bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1057: BRMS1 restoration of gap junctional intercellular communication involves modulation of KATP channels and downregulation of phosphoinositide 3-kinase (PI3K)
Authors: Bodenstine Thomas M. , Vaidya Kedar S. , Ismail Aimen , Cook Leah M. , Welch Danny R.
Published: February 2011

Metastatic disease remains the most lethal aspect of cancer, accounting for over 90% of cancer related mortality. Breast Cancer Metastasis Suppressor 1 (BRMS1) specifically inhibits the ability of tumor cells to metastasize to secondary sites while having little or no affect on primary tumor growth. Clinically, a loss of BRMS1 mRNA expression in breast cancer patients is correlated with poor prognosis and a decrease in BRMS1 protein levels predicts reduced disease free survival. The mechanisms of BRMS1 remain unclear; however expression of BRMS1 significantly decreases the PI3K signaling pathway, a pathway that is often over activated in cancer cells. PI3K is a lipid kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) to PtdIns(3,4,5)P3, leading to downstream signaling. BRMS1 acts to reduce the substrate of PI3K, PtdIns(4,5)P2, by >90% when expressed in metastatic cells. Re-expression of BRMS1 in cancer cell lines also restores gap junctional intercellular communication (GJIC), a process that mediates cellular homeostasis and is often lost during neoplastic progression. GJIC has been shown to be affected by PI3K; thus we hypothesized that inhibition of PI3K signaling by BRMS1 might be responsible for the restoration of GJIC. In support of this, treatment with the PI3K inhibitor LY294002 (10µM) in vector control and parental cell lines from multiple cancer cell types, mimicked the effects of BRMS1 on GJIC. Interestingly, inhibition of signaling molecules further downstream of PI3K (AKT, mTOR) did not produce the same effect, suggesting the mechanism of action may directly involve the ratio of PtdIns(4,5)P2/PtdIns(3,4,5)P3. We show that alterations in an ATP dependent potassium channel, KATP, regulated by phosphoinositides, is responsible for the changes in GJIC observed by BRMS1 expression. Delineation of this mechanism will allow for in vivo analysis to determine if changes in GJIC are causative for the BRMS1 mediated suppression of metastasis. Taken together, our data suggest an inhibitory mechanism on gap junctional communication mediated by PI3K, that is relieved by restoration of BRMS1 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1057.
Abstract 1061: Glutathione S-transferase P1 is required for growth and survival of human glioma cells
Authors: Ali-Osman Francis , Liu Jiewu
Published: February 2011

Overexpression of glutathione S-transferase p1 (GSTP1), a protein involved in Phase II metabolism and cell signaling, is a hallmark of diverse human malignancies, including gliomas, and is associated with resistance to therapy, aggressive tumor growth and poor patient survival. Although, the function of GSTP1 in drug metabolism and detoxification is well documented, its role in regulating tumor cell growth remains unclear. Here, we report that in human glioma characterized by high GSTP1 expression, GSTP1 is obligately required for the growth and survival of the tumor cells. For this, we stably infected cells of the MGR3 glioblastoma multiforme (GBM) cell line that expresses high de novo levels of GSTP1, with a lentiviral vector that either expresses a short hairpin RNA (shRNA) specifically targeting to GSTP1 or a control shRNA. We isolated two clones, 1 and 2 and showed by western blotting that the GSTP1 specific shRNA mediated decreases in GSTP1 expression of 92% and 100%, respectively, relative to controls while infected with eGFP shRNA. Growth kinetic analysis showed the in vitro growth rates of both clones 1 and 2 to be significantly lower than that of the parental or control clone, with population doubling times of 108.9 ± 17.6 hr for clone 1 and 94.8 ± 4.3 hr for clone 2, compared with 40.6 ± 2.6 hr for the parental cells and 44.7 ± 2.0 hr for the control transfected cells. Cell cycle analysis showed that the GSTP1 knockdown induced a significant almost 2-fold increase in the sub-G1 (apoptotic) population that correlated with an increase in caspase 3/7 activation. Western blotting showed no significant changes in total protein levels of ERK, JNK and p38 MAPK following GSTP1 knockdown, however, phosphorylation levels of both ERK1/2 and JNK1/2 increased significantly, while phospho- p38 MAPK levels did not change. JNK downstream signaling, viz., phosphorylation of c-Jun, increased dramatically in cells of the two clones. These alterations in MAPK signaling in the tumor cells associated with GSTP1 silencing correlated with the decreased survival and growth kinetics and corresponding changes in apoptosis/survival signals, including, decreased bcl2 and increased bax levels in the tumors. Our results show that GSTP1 is required for cell survival and growth of human GBM cells that naturally over-express high GSTP1 levels. This is consistent with the aggressive tumor growth and poor survival of patients whose tumors contain high levels of GSTP1 and provide a rationale for targeting GSTP1 as a potential therapeutic strategy for malignant gliomas.

Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA).

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 1061.
Abstract 1078: Rosiglitazone inhibits breast cancer growth and proliferation via cyclin D1 inhibition.
Authors: Oberlick Elaine , Taliaferro-Smith LaTonia D. , Harris Wayne , Knight Brandi B. , Nagalingam Arumugam , Waller Edmund , Sharma Dipali
Published: February 2011

Breast cancer ranks as the most commonly diagnosed and second leading cause of cancer death in North American women. Thiazolidinediones (TZDs) are a class of anti-diabetic drugs primarily used to treat type 2 diabetes mellitus by increasing insulin sensitivity; these compounds are also known to possess anti-tumor effects in several cancer cells including lung and breast cancer. The present study was designed to evaluate the antiproliferative effects of Rosiglitazone on triple negative breast cancer cells. MDA-MB-231 breast cancer cells were treated with Rosiglitazone and compared with negative control (without treatment) for modulation of molecules involved in cell-cycle regulation and apoptosis, including cyclin D1, caspase-3 and −9, Bad, and Bax. Our results show that Rosigliatzone suppresses the proliferation of highly-metastatic human breast cancer MDA-MB-231 cell line by increasing the number of cells in G1 and reducing the number of cells progressing through the S-phase of the cell cycle. Further investigation revealed that the mechanism by which Rosiglitazone arrested cells in the G1-phase was via inhibition of cyclin D1 (a cell cycle regulatory protein that is essential for progression through the G1-phase of the cell cycle and has an central pathogenetic role in breast cancer). Our lab has previously shown an essential role of cyclin D1 upregulation in breast cacner growth and proliferation. Collectively, our results suggest that Rosiglitazone inhibits cancer cell proliferation by modulating expression of cyclin D1 and may provide a novel role for the use of Rosiglitazone as a therapeutic antitumorigenic agent.

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 1078.
Abstract 1084: Honokiol induces G1 phase cell cycle arrest in human epidermoid carcinoma A431 cells
Authors: Guillermo Ruth F. , Chilampalli Shivani , Chandrasekhar Gudiseva , Kaushik Radhey S. , Fahmy Hesham , Dwivedi Chandradhar
Published: February 2011

Recent studies from our laboratory have indicated that pre applications of honokiol (one hour before) prevent UVB- induced skin cancer development in female SKH-1 mice.

Honokiol reduced significantly the cell viability of A431, as assessed by MTT (IC50 60 μM) and BrdU incorporation assay.

The aim of this study is to investigate the effects of honokiol on the cell cycle of human epidermoid carcinoma A431 cell line and to investigate the influence of honokiol in the A431 cell cycle regulator proteins expression.

A431 cells were treated at different concentrations with honokiol for specified periods of time (8, 16, 24, 32, 40 and 48 hours). Cells were harvested and fixed in 70% ethanol and stored at −20 °C. The cell cycle phase analysis was performed by using a Beckton Dickinson Facstar flow cytometer and CellQuest software. Cell lysates were prepared from A431 cells treated with honokiol in similar conditions; proteins were analyzed by Western blotting.

Treatment with honokiol at concentration 50 μM and time periods of 24, 32, 40 and 48 hours blocked the A431 cell cycle in G1 phase significantly (P<0.05) and decreased the percentage of cells in the S and G2/M phase.

Honokiol affects the expression of proteins involved in the A431 cell cycle regulation, down-regulating the expression of cyclin A, CDK2, CDK4 and CDK6 proteins and up-regulating the expression of p21 and p27 proteins which are CDKs inhibitors.

These findings indicate that honokiol causes cell cycle arrest at G1 phase and alteres the expression of various proteins involved in the G1 phase of A431 cell cycle.

(Supported by Translational Cancer Center Research Grant, funded as 2010 Research Initiative Center by the State of South Dakota).

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 1084.
Abstract 1087: The expression and activity of actin-regulating protein cofilin are associated with the cell cycle progression
Authors: Chiang Pei-Hsun , Hung Shih-Chieh , Lee Yi-Jang
Published: February 2011

Cofilin is a member of actin depolymerizing factor (ADF)/cofilin family that can regulates actin dynamics for cancer growth and invasion/metastasis. Several lines of evidence have demonstrated that cofilin is associated with the cell cycle regulation, while the underlying mechanisms remain to be addressed. To better understand the involvement of cofilin in cell cycle progression, we dissected the cofilin expression and activity in different phases of the cell cycle. We synchronized human non-small lung A549 cancer cells in G1, S and G2/M phase by serum starvation, double thymidine blockage and nocodazole treatment, respectively. It was found that the expression of endogenous cofilin was weakest in G1 phase, while it was strongest in S phase and was modest in G2/M phase. This observation was further confirmed by detecting the down-regulation of highly expressed cofilin in S phase-synchronized cells entering mid-late S, G2/M and G1 phase of the cell cycle. Interestingly, it also showed that inactive form of cofilin (ser-3 phosphorylated) weakly expressed in S phase and gradually increased when cells progress through G2/M and G1 phases. These results suggest that cofilin expression and activity are coordinately regulated during the cell cycle progression. It is also consistent with our previous findings that enforced expression of cofilin results in G1 phase arrest but not S and G2/M phases. Moreover, the level of cofilin transcripts was not significantly altered in different phases of cell cycle. On the other hand, pretreatment of the proteasome inhibitor MG132 resulted in increase of cofilin in G1 phase, suggesting that the fluctuation of cofilin during cell cycle progression is associated with the protein stability. In addition, transduction of undegradable Y68F form of mutant cofilin potentially led to increase of the G1 percentage. Taken together, precisely regulation of cofilin expression and activity is important for cell cycle progression. Targeting on cofilin may provide alternative therapeutic strategy for cancer treatment.

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 1087.
Abstract 1096: Arf-independent activation of p53 by the Dmp1 tumor suppressor
Authors: Kai Fumitake , Kendig Robert D. , Frazier Donna P. , Taneja Pankaj , Maglic Dejan , Sugiyama Takayuki , Fry Elizabeth A. , Willingham Mark C. , Inoue Kazushi
Published: February 2011

Cyclin D binding myb-like protein 1 (Dmp1; Dmtf1) is a tumor suppressor that is deleted in ∼40 % of human non-small cell lung carcinomas. The Dmp1 promoter receives signals from oncogenic Ras and the protein shows its activity as a tumor suppressor by directly binding and transactivating the Arf promoter, and thereby inducing Arf-, p53-dependent cell cycle arrest. Both Eµ-Myc and K-rasLA-induced tumor development was accelerated in Dmp1+/− and Dmp1−/− mice, suggesting that Dmp1 is haplo-insufficient for tumor suppression. In Eµ-Myc lymphomas, the combined frequencies of p53 mutation and Arf deletion in the Dmp1+/− or Dmp1−/− mice were significantly lower (∼10 %) than that in Dmp1+/+ littermates (∼50 %), indicating that Dmp1 is a physiological regulator of the Arf-p53 pathway in vivo. We recently found that the frequency of p53 mutation (∼40 %) was significantly decreased in both Dmp1+/− and Dmp1−/− backgrounds (< 10 %) in the K-rasLA lung cancer model where the Ink4a/Arf involvement is very rare. Moreover, our recent data show that Dmp1 physically interacts with p53 to neutralize the activity of Hdm2. To demonstrate the Arf-independent function of Dmp1 on p53 activation in vivo, we injected doxorubicin into wild-type, Arf-null, and Dmp1-null mice, harvested the thymus at 2 hr intervals, and studied the expression of the p53 targets, p21cip1 and bbc3, by real-time PCR and immunohistochemistry. Thymus was chosen as a target tissue since Dmp1 is highly expressed in the medulla in our histochemical studies. Western blotting analyses with DO-1 and phosphoserine-specific antibodies verified p53 activation by doxorubicin. Cleaved caspase 3 was detectable in the thymic medulla of wild-type mice 6 hrs after tail injection of doxorubicin, indicating the induction of apoptotic cell death by the genotoxic drug. Cleaved caspase 3 staining was significantly decreased in the thymus from Arf−/− mice as compared to wild-type mice, but the staining was even lower in Dmp1−/− mice. Consistent with these findings, the p21cip1 mRNA induction (∼23-fold increase in wild-type mice) was more significantly compromised in Dmp1−/− mice (∼7-fold) than in Arf−/− mice (∼15-fold). Bbc was undetectable in Dmp1−/− thymus even 4-6 hrs after injection of doxorubicin while it was significantly induced (∼3-fold) in both wild-type and Arf−/− mice. These mRNA data were confirmed by immunohistochemical staining of thymic tissues with p21cip1 and bbc3-specific antibodies. Together, our data demonstrate the Arf-independent activation of p53 by Dmp1 in response to genotoxic stress in vivo. We are currently conducting the same series of experiments in the mouse lung.

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 1096.
Abstract 1100: p53 blocks TGFβ-induced breast cancer metastasis by downregulating angiopoietin-like 4
Authors: Elangovan Selvakumar , Prasad Puttur D. , Ganapathy Vadivel , Thangaraju Muthusamy
Published: February 2011

Transforming growth factor β1 (TGFβ1) is a multifunctional growth factor with a complex dual role in tumorigenesis. During the early stages of tumor formation, TGFβ1 acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis in tumor cells. In advanced stage of cancer, many tumor cells become unresponsive to the growth-inhibitory actions of TGFβ1 and become more motile, more invasive, and resistant to apoptosis. The molecular mechanism responsible for the conversion of TGFβ1-associated tumor suppression into TGFβ1-associated pro-oncogenic and pro-metastatic signaling is not known. Thus, identification of the signaling pathway responsible for this process would have a potential impact in developing novel breast cancer therapy. Recent studies have shown that TGFβ1 induces ANGPTL4 expression, which leads to lung metastasis of breast cancer through disruption of the endothelial barrier. ANGPTL4, also known as fasting-induced adipose factor (FIAF), is a circulating protein; it plays a role in adipogenesis, angiogenesis, carcinogenesis and metastasis. The expression of ANGPTL4 is dramatically induced in human metastatic breast cancer cell line MB231 by TGFβ1. While studying the molecular mechanisms that control the TGFβ1-associated ANGPTL4 induction, we found that p53 functions as a potent negative regulator of TGFβ1 dependent induction of ANGPTL4 expression. In human breast cancer cells, TGFβ1 induces ANGPTL4 expression only in p53-null cell lines like MB453 and MB468, and in cell lines which express non-functional p53 like T47D and MB231. TGFβ1 is unable to induce ANGPTL4 expression in cells expressing the functional p53. To identify the mechanism by which p53 inhibits ANGPTL4 expression, we cloned 2.2 kb human ANGPTL4 promoter fragment into luciferase (pGL3-Luc) and GFP (pU3RII-pEGFP) reporter plasmids, and we used these constructs for reporter assays in p53 inducible (p53-Tet-On) MB453 cell line. The results showed that functional expression of p53 in MB453 cells effectively blocks the ability of TGFβ1 to induce the transcriptional activation of ANGPTL4, and suggest that p53 serves as a molecular dictator to block the tumor-suppressive and pro-metastatic actions of TGFβ in breast tissue. These studies describe the molecular processes involved in the regulation of ANGPTL4 expression in breast cancer, and will be helpful in the design and development of novel therapeutic strategies for the treatment of breast 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 1100.
Abstract 1101: Reexpression of hSNF5 regulates the transcriptional activity of the p21 promoter through p53 dependent or independent mechanisms in malignant rhabdoid tumors
Authors: Kuwahara Yasumichi , Charboneau Aubri , Knudsen Erik S. , Wissman Bernard E.
Published: February 2011

Malignant rhabdoid tumor (MRT), a highly aggressive cancer of young children, displays inactivation of the hSNF5 gene in primary tumors and cell lines. We have previously reported that reexpression of hSNF5 in MRT cell lines causes a G1 arrest via p21WAF1/CIP1 (p21) mRNA induction. However, the mechanisms of p21 promoter activation by hSNF5 remain unclear.

Because p21 is a transcriptional target of the p53 tumor suppressor gene, we determined the role of p53 in hSNF5-induced p21 activation. We reexpressed hSNF5 in the A204 and TTC642 MRT cell lines, with or without stable knockdown of p53 expression, using adenoviral vectors expressing either hSNF5 and GFP or GFP. While loss of p53 expression significantly inhibited p21 transcriptional activity induced by hSNF5 in A204 cells at 24 hours, it did not alter its activity by hSNF5 after 24 hours in the TTC642 cells. These results suggested that the up-regulation p21 transcription by hSNF5 operated through p53-dependent and -independent mechanisms in the A204 and TTC642 cell lines, respectively. Next, we used chromatin immunoprecipitation (ChIP) analysis of the p21 promoter to determine where hSNF5 appeared at the p21 promoter and whether its recruitment altered binding of other transcription factors or the chromatin landscape. Our results indicated that reexpressed hSNF5 binds within 1 kb of transcript start site (TSS), with maximal enrichment at the TSS in both cell lines. Furthermore, histone acetyltransferases (HATs), RNA polymerase II (RNAPII), and BRG-1 are assembled with p53 and CDK8 (co-activator of p53 transcriptional program) by reexpression of hSNF5 in A204 cells. In contrast, while p53 and CDK8 occupancy did not change after hSNF5 reexpression in the TTC642 cells, HATs, RNAPII, and BRG-1 levels increased at the p21 promoter. These findings correlated with the results of p53 knock-down experiments. Furthermore, histone H3K36 tri-methylation increased downstream of the TSS in both cell lines. These results suggested hSNF5 regulates the initiation activity of the p21 promoter by either itself or p53 recruitment, resulting in the mRNA elongation. Our findings demonstrate that while induction of p21 transcription by hSNF5 in A204 cells depends on p53, it occurs independently of p53 in TTC642 cells. The lack of dependence upon p53 appears to extend to other MRT cell lines tested in our laboratory. Furthermore, our results suggest that hSNF5 may alter p21 transcriptional initiation by itself or through the recruitment of other uncharacterized transcription factors.

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 1101.
Abstract 1106: CEACAM1 expression dependent on p53 after DNA damage
Authors: Shin jae sik , Jin dong-hoon , Hong seung-woo , Kim jee-eun , Jung da-jung , Kang jae-seung , Hwang young-il , Lee wang-jae
Published: February 2011

The glycoprotein carcinoembryonic-antigen-related cell adhesion molecule 1 (CEACAM1) is a membrane-bound protein found in microvilli of the apical colon cell surface. Recently, It was reported that signal transduction through the cytoplasmic domains appears to be important for the tumor suppressor function of CEACAM1 and also apoptosis induction in early tumor development. However, the mechanisms that control CEACAM1 expression are not clear. In this study, we show that CEACAM1 expression is induced in a p53-dependent manner. More importantly, alternative splicing of CEACAM1 to the long form (CEACAM1-4L) and the short form (CEACAM1-4S) is regulated by p53 in human colon carcinoma cells, Hct116. Treatment with etoposide induced CEACAM1 expression in cells that have p53 function, but not in cells lacking p53 function. In addition, knockdown of endogenous wild-type p53 using RNA interference and p53 overexpression by adenoviral vector were paralleled with CEACAM1 expression in Hct116 cells. These results suggest that induction of CEACAM1 in the context of tumor suppression is regulated in a p53-dependent manner and implicate CEACAM1 as a key determinant of cell fate.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1106.
Abstract 1109: Identification of a novel MDM2 splice transcript that lacks nuclear transport signals but harbors the p53 binding motif in human glioma cells
Authors: Antoun Gamil , Ali-Osman Francis
Published: February 2011

MDM2 a nuclear phosphoprotein binds with E3 ubiquitin ligase activity, binds and targets p53 for proteosomal degradation, hence is a negative regulator of p53. Consequently, over-expression of MDM2 results in the loss of p53 tumor suppressor function even in the absence of inactivating mutations in the p53 gene. To date, more than 40 different alternative spliced MDM2 transcript variants have been reported in both normal and tumor cells, with most of these lacking, in part or in full, the p53 binding domain of MDM2. In this study, we examined the levels of MDM2 gene expression in a panel of glioblastoma (GBM) cell lines and in vivo primary tumor xenografts by quantitative PCR /RT-PCR and examined the full length MDM2 cDNAs for the presence of MDM2 splice transcript variants using the nested PCR technique and a combination of direct sequencing and TA vector sequencing. The results showed a significant heterogeneity in MDM2 expression and in MDM2 splice variants among the tumors. Importantly, we identified a novel splice MDM2 transcript that was consistently associated with low MDM2 transcript levels in the tumors. Sequence analysis of this MDM2 spliced transcript showed it to harbor an internal deletion of three exons, namely 7, 8, and 9, containing the nuclear localization signal (NLS), the nuclear export signal (NES) and a portion of the acidic domain (AD). The p53 binding domain was fully conserved. The deletion caused a shift in the MDM2 open reading frame that created a stop codon (TGA) at codon 316. The spliced MDM2 transcript encoded a truncated MDM2 protein that harbors the functional p53 binding motif capable of interacting with the p53 protein but without the ability to localize to the nucleolus. Overall, our results with the 15 tumors showed no strong correlation between the presence of this novel splice MDM2 transcript and the expression levels of p53in the tumors. Studies are ongoing to determine the functional consequences of this novel MDM2 spliced variant on p53 downstream function in glioma cells.

Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA).

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 1109.
Abstract 1114: Linker region of the BRCA2 protein increases chemoresistance to cisplatin: Screen for the characterization of cancer-associated variants
Authors: Warren Curtis R. , Farach-Carson Mary C. , Catts Zohra Ali-Khan
Published: February 2011

Screening for mutations in the BRCA1 and BRCA2 genes is standard for patients with family histories suggesting Hereditary Breast and Ovarian Cancer Syndrome (HBOC). Often, patients present with a familial cancer pedigree and a BRCA sequence containing an uncharacterized missense mutation. Such mutations are termed “Variants of Unknown Significance” (VUS) because their ability to alter function of the protein in vivo is unknown [1]. VUS complicate the genetic counseling process as the genetic counselor cannot advise patients if their VUS predisposes them to cancer. We discovered a novel VUS that occurred in the BRCA2 gene in a cancer registry patient in Delaware. Further mining of this registry identified two novel VUS occurring in the same region of the BRCA2 gene encoding the linker region of the BRCA2 protein, a poorly understood functional region of the protein. The two variants were R2341C and N2452D, both carried by breast cancer patients whose family histories were indicative of HBOC. The Breast Cancer Information Core (BIC)[2] in the National Institutes of Health National Human Genome Research Institute catalogues cancer-associated mutations in BRCA1 and BRCA2, including over 500 mutations reported to occur in the linker region; over 300 of these are VUS's. Our lab-based study focused on the linker region peptide encoded by exons twelve through fourteen of the BRCA2 gene. To develop a new method to characterize VUS in the BRCA2 gene, we chose 13 VUS occurring on evolutionarily conserved residues with varying frequencies. One, I2285V, was reported 79 times, whereas several others were reported only once. To determine if these VUS represent polymorphisms or if they change the function of the BRCA2 protein, we constructed expression plasmids encoding each mutant peptide for use in stable transfection experiments in T47D cells. Four mutant peptide expression clones and a wild-type linker region expression clone were studied by challenging the cell lines with the DNA crosslinking compound cisplatin. Several of the mutants increased survival in transected cell lines, indicating that the linker region peptide may interact directly or indirectly with a protein or DNA partner in the DNA damage repair pathway. In this case, the reported VUS mutations may change the activity of the BRCA2 linker region. We conclude that this may be a good method for screening the effect of VUS on protein function. Further study is required to create a high-throughput, standardized assay for characterization of VUS which would benefit patient and clinician alike.

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 1114.
Abstract 1116: Epigenetic control of the BRCA1 expression by a metabolic switch
Authors: Di Lijun , Fernandez Alfonso , Gardner Kevin
Published: February 2011

Though deletion or loss of BRCA1 function is the major cause of most familial forms of breast cancer, decreased expression of non-mutated BRCA1 alleles is often found in sporadic breast cancer, suggesting that alterations in BRCA1 transcriptional regulation may contribute to non-inherited forms of breast cancer. In this study, we show that BRCA1 expression is controlled by a tightly regulated dynamic balance between the association of transcriptional co-activators and co-repressors that regulate histone acetylation and chromatin accessibility at the BRCA1 bi-directional promoter to enforce unidirectional transcriptional activation in response to estrogen stimulation. A central regulatory step in the control of the BRCA1 promoter is the eviction of the co-repressor and metabolic sensor C-terminal-binding protein1 (CtBP1). Loss of CtBP1 from the BRCA1 promoter induced by estrogen, CtBP1 RNAi depletion or increasing NAD+/NADH ratio results in elevated histone acetylation, HDAC1 dismissal from the BRCA1 promoter and increased BRCA1 transcription. Notably this level of regulation is lost in the absence of physiological chromatin structure at the BRCA1 promoter. These findings indicate that BRCA1 expression is controlled by a metabolic switch that dynamically influences the localized deposition of epigenetic marks and chromatin structure at the BRCA1 promoter and suggests that caloric intake may influence the levels of tumor suppressor function in mammary tissues.

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 1116.
Abstract 1129: FLCN-putative tumor suppressor
Authors: Seabra Laurence , Nahorski Michael , Rienmann Anne , Lu Xiaohong , Lim Derek , Maher Eamonn
Published: February 2011

Birt-Hogg-Dubé (BHD) is an autosomal dominantly inherited genodermatosis associated with the development of renal neoplasia, spontaneous pneumothorax and benign fibrofolliculomas. The BHD gene has been maps to chromosome 17p11.2 and encodes a protein called folliculin (FLCN). Although familial forms of renal cell carcinoma account for only about 3% of all cases, the elucidation of the molecular mechanisms of familial RCC syndromes has provided seminal insights into the pathogenesis of sporadic RCC. This is exemplified by the role of VHL tumor suppressor gene (TSG) inactivation in sporadic clear cell RCC. The FLCN TSG product has been linked to the mTOR/AMPK signaling pathways but the function of folliculin is much less well characterised than for other renal TSGs such as VHL. Among the multiple functions that have been associated with the VHL TSG product is a role in the regulation of apoptotic cell death. We have investigated whether folliculin also has a role in apoptosis. Comparison of isogenic FLCN null and replete cell lines demonstrated increased cell death in the latter. Flow cytometry revealed a 30 % shift in apoptotic index as determined by Annexin V vs PI staining which was demonstrated by a shift in the cell population towards an early apoptotic state. Expression of apoptosis-related proteins was investigated in FLCN null and replete cell lines using proteome array profiler. This demonstrated increased expression of SMAC, claspin HTRA2 and XIAP in FLCN replete cells. Further investigations to determine the precise role of folliculin in the regulation of apoptosis and the relationship between FLCN-induced apoptosis and p53 status are in progress.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1129.
Abstract 1143: Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)
Authors: Kudo Kenji , Xi Yaguang , Wang Yuan , Song Bo , Chu Edward , Ju Jingyue , Russo James J. , Ju Jingfang
Published: February 2011

We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil (5-FU) treatment as assessed by real time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.

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 1143.
Abstract 1148: Array comparative genomic hybridization of diffuse intrinsic pontine glioma samples obtained from formalin fixed paraffin embedded autopsy specimens
Authors: Suuriniemi Miia , Warren Katherine , Killian Keith , Wang Yonghong , Quezado Martha , Walker Robert L. , Meltzer Paul S.
Published: February 2011

We have recently optimized the method of formalin fixed paraffin embedded (FFPE) tissue processing for high resolution array comparative genomic hybridization (aCGH). This will facilitate the copy number assessment of number archival clinical surgical pathology specimens as well as biopsies with debated utility. Molecular characterization of archival specimens can potentially clarify the biology of diseases with unclear etiology and poor prognosis such as diffuse intrinsic pontine glioma (DIPG). In the case of DIPG, which is seldom biopsied, at present autopsy samples provide the only practical approach to studying this disorder. Here we performed aCGH on FFPE autopsy specimens obtained from children with DIPG.

DNA was extracted from FFPE blocks obtained from autopsy on 14 pediatric DIPG patients using a modified Qiagen DNeasy kit. Non-enzymatic Universal Linkage System (ULS) chemical labeling was used to directly couple fluorescent dyes with the sample and reference DNAs (1ug each), which were then hybridized to 105k oligonucleotide CGH microarrays (Agilent). Arrays were scanned and data analyzed in Nexus Copy Number software.

Twenty-one samples from 14 subjects were available for the study from which CGH was successfully performed on 18 samples representing 12 patients. All samples demonstrated DNA aberrations. Frequent DNA copy number changes included gain of 1q, 7p, and loss of 10q. High copy number amplification of known oncogenes was observed, including PDGFRA, EGFR, CCND1, and MYCN. Homozygous deletions of known tumor suppressor genes included PTEN and CDKN2A.

Our tissue processing method demonstrates the feasibility of successfully using DNA derived from FFPE autopsy material for aCGH. The pediatric DIPG sample set was highly informative with the majority of specimens showing at least 1 abnormality related to a known cancer gene. Significant heterogeneity among DIPG patients as well as aberrations in candidate drug targets (KIT and EGFR) were observed.

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 1148.
Abstract 1149: Genetic analysis of single cells
Authors: Fan Jian-Bing , Chen Jing , April Craig , Klotzle Brandy , Royce Thomas , Cann Gordon , Talasaz AmirAli H. , Islam Saiful , Kjallquist Una , Linnarsson Sten
Published: February 2011

We are developing highly sensitive and quantitative genomic technologies for genetic and expression analysis of single cells or trace amount of DNA/RNA materials, using microarray and next-generation sequencing platforms. We have tested various Multiple Displacement Amplification (MDA)-based protocols under a variety of reaction conditions. With our current protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 cell and 5 cells, respectively. We are also developing RNA amplification methods for high-throughput expression profiling of single cells. With our current protocol, we were able to generate reproducible expression profiles, R2 = 0.73 and 0.77, using 10 pg and 50 pg total RNA input, respectively. In addition, the profiles correlated well with those obtained with standard 100 ng total RNA input, R2 = 0.61 and 0.77, respectively. Our data show that sequencing of single-cell transcriptomes can clearly distinguish embryonic stem cells from embryonic fibroblasts and tumor cells. We are currently using these technologies to study medical specimens such as circulating tumor cells and cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1149.
Abstract 1151: HER2- and EGFR-specific Affiprobes - Novel recombinant optical probes for cell imaging
Authors: Lyakhov Ilya , Kuban Monika , Zielinski Rafal , Kramer-Marek Gabriela , Capala Jacek
Published: February 2011

The human epidermal growth factor receptors EGFR (Her1, ErbB1) and HER2 (HER2/neu, ErbB2, or c-erb-b2) are members of EGFR (ErbB) family of cell-surface receptors/tyrosine kinases: EGFR, HER2, HER3, and HER4. There are several identified ligands for these receptors (except HER2), including EGF and TGF-alpha, that induce receptor dimerization followed by tyrosine autophosphorylation, which leads to cell proliferation. EGFR and HER2 are found overexpressed in a number of cancer types, such as breast, ovarian, salivary gland, stomach, kidney, colon, prostate, and non-small cell lung cancer. Research has shown that EGFR- or HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic function, its internalization mechanism, and subcellular trafficking are not fully understood, partially because the availability of imaging reagents and tools for detection of EGFR and HER2 is still restricted to medium/low affinity antibody conjugated to small-molecule dyes. We have created photo-stable and relatively simple-to-prepare imaging probes for in vitro staining of EGFR and HER2. These new reagents, called Affiprobes, consist of the targeting moiety, a HER2- or EGFR-specific Affibody® molecule, and a fluorescent detection moiety mCherry (Red), or EGFP (Green). These fluorescent proteins were chosen due to their high stability and solubility and low toxicity for eukaryotic cells. Furthermore, HER2 and EGFR receptors can be detected simultaneously using corresponding optical filter sets. Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio for the Affiprobes. This type of optical probe can be easily extended to targeting other cell-surface antigens/receptors using different Affibody® molecules and fluorescent proteins.

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 1151.
Abstract 1155: Quantification of circulating tumor cells in peripheral blood using EGFR-targeting gold nanoparticles
Authors: Wang Xu , Qian Ximei , Beitler Jonathan J. , Chen Zhuo Georgia , Nie Shuming , Shin Dong M.
Published: February 2011

Background: The prognostic significance of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (HNSCC) is of great clinical interest. HNSCCs are overwhelmingly EGFR positive. Objective: Our goal was to pilot nanotechnology to identify CTCs in human xenograft mouse model as a prelude to clinical use. Methods: Surface-enhanced Raman Scattering (SERS)-gold nanocrystals (60 nm) were prepared by spectral encoding with a Raman reporter molecule and then stabilized and protected by low molecular weight polyethylene glycols (PEGs). Heterofunctional PEG was covalently conjugated to epidermal growth factor (EGF) peptide, a ligand that binds to EGFR with high specificity and affinity. To identify CTC in normal mouse blood, varying quantities of Tu212 cells were added into mice blood samples, then our nanoparticle was added to the sample and the presence of Tu212 cells were detected in vitro using laser excitation of the Raman reporters. Different types of cancer cells with variable EGFR expression were also examined in blood to confirm the specificity of the EGF-targeted SERS nanoparticles. Normal mouse blood served as the experimental control to account for non-specific binding. Results: The cellular uptake of the EGFR-targeting nanoparticle in high EGFR expression Tu212 cells was about 10-fold higher than that in low EGFR expression H460 cells in vitro, suggesting a specific EGFR targeting of the gold nanoparticle. Tu212 cells could be selectively labeled and quantified (5.6 × 104 nanoparticles/cell) at a concentration of approximately 10 cells per ml of normal mouse blood. Comparing the labeling of Tu212 cells by our nanoparticles with H460 cells in the mouse blood samples showed that the signal intensity in Tu212 cells was about 9-fold higher than that in H460 cells ((3750 ± 230 vs. 350 ± 120, respectively, p <0.05). These results indicated high sensitivity and specificity of using SERS-gold nanoparticles for detection of CTCs. Conclusion: We have demonstrated the ability to use targeted surface-enhanced Raman gold nanoparticles to quantify CTCs with EGFR expression (This research was supported by the National Cancer Institute award 5 P50 CA128613 and U54CA119338-04).

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 1155.
Abstract 1157: Ubiquitin-proteolytic control of HOX homeodomain proteins in normal and malignant hematopoiesis
Authors: Zhou Pengbo , Lee Jennifer , Zhang Jianxuan , Liu Liren , Shieh Jae-Hung , Moore Malcolm A. S.
Published: February 2011

The HOX homeodomain proteins are key regulators of hematopoiesis. HOX genes are expressed in primitive hematopoietic cells, and their prompt downregulation is associated with hematopoietic differentiation and maturation. Although transcriptional inactivation of HOX genes during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOX proteins. We have previously shown that the CUL4A ubiquitin ligase controls the stability of HOXA9 by promoting its ubiquitination and proteasome-dependent degradation. Interfering with CUL4A biosynthesis resulted in alterations of the abundance of HOXA9, mirrored by impairment of the myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. Here we show that other HOX family members are also subjected to CUL4A-mediated ubiquitination and destruction, and defined the degradation signal (degron) for these HOX proteins. A degradation-resistant recombinant HOX protein was engineered and affinity-purified, and when directly transduced into G-CSF-mobilized CD34+ adult hematopoietic cells, demonstrated significant enhancement of promoting ex vivo expansion of these primitive hematopoietic progenitors. These results revealed a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis, and opened up new avenues for ex vivo expansion of hematopoietic stem cells for therapeutic use.

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 1157.