bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2872: Induction of autophagy in hypoxic HT29 cells requires functional JNK1
Authors: Vasilevskaya Irina A. , Roberts David , O'Dwyer Peter J
Published: June 2012

BACKGROUND: Hypoxic HT29 colon cancer cell lines exhibit higher sensitivity to oxaliplatin when SEK1 function is impaired and higher resistance when MKK7 is down regulated (which in turn, cause more profound inhibition of JNK activation). We observed differential effects of JNK1/JNK2 modulation on oxaliplatin sensitivity of hypoxic HT29 cells to oxaliplatin treatment that implied a pro-survival function for JNK1. Further, we have shown that both oxaliplatin- and hypoxia-induced autophagy in these cells is diminished in the presence of JNK inhibitor SP600125, and that the cells are sensitized thereby.

PURPOSE: Here we investigate further how modulation of signaling through JNK influences autophagy in hypoxic HT29 cells treated with oxaliplatin, and expand the cellular model to additional colon cancer cell lines. We created a panel of HT29-derived cell lines stably expressing dominant negative constructs for SEK1 (HTS13), MKK7 (HTM9), JNK1 (HTJ1.3) and JNK2 (HTJ2.2). We also utilized 6 colon cancer cell lines (HCT116, LoVo, RKO1, SW480, DLD1 and HCT15) in which a dominant negative construct for JNK1 was introduced by viral delivery.

RESULTS: Our data demonstrate that in HTJ1.3 cells, hypoxic induction of autophagy is impaired. Silencing of JNK2 in these cells by viral delivery of shRNA does not affect either autophagy induction, or sensitivity to oxaliplatin. Downregulation of up-stream activators of JNK, as in the dnSEK1 or dnMKK7 derivatives, similarly does not affect autophagy induction, while still demonstrating differential effects on activation of JNK isoforms and its major target, c-Jun. Finally, we show that colon cancer cell lines differ in their ability initiate autophagy under hypoxia. Correspondingly, the introduction of dominant negative JNK1 causes some enhancement of oxaliplatin cytotoxicity in LoVo, RKO1 and HCT15 cell lines, which all demonstrate a greater propensity to autophagy under these circumstances.

CONCLUSIONS: Our data demonstrate that JNK1 plays a central role in autophagy induction under hypoxic conditions. Knockdown of JNK1 renders HT29 cells more sensitive to hypoxia due to impaired autophagy induction, and JNK2 is unable to substitute for JNK1 under these conditions. Our results also imply involvement of alternative JNK1 activators in autophagy induction by hypoxia. Finally, induction of autophagy and enhancement of oxaliplatin cytotoxicity, by this mechanism, appear to be model-specific in colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2872. doi:10.1158/1538-7445.AM2011-2872
Abstract 2875: Ceramide suffocates cancer cells to death: Regulation of mitophagy by ceramide signaling
Authors: Ogretmen Besim , Sentelle David , Senkal Can E.
Published: June 2012

Sphingolipid ceramide is a bioeffector molecule involved in the regulation of cell death. Endogenous ceramides contain different fatty acid chain lengths varying between C12-C26-ceramides. Ceramide synthase 1 (CerS1) selectively regulates the generation of C18-ceramide, which is down-regulated in the majority of tumors obtained from patients with head and neck squamous cell carcinomas (HNSCC). Reconstitution of C18-ceramide generation using a tetracycline inducible vector system for stable expression of CerS1 inhibits the growth of HNSCC cells and xenografts in culture and SCID mice, respectively. In this study, our goal was to determine mechanisms by which CerS1/C18-ceramide inhibits HNSCC tumor growth. Data revealed that reconstitution of C18-ceramide generation by wt-CerS1 induction, but not its catalytically inactive mutant, induces autophagy as detected by various techniques in HNSCC cells. Interestingly, soft agar studies showed that CerS1/C18-ceramide induction inhibits HNSCC cell growth, which is prevented by addition of the autophagy inhibitor 3-methyladenine. In addition, knockdown of Atg3 and Atg7 in HNSCC cells protected against cell death induced by exogenous addition of the C18-ceramide therapeutic analog, C18-pyridinium-ceramide (C18-Pyr-Cer). Additionally, Atg5-/- MEFs showed resistance to cell death when compared to wild type MEFs upon C18-Pyr-Cer exposure. Taken together these results suggest that autophagy is required for cell death. Electron microscopy showed that addition of C18-Pyr-Cer to UM-SCC-22A cells induced autophagosome formation and fusion with mitochondria. Mechanistically, our studies showed that CerS1/C18-ceramide-induced cell death/inhibition of tumor growth are mediated by mitochondrial damage and inhibition of respiration via induction of mitophagy. Inducible expression of CerS1/C18-ceramide and C18-Pyr-Cer treatment resulted in significant decreases in mitochondrial oxygen consumption rates. Furthermore, live and fixed cell imaging with mitochondrial markers MitoTracker Red colocalized with lysosomal marker LysoTracker Red upon C18-Pyr-Cer treatment and CerS1/C18-ceramide induction indicating mitochondrial fusion with lysosomes. Conversely, ceramide-mediated mitophagy was inhibited using LC3B siRNA, suggesting that mitochondria might be targeted to autophagosomes by ceramide signaling. In conclusion, we provide novel evidence here which suggest that C18-ceramide targets autophagosomes to mitochondria (mitophagy), which results in the modulation of respiration/oxygen consumption, leading to HNSCC growth inhibition and tumor suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2875. doi:10.1158/1538-7445.AM2011-2875
Abstract 2888: SOCS3-deficient cells are defective in the DNA damage response
Authors: Cacalano Nicholas A. , Jiang Sisi
Published: June 2012

The SOCS family of proteins are negative feedback inhibitors of cytokine and growth factor signal transduction, targeting primarily the STAT family of transcription factors. Several SOCS genes, including SOCS1 and SOCS3 are hypermethylated and silenced in solid tumors, which is believed to impart a growth advantage via hyperactivation of the STAT pathway. Despite the growing role of SOCS in the pathogenesis of cancer, very little is known of their role in modulating tumor cell responses to therapy. We recently identified an unexpected function for SOCS3 in the context of the DNA damage response. We found that cells lacking SOCS3 are sensitive to ionizing radiation (IR) in a clonogenic survival assay due to impaired p21 (Cip1/Waf-1) upregulation, which results in failure of the radiation-induced G1-S checkpoint. Normal cell cycle arrest and p21 upregulation could be restored in SOCS3-/- MEFs by overexpression of a phosphorylation-deficient, dominant-negative (DN) mutant of STAT3. Two STAT3-dependent genes known to suppress p21 expression, c-Myc and BCL6 are overrepresented in SOCS3-deficient MEFs. Likewise, human tumor cells that repress SOCS3 expression by epigenetic silencing are also more radiosensitive than their SOCS3+ counterparts. In agreemwnt with our data in MEFs, c-Myc and BCL6 are overexpressed in human tumor cells that silence SOCS3 expression by promoter hypermethulation, suggesting that tumors lacking SOCS3 may be more vulnerable to IR-based therapies due to impaired cell cycle arrest and DNA repair. Our data suggest that therapeutic targeting of SOCS3 in tumor cells could sensitize cells to IR and improve the effectiveness of radiotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2011-2888
Abstract 2895: The epithelial to mesenchymal transition is associated with alterations in the mitogenic signaling pathways that drive cancer cell proliferation
Authors: Corsino Patrick E. , Law Mary , Jahn Stephan , Davis Brad , Law Brian
Published: June 2012

The Epithelial to Mesenchymal Transition (EMT), by facilitating the metastatic process, is an important step in tumor progression for many types of cancer. We have developed a mouse tumor model, driven by the HER2/neu oncogene, whereby this process can be studied robustly both in vivo and in cell culture. This system has revealed significant changes in mitogenic signaling networks that occur during EMT. Post-EMT cancer cells acquired increased responsiveness to HGF, PDGF, and LPA. This change in responsiveness was accompanied by a decrease of Her2, c-Src and IRS-1 levels, as well as an increase in Axl, PDGFR and c-Met levels. Perturbations in these signaling cascades results in the differential sensitivity to certain anti-cancer therapeutics. Post-EMT cells were less sensitive than Pre-EMT cells to the MEK inhibitor U0126. Furthermore, while Pre-EMT cells exhibited cell cycle arrest in response to rapamycin, Post-EMT cell proliferation was stimulated. These observations may have significant bearing on the efficacy of targeted therapeutic agents in patients with metastatic breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2011-2895
Abstract 2902: Phosphorylation of 12 lipoxygenase at Tyrosines 19 and 614 regulates its activity during beta4 integrin signaling
Authors: Dilly Ashok Kumar , Tang Keqin Tang , Guo Yande Guo , Joshi Sangeeta , Ekambaram Prasanna , Maddipati Krishna Rao , Cai Yinlong , Tucker Stephanie , Honn Kenneth
Published: June 2012

12-Lipoxygenase (12-LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE, an eicosanoid implicated in tumor angiogenesis, growth, and metastasis. We have demonstrated previously that 12-LOX interacts with integrin beta4 cytoplasmic domain by using yeast two hybrid system. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. Increased expression of 12-LOX and beta4 integrin have been documented in a number of carcinomas. The purpose of this study is to find out the mechanisms underlying the signaling pathways in 12-LOX mediated cancer progression. The phosphorylation of beta4 integrin, Src, and 12-LOX were analyzed using specific antibodies against phospho-tyrosine, phospho-beta4 integrin, Phospho-Src (pSrc). c-Src activity and association was determined in beta4 integrin immunoprecipitate. The HEK293 cells transiently transfected with wild type or dominant negative (DN) Src were used as in vitro models to study the regulation of 12-LOX activity. The 12-LOX product 12(S)-HETE was analyzed by using an analytical liquid chromatography-tandem mass spectrometry.12-LOX tyrosine phosphorylation sites mutants viz., 19, 295, and 614 were used to study the effect on its activity upon beta4 ligation. We show here that the cytoplasmic domain of beta4 integrin, can interact specifically with pSrc after beta4 integrin ligation in A431 cells. Herein we report that beta4 integrin ligation resulted in significant increase in 12(S)-HETE level in A431 cells, compared to that of unligated beta4 integrin. An increase in Src kinase activity was found in beta4 integrin ligation of A431 cells and inhibition of Src kinase by PP2 or DN Src significantly reduced 12(S)-HETE production. The ligation of beta4 integrin induces src activation, which resulted in tyrosine phosphorylation of 12-LOX. The residues that are important for 12-LOX activity and tyrosine phosphorylation were mapped to Y19 and Y614. We examined that 12-LOX mutants (Y19F and Y614F), showed 70% less enzymatic activity, compared to wild type 12-LOX. Furthermore, we showed that the 12-LOX activity modulated by these residues impacts migration in HUVEC cells. To our knowledge, this is the first report to show that Src kinase activity is required for beta4 integrin mediated regulation of 12-LOX, which may in turn regulate metastatic potential of cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2011-2902
Abstract 2924: IGF-IR inibition induces MAPK signaling in prostate carcinoma cells
Authors: Morris Brandon L. , Young Talearia , van Golen Cynthia M.
Published: June 2012

Prostate carcinoma, the most common malignancy in men, metastasizes primarily to bone. Multiple factors, including integrins, chemokines, and growth factors are implicated in the regulation of this process. In our laboratory, we are interested in the role of the insulin-like growth factor (IGF) family of proteins in the regulation of tumor metastasis to bone. Metastatic prostate carcinoma cells express the type I IGF receptor (IGF-IR) and are responsive to IGF ligands. Therefore, it has been suggested that inhibitors against the IGF-IR may be effective against prostate carcinoma bone metastasis.

Our hypothesis is that IGF-IR inhibitors will prevent prostate carcinoma growth and survival signaling. PC-3, LNCaP, and bone-adapted C42B prostate carcinoma cells were plated in a 96-well growth assay and treated with/without IGF-I and/or neutralizing antibody against the IGF-IR. Predictably, IGF-I treatment leads to an increase in cell number over a period of 4 d; however, inhibition of the IGF-IR enhances, rather than diminishes, this effect on growth. To further understand this paradoxical finding, Western immunoblotting was performed to analyze the effect of IGF-IR inhibition on IGF-IR expression and signaling. PC-3 cells were treated with increasing concentrations of a neutralizing antibody against the IGF-IR. With increasing concentrations of the neutralizing antibody, IGF-IR and Akt expression levels remain unchanged. In contrast, p44/p42 MAPK expression and activation increases with increasing concentrations of the IGF-IR neutralizing antibody. These data suggest that IGF-IR inhibition induces an upregulation of the p44/p42 MAPK pathway in PC-3 prostate carcinoma cells.

The data collected indicate that our original hypothesis was incorrect. Our current hypothesis is that treatment of prostate carcinoma cells with the IGF-IR neutralizing antibody induces receptor internalization supporting prolonged signaling rather than lysosomal degradation. We are currently investigating this hypothesis using immunofluorescence and confocal microscopy.This work was supported by NIH/NCRR 2 P20 RR016472-09 (INBRE) and the Children's Neuroblastoma Cancer Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2924. doi:10.1158/1538-7445.AM2011-2924
Abstract 2927: Regulation of Na+/H+ exchanger 1 (NHE1) expression by hyperactivation of Akt in PTEN-null mouse embryonic fibroblasts
Authors: James Joanne , Kumar Jaspal Kaur , Clement Marie Veronique
Published: June 2012

The serine/threonine kinase, Akt, plays a key role in regulating the signaling of a multitude of biological processes including cell proliferation, differentiation, migration and survival. In addition, many human cancers commonly display aberrant hyperactivated Akt survival pathway, a phenomenon that is frequent in tumours that have impaired or deletion of the PTEN tumour suppressor gene. Interestingly, we have recently demonstrated that an increase in intracellular O2− could replace the removal of growth factors resulting in the activation of Akt through the inhibition of the tumour suppressor PTEN and inducing an increased expression of the Na+/H+ exchanger 1 (NHE1), mimicking the phenotype of PTEN-null mouse embryonic fibroblasts. The Na+/H+ exchanger 1 (NHE1) is a membrane antiporter that has been described to promote cell survival via a dual mechanisms- defending cell volume and pHi through Na+/H+ exchange and by functioning as a scaffold for the recruitment of a signalplex involving, PI3-Kinase, ERM and Akt. In the present study, we asked whether the hyperactivity of Akt found in cells that have lost the expression of PTEN could influence the expression of NHE-1. We show that the PTEN-null mouse embryonic fibroblast model displayed an increased expression of Na+/H+ exchanger 1 (NHE1) and hyperactivated Akt compared to its PTEN wildtype counterpart. Moreover the half-life of NHE-1 was shown to increase in PTEN-null fibroblasts compared to the wildtype mouse embryonic fibroblasts. Hence, we hypothesized that Akt hyperactivation could influence the stability of NHE-1 expression. Indeed, using drugs such as LY294002 and Wortmannin to inhibit the activation of AKT, we showed that activated Akt had an effect on NHE-1 expression as the decrease in NHE-1 expression correlated with the dephosporylation of Akt, both at the mRNA and protein levels. Similarly, by silencing Akt gene expression, a more prominent decrease in NHE-1 protein expression was observed. Taken together, our data suggest that hyperactivated Akt may play a crucial role in the stability of NHE-1 in PTEN null fibroblasts, a phenomenon not observed in wildtype fibroblasts. Results from these studies could establish that Akt may be involved in the regulation of NHE-1 expression in tumours that have lost the expression of PTEN.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2927. doi:10.1158/1538-7445.AM2011-2927
Abstract 293: Characterization of renal tumor-initiating cells in human renal carcinoma cell lines
Authors: Wang Lin , Park Paul , Zhang Huina , Marca Frank La , Lin Chia-Ying
Published: June 2012

High aldehyde dehydrogenase activity (ALDH) has been used as a putative marker for the tumor- initiating subpopulation in many cancer types. We previously showed that tumor-initiating cells in human osteosarcoma OS99-1 cell line can be identified using ALDH. In the current study we sought to determine whether ALDH can be also used as a marker to isolate tumor-initiating populations from human renal carcinoma cell lines. By using a xenograft model in which cells from human renal carcinoma cell line ACHN and Caki-2 were grown in NOD/SCID mice, we identified a subset of renal cells that were highly tumorigenic based on the high aldehyde dehydrogenase activity. Isolated cancer cells with relatively high aldehyde dehydrogenase activity (ALDHbr cells) revealed a greater colony-forming efficiency, higher proliferative output, the ability to grow in non-adhesive spheroids, the capability to undergo adipogenic and osteogenic differentiation, and increased expression of stem cell marker genes Oct3/4A, Nanog and Pax-2. In vivo experiments showed that ALDHbr cells were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDHlo cells), generating new tumors with as few as 25 cells. The isolation of renal tumor-initiating cells by their high ALDH activity may lead to a new insight into the study of how renal cell carcinoma may take place, and may lead to effective prognostic and therapeutic strategies for the treatment of patients with kidney cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 293. doi:10.1158/1538-7445.AM2011-293
Abstract 2956: Prognostic value of PCNA expression in salivary gland cancer in consideration of different histological subtypes
Authors: Stenner Markus , Demgensky Ariane , Molls Christoph , Hardt Aline , Klussmann Jens P.
Published: June 2012

Background: The expression of the cell proliferation-related marker Proliferating Cell Nuclear Antigen (PCNA) has shown to be a significant prognostic indicator in various human cancers. Its role in malignant tumors of the major salivary glands remains unclear.

Methods: We analysed the expression of PCNA in 159 patients with parotid gland cancer (12 different histological subtypes) by means of immunohistochemistry. Patients were divided into two groups due to the median PCNA expression level. The experimental findings were correlated with clinicopathological and survival parameters. The mean follow-up time was 56.6 months.

Results: Cytoplasmic expression of PCNA was found in 89% of cases. A high cytoplasmic expression of PCNA was found in 47% of the examined tumors. High cytoplasmic PCNA expression was an independent prognostic indicator for a significant poorer 5-year disease-free survival rate (50.6% versus 73.8%, p=0.036) as well as overall survival rate (57.0% versus 78.8%, p=0.006).

Conclusion: The correlation between cytoplasmic PCNA expression and survival probabilities of salivary gland cancer might make this an effective tool in patient follow-up, prognosis and therapy in future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2011-2956
Abstract 2963: Pharmacologic Chk2-inhibition promotes increased killing of human colorectal cancer cells and protects from gastrointestinal toxicity following Irinotecan/trail death-receptor agonists (TDRA)
Authors: Finnberg Niklas K. , Arunasalam Navaraj , Lang Krystle , Cerniglia George , Motoyama Noboru , Zhou Bin-Bing , El-Deiry Wafik S.
Published: June 2012

Dose-limiting gastrointestinal toxicity following systemic chemotherapy-based treatment regimen is associated with increased cost and reduced cure-rate. Pharmacologic inhibition of Chk2 has the potential to sensitize cancer cells to cancer therapy while at the same time protect normal tissues from toxicity. Cell death in normal tissues and cells is blocked by inhibition or loss of chk2 in a DNA damage-specific manner whereas tumors cells with check-point aberrations are sensitized to death. We show that inhibiting Chk2-by the use the pharmacologic inhibitors of the Chk2-kinase “Chk2 Inhibitor II” (CI-II) and PV-1019 has the potential to both protect the GI mucosa and improve cell-kill of colorectal cancer cells when Irinotecan is combined with trail death-receptor agonists (TDRA). HCT116-Chk2-/- cells were increasingly sensitive to Irinotecan, TDRA and the combination thereof and PV-1019 had single agent activity towards SW620 cells at 10µM and caused sensitization to Irinotecan at lower doses. Gastrointestinal (GI) toxicity in mice following treatment with the TDRA-antibody MD5-1 in combination with high-dose Irinotecan is completely mediated by chk2 since Chk2-deficient mice are protected and pharmacologic inhibition of Chk2 using CI-II following Irinotecan/TDRA reduced the extent of weight loss and lethality associated with GI-toxicity in the mice. Interestingly, chk2-inhibition only conferred significant protection following Irinotecan/TDRA but not fluorouracil/TDRA, suggesting an Irinotecan-specific protection from GI-toxicity. Concomitant pharmacologic inhibition in conjunction with Irinotecan-based therapy may improve the therapeutic window by reducing GI-toxicity and increasingly promote colorectal cancers cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2011-2963
Abstract 2965: hSSB1 binds and protects p21 from ubiquitin-mediated degradation and positively correlates with p21 in human hepatocellular carcinomas
Authors: Kang Tiebang , Xu Shuangbing , Zeng Yi-Xin
Published: June 2012

Down-regulation of hSSB1, a single-stranded DNA-binding protein, causes increased radiosensitivity, defective checkpoint activation and genomic instability. However, the mechanisms of hSSB1 function in these responses remain to be uncovered. Here, we present evidence that hSSB1 and p21 interact through their N-terminal domains and that this interaction may prevent p21 from ubiquitin-mediated degradation. Furthermore, both promotion of the G1/S transition and abrogation of the G2/M checkpoints induced by hSSB1 knockdown are substantially rescued by overexpressing p21. Most importantly, hSSB1 and p21 levels are positively correlated in human hepatocellular carcinomas, as determined by immunostaining. Therefore, hSSB1 may positively modulate p21 to regulate cell cycle progression and DNA damage response, implicating hSSB1 as a novel, promising therapeutic target for cancers such as hepatocellular carcinomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2965. doi:10.1158/1538-7445.AM2011-2965
Abstract 2969: A Wee1 kinase inhibitor, MK-1775, sensitizes cervical carcinoma cell lines to cisplatin and topotecan
Authors: Shumway Stuart D. , Kubica Jamie L. , Guertin Amy D. , Hurd Melissa S. , Wexler Roseanne S. , Demuth Tim , Toniatti Carlo
Published: June 2012

The Wee1 tyrosine kinase plays a key role in checkpoint arrest by phosphorylating and inhibiting cyclin-dependent kinases 1 and 2, two CDKs that drive S-phase and G2- to M-phase transitions, respectively. Interfering with Wee1 leads to elevated CDK1 and 2 activity, which in turn overcomes checkpoint arrest and causes cells to progress through the cell cycle in the presence of DNA damage. Wee1 inhibitors are therefore expected to sensitize cancer cells to DNA damaging chemotherapeutics. In particular, p53-deficient cells are postulated to be particularly sensitive to Wee1 inhibition because they lack a G1-checkpoint and rely more on S- and G2-phase checkpoints where Wee1 activity is critical. The p53 pathway is frequently inactivated in cervical carcinoma either through TP53 mutation or through functional inactivation by Human PapillomaVirus (HPV) gene products. We thus investigated the capability of MK-1775, a selective and potent inhibitor of Wee1, to sensitize cervical cancer cell lines to cisplatin and topotecan. We have evaluated the potential of the Wee1 inhibitor MK-1775 to sensitize cervical cancer cells to standard-of-care treatments both in vitro and in vivo. Using a panel of cervical cancer cell lines, we demonstrated that MK-1775 at clinically relevant concentrations potentiates the cytotoxic activity of cisplatin and topotecan in vitro as indicated by the average 3-fold and 2-fold shift in the CC50s for cisplatin and topotecan, respectively. Chemosenstization was observed in both HPV-negative/p53 mutant and HPV-positive p53/wild-type cervical cancer cell lines. Finally, we have demonstrated that MK-1775 significantly increases the in vivo efficacy of a cisplatin-topotecan combination therapy in xenograft models of HPV+ cervical carcinoma. Based on these data, MK-1775 is currently undergoing clinical evaluation in a phase 1b/2 trial in platinum combination for the treatment of cervical carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2011-2969
Abstract 2974: Regulation of cyclin dependent kinase 7 through a phosphotidylinositol 3-kinase/PKC-ι mediated cell proliferation cascade
Authors: Desai Shraddha , Acevedo-Duncan Mildred
Published: June 2012

PKC-ι is highly over expressed in the transformed phenotype of human glioma and benign and malignant meningioma however little is understood about its role in glioma cell proliferation. Several upstream molecular aberrations and/or loss of PTEN have been implicated that lead to constitutive activation in phosphotidylinositol (PI) 3-kinase pathway. PKC-ι is one of the important downstream targeted mediators which are often up regulated by PI3-kinase. Cyclin dependent kinase activating kinase (CAK) which comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator which phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. We studied a potential function of PKC-ι in cell cycle progression and proliferation in two human glial tumor derived cells T98G and U87MG. Results show that PKC-ι directly associate and phosphorylate Cdk7 at Thr-170, which is required for Cdk activation and G1/S cell cycle transition. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at Thr-160. Purified PKC-ι was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-ι knockdown with silencing RNA (siRNA) as well as PI3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. Collectively, these findings suggest presence of a novel PI3-kinase/PKC-ι/Cdk7/cdk2 cell proliferation pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2011-2974
Abstract 2985: CHD1L upregulates TCTP and induces mitotic defects and chromosome missegregation through decreasing Cdk1 activity by Cdc25C ubiquitination
Authors: Chan Tim Hon Man , Chen Leilei , Guan Xin-Yuan
Published: June 2012

CHD1L is a target oncogene responsible for the 1q21 amplification event in hepatocellular carcinoma (HCC). Here, TCTP was found to be a CHD1L target gene. CHD1L protein directly binds to the promoter region (−733/-1027) of TCTP and upregulates the latter's expression in both cell lines and clinical specimens. Overexpression of TCTP, detected in 41.9% of human HCCs, was significantly associated with heavy liver cirrhosis and advanced tumor stage. During mitosis, the elevated expression of TCTP could accelerate the ubiquitin-proteasome degradation of Cdc25C, which in turn led to the failure in the dephosphorylation of Cdk1 on Tyr15 and the decreased Cdk1 activity, finally causing a faster mitosis exit and chromosome missegregation. Depletion experiments confirmed that TCTP accelerates M phase progression and induces tumorigenicity. Overall, we reveal a novel molecular pathway (CHD1L)-(TCTP)-(Cdc25C)-(Cdk1) during hepatocarcinogenesis which causes the malignant transformation of hepatocytes with the phenotypes of accelerated mitotic progression and the production of aneuploidy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2985. doi:10.1158/1538-7445.AM2011-2985
Abstract 2991: ATM is activated in mitosis and required for the spindle checkpoint
Authors: Yang Chunying , Tang Xi , Guo Xiaojing , Wong Stephen , Fu Li , Xu Bo
Published: June 2012

The ATM kinase, mutation of which leads to the human autosomal recessive disorder Ataxia-Telangiectasia (A-T), plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA double strand breaks and essential for cell cycle checkpoints. However, ATM function outside the DNA damage response is less clear. Here we report that ATM is activated in mitosis in the absence of DNA damage and is required for the spindle assembly checkpoint. We generated stable cell lines expressing either control shRNA or ATM shRNA and monitored chromosome dynamics and the timing of mitosis by time-lapse video microscopy. The results reveal that ATM-depleted cells proceed into anaphase despite the presence of maloriented and unaligned chromosomes; whereas control cells do not proceed into anaphase until the completion of chromosome alignment on the metaphase plate. We also find ATM-depleted cells exit mitosis much earlier than control cells. In addition, we found ATM-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) defect. Enzymatic assays indicate that ATM kinase activity is enhanced during mitosis. Further, mitotic ATM activation is mechanistically distinct from that induced by ionizing radiation. Next we demonstrate that mitotic-dependent ATM activation relies on the Aurora-B kinase, a chromosome passenger protein, which is implicated in chromosomal alignment, spindle checkpoint and cytokinesis. Aurora-B phosphorylates ATM at Serine 1403 both in vitro and in vivo. Immunofluorescence experiments show that ATM-Ser1403 phosphorylation has a similar localization pattern to Aurora B during mitosis. More interestingly, we find that ATM Ser1403 phosphorylation is evident in malignant breast cancer tissues and is highly correlated with the mitotic index (p=0.001, Pearson Correlation Test). Cells expressing the mutation of Serine 1403 to Alanine show a defective SAC and accelerated mitotic progression, similar to that of ATM-depleted cells. Collectively, our data demonstrate the functional significance of ATM activation in mitosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2011-2991
Abstract 3006: A novel method for locus specific detection of 5-hydroxymethylcytosine
Authors: Yen James L. , Choi Stella , Van Eden Marc E. , Jia Xi Yu
Published: June 2012

Epigenetic mis-regulation leading to diseases such as cancer is classically understood in terms of aberrant DNA methylation patterns. For decades, 5-methylcytosine (5-mC) has been the focus of epigenetic DNA modification studies. But recently, a novel epigenetic DNA modification, 5-hydroxymethylcytosine (5-hmC), has been shown to be present in mammalian brain and ES cells. Current tools available to map DNA methylation (e.g. bisulfite conversion) cannot distinguish between 5-mC and 5-hmC. Here we describe a new method for locus specific interrogation of 5-hmC within DNA.

Treatment of DNA with a 5-hmC glucosyltransferase enzyme specifically adds a glucose moiety onto preexisting 5-hmC residues, yielding glucosyl-5-hydroxymethylcytosine (glu-5-hmC). Subsequent digestion of DNA with glu-5-hmC sensitive restriction endonucleases effectively cleaves DNA with cytosine, 5-mC, or 5-hmC within their recognition site. But when glu-5-hmC is present, DNA is not able to be cleaved. Exploiting this difference in “cleavability”, 5-hmC can be detected at loci of interest using qPCR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2011-3006
Abstract 3008: Genome-wide siRNA screening identifies epigenetic silencing factor networks as potential targets for cancer therapy
Authors: Poleshko Andrey S. , Einarson Margret B. , Skalka Anna Marie , Katz Richard A.
Published: June 2012

Epigenetic processes control the binary on-off states of specific gene sets, thereby creating heritable transcription patterns that drive development, and maintain cellular identity. The prominent epigenetic regulatory marks on eukaryotic chromatin are histone modifications and DNA cytosine methylation (5meCpG). These marks are placed by protein complexes, including members of the histone modifying and DNA methyltransferase (DNMT) enzyme families. It is hypothesized that errors in placement, removal, or reading of epigenetic marks can cause human disease through inappropriate silencing of specific genes. As the epigenetic marks that mediate gene silencing are reversible, there is interest in devising therapeutic strategies to reactivate epigenetically silent genes. Inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme families can reverse epigenetic silencing and produce anti-tumor effects, possibly through reactivation of silent tumor suppressor genes (so-called epigenetic therapy). As these inhibitors show limited specificity within enzyme families, their precise mechanisms of action are not well understood.

To identify cellular factors involved in maintenance of epigenetic silencing, we constructed a population of human cells harboring epigenetically silent GFP reporter genes. Using this cell population we have implemented a GFP reporter-based siRNA knockdown screen to identify novel factors and networks that maintain epigenetic silencing in human cells. We have now completed a genome-wide, high throughput siRNA-based screen (21,122 siRNA targets). The screen has produced 128 gene hits that have satisfied several validating criteria. The output of this screen was of high quality, as several of the factors were identified in an earlier independent screen. Among the newly identified hits, there was a significant enrichment for factors that mediate SUMOylation, as well as 25 factors corresponding to novel genes, including 9 with no predicted functions.

This screen has the potential to identify novel cellular pathways and reveal new targets for epigenetic therapy of cancer and other diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2011-3008
Abstract 3009: Genetic and epigenetic variations resulting from Alu retrotransposition
Authors: de Andrade Alexandre , Xie Hehuang , Wang Min , Bonaldo Maria F. , Soares Marcelo B.
Published: June 2012

Alu repeats are the most prevalent retrotransposons, comprising approximately 10% of the human genome. Alu retrotransposons have amplified during primate evolution by an RNA-mediated copy and paste mechanism. Among the Alu subfamilies, AluY elements and its variants exhibit the highest rates of retrotransposition. De novo retrotransposition of active Alu elements have given rise to insertion polymorphisms in human populations. While generating methylation maps of Alu elements in human normal cerebellum and in ependymoma tumors, Xie and colleagues (2009, 2010) also identified putative new Alu element insertions. In this study, we explored these Alu libraries to identify recent Alu element insertions and to understand the (epi)genetic variations that result from such events. This study may also enable identification of DNA structural variation that may occur at the onset of tumorigenesis and/or during tumor progression.

A total of 327 putative recent Alu element insertions represented by 1,762 sequence reads were identified, including 316 events that have not yet been documented in either dbSNP or dbRIP. Forty-seven out of a total of forty-nine randomly selected events representing nineteen genomic loci were sequence-verified. Alu element insertions remained heterozygous for sixteen out of nineteen genomic loci in one or more individuals. The polymorphic Alu elements are enriched for young Alu families and they present some characteristic sequence features, such as longer poly(A) tails. In addition, they also present the TT/AAAA consensus sequence at their 3’-flanking regions, and AT-rich target site duplications. Despite being homozygous or heterozygous insertions, these Alu elements are heavily methylated. Further methylation analysis on two genomic loci revealed no methylation difference in two heterozygous alleles and also no methylation difference at CpG dinucleotides flanking the Alu insertion sites. These results indicate that the recently integrated Alu elements have not influenced the methylation status of their neighboring CpG dinucleotides.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2011-3009
Abstract 301: 67-kDa laminin receptor as prognostic marker and therapeutic target in epithelial ovarian carcinoma
Authors: Lee Jeong-Won , Cho Young-Jae , Choi Chel-Hun , Park Young Ae , Choi Jung-Joo , Jeon Hye-Kyeong , Kim Byoung-Gie , Bae Duk-Soo
Published: June 2012

Objectives: The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor with high affinity for laminin, which plays a key role in tumor invasion and metastasis.

We investigated the clinical significance of 67LR expression including roles of prognostic marker or therapeutic target in epithelial ovarian carcinoma.

Methods: The expression of 67LR was evaluated by immunohistochemistry in 71 patients with ovarian carcinomas including 54 serous, 13 endometrioid and 4 mucinous types. We assessed the correlation of 67LR expression with clinical characteristics including histology, tumor grade, FIGO stage, and survival. In vitro experiment was performed for 67LR with inhibition using siRNA to evalute its role in cell growth control and sensitivity to chemotherapeutic agents in ovary carcinoma cell line.

Results: The expression of 67LR protein with mainly membranous location was observed in majority (45/62, 73 %) of the EOCs but not in normal ovarian tissues (P<0.001). Thirty (48.4%) of 62 specimens were scored as cytoplasmic-67LR-positive. The 67-LR membranous expression was correlated with poorer progression-free survival (HR = 2.14, 95% CI 1.08-4.23, P = 0.028), though it was not an independent prognostic factor. And interestingly, cytoplasmic expression without membranous expression was correlated with poorer overall survival (P = 0.002). When 67-LR siRNA was transfected to several ovarian cancer cells, there was significant in vitro effect on cell survival and proliferation assessd by MTT assay.

Conclusion: These finding suggest that 67LR expression might be a prognostic marker for survival and it may play possible target for experimental treatment of epithelial ovary carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 301. doi:10.1158/1538-7445.AM2011-301
Abstract 3011: Evaluation of a high throughput platform for chromatin immunoprecipitation
Authors: Li Zirong
Published: June 2012

Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated

Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously.

Recently, we have developed a simple chromatin immunoprecipitation protocol, which utilizes high capacity protein A/G coated magnetic beads and 96 well plate. This protocol requires fewer steps, little hands-on time and is compatible with multi-channel pipetting and liquid handling robot. This protocol allows genome wide mapping of transcription regulatory network in a high throughput manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3011. doi:10.1158/1538-7445.AM2011-3011
Abstract 3021: An in vitro investigation of metabolically sensitive epigenetic biomarkers in breast cancer progression
Authors: Simpson Natalie E. , Tryndyak Volodymyr P. , Beland Frederick A. , Pogribny Igor P.
Published: June 2012

Cancer cells, including breast cancer cells, display unique alterations in major cellular metabolic pathways; therefore, it is widely believed that a better understanding of molecular cancer cell metabolism may reveal new approaches to aid in the diagnosis, treatment, and prevention of breast cancer. Results from recent in vivo and in vitro studies have demonstrated substantial metabolic differences between non-invasive and invasive breast cancers. Epigenetic marks, including histone post-translational modifications and DNA methylation, are emerging as biomarkers for most of the major human cancers. Additionally, the histone modifying enzymes (HME) (i.e., histone acetyltransferases, deacetylases, methyltransferases, and demethylases) that regulate these epigenetic changes are metabolically sensitive. In light of these considerations, the goal of the current study was to determine if any metabolic differences between non-invasive epithelial and invasive mesenchymal breast cancer cells are associated with the differences in histone modifications identified previously as being biomarkers of breast cancer progression. By comparing gene expression profiles from non-invasive breast cancer cells with an epithelial phenotype (i.e., MCF-7, MDA-MB-361, and T-47D cells) to invasive cells with an aggressive mesenchymal phenotype (i.e., Hs-578T, BT-549, and especially MDA-MB-231 cells), we determined that many metabolically regulated HMEs are differentially expressed in these cancer cells. We further demonstrated reduced acetylation of histone H3 lysine 9/18, reduced acetylation of histone H4 lysine 16/20, and reduced tri-methylation of histone H4 lysine 20 in breast cancer cells with a mesenchymal phenotype as compared to cells with an epithelial phenotype. Interestingly, the observed histone modification profile of invasive breast cancer cell lines is similar to what is found in individuals who exhibit disease recurrence and have a poor prognosis. These results suggest that metabolically sensitive histone modifications and their corresponding HMEs may be used as diagnostic and prognostic biomarkers. This makes them attractive targets for chemopreventive and chemotherapeutic strategies in breast cancer management.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2011-3021