues or neoplasias show an increasing expression of EpCAM. Since it is found in large quantities in carcinomas, it seemed to be useful as novel therapeutic target for monoclonal and bispecific antibodies raised against the protein. Due to lack of data in gastric carcinomas, we investigated these tumors and their corresponding metastases. Therefore we evaluated the immunohistochemical reactivity of 104 gastric cancers, and their lymph node as well as distant metastases, using the mouse monoclonal antibody ESA by use of a previously defined scoring system for overexpression. Most of the tumour cells showed distinct membraneous reactivity, while normal gastric epithelium as internal control was EpCAM negative. 104 cases presented with an overexpression in 65,39%, without significant correlation to tumour stage or grade (p=0,339), no significant difference to histological growth patterns (according to Lauren) was found (p=0,186). 46,15 % of 26 mucinous carcinomas showed an overexpression, without mucinous parts they had an overexpression of 68 %, resulting in highly significant differences (p = 0.039 - two tailed) in these two groups. 54 primary tumors with metastases were evaluated. 62.96 % of these primary tumours had an EpCAM overexpression. 52 cases with lymph node metastases revealed a positive correlation with high significance (Pearson, p < 0.05). The paired-samples T-test no significance was found (p = 0.283 - two tailed). Comparing primary tumors with distant metastases a proportional relationship between the values was found (Pearson). The t-test brought no significant differences (p = 0.120). Between the lymph node metastases and distant metastases a positive correlation could be seen (Pearson, p = 0.192), without significance in the t-test (p = 0.121). Our study shows an EpCAM overexpression already in early stages of gastric cancers and a significant correlation between the primary tumors and metastases. So it seems to be possible to conclude from the EpCAM evaluation in available tissue of the primum to its metastases, facilitating clinical use of EpCAM-based immunotherapies and provide higher efficiency in the fight against all kinds of metastasizing gastric carcinomas. Recently designed new bispecific monoclonal antibodies are exerting improved therapeutic effects through antibody-dependent cellular cytotoxicity or complement. Also promising results were described for experimental gene therapy using EpCAM-targeted adenoviral vectors in human gastric and oesophageal carcinomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5333. doi:1538-7445.AM2012-5333
Abstract 5336: Distinct liver cancer stem cells defined by EpCAM and CD90 in human hepatocellular carcinoma
Authors: Yamashita Taro , Honda Masao , Kawaguchi Kazunori , Takatori Hajime , Sunakozaka Hajime , Wang Xin W. , Kaneko Shuichi
Published: August 2012

Recent evidences suggest that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells: CSCs). Although CSCs have been identified using several CSC markers and are now considered a pivotal target for the eradication of HCC, little information is known about the characteristics of marker-positive cells. Here we explored the expression of CSC markers CD90 and EpCAM in primary HCCs and cell lines to investigate the molecular characteristics of each cell type. Gene expression profiling and immunohistochemistry analyses were used to characterize 340 surgically resected HCC tissues and 11 advanced HCC tissues with distant organ metastasis obtained from autopsy. Magnetic-activated cell sorting was used to isolate EpCAM+ or CD90+ cells from surgically resected 12 fresh HCC specimens, which were tested for hepatic stem cell properties including their tumor initiating capacity in NOD/SCID mice. In primary HCC, EpCAM+ and CD90+ cells resided distinctively, and gene expression analysis of sorted cells suggested that EpCAM+ cells had the features of epithelial stem cells whereas CD90+ cells had those of vascular endothelial cells. Clinicopathological analysis indicated that presence of EpCAM+ cells was associated with poorly differentiated morphology and high serum AFP with a tendency of portal vein invasion, while the presence of CD90+ cells was associated with high incidence of distant organ metastasis within two years after surgical resection and advanced TNM stages. Serial xenotransplantation of EpCAM+/CD90+ cells in NOD/SCID mice revealed the rapid growth of EpCAM+ cells in the subcutaneous lesion and highly metastatic capacity of CD90+ cells into the lung. Xenotransplantation experiments of cell lines further demonstrated that EpCAM+ cells had the features of hepatic CSCs with marked tumorigenic capacity whereas CD90+ cells had those of vascular endothelial cells with highly metastatic features. Markedly, CD90+ cells showed the abundant expression of c-Kit and VEGFR1 and chemosensitivity to imatinib methylate (c-Kit inhibitor) and axitinib (VEGFR1/2 inhibitor). Taken together, our data strongly suggest the discrete natures of EpCAM+ and CD90+ cells with specific gene expression patterns and chemosensitivity to the molecularly targeted therapy. The presence of distinct CSCs may determine the clinical outcome of HCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5336. doi:1538-7445.AM2012-5336
Abstract 534: Protein kinase c ≤ cooperates with Pten deficiency in prostate cancer progression
Authors: Garg Rachana , Blando Jorge , Perez Carlos J. , Benavides Fernando , Kazanietz Marcelo G.
Published: August 2012

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related deaths among men. Protein kinase C epsilon (PKC∈), a member of the PKC family of phorbol ester/diacylglycerol receptors, has been shown to be up-regulated in human prostate cancer specimens. We recently demonstrated that mice that specifically overexpress PKC∈ in prostate under the control of the probasin promoter (PB-PKC∈) develop hyperplasia and PIN lesions. However, PKC∈ overexpression was insufficient to drive neoplastic changes in the mouse prostate. In the present study our goal was to determine whether PKC∈ overexpression can cooperate with Pten deficiency, one of the most common genetic alterations in human prostate cancer. We stably overexpressed PKC∈ in two pairs of isogenic mouse prostate epithelial lines that are either heterozygous (P2, P8) or homozygous (CaP2, CaP8) for Pten deletion. PKC∈ overexpression led to significant enhancement in cell proliferation compared to the respective control cells and this effect is more pronounced in CaP2 or CaP8 cells. Moreover, assays of colony formation in liquid medium revealed that anchorage-dependent growth was augmented in PKC∈-overexpressing cells relative to control cells. A significant elevation in phospho-Akt levels was observed in PKC∈ overexpressing cells relative to respective controls. To further elucidate the cooperativity between PKC∈ overexpression and Pten loss we intercrossed the PB-PKC∈ transgenic mice and Pten+/− mice. Strikingly, the resulting compound mutant mice overexpressing PKC∈ and haploinsufficient for Pten (PB-PKC∈; Pten+/−) developed fully invasive adenocarcinoma at 12 months of age. Very strong phospho-Akt, phospho-S6 and phospho mTOR staining could be detected in adenocarcinomas from PB-PKC∈; Pten+/− mice. Furthermore, we detected enhanced p-Erk, Cox-2, STAT3, NF-κB and cyclin D1 signaling in adenocarcinomas from PB-PKC∈;Pten+/− mice than in lesions from each monogenic model. These data thus suggest that PKC∈ overexpression cooperates with a defined oncogenic input to promote prostate tumorigenesis. These findings also suggest a crucial role of PKC∈ in survival.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 534. doi:1538-7445.AM2012-534
Abstract 5343: Identification of brain tumor initiating cells using the stem cell marker aldehyde dehydrogenase 1
Authors: Choi Seung Ah , Lee Ji Yeoun , Phi Ji Hoon , Wang Kyu-Chang , Park Chul-Kee , Park Sung-Hye , Kim Seung-Ki
Published: August 2012

Aldehyde dehydrogenase 1 (ALDH1) has been identified in stem cells from both normal and cancerous tissue. This study aimed to evaluate the potential of ALDH1 as a universal brain tumor initiating cell (BTIC) marker applicable to primary brain tumors and their biological role in maintaining stem cell status. Cells from various primary brain tumors (24 pediatric and 6 adult brain tumors) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH1 expression on BTIC characteristics in vitro and on tumorigenic potential in vivo. Primary cultured brain tumor cells showed universal expression of ALDH1, with 0.3 to 28.9% of the cells in various tumors identified as ALDH1+. ALDH1+ cells generate neurospheres with high proliferative potential, express neural stem cell markers (nestin and musashi) and differentiate into multiple nervous system lineages. ALDH1+ cells are phenotypically distinct from CD133+ cells, and they show high expression of induced pluripotent stem cell-related genes. Notably, targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After three months, ALDH1+ cells gave rise to tumors in 93% of mice whereas ALDH1- cells did not. The characteristic pathology of mice brain tumors from ALDH1+ cells was similar to that of human brain tumors, and these cells are highly proliferative in vivo. Our data suggest that primary brain tumors contain distinct subpopulations of cells that have high expression levels of ALDH1 and BTIC characteristics. ALDH1 might be a potential therapeutic target applicable to primary brain tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5343. doi:1538-7445.AM2012-5343
Abstract 5346: Tumor cell heterogeneity and chromosomal abnormality in malignant pleural effusion (MPE) samples of lung cancer
Authors: Basak Saroj Kumar , Rao Nagesh P. , Oh Scott , Mysore Veena S. , Srivatsan Eri S. , Batra Raj K.
Published: August 2012

Tumor heterogeneity is a hallmark of cancer and different types and stages of cancer express various cell surface markers and gene expression profiles. To understand tumor cell heterogeneity in lung cancer, we have developed a model using primary cells from malignant plural effusion (MPE) from lung cancer patients. Using this system we have identified CD44high expressing primary tumor cells within the MPE that form colonies in soft agar in vitro, and tumors in SCID/NOD mice. RT-PCR analysis revealed higher expression of cancer stem cell markers, Bmi-1, hTERT, EZH2, and OCT-4 in CD44high cells than the CD44low cells. Thus, our results indicate that primary tumor cells of MPE derived from lung cancer patients show heterogeneity with respect to their tumorigenic potential. To determine whether we could detect cytogenetic abnormalities in these samples, we performed karyotypic analysis of the MPE samples. Fibroblast cell line GM 05399 served as a control for these studies. Hyperdiploidy was observed in all three MPE samples and in the control cell line H 2122. Abnormalities of chromosome 1p were observed seen in all the samples that led us to perform FISH (fluorescence in situ hybridization) studies using a dual color 1p36 and 1q25 probe (control). We found LOH (loss of heterozygosity) at 1p36 in two MPE samples and rearrangements of both 1p/1q regions in the third MPE sample. Cell line H 2122 contained one normal chromosome 1 and an unbalanced translocation of unknown origin at 1p36 consistent with deletion of 1p. Our studies therefore suggest that this 1p36 deletion could result in the inactivation of a tumor suppressor gene and other sequences that include miRNA 34a also mapped to this locus. Chromosomal rearrangements further indicate that cells with multiple phenotypes including those related to metastasis, and chemo-radiation resistance might be present in the MPE samples. These studies provide a good basis suggesting that MPE would serve as a useful model system for the identification of genes related to the progression of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5346. doi:1538-7445.AM2012-5346
Abstract 5347: Identification of differentially expressed proteins in breast cancer stem cells using proteomic profiling
Authors: Kanojia Deepak P. , Zhou Weidong , Zhang Jiajia , Lo Pang-Kuo , Zhou Jun , Chen Hexin
Published: August 2012

Cancer stem cells (CSCs) are now recognized as pivotal entity in the development and progression of breast cancer. The CSCs are believed to be responsible for recurrence/relapse of cancer because of their ability to resist chemotherapeutic drugs. The present drugs in the clinics predominantly target the highly proliferating cells sparing the slow dividing and resistant cells. If the drugs targeting the cancer stem cells evolve, there is a possibility of eradication of cancer. In this direction it is imperative to understand the fundamental difference between the cancer stem cells and the non cancer stem cells. Hence, a basic understanding of genes expressed in CSCs vs non-CSCs needs to be explored. A large number of high throughput and extensive Micro array studies have been carried out to understand the normal and various breast cancer subtypes however, the RNA profiles does not necessarily match the protein profile, Therefore determination of protein levels is the key to identify the novel targets for cancer stem cells. To address this issue and to identify meaningful targets for breast cancer we have characterized breast cancer stem cells from primary cultures of Her2/Neu transgenic mice. We identified the altered proteins through LC-MS/MS using an LTQ-orbitrap mass spectrometer and confirmed those results using quantitative RT-PCR. We further performed insilico analysis, using microarray datasets, for identification of group of genes, which in combination can provide prognostic importance. We identified PTMA, S100A4, S100A6, TNXRD1, COX-1, COX-2 and FTH1 as a group of genes, which in combination were significantly associated with poor clinical outcome of breast cancer patients. Among these genes FTH1 was identified as a novel protein associated with prognosis of breast cancer, which is overexpressed in enriched breast CSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5347. doi:1538-7445.AM2012-5347
Abstract 5348: Transduction of tumors in vivo with choline kinase shRNA downregulates breast cancer stem cell markers
Authors: Krishnamachary Balaji , Penet Marie-France , Gadiya Mayur M. , Mori Noriko , Mironchik Yelena , Glunde Kristine , Bhujwalla Zaver M.
Published: August 2012

Increased phosphocholine is one of the metabolic signatures of cancer, and is closely associated with malignant transformation, invasion and metastasis. Increased phosphocholine is primarily due to increased expression of choline kinaseα, the enzyme that converts choline to the membrane precursor phosphocholine. Stem-like breast cancer cells (SBCCs) are drug resistant, invasive, and likely to lead to recurrence and repopulation. High CD44 adhesion molecule expression and high expression of the drug transporter ABCG2 are two markers associated with populations enriched with SBCCs [1]. Here we have examined the relationship between choline kinase and these two markers of SBCCs. We have determined the relationship between choline kinase downregulation and two markers of SBCCs, CD44 and ABCG2 in MDA-MB-231 breast cells in vitro and tumors in vivo upon lentiviral transduction of shRNA targeting choline kinase [2]. CD44 and its variant isoforms CD44v6 and CD44v8, together with ABCG2 showed a significant reduction in mRNA (30% for CD44 total; 80% for CDv6 and v8, and 45% for ABCG2) and protein expression levels following transduction of MDA-MB-231 cells with lentivirus expressing shRNA against choline kinase. Similar decreases were also observed following systemic intravenous injections of lentivirus expressing choline kinase shRNA delivered over four weeks. These studies provide insight into potential metabolic targeting of SBCCs. 1. Al-Hajj, M et al., Proc Natl Acad Sci U S A, 2003. 2. Krishnamachary, B et al., Cancer Research, 2009. This work was supported by NIH R01CA136576 and P50 CA103175.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5348. doi:1538-7445.AM2012-5348
Abstract 535: The role of high mobility group box 1 (HMGB1) in prostate carcinogenesis
Authors: Patel Purvi , Zheng Michelle , Bosland Maarten C. , Kajdacsy-Balla André , Munirathinam Gnanasekar
Published: August 2012

Prostate cancer is the most common cancer among older men and the second leading cancer-related cause of death in them. It is responsible for nearly 30,000 deaths per year in the United States. The causes for the development of prostate cancer are not clearly known although age and genetic factors play a key role in the etiology of this cancer. Epidemiological and experimental studies suggest that deregulated androgen levels and chronic inflammation also predispose to prostate cancer. However, the underlying molecular mechanisms which result in prostate cancer are not yet well known. Therefore, this study focuses on characterizing the role of High Mobility Group Box 1 protein (HMGB1), a co-activator of sex hormone receptors and a pro-inflammatory cytokine in the development of prostate carcinogenesis. In this study, we used an immortalized normal prostate cell (RWPE-1) to test if expression of HMGB1 in these cells will activate oncogenic signaling and transform them to a malignant phenotype. To achieve this, RWPE-1 cells were transfected with HMGB1 gene; stable lines were constructed and designated as RWPE-HMGB1. Cell cycle analysis by flow cytometry showed that majority (73%) of RWPE-1 cells over expressing HMGB1 was in the G2/M phase followed by polyploidy state (25%). Importantly, RWPE-HMGB1 cells when exposed to R1881 (synthetic androgen) treatment proliferated significantly in a time dependent manner compared to control RWPE-1 cells. Interestingly, the proliferative effects of HMGB1 in RWPE-1 cells was inhibited by anti-androgenic treatment (casodex) implicating HMGB1 to be a co-activator of androgen receptor in prostate cells. Further mechanistic studies revealed that expression of HMGB1 in RWPE-1 cells stimulated androgen receptor expression and PSA secretion in these cells. In addition, our studies also identified HMGB1 as an activator of two key extracellular (Extracellular signal-regulated kinase [Erk] and Toll-like receptor-4 [TLR-4]) pathways in RWPE-1 cells resulting in the inhibition of caspase-3 activation and prevention of PARP-1 cleavage suggesting HMGB1 is an inhibitor of apoptosis in prostate cancer cells. Overall, results of our study have identified a potential role of HMGB1 in prostate cancer which may help design drugs to target HMGB1 in preventing prostate carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 535. doi:1538-7445.AM2012-535
Abstract 5350: Aldehyde dehydrogenase 1 is useful for identifying some subtypes of non-basal triple negative breast cancer
Authors: Ishikawa Takashi , Ichikawa Yashushi , Shimizu Daisuke , Yamada Akimitsu , Tanabe Mikiko , Sasaki Takeshi , Kida Kumiko , Kimura Mariko , Ota Ikuko , Chishima Takashi , Endo Itaru
Published: August 2012

BACKGROUND Triple negative breast cancer (TNBC) is heterogeneous and consists of tumors associated with basal-type and cancer stem cell (CSC) phenotypes. Identifying these specific phenotypes is important for treating TNBC. The expression of aldehyde dehydrogenase 1 (ALDH-1), a biomarker of CSC phenotype, is observed more frequently in TNBC than non-TNBC and is associated with biologically aggressive features. AIM In order to categorize types of breast cancer, especially TNBC, ALDH-1 was examined in terms of 1) its association with pathological factors and 2) the genes significantly expressed in ALDH-1 positive cells. METHODS 1) The expression of ALDH-1 and its association with the other pathological factors of ER, PgR, HER2, p53, Ki-67, CK5/6, EGFR were examined immunohistochemically in 87 breast cancer samples. Histological grade was assigned to each of the samples.2) ALDH-1 positive and negative cells were collected by microdissection from formalin-fixed paraffin- embedded samples of 5 ALDH-1 positive breast cancer cases. mRNA was extracted and the expression profile of approximately 5,000 genes was assessed by cDNA microarray (Affymetrix GeneChip®, Human Genome U133 Plus 2.0 Array). By comparing gene expressions of ALDH-1 positive and negative cells in the same sample, characteristic genes related to CSC were identified. RESULTS 1) ALDH-1 was inversely correlated with ER (p=0.0006) and PgR (p=0.0054), while positively correlated with Ki67 (p=0.0023) and TopoII (p=0.0014). No association was found between ALDH-1 with CK5/6 or with EGFR. 2) Approximately 800 annotated genes were significantly up- or down- regulated. Several characteristic genes related to ALDH-1 have been presently confirmed by other methodologies. CONCLUSION CK5/6 and EGFR are currently used to identify basal-type TNBC. Because ALDH-1 was not associated with CK5/6 or EGFR, ALDH-1 and related genes may be helpful in identifying some non-basal types of TNBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5350. doi:1538-7445.AM2012-5350
Abstract 5352: Investigation of stem-cell markers in pleomorphic adenoma: Immunohistochemical and molecular study
Authors: Ianez Renata , Buim Marcilei , Coutinho-Camillo Cláudia , Soares Fernando , Lourenço Silvia
Published: August 2012

Background: Salivary gland neoplasms are originated from the various salivary gland compartments and are histologically related to these structures. Several protein and molecular markers are employed in research and diagnosis in order to classify and understand histogenesis and other aspects of salivary gland neoplastic lesions. Pleomorphic adenoma is a benign salivary gland neoplasm composed by epithelial and myoepithelial cells and a complex stroma. Its varied structural and architectural aspects suggest the participation of stem cells in its composition. Additionally, pleomorphic adenoma originates from intercalated duct of salivary glands, a region reputed to host the regenerative/ stem-cell compartment of these glands. The present work investigated stem-cell markers (CD24, CD44) in pleomorphic adenoma and in specimens of developing human salivary glands using immunohistochemistry and real-time RT-PCR. Material and Methods: 101 cases of pleomorphic adenomas were used - 55 cases were FFPE and 60 specimens were fresh frozen tissue. From the total, 14 were pared (FFPE and frozen tissue). Salivary gland specimens dissected from 20 human foetuses were also included. Results: All cases of pleomorphic adenomas were positive for the stem-cell markers studied. Neoplastic luminal structures were positive for CD24. Modified myoepithelial cells were positive for CD44. In foetal salivary glands, these markers were restricted to the intercalated duct region. Increased expression of CD44 stem-cell marker was observed in pleomorphic adenoma specimens using real-time RT-PCR technique when compared to normal salivary gland controls. An specific expression pattern was not observed for CD24 when compared to the normal salivary gland tissue - in some cases of pleomorphic adenoma there was an increased expression of the protein, whilst in other cases the expression was decreased or normal-like. Conclusion: Pleomorphic adenoma cells share similar markers with stem-cells; it is not possible to confirm that neoplastic cells bear the same characteristics of multipotency.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5352. doi:1538-7445.AM2012-5352
Abstract 5354: Nuclear spheres in developmental biology and diagnosis
Authors: Nieburgs Herbert E.
Published: August 2012

Introduction: The nuclear content of individual spheres with identical luminal diameters in buccal mucosa cells of patients with a malignant neoplasm at a distant site was first described by the author in 1962. Their biologic etiology remains unknown however. Recent studies revealed data on their correlation with nuclear spheres of cancer stem cells in asymmetric division. Materials and Methods: Observation of nuclear micro spheres was made by means of the highest magnification of the light microscope using a 100x objective and 10x ocular with a microscopic tube length of 1.25. The hematoxylin-eosin stain was applied for histological sections and cytological specimens. Buccal mucosa spreads of 145 cases were examined in correlation with cancer stem cells. Nuclear spheres in buccal mucosa cells were compared to those in self-renewal daughter cells of cancer stem cells in asymmetric division. Results: In asymmetric division of cancer stem cells the self-renewal daughter cell nucleus consisted entirely of spheres with identical luminal diameters of 0.33 to 0.5 µm. The other daughter cell was well differentiated with a nucleus devoid of spheres. In the buccal mucosa specimens with a distant malignant neoplasm, the nuclear content of spheres with luminal diameters of < 0.5 µm were noted in 112 of 145 cases with a sensitivity of 77% and specificity of 86%. The primary tumors were diverse in type and location. Discussion: The scientific literature is silent as to the rationale for nuclear spheres that appear with their identical luminal diameters in the buccal mucosa cells distant from a primary malignant neoplasm and identical to those of self-renewal daughter cells in asymmetric stem cell division. Nuclear spheres in cells distant from the primary tumor may occur as a result of circulating blood DNA or through blood cell migration to the buccal mucosa. The systemic manifestation of nuclear spheres at distant sites from a primary tumor is in keeping with the systemic involvement of cancer described by numerous investigators including the teams led by M. Us-Krasovic and B. Palcic (2005) and D.S. Alberts and P.H. Bartels (2007). The identification of nuclear spheres as a result of an overt or occult cancer offers the potential of a non-invasive cancer test.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5354. doi:1538-7445.AM2012-5354
Abstract 5358: Comparison of tumor-forming ability in different fractions of pancreatic ductal adenocarcinoma cells
Authors: Hagio Masahito , Matsuda Yoko , Kawamoto Yoko , Yanagisawa Yuji , Makino Hiroshi , Naito Zenya , Ishiwata Toshiyuki
Published: August 2012

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is associated with a high mortality rate due to its aggressive growth and high metastatic rate. Recently, cancer stem cells have been reported to play a central role in the proliferation, metastasis, and drug resistance of cancer. Although cancer stem cell-like cells seem to be included very loosely in cancer cells, information is still limited. Furthermore, the appropriate way of detecting cancer stem cells has remained controversial. In the present study, we tried to detect and collect a side population (SP) fraction as pancreatic cancer stem cells using Hoechst staining, and compared the in vivo tumor-forming abilities between the major population (MP) and SP of PDAC cells. Methods: A human pancreatic cancer cell line, KLM-1, was stained with Hoechst 33342. We distinguished between MP and SP of cells by the positivity of Hoechst, and then each fraction was isolated using a flow cytometer. Different numbers (1.0×102, 103, 104, and 105) and fractions (MP and SP) of cells were subcutaneously injected into the left and right sides of the back of NOD.CB17-Prkdcscid/J (NOD/SCID) mice. During a 5-week test period, the tumor size was measured regularly. Results: After 5 weeks, tumors were formed in mice injected with above 1.0×104 cells of both fractions. Especially, with the injection of 1.0×105 cells, the tumor-forming rate was 100% in the SP group, whereas it was 66.7% in the MP group. Furthermore, tumors were generated at about 1 week after the injection of the SP fraction (1.0×105 cells); however, in the MP group, it took about 3 weeks to generate a tumor after the injection of the same number of cells. Totally, the tumor volumes of the SP group were bigger than those of the MP group. Even with the injection of 1.0×104 cells of the SP fraction, the tumor volume was bigger than that on the injection of 1.0×105 cells of the MP fraction. Subcutaneous tumor tissues derived from SP and MP cells showed similar histological features. Conclusion: The malignancy of the SP fraction in PDAC cells was confirmed. This fraction might include cancer stem like-cell properties, and may be a candidate for therapeutic target in malignant PDAC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5358. doi:1538-7445.AM2012-5358
Abstract 5359: Gene expression characterization of different cancer stem cell isolation methods in head and neck cancer
Authors: Thibodeau Bryan , Geddes Timothy , Galoforo Sandra , Akervall Jan , Wilson George
Published: August 2012

Background: Head and neck squamous cell cancer (HNSCC) is a heterogeneous cancer that continues to pose major clinical problems. There is growing evidence that cancer stem cells (CSCs) may be an important factor in disease progression and resistance to current treatments. Establishing that stem cells are present in HNSCC and characterizing their global gene expression patterns provides the rationale for this study. Methods: A series of 5 HNSCC cell lines that have been maintained at low passage number such that they maintain phenotypic and morphological characteristics similar to the primary tumor were used in this study. Three different strategies were used to isolate stem cells by flow cytometry sorting: 1) Hoechst 33342 “side” population, 2) Aldefluor, and 3) CD44 bright cells. Global gene expression for each isolated CSC-enriched population and its associated CSC-depleted population was analyzed using Affymetrix Human Exon 1.0ST arrays. Gene set enrichment analysis was performed using Ariadne Pathway Studio. Results: The different methods of identifying CSCs produced widely different populations. In side population cells, there were 77 differentially expressed genes (p<0.05 and 1.5-fold cutoff) while only 59 and 17 genes were differentially expressed in the CD44 bright and Aldefluor bright populations, respectively. Not a single gene was commonly altered between any two of the isolation methods. Gene set enrichment analysis, however, was able to identify categories that differed between the CSC-enriched and CSC-depleted populations. Biological processes of cell cycle, cell division, mitosis, translational elongation, and spindle assembly were highly regulated. Microtubule binding/microtubule plus-end binding and structural constituent of ribosome were commonly altered molecular functions. Conclusions: This study suggests that current methods for isolating putative stem cells in HNSCC result in diverse populations with differing expression of specific genes. However, when examined for global expression patterns, several important categories of genes are commonly altered between the CSC-enriched and CSC-depleted populations. Some of the known characteristics of stem cells such as asymmetric division and unlimited growth potential are suggested by the highly regulated categories of cell division and spindle assembly. From these studies we hope to gain greater insight into the importance of stem cells in HNSCC as well as improve the capability of identifying these potentially important new targets for treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5359. doi:1538-7445.AM2012-5359
Abstract 5362: Multidrug-resistance (MDR-1) gene expression in immature glioma cells
Authors: Shimizu Keiji , Ishida Eri , Higashi Yoichiro , Fujimoto Yasunori , Yawata Toshio
Published: August 2012

Cancer stem cell hypothesis suggests that small populations of stem-like cells exist in the tumor mass and harbor drug resistance and low radio-sensitivity, and thus resulting in tumor recurrence after various treatments. In this study, we investigated the relationship between cell differentiation and drug resistant gene expression in glioblastoma. Glioma stem-like cells freshly isolated from surgical specimens were enriched in tumor sphere (TS) culture and were more resistant to anti-cancer drugs compared with their differentiated cells in cell viability assay. These cells were also tested for phosphorylation of histone H2AX at Ser139, a biomarker of DNA lesions including DNA double-strand and single-strand breaks. Differentiated cells were much more susceptible to anti-cancer drug-induced DNA damage, compared with TS cells. The expression of drug resistance genes, MDR-1, MRP-1 and MRP-2 in TS cells was more than those in their differentiated cells by RT-PCR analysis. However, there was no correlation between drug resistance genes and stem cell marker CD133 expression. It is possibly caused by another cell population expressing drug resistance genes, therefore, we sought to define the existence of progenitor cells in glioma by detection of glial progenitor marker, A2B5 using flow cytometric analysis. CD133 and A2B5 positive cells were more abundant in TS cells and were decreased during induction of differentiation. The expression of MDR-1 was frequently observed in CD133+/A2B5−, CD133+/A2B5+ and CD133−/A2B5+ cells. Reduction of MDR-1 expression concomitantly occurred with the differentiation of these immature glioma cells. Thus, it is revealed that CD133 negative cells expressing MDR-1 are A2B5-positive glioma cells. These results suggest that drug resistance genes are expressed not only in tumor stem cells but also in progenitor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5362. doi:1538-7445.AM2012-5362
Abstract 5363: Aggregation rather than monoclonal expansion explains ovarian cancer spheroid formation
Authors: Huang Zhiqing , Lowery William J. , Berchuck Andrew , Murphy Susan K.
Published: August 2012

Purpose: It is thought that a subpopulation of ovarian cancer cells possess stem cell like properties and are responsible for both the emergence of cancer and for subsequent chemoresistance and recurrence. A common approach used to enrich for cancer stems cells in vitro involves culture in selective media on low attachment plates resulting in non-attached multicellular spheres. Spheroids are present in ascites fluid of women with ovarian cancer, and may contain stem cells responsible for metastasis and recurrence. Our objective was to address whether spheroids are truly monoclonal in origin and arise from expansion of a single common progenitor cell. Procedures: We cultivated 5 ovarian cancer cell lines (DOV13, HEYC2, OVCAR2, OVCAR3, and PEO4) to confluence in monolayer cultures. The cell lines were chosen for their ability to form spheroids as well as variation in expression of the stem cell marker CD133 (OVCAR2, OVCAR3 CD133+, others negative). The cells were trypsinized to form single cell suspensions and plated at densities ranging from 100 to 5000 cells per plate in stem cell-selective media in ultra-low attachment culture dishes. A Zeiss Axio Observer microscope was used for time-lapse photography of the plated cells at 5-minute intervals over a 24-hour period. In addition, single cell suspensions were subjected to flow activated cell sorting to plate one cell in each well of 96-well Costar ultra-low cluster plates to monitor clonal formation of spheroids. Results: Cells were dissociated and plated under stem cell-selective conditions and 288 images of each cell line were recorded over 24 hours. The cells were found to begin to aggregate within two to four hours, regardless of cell numbers plated. By 24 hours post-plating, there was pronounced aggregation in 5/5 (100%) cell lines with formation of tightly compacted spheroid structures in 4/5 (80%) cell lines. Single cells in the 96-well plates showed no evidence of spheroid formation over a 14-day observation period. Conclusion: We observed spheroid formation in both CD133 positive and negative ovarian cancer cell lines. Formation occured due to cellular aggregation rather than clonal expansion from a single progenitor with stem cell properties. Although cellular aggregation may be relevant to ovarian cancer biology, these data suggest that spheroid formation should be viewed with caution if used as a proxy for monoclonal expansion of ovarian cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5363. doi:1538-7445.AM2012-5363
Abstract 5372: Dendritic-cell based vaccine for advanced pancreatic cancer: A report of 39 cases treated in Seren Clinic Nagoya
Authors: Kobayashi Masanori , Sakabe Tomoyo , Kure Shigehiro , Okamoto Masato , Yokokawa Kiyoshi
Published: August 2012

Objective: In the current study, we retrospectively assessed the efficacy of dendritic cell (DC)-based vaccine in advanced pancreatic carcinoma (PC). Patients: From Jan 2009 to April 2010, 39 patients (19 males and 20 females; aged 32-78 years, median 64 years) with advanced PC were treated with DC-based vaccine, and 38 patients also received GEM and/or S-1. Methods: Patient-derived peripheral blood mononuclear cells were obtained by leukapheresis (400ml×13 cycles), then adherent cells were cultured with GM-CSF, IL-4, OK-432 and PGE2 to generate DCs. The DCs pulsed with tumor specific peptides (WT1/MUC1) were injected at 2-week intervals (mean=8.3, max= 16 vaccinations). Results: The mean observation period was 257.6 days. None of the 39 patients showed CR, but we have found out 9 cases of PR and 9 cases of SD in accordance with RECIST. Response rate was 55% in the patients in combination with S-1+GEM and 9% with GEM alone. Disease control rate was 77.8% with S-1+GEM and 38% with GEM alone. Conclusions: DC-based vaccination in combined with GEM/S-1 was shown to be safe and possibly effective in patients with advanced pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5372. doi:1538-7445.AM2012-5372
Abstract 5400: Investigating the role of IDO in MUC1 expressing breast cancers
Authors: Besmer Dahlia , Kidiyoor Amritha , Nath Sritama , Roy Lopamudra Das , Curry Jennifer , Mukherjee Pinku
Published: August 2012

Breast cancer is the second most common cancer in the U.S. and contributes to 40,000 deaths a year. Breast cancer vaccines are currently being considered as a clinical intervention that may reduce the chance of metastasis and recurrence, and perhaps even function to be effective in cancer prevention. MUC1, a membrane tethered mucin glycoprotein, is over expressed in >90% of breast cancers, and therefore has been recently described as the second most targetable tumor antigen by the National Cancer Institute. There are currently ongoing trials using the MUC1 vaccine in patients with metastatic breast cancer. However, immunotherapy has had limited success because tumors have the ability to undergo immune evasion tactics. This includes expression of Indoleamine 2,3-dioxygenase (IDO) immunosuppressive enzymes. IDO was first identified in maintaining maternal tolerance towards the antigenically foreign fetus during pregnancy. Its activity is increased under pathological conditions, including during tumor development. IDO is emerging as a key player in T cell suppression and in the induction of immune tolerance to tumors. The present study is focused on understanding the role of IDO enzymatic activity on tumor development and immune function. In this study, we injected two cell lines that express IDO, and one cell line that is IDO null (MTAG, MTAG.MUC1 and IKOM, respectively). These cell lines were injected into either IDO null mice, or control c57/bk6 mice (n=3 each). We hypothesized that IDO expression in the tumor microenvironment of mice creates a pathological state of immune suppression resulting in altered tumor progression and immune function. We show that mice (whether they be IDO null or blk6) injected with IKOM cells have significantly slower tumor progression and rates of secondary metastasis as compared to those injected with IDO expressing tumor cells (p<0.05). In vitro, IKOM cells had significantly lower proliferative rates than the IDO expressing cells. Furthermore, mice injected with IKOM cells had a greater percentage of CD4+ and CD8+ T cells as compared to those mice injected with IDO expressing tumor cells. Mice injected with IDO expressing cells had a greater percentage of MDSC's characterized by the expression of gr1 and cd11b. Future studies would investigate the role of MUC1 based vaccines in combination with an IDO inhibitor, with the goal of reducing metastasis and increasing survival in patients with breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5400. doi:1538-7445.AM2012-5400