bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2212: C-terminus-truncated hepatitis B virus X protein enhances cell invasiveness in hepatocellular carcinoma
Authors: Sze Karen Man-Fong , Chu Glanice Kin-Yan , Ng Irene Oi-Lin
Published: August 2012

Hepatocellular carcinoma (HCC) is the fifth of major malignancies worldwide and chronic hepatitis B virus (HBV) infection is one of its well known risk factors. Random integration of the HBV DNA into host genome is frequent in human HCCs and this leads to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). In this study, we investigated the frequency of this C-terminal truncation of HBx in human HCCs and its functional significance in HCC cells. In the 51 HBV-positive patients with HCC, full-length HBx was detected in all of the non-tumorous livers. Interestingly, natural COOH-truncated HBx was found in 47% tumours. Upon clinicopathological analysis, presence of natural COOH-truncated HBx significantly correlated with the presence of venous invasion, a hallmark of metastasis (p < 0.01). Doxycycline- inducible stable expression of the full-length HBx and C-terminal truncated forms of HBx in HepG2 cells was employed for in vitro cell invasion assay and cell signalling analysis involved in cell invasiveness. Inducible expression of COOH-truncated HBx protein (with 24 amino acids truncated at C-terminal end) in HepG2 cells enhanced cell invasiveness, and C-Jun transcriptional activity, and increased MMP10 transcription production, as compared with the full-length HBx. The activation of the MMP10 promoter by the COOH-truncated HBx was abolished when the AP-1 binding sites were mutated. Our data suggest that COOH-truncation of HBx may play a significant role in enhancing cell invasiveness and metastasis in HCC, possibly via activation of the MMP10 through C-Jun signaling pathway. (This study was supported by a Hong Kong Government RFCID grant 05050022)

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 2212. doi:1538-7445.AM2012-2212
Abstract 2215: Glucocorticoid receptor function is essential for SOCS2-mediated negative regulation of hepatic GHR signaling
Authors: Mueller Kristina M. , Kornfeld Jan-Wilhelm , Schuetz Guenther , Moriggl Richard
Published: August 2012

GHR signaling plays an essential role in somatic growth and development as well as in the maintenance of metabolic homeostasis. Target tissue responsiveness to GH is dependent on postreceptor mechanisms to circumvent unphysiological and off-target effects due to prolonged GHR activation. GHR signals amongst others through the JAK2/STAT5 pathway. SOCS2, a GH-STAT5-regulated gene, is a key negative regulator of GHR sensitivity. Transcription of distinct GH-STAT5-dependent gene subsets requires coactivator function of the glucocorticoid receptor (GR). This mechanism was shown to involve direct binding of GR to the STAT5 N-terminus. Earlier studies have implied that regulation of SOCS2 also requires synergistic action of STAT5 and GR. Therefore, our aim is to investigate the molecular interaction between STAT5 and GR to regulate SOCS2 expression. Hepatocyte-specific deletion of the GR causes reduced hepatic Socs2 mRNA expression approximately to the same extent as STAT5-deficient livers. Furthermore, STAT5 activity was increased and prolonged in GR-deficient livers upon exogenous GH administration. Genes which are dependent on functional STAT5-GR protein interaction often harbor potential STAT5 oligomer binding sites in their promoter regions. Using bioinformatic screening, we found three STAT5 oligomer binding sites in the murine distal Socs2 promoter region. First, binding of activated STAT5 to the putative binding sites was confirmed by DNA binding assays. Subsequently, we performed transactivation studies using a luciferase reporter construct containing the relevant region of the Socs2 promoter and an additional 3′ region to assess the requirement for putative enhancer sequences. Maximum luciferase activity was observed in the presence of activated STAT5b and GR only. However, this pronounced transactivation was severely impaired upon transfection of the aminoterminally truncated STAT5 isoform (STAT5ΔN). Additionally, direct physical interaction of activated STAT5 and GR on the Socs2 promoter was detected in GH/dexamethasone-treated control livers by re-ChIP assays. We demonstrate that full Socs2 transactivation requires STAT5-GR protein-protein interaction in hepatocytes. As the liver represents a primary site of GH-mediated STAT5 activation, deregulated GR signaling could lead to imbalanced liver GH sensitivity due to aberrant SOCS2-mediated feedback inhibition.

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 2215. doi:1538-7445.AM2012-2215
Abstract 2219: Mechanism of trancriptional repression by EWS/FLI in Ewing's sarcoma
Authors: Sankar Savita , Lessnick Stephen L.
Published: August 2012

Objective: Ewing's sarcoma is a highly aggressive bone-associated malignancy affecting children and young adults with most cases harboring a somatic translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant transcription factor. Most studies indicate that EWS/FLI functions as a transcriptional activator. However, recent genome-wide transcriptional studies have raised the possibility that EWS/FLI may also repress target genes, although the mechanistic basis for this repression is unknown. We sought to test the hypothesis that EWS/FLI functions as a direct transcriptional repressor at some genetic loci, and that transcriptional repression is required for the oncogenic function of EWS/FLI. Furthermore, we sought to define relevant domains and corepressors that are involved in this activity. Methods: RNAi based microarray combined with genome-wide ChIP-Chip analyses were used to identify direct repressed target genes of EWS/FLI. Soft agar and xenograft tumor formation assays were performed to assess oncogenic transformation. Deletion mutants of EWS/FLI were constructed and used to map the repression domain. Co-immunoprecipitations were performed to validate the interaction of EWS/FLI with transcriptional co-repressors. Luciferase reporter assays and gel shift assays were utilized to characterize the binding site for EWS/FLI at repressed gene promoters. Results: We identified a subset of EWS/FLI-repressed target genes that are directly bound by the protein. Using these target genes, we identified domains in EWS/FLI necessary and sufficient for repression. Interestingly, these repression domains are also necessary for oncogenic transformation. We identified two HDACs that are involved in this repression, and identified a likely candidate transcriptional repression complex that binds EWS/FLI and contributes to its repressive function. Finally, we found that transcriptional repression is absolutely required for the oncogenic function of EWS/FLI, as two downregulated targets function as suppressors of Ewing's sarcoma oncogenesis. Conclusion: Transcriptional deregulation by EWS/FLI is a key oncogenic event in Ewing's sarcoma pathogenesis. Our data demonstrated that, in addition to transcriptional activation, EWS/FLI mediated transcriptional repression is also critical in Ewing's sarcoma oncogenesis. This is the first time that a detailed molecular mechanism of EWS/FLI mediated direct repression is reported, and has important implications for therapeutic targeting of EWS/FLI in Ewing's sarcoma.

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 2219. doi:1538-7445.AM2012-2219
Abstract 2231: The over-activation of Akt1 oncogene heightens the Glypican-3 (GPC3) effects on metastatic progression of murine mammary tumor cells
Authors: Romero Silvina A. , Huvelle Maria A. Lago , Peluffo Guillermo D. , de Kier Joffe Elisa Bal , Urtreger Alejandro J. , Peters Maria G.
Published: August 2012

It is known that Akt kinases 1, 2 and 3 have critical roles in regulating survival, metabolism and other cellular activities. However, their functions on migratory potential and metastasis development are less clear. GPC3 is a proteoglycan downregulated in breast tumors. We have previously shown that GPC3 reexpression inhibits metastatic capacity in vivo of the LM3 murine mammary adenocarcinoma cells. Moreover, this phenomenon was associated with a partial reversion of the epithelial-to-mesenchymal transition as shown in vitro. On the other hand, we have also determined that GPC3 modulates Akt signaling pathway in LM3 cells. The aim of this work was to investigate the role of Akt1 on the invasive/metastatic behavior of GPC3 reexpressing cells. First, we have confirmed that LM3-GPC3 cells present higher Akt1 levels than LM3-vector ones by western blot. To elucidate the function of this isoform, LM3-GPC3 clones were infected with viruses carrying a constitutively active variant of Akt1 (CA Akt1). Although there were no changes in the actin cytoskeleton organization, we could determine by a wound healing assay that LM3-GPC3-CA Akt1 cells were less migrant than their controls (Wound coverage: LM3-GPC3 control=50% vs. LM3-GPC3-CA Akt1=30%, p<0.05). To address an in vivo effect, different clones were injected s.c. into syngeneic female BALB/c mice. We found that these tumors did not differ in their growth rate, reaching a similar size at 30 days post inoculation (LM3-vector= 189±25, LM3-GPC3= 181±40, LM3-GPC3-CA Akt1= 200±51 mm3). No differences were found in either tumor incidence or in tumor latency. In order to study the experimental metastatic ability, all cell lines were i.v. inoculated. We observed that LM3-GPC3-CA Akt1 tumors developed fewer lung metastases than controls (Median [Range]: LM3-GPC3 control=9 [3-29] vs. LM3-GPC3 CA Akt1=0.5 [0-10], p<0.05). Moreover, the LM3-GPC3-CA Akt1 metastatic nodes were also of a smaller size (<0.5 mm). In sum, our results suggest that Akt1 expression and signaling could be mediating GPC3 inhibitory effect on the metastatic dissemination. The over-activation of this oncogene would exacerbate or heighten GPC3 effects, preventing the metastasis progression through the reduction of cell migration.

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 2231. doi:1538-7445.AM2012-2231
Abstract 2239: The effect combining the KIT inhibitor Imatinib with the PI3K inhibitor BKM120 or the dual PI3K/mTOR inhibitor BEZ235 on the proliferation of gastrointestinal stromal tumor cell lines
Authors: Li Fang , Growney Joseph , Battalagine Linda , Qiu Shumei , Manley Paul , Monahan John
Published: August 2012

Gastrointestinal stromal tumors (GIST) are the most common type of sarcoma found in the gastrointestinal tract, with 5,000-6,000 cases per year diagnosed in the United States. Activating mutations in KIT, or occasionally in PDGFR are present in up to 90% of GISTs. The PI3K/AKT pathway has been demonstrated to be a crucial survival pathway in imatinib-resistant GISTs. In this study, we evaluated single agent activities of the KIT inhibitor imatinib, the PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitor BEZ235, as well as the synergistic activity of the combination of imatinib with BKM120 and imatinib with BEZ235 in a panel of GIST cell lines. For example, when the antiproliferative effects of imatinib and BKM120 were evaluated in combination at concentrations from 0.37 µM to 3.3 µM, growth suppression was observed in excess of the percent inhibition achieved by imatinib or BKM120 single agent treatment in the GIST882 and GIST430 cell lines. Our results suggest that combining imatinib with BKM120 or BEZ235 may provide a therapeutic strategy for imatinib-resistant GISTs.

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 2239. doi:1538-7445.AM2012-2239
Abstract 2245: The role of STAT3α in Pten-dependent hematopoietic malignancy
Authors: Mizutani Tatsuaki , Casanova Emilio , Stoiber Dagmar
Published: August 2012

Dysregulation of the Janus kinase (Jak) - signal transducer and activator of transcription (Stat) signaling pathway is often associated with cancer. Persistent Stat3 activation has been described in many hematological malignancies. Two isoforms due to alternative splicing have been described for Stat3: Stat3α and Stat3α. Both isoforms exhibit overlapping but distinct biochemical properties. Interestingly, Stat3α has been found in the bone marrow of patients with acute myeloid leukemia (AML) and in several AML cell lines. The role of Stat3α in leukemia development remained elusive so far. To test for an impact of Stat3α in leukemia formation, transgenic mice overexpressing Stat3α have been generated. The expression of the transgene depends on Cre-recombinase activity therefore the mice were intercrossed with Mx-Cre transgenic animals, which enables activation of Cre recombinase in the hematopoietic compartment. This was verified by detection of the reporter protein eYFP in bone marrow cells upon treatment with poly I/C. The Stat3αtgMx-Cretg compound mice were further bred with Ptenfl/fl mice, since loss of Pten in hematopoietic stem cells of mice was reported to lead to myeloproliferative disorder (MPD) that later on progresses to an AML or ALL like disease. Upon poly I/C treatment Stat3αtgMx-CretgPtenfl/fl compound mice diseased with significantly enhanced latency when compared to Mx-CretgPtenfl/fl mice. Accordingly, 20 days after poly I/C injection Stat3αtgMx-CretgPtenfl/fl animals displayed also far less Mac1+Gr1+ myeloid cells than Mx-CretgPtenfl/fl mice. Taken together, our findings suggest that Stat3α acts as a tumor suppressor to prevent Pten dependent MPD/leukemia.

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 2245. doi:1538-7445.AM2012-2245
Abstract 2260: Novel molecular therapy targeting Mcl-1 and modulating autophagy in castration-resistant prostate cancer
Authors: Tang Wenhua , Qu Yang , Lan Lan , Wu Daocheng , Xu Liang
Published: August 2012

Chemo/radioresistance markedly impair the efficacy of cancer therapy. Anti-apoptotic Bcl-2 family proteins such as Bcl-xL, Bcl-2 and Mcl-1 are overexpressed in castration-resistant prostate cancer (CRPC) and contribute to prostate tumor initiation, progression and resistance to radiotherapy. A natural BH3-mimetic small molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing Phase II clinical trials for human prostate cancer. We have recently shown that (-)-gossypol preferentially induces autophagy in CRPC cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent cells with low Bcl-2 and sensitive to apoptosis. We have explored a series of gossypol derivatives and identified two novel BH3-mimetic lead compounds that are more potent than the parental compound in inducing autophagic cell death in CRPC. A novel pan-Bcl-2 inhibitor, apogossypolone (ApoG2), a derivative of (-)-gossypol, has a better binding affinity to Mcl-1 and an improved antitumor activity, and shows promising efficacy on a variety of cancers in vitro and in vivo. Treatment with the ApoG2 prior to radiotherapy enhances cell death via autophagic pathway in cancer cells which have high Mcl-1 and resistant to apoptosis. In addition to abrogating Bcl-2/Mcl-1–Beclin1 interactions, ApoG2 activates the autophagic pathway Atg5-dependently which synergizes with the effect of radiation. We further investigated the precise mechanism governing the influence of ApoG2 on IR-induced cell death. Our data suggest that, ApoG2 pretreatment suppresses IR induced Akt activation and mTOR phosphorylation. In this way, it serves as a potent radiosensitizer to affect autophagic cell death in cancer cells. Administration of ApoG2 prior to IR further delayed tumor growth of castration resistant prostate cancer xenograft model and increased autophagic cell death in vivo. Together, our data demonstrate a novel mode of action of ApoG2 as an enhancer of radiation induced autophagic cell death and provide mechanistic details of its tumor cell killing activities. These new insights into different modes of cell death induced by Mcl-1 inhibitors may facilitate the rational design of clinical trials by selecting patients most likely to benefit from the Mcl-1-targeted molecular therapy.

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 2260. doi:1538-7445.AM2012-2260
Abstract 2266: Beclin-1 regulation of autophagy in endocrine resistant breast cancer
Authors: Facey Caroline O. B. , Shajahan Ayesha N. , Clarke Robert
Published: August 2012

Resistance to endocrine therapy occurs in nearly 50% of all patients diagnosed with estrogen receptor ≤ positive breast cancer, thus elucidating mechanism(s) of endocrine resistance is of major importance. To provide a model, we selected MCF7 variants that are estrogen independent and either sensitive (LCC1) or resistant (LCC9) to antiestrogens: Faslodex (FAS; ICI 182, 780) and Tamoxifen (TAM). Antiestrogens induce endoplasmic reticulum stress which, depending on cell type, stimulus, and duration, may subsequently activate the unfolded protein response (UPR), autophagy, and/or apoptosis. Gene microarray analysis of LCC9 cells show high expression levels of X-box binding protein 1 (XBP1), implicating the unfolded protein response (UPR) as a pro-survival mechanism of resistance. In addition to an elevated UPR, autophagy is substantially elevated in response to antiestrogens. To better understand the role of autophagy in antiestrogen responsiveness, we investigated the consequence of knocking down BECLIN-1 in LCC1 and LCC9 cells treated with FAS and TAM. Preliminary results indicate a time-dependent change in autophagy following knock-down of beclin-1 in response to short-term (48h) and long-term (6 day) treatment. At 48h, sensitive LCC1 cells respond to antiestrogens with high levels of autophagy measured by increased LC3BII to LC3BI ratio and decreased expression of p62. Relative cell density (measured by crystal violet assay) decreases by 20% at 48h and by 80% at 6 days in LCC1 cells however, LCC9 cells are minimally affected by antiestrogen treatment. The level of autophagy remains high at 6 days in sensitive cells however levels remain constant in resistant cells. Autophagy is significantly lowered by reduced BECN1 in both sensitive and resistant cells however there is an increase in cell density in sensitive cells transfected with BECN1- shRNA at 6 days, indicating an autophagy associated programmed cell death mechanism in response to antiestrogen treatment. Protein expression of an active form of caspase −12/4 (an initiator caspase activated during endoplasmic reticulum stress) is elevated in sensitive cells at 6 days in response to antiestrogen treatment, also indicating the onset of apoptosis at this time. Elevated expression of Bcl-2 family members in resistant cells may play a more substantial role than BECN1 alone, as the combination of Bcl-2 family protein inhibitors and BECN1-shRNA sensitizes resistant cells to a greater extent. Thus, Bcl-2 family members in resistant cells may play a critical role in managing the balance between cell survival (moderate autophagy) and programmed cell death (excessive autophagy and apoptosis) in response to antiestrogen 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 2266. doi:1538-7445.AM2012-2266
Abstract 2267: Inhibition of autophagy in hypoxic HT29 cells reverses hypoxia-induced resistance to oxaliplatin
Authors: Vasilevskaya Irina A. , Selvakumaran Muthu , Roberts David , O'Dwyer Peter J.
Published: August 2012

BACKGROUND: We have shown previously that inhibition of JNK1 renders HT29 colon adenocarcinoma cells more sensitive to hypoxia and/or oxaliplatin due, in part, to impaired autophagy induction. We also showed that the autophagy inhibitor chloroquine sensitizes HT29 to oxaliplatin in vitro and in mouse xenograft model. PURPOSE: Here we investigate further by molecular approaches, including modulation of signaling through JNK, how inhibition of autophagy affects sensitivity of hypoxic colon cancer cells to oxaliplatin. We employed a panel of HT29-derived cell lines: lines stably expressing dominant negative constructs for either JNK1 (HTJ1.3) or JNK2 (HTJ2.2) singly, derivatives of these lines in which the other JNK gene is silenced by viral delivery of shRNA construct (HTJ1s2 and HTJ2s1), as well as HT29-derived lines with down-regulated BclX (HTBX) or Beclin-1 (HTB). RESULTS: We found that down-regulation of JNK1 leads to inhibition of autophagy accompanied by increase in apoptotic (versus necrosis) cell death. Clonogenic assays revealed higher resistance of HTJ1s2 cells to oxaliplatin under hypoxia, whereas HTJ2s1 cells demonstrated slight increase in sensitivity to the drug in this setting. Down-regulation of either Beclin-1 or BclX resulted in significant sensitization to oxaliplatin in oxic and, even more so, in hypoxic conditions (three-folds on average), while exerting differing effects on autophagy induction. CONCLUSIONS: Our data demonstrate that JNK1 plays an important role in autophagy induction under hypoxic conditions in the HT29 colon adenocarcinoma cell line and that its inhibition increases apoptotic cell death in this setting. Inhibition of autophagy induction by down-regulation of JNK1 and knock-down of Beclin-1 or BclX resulted in reversal of oxaliplatin resistance in hypoxic HT29 cells. These findings suggest that targeting these proteins may be of value in colon cancer 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 2267. doi:1538-7445.AM2012-2267
Abstract 2284: MIR-200 family as EMT related microRNA in renal cell carcinoma
Authors: Enokida Hideki , Yoshino Hirofumi , Yamasaki Takeshi , Hidaka Hideo , Chiyomaru Takeshi , Nohata Nijiro , Seki Naohiko , Nakagawa Masayuki
Published: August 2012

(Background) Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC represents the most common renal cancer histology. However surgical treatment is provided for localized disease, relapse or metastasis of the patient is caused in a considerable ratio. At present, metastatic RCC is difficult to treat and the process of metastasis is not well understood. Therefore, it is crucial to find molecular mechanisms based on recent genome analysis in RCC oncogenesis and metastasis. Based on the microRNA (miRNA) expression signature of RCC revealed that miR-200 family significantly reduced in RCC cells. The miR-200 family (miR-200b, miR-200a and miR-429 are encoded by single polycistronic transcript on chromosome 1p36.33 and miR-200c and miR-141 are cluster on chromosome 12 p13.31) of miRNA plays a major role to epithelial to mesenchymal transition (EMT) by targeting transcription repressors, zinc-finger E-box binding homeobox (ZEB1) and ZEB2. In this study, we investigated the functional significance of miR-200 family and identified the novel cancer pathways in RCC. (Methods) Cell proliferation and invasion assay was performed by restoration of mature miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) in RCC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-200 family by microarray analysis. (Results) The mRNA expression levels of ZEB1 and ZEB2 were significantly decreased by miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) transfectantion in RCC cells. Restoration of each miRNAs significantly inhibited cell proliferation and invasion in RCC cells. Interestingly, the morphological changes were recognized by miR-200 family transfection in RCC cell lines. Gene expression analysis showed more than ten candidate genes were searched for miR-200 family targets and these genes were up-regulated in RCC clinical specimens. (Conclusions) Our data suggest that miR-200 family function as tumor suppressors in RCC. EMT-related tumor suppressive miR-200 family mediates novel molecular targets provide new insights into the potential mechanisms of RCC oncogenesis and metastasis.

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 2284. doi:1538-7445.AM2012-2284
Abstract 2286: p53-miR-dependent post-transcriptional circuits: mechanisms, targets and inter-individual variation
Authors: Ciribilli Yari , Bisio Alessandra , Sanctis Veronica De , Vescovo Valerio Del , Lion Mattia , Jegga Anil G. , Denti Michela A. , Inga Alberto
Published: August 2012

Using bioinformatics approaches, we identified a group of candidate microRNAs (miRs) for direct p53 transcriptional control. To validate p53 family-mediated control of the newly predicted target miRs we evaluated the potential for wild type p53, p63α and p73α to transactivate from p53 response elements (REs) mapped at the miR promoters. Results obtained with an established yeast-based assay indicated that the REs found at miR-10b, -23b, -106a, -151, -191, -198, -202, -221, -320, -1204, -1026 genes were responsive to p53 of which 8 were also responsive to p63α or p73α. ChIP assays in doxorubicin-treated HCT116 cells -p53+/+ vs p53-/– revealed moderate p53 occupancy at the miR-202, -1206 sites, and weak occupancy at miR-10b, -191. RT-qPCR analyses in HCT116 and MCF7 cells showed modest doxorubicin- and/or p53-dependent regulation for miR-23b, -151, -191. We then focused on miR-191 and its potential impact on MDM4, an important modulator of p53. The A>C SNP (rs4245739) in the proximal portion of the MDM4 3′UTR occurs in a predicted binding site for miR-191, with the A allele resulting in a mismatch at the predicted miR-191 seed. This SNP is in strong association with the tag-SNP (rs2369244) within the MDM4 haplotype that was reported to impact on cancer risk. Using MCF7 and HCT116 cells that are heterozygous for rs4245739, we examined at the endogenous MDM4 gene level the impact of varying the expression of miR-191. A slight allelic imbalance was observed by allele-specific qPCR when miR-191 was over-expressed, and the co-transfection of miR-34a, for which seed sequences are predicted in the distal portion of the MDM4-3′UTR, had an additive effect, leading to ∼20% reduction of the C-allele. Western blot analyses indicated that steady-state MDM4 protein levels were reduced by miR-191 and/or miR-34a overexpression. Our study reveals additional miRs that could be directly regulated by the p53-family of transcription factors and could contribute to the tuning of p53-induced responses with the possibility of inter-individual variations due to functional SNPs.

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 2286. doi:1538-7445.AM2012-2286
Abstract 2312: A genomics approach to determining the role of DICER1 in tumorigenesis: Interferon responses in the cancer cell
Authors: Chiappinelli Katherine B. , Haynes Brian C. , Brent Michael R. , Goodfellow Paul J.
Published: August 2012

DICER1 is required for generation of mature microRNAs (miRNAs) and other short noncoding RNAs. Reduced DICER1 levels are associated with adverse outcomes in a variety of cancers and multiple lines of evidence suggest DICER1 is a tumor suppressor. The effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. To determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels had no effect on cell proliferation but were associated with enhanced cell migration and increased growth in soft agar. MiRNA profiling for the KLE cancer cell line DICER1 knockdowns (two different DICER1 shRNA constructs) revealed that miRNA levels were decreased overall. Not surprisingly, many mRNA transcripts were upregulated in the DICER1 knockdowns. A striking number of interferon stimulated genes (ISGs) showed upregulation. Upregulation of six ISG transcripts was validated by qRT-PCR in three independent knockdowns of DICER1 in KLE cells. IFNα, a key upstream regulator of the interferon response, was significantly increased in DICER1 knockdowns (KLE and additional endometrial cancer cell lines). IFNα protein secreted in media of DICER1 knockdown cells activated an interferon response in HT29 colon carcinoma cells. The majority of ISGs with increased transcript levels lacked miRNA target sequences and as such it seemed unlikely that the increase in expression levels was secondary to reduced miRNA levels. We tested the hypothesis that defects in miRNA processing could trigger an interferon response. We showed that DICER1 knockdowns in which miRNA processing is reduced have increased levels of pre-microRNAs in the cytoplasm. Pre-microRNAs are double-stranded molecules and long enough (60-100 nt) to activate the dsRNA sensors in the cytoplasm, such as Mda5/IFIH1, that typically sense viral dsRNA (> 30 nt) and stimulate the interferon response. Knockdown of DROSHA, necessary for processing of pri-miRNAs to pre-miRNAs, did not result in accumulation of pre-miRNAs and did not cause an interferon response. Our findings suggest elevated pre-microRNA levels trigger the interferon response to double stranded RNA (incompletely processed miRNA and other species). Rescue experiments (restoring DICER1 function) are currently underway to shed additional light on how reduced double-stranded RNA processing might elicit an interferon response. Work is ongoing to determine the relationship between the observed interferon response and the migration and soft agar phenotypes.

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 2312. doi:1538-7445.AM2012-2312
Abstract 232: Protein kinase C b1 (PKCb1) inhibits the malignant phenotype of the human pancreatic tumor-derived cell line PANC1
Authors: Cirigliano Stéfano M. , Mauro Laura V. , Grossoni Valeria C. , Joffe Elisa D. Bal De Kier , Colombo Lucas L. , Puricelli Lydia I. , Urtreger Alejandro J.
Published: August 2012

Pancreatic cancer is a deadly disease with a 5% overall survival after five years. Different members of the PKC family have been described as modulators of pancreatic cancer progression but, till nowadays, the relevance of each PKC isoform in this process is unclear. The aim of the present work was to study the effect of PKCb1 overexpression, on several critical determinants of tumor progression using the human pancreatic tumor-derived cell line PANC1. Cells were stably transfected with an expression vector containing the full-length PKCb1 gene (PKCb1-PANC1) or with the empty vector, as control (pMTH-PANC1). While no morphologic alterations could be detected in vitro, PKCb1 overexpression induced a significant reduction in the proliferative capacity of PANC1 cells. The reduction in this capacity could be associated with a decrease in Cyclin D1, Cyclin E and phosphorylated ERK1/2 levels, and an increase in p21 expression levels, as measured by Western blot. In addition PKCb1-PANC1 cells showed an increased resistance to cell death induced by serum starvation (% viability: 58.54±4.1 vs 39.29±4.2 in pMTH-PANC1, p<0.05). Using flow cytometry we could determine that serum deprivation promoted PKCb1-PANC1 arrest in the G1 phase of the cell cycle (cells in G0/G1: 86.7±1.0% vs 45.5±0.8% in pMTH-PANC1, p<0.05). Moreover, the overexpression of PKCb1 reduced the in vitro invasive capacity using Matrigel coated transwells (cells x 10 microscopic x400 fields: 2.22±0.9 vs 10.10±2.6 in pMTH-PANC1, p<0.05). This behavior could be associated with a significant inhibition of secreted proteases activity (uPA: 4.11±0.8 vs 12.32±4.1 IU/mg/24h in pMTH-PANC1, p<0.05 and MMP-9: 15.24±0.03 vs 24.89±2.6 AU/mg in pMTH-PANC1, p<0.05). When control and PANC1-PKCb1 cells were s.c. inoculated into nude mice it was observed that PKCb1 cells were almost unable to grow and form tumors while vector-transfected cells retained this ability. In sum, our results suggest that PKCb1 overexpression in human pancreatic tumor cells PANC1 induced a less malignant phenotype both in vivo and in vitro.

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 232. doi:1538-7445.AM2012-232
Abstract 233: Polo-like kinase 1 facilitates loss of Pten-induced prostate cancer formation
Authors: Liu X. Shawn , Song Bing , Elzey Bennett D. , Ratliff Timothy L. , Konieczny Stephen F. , Cheng Liang , Liu Xiaoqi
Published: August 2012

Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the molecular mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress, and that nuclear function of Pten, but not its phosphastase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a key regulator of the cell cycle. We found that elevated Plk1 is critical for Pten-depleted cells to adapt to mitotic stress for survival, and that re-introduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. In order to evaluate the clinical relevance of our finding, we examined the expression level of Plk1 in human prostate cancer tissue assay which includes normal tissue samples, prostate hyperplasia samples and malignant tumor samples. A correlation between overexpression of Plk1 and genetic inactivation of Pten was observed during prostate neoplasia formation. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.

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 233. doi:1538-7445.AM2012-233
Abstract 2339: Specific binding of modified ZD 6474 (Vandetanib) monomer and its dimer with VEGF receptor 2
Authors: Kim Yoo-Shin , Li Feng , O'neill Brian Edward , Li King CP , Li Zheng
Published: August 2012

Introduction: Tumor angiogenesis is an important component of cancer biology driven in part by the thesis that tumor vessel growth is a necessary requirement for tumor growth. Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF). A significant portion of research on tumor angiogenesis has focused on VEGF and its blockage with the expectation that dramatic results would be demonstrated in cancer patients as previously documented in animal models. Vandetanib, also known as ZD6474, is an antagonist of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and the epidermal growth factor receptor (EGFR or HER1 or ErbB1). It is an orally bioavailable small molecule tyrosine kinase inhibitor of multiple growth factors, and is now available in the US for the treatment of metastatic medullary thyroid cancer (MTC). Methods: ZD 6474 monomer was purchased modified to add a fluorescent dye (6-FAM). Its coupled ZD 6474 dimer was also prepared using a PEG linker, similarly with FAM fluorescent dye. The two compounds were compared in a series of in vitro studies using human endothelial cell (HUVECs) and several cancer cell lines. We first looked at VEGFR binding affinity and specificity with cell uptake and blocking study. Then, the effects on cellular viability and angiogenesis were compared using with tubular formation of HUVECs in vitro, also matrigel plug assay, and bio-distribution test on VEGF over expressed U87 human glioma tumor model in vivo. Results: Imaging of ZD 6474 dimer showed much better binding and uptake in HUVECs and other VEGFR expressing cells than ZD 6474 monomer, including during systemic delivery to tumors. However, the therapeutic and anti-angiogenic effects were mixed, with most tests showing the dimer to have same or weaker effects than the monomer. Conclusion: This study provides a rational for the evaluation of dimeric ZD 6474 as a potential imaging agent. Paradoxically, it also demonstrates that the enhanced receptor binding achieved by multivalency does not always translate into enhanced therapeutic effectiveness. Keyword: Vandetanib (ZD 6474), anti-angiogenesis, VEGFR

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 2339. doi:1538-7445.AM2012-2339
Abstract 2351: Identification of somatic mutations in esophageal squamous cell carcinoma and corresponding xenograft by next-generation sequencing
Authors: Fang Douglas D. , Gao Yibo , Xu Qiang , Sun Hongye , Zhang Bin , Chen Zhaoli , Chan Chi-Chung , He Jie
Published: August 2012

The genomes of all cancer cells carry somatic mutations, including both passenger and driver mutations. Driver mutations, which are implicated in oncogenesis, are most likely preserved in xenografted tumor cells in immunedeficient mice. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas in the gastrointenstinal tract. It is prevalent in the developing world and associated with substantial morbidity and mortality. Molecular mechanisms contributing to initiation and progression of ESCC are still poorly understood. Systematic identification of genetic variations and putative driver mutations may lead to understanding of pathogenesis of the disease. In this study, using next-generation sequencing we performed exome sequencing of eight paired Chinese esophageal squamous cell carcinoma and xenograft. Comparing with control genomes obtained from case-matched peripheral blood cells, we identified a wide variety of somatic variations in both primary and xenograft tumors, including TP53, RGMA, MUC4, TCEAL6, PRSS3, etc. Moreover, we found that mouse sequences accounted for a significant fraction of sequence reads from xenograft samples. Excluding these mouse sequences significantly improved the accuracy of mutation detection in xenograft samples. Overall, our findings shed a light on important somatic mutations involved in ESCC. In addition, comparison between primary and xenograft tumors may lead to identification of driver mutations that confer growth advantage. These mutations represent therapeutic opportunities for development of targeted therapy.

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 2351. doi:1538-7445.AM2012-2351
Abstract 237: Novel applications of pSIVA, a new polarity sensitive phosphatidylserine (PS) apoptosis/cell death probe
Authors: Stein Lisa S. , Quintel Payton D. , Krajewski Stan , Rosenberg Jonathan S. , Singh Sujay K.
Published: August 2012

pSIVA is a newly developed Annexin B12 based polarity sensitive probe for monitoring exposed PS, a hallmark of apoptosis. A unique feature of pSIVA is that it fluoresces (green channel) only when bound to PS, and hence no wash steps are needed to remove unbound probe. This attribute is enabling the development of new methodologies not previously possible with classical Annexin V conjugates, which fluoresce irrespective of whether or not they are bound to PS. In this study we developed a flow cytometry protocol whereby pSIVA is added directly to cell culture media, avoiding the use of buffers. The protocol eliminates unforeseen perturbation or negative effects that standard buffers can have on sensitive cell types. Multicolor methodology that employed pSIVA-labeled cells along with intracellular markers was developed for flow cytometry and microscopy platforms. A model using primary mouse neurons labeled with pSIVA, DAPI and MAP2 (neuronal marker) indicated pSIVA stained the cell body of spontaneously dying neurons but stained both the cell body and degenerating axons in staurosporine treated neurons. These results suggest that mechanisms of PS exposure may vary between different apoptotic processes, including spontaneous versus induced apoptosis. They also suggest pSIVA has potential for elucidating new pathways of PS exposure heretofore not possible with existing reagents.

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 237. doi:1538-7445.AM2012-237
Abstract 2380: Detecting human circulating endothelial cells using the acoustic focusing cytometer
Authors: Anderson April , Haataja Kristi , Bradford Jolene A.
Published: August 2012

Circulating endothelial cells (CECs) are mature cells shed from blood vessel walls during the natural process of endothelial cell turnover. Elevated levels of CECs have been reported in a host of pathological conditions including cardiovascular disorders, infectious diseases, immune disorders, post-transplantation analysis, and cancer. In cancer research, CECs have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence and metastasis formation during or after chemotherapy. In healthy individuals, CECs are reported to be present in very low numbers: 0.01%-0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of CECs, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic-focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing CECs. Here we present a method for detecting human circulating endothelial cells using the Attune® Acoustic Focusing Cytometer. The method includes a number of conventional ways to improve rare event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1000 µL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic-focusing cytometer would be 10-12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method; details and specific panels designed for both the blue/violet and blue/red Attune® cytometer configurations are outlined.

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 2380. doi:1538-7445.AM2012-2380
Abstract 239: A novel multimeric protein scaffold stimulates apoptotic signaling through TRAILR2
Authors: Chen Hong , Swers Jeffery , Inigo Ivan , Carrasco Rosa A. , Grinberg Luba , Wang Lin , Leow Ching-Ching , Xiao Zhan , Wu Herren , Hollingsworth Bob , Baca Manuel , Tice David A.
Published: August 2012

TRAILR2 (DR5, TNFRSF10B) is a member of the TNF-receptor superfamily that mediates programmed cell death following stimulation with its ligand. Binding of homotrimeric ligand TRAIL to TRAILR2 induces oligomerization of the receptors and triggers the apoptotic pathway through induction of caspases. Although TRAILR2 is found to be expressed in a number of normal cell types it is routinely found at high levels in many cancer types. Since TRAIL has been shown to preferentially induce apoptosis in cancer cells versus normal cells, several TRAIL receptor agonists including monoclonal antibodies as well as the ligand itself, are currently in clinical development. Here, we describe a novel protein scaffold (Tn3) based on a fibronectin type-III domain from the protein Tenascin that was engineered to specifically agonize TRAILR2. Connecting TRAILR2-binding Tn3 domains together significantly improved the in vitro potency of the molecule in several cancer cell models. For example, a hexameric TRAILR2-binding Tn3 construct has an EC50 of 0.5 pM in a 72 hour cell viability assay with Colo-205 cells. This is approximately 2 logs better than TRAIL. Furthermore, a subset of TRAIL-resistant cancer cell lines derived from multiple tissues of origin is sensitive to the multimeric Tn3. The improved potency over TRAIL was demonstrated in colorectal cancer xenograft models. In addition, tumor regression was also observed at doses as low as 0.3 mg/kg. These results suggest that a multimeric Tn3 protein scaffold targeting TRAILR2 has more potent anti-tumor activity in vitro and in vivo compared than the natural ligand.

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 239. doi:1538-7445.AM2012-239