bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3143: Sodium butyrate induced cellular senescence, inhibited invasion and modulated cellular metabolism in glioblastoma cells
Authors: Nakagawa Hidemitsu , Shindou Masahiro , Sasagawa Satoru , Itoh Kazuyuki
Published: May 2015

Sodium butyrate (SB) -C-4 saturated fatty acid (short-chain fatty acids) present in the human bowel membrane in the quite high concentration (2 mM) as food metabolites. It was previously reported that SB showed anti-tumor effect as HDAC inhibitor, however, the precise mechanism has not been elucidated yet. Here, we focused on the role of SB on cancer cell growth, motility and invasion. Physiological concentration of SB (0.25-4 mM) induced inhibition of cell growth, colony formation in soft agar, in vitro motility and invasiveness with Boyden chamber and wound healing assay in a dose-dependent fashion using human glioblastoma (A172) cells and rat mammary Walker 256 cells. SB also affected the morphology of both cells, namely spread out with marked stress fibers bridging focal adhesions. Phosphorylation levels of focal adhesion kinase (Y577 and Y397 sites) were increased but that of myosin light chain (S19 site) was not altered. All of these biological effects of SB were reversible, and recovered after withdrawal. In contrast, HDAC inhibitor trichostatin A inhibited cancer cell growth irreversibly but not affected motility, invasiveness and morphology. In addition, A172 cells treated with SB showed positivity for senescence-associated β-gal (SA-β gal) staining with elevated expression levels of p53 and p21 proteins in a time and dose-dependent manner. In contrast, mRNA level of p21 was sharply declined after the treatment with SB. Knocking down of p21 protein expression by siRNA reversed both the growth inhibition and positivity for SA-β gal staining but not the inhibition of invasiveness. Thus p21 was required for cellular senescence but not for the decrease in motility induced by SB. Further, SB increased the expression of isocitrate dehydrogenase 1 (IDH1), critical enzyme for glioblastoma progression and metabolism. Finally, intrathecal administration of SB effectively inhibited the invasion of Walker256 cells into the brain and elongated the animal (rat) survival in vivo. Collectively, SB inhibited growth with induced cellular senescence, inhibited invasion and modulated the expression of IDH-1, and thus would be a good candidate for anti-invasive therapy of brain tumor without severe adverse effects.

Citation Format: Hidemitsu Nakagawa, Masahiro Shindou, Satoru Sasagawa, Kazuyuki Itoh. Sodium butyrate induced cellular senescence, inhibited invasion and modulated cellular metabolism in glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2014-3143
Abstract 3145: eIF4E, an adverse prognostic marker of melanoma patient survival, increases melanoma cell invasion
Authors: Khosravi Shahram , Ardekani Gholamreza Safaee , Martinka Magdalena , Ong Christopher John
Published: May 2015

Human cutaneous melanoma is a life-threatening skin cancer due to its invasive nature and high metastatic potential, leading to poor prognosis for melanoma patients. However, the mechanisms for melanoma invasion and metastasis are poorly understood. Human eukaryotic translation initiation factor 4E (eIF4E) has been shown to be associated with tumor progression in multiple cancer types. However, the role of eIF4E in melanoma progression is not very well known. We examined eIF4E expression in 448 melanocytic lesions at different stages using tissue microarray and found that positive eIF4E staining was significantly increased in primary melanomas compared to dysplastic nevi (P<0.001), and further increased in metastatic melanomas compared to primary melanomas (P=0.008). EIF4E expression was correlated with melanoma thickness (P<0.001) and was inversely correlated with overall and disease-specific 5-year survival of all and primary melanoma patients especially those with tumor ≥4mm thick combined with metastatic melanoma patients. Furthermore, positive eIF4E expression was significantly higher in AJCC stage I-II melanomas compared to stage III-IV melanomas. Multivariate Cox regression analysis also revealed that eIF4E is an independent prognostic marker. FACS analysis and sulforhodamine B (SRB) cell proliferation assay showed that eIF4E knockdown in melanoma cells resulted in a significant increase and decrease in apoptosis and cell proliferation, respectively. EIF4E knockdown in melanoma cells also resulted in a down regulation of mesenchymal markers such as vimentin, N-cadherin, α-smooth muscle actin (α-SMA) and the EMT inducing transcription factor Twist. Additionally eIF4E knockdown in melanoma cells led to a decrease in the expression of c-Myc and Bcl2 and an increase in the expression of cleaved PARP and cleaved Caspase3. Moreover, down regulation of eIF4E resulted in a decrease in both melanoma cell invasion and MMP-2 activity. Taken together our data suggests the eIF4E may promote melanoma cell invasion and metastasis by inducing EMT and preventing apoptosis and also by increasing MMP-2 activity. EIF4E may also serve as a promising prognostic marker and a potential therapeutic target for melanoma.

Citation Format: Shahram Khosravi, Gholamreza Safaee Ardekani, Magdalena Martinka, Christopher John Ong. eIF4E, an adverse prognostic marker of melanoma patient survival, increases melanoma cell invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3145. doi:10.1158/1538-7445.AM2014-3145
Abstract 3149: Targeting ERBB2-induced, lysosome-mediated invasion
Authors: Kallunki Tuula , Brix Ditte M. , Rafn Bo , Bundgaard Clemmensen Knut Kristoffer , Andersen Sofie Hagel , Ambartsumian Noona , Jäättelä Marja
Published: May 2015

ErbB2 overexpression is strongly associated with invasive cancer with high recurrence and poor prognosis. Similarly, increased expression of lysosomal proteases, cysteine cathepsins, is observed in several cancers and correlates with enhanced angiogenesis, invasion and metastasis. We recently showed that ErbB2 level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. The increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and is mediated by novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, MZF1 and ETS1.

With a pharmacological protein kinase inhibitor screen, using the ErbB2 inhibitor Lapatinib as positive control, we have now identified three inhibitors putatively targeting this novel ErbB2-induced signaling network. Our data shows that the treatment of ErbB2-positive, highly invasive breast or ovarian cancer cells with any of these inhibitors specifically reduces cysteine cathepsin expression, activity and invasion of these cancer cells in 3D Matrigel cultures. It also reverses lysosomal distribution from the invasion supporting pericellular position to less-invasive perinuclear position, where the lysosomal exocytosis and secretion of lysosome contents to extracellular space to assist extracellular matrix proteolysis becomes less likely. In this study we have identified novel inhibitors for ErbB2-induced invasion and discovered a novel, function for clinically used ErbB2 inhibitor Lapatinib in the regulation of lysosomal function and trafficking.

Citation Format: Tuula Kallunki, Ditte M. Brix, Bo Rafn, Knut Kristoffer Bundgaard Clemmensen, Sofie Hagel Andersen, Noona Ambartsumian, Marja Jäättelä. Targeting ERBB2-induced, lysosome-mediated invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3149. doi:10.1158/1538-7445.AM2014-3149
Abstract 3155: Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma
Authors: Iizuka Shinji , Gould Christine M. , Buschman Matthew D. , Begoña Diaz , Abdullah Christopher , Courtneidge Sara
Published: May 2015

Invadopodia are actin-based proteolytic structures which facilitate cancer cell invasion and metastasis. While it was previously believed that invadopodia are not required for tumor cell growth, based on studies in monolayer cell culture, more recently we have noted a role for invadopodia proteins in growth in more physiological 3-dimensional (3D) extracellular matrix contexts. From previous work in our lab, the Tks adaptor proteins with 5 (Tks5) and 4 (Tks4) SH3 domains, respectively, were identified as Src kinase substrates and both are essential molecules for functional invadopodia formation in mouse and human cancer cells.

Here, we assess the roles of the Tks adaptors in melanoma tumor growth and invasion both in vivo and in vitro. To address the mechanism as to how the Tks adaptors control tumor growth and invasion, in vitro 3D growth assays in collagen I were performed. Knockdown of either Tks4 or Tks5 in C8161.9 or A375 melanoma cells decreased 3D growth in collagen I, a process that also appeared to be dependent upon metalloproteinase (MMP) activity. Interestingly, knockdown of either Tks4 or Tks5 in A375 melanoma cells decreased cell-surface expression of MT1-MMP, a key protease involved in tumor growth and metastasis. In vivo tumor growth and metastasis assays in mice using human and mouse melanoma cells depleted of Tks4 or Tks5 by shRNA revealed roles for the Tks adaptors in both the size and the number of lung metastases. Finally, analysis of a human melanoma tissue array with a Tks5 antibody, and RNAseq data, indicates that Tks5 expression is increased in lymph node and other metastases in comparison to nevi and primary sites.

These data suggest that the Tks adaptors control melanoma tumor growth and invasion by regulating invadopodia formation and perhaps also cell surface expression of MT1-MMP, and thus contribute to melanoma progression.

Citation Format: Shinji Iizuka, Christine M. Gould, Matthew D. Buschman, Diaz Begoña, Christopher Abdullah, Sara Courtneidge. Tks adaptor proteins and invadopodia formation in the growth and metastasis of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3155. doi:10.1158/1538-7445.AM2014-3155
Abstract 3156: Autocrine signaling through release of ATP and activation of P2X7 receptor promotes migration of human lung cancer cells
Authors: Takai Erina , Tsukimoto Mitsutoshi , Harada Hitoshi , Hiasa Miki , Moriyama Yoshinori , Kojima Shuji
Published: May 2015

Cell migration is essential step of cancer metastasis. TGF-β1 plays a key role in cancer progression through induction of various biological effects, including cell migration. Extracellular nucleotides, such as ATP, released from cells play a role in signaling through activation of P2 receptors. However, it remains poorly understood whether ATP-P2 receptor signaling is involved in cancer progression. We show here that release of ATP followed by activation of P2X7 receptors plays a key role in migration of lung cancer cells. Treatment with TGF-β1 facilitated migration of human lung cancer A549 cells, which was blocked by pretreatment with ecto-nucleotidase and P2 receptor antagonists in a wound healing-based assay and Transwell assay. ATP and P2 agonists facilitated cell migration. TGF-β1-induced actin remodeling, which contributes to cell migration, was also suppressed by pretreatment with ecto-nucleotidase and P2 receptor antagonists. Knockdown of P2X7 receptor suppressed TGF-β1-induced migration and actin remodeling. These results indicate the involvement of TGF-β1-induced ATP release in cancer cell migration, at least in part, through activation of P2X7 receptors. We found that TGF-β1 caused release of ATP from A549 cells within 10 minutes. Both ATP-enriched vesicles and expression of a vesicular nucleotide transporter (VNUT) SLC17A9, which is responsible for exocytosis of ATP, were found in cytosol of A549 cells. TGF-β1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-β1-induced cell migration and actin remodeling were also decreased in SLC17A9-knockdown cells. These results suggest the importance of exocytosis of ATP in cell migration. In addition, treatment with specific antagonist of P2X7 receptor (A438079) suppressed TGF-β1-induced migration of another human lung cancer H292 cells, but not of non-cancerous BEAS-2B cells. H292 cells released ATP in response to TGF-β1 stimulation, while BEAS-2B cells did not. Furthermore, in high-motile human lung cancer PC-9 cells, a large amount of ATP was released under standard cell culture condition, and P2X7 receptor was constitutively activated. Blockade of P2X7 receptor significantly suppressed motile activity of PC-9 cells. Taken together, these results suggest that autocrine signaling through release of ATP and activation of P2X7 receptor play a role in migration of human lung cancer cells.

Citation Format: Erina Takai, Mitsutoshi Tsukimoto, Hitoshi Harada, Miki Hiasa, Yoshinori Moriyama, Shuji Kojima. Autocrine signaling through release of ATP and activation of P2X7 receptor promotes migration of human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2014-3156
Abstract 3157: An Rgnef (p190RhoGEF/Arhgef28) signaling axis regulates ovarian cancer progression
Authors: Miller Nichol L. G. , Tancioni Isabelle , Uryu Sean , Kleinschmidt Elizabeth G. , Connolly Denise C. , Schlaepfer David D.
Published: May 2015

Ovarian cancer is a leading cause of cancer death in women, is usually diagnosed in late stage, and currently has insufficient effective targeted therapies. The primary mode of metastasis is unique in that dissemination occurs via tumor cell shedding into the peritoneal cavity, survival and growth within ascites, and re-adhesion and proliferation of tumor cells at sites within the abdomen. Therefore, molecules and pathways involving cell adhesion and survival in non-adherent environments are of particular interest. Rgnef (p190RhoGEF/Arhgef28) is a Rho family guanine exchange factor that associates with focal adhesion kinase (FAK) and functions both as a downstream target of FAK tyrosine kinase activity and a regulator of FAK activity following integrin stimulation. FAK regulates cell adhesion, migration, and survival. Expression of FAK is associated with poor clinical outcome, its activity (as measured by FAK Y397 phosphorylation) is frequently increased in serous ovarian carcinomas, and it is currently under investigation as a therapeutic target in ongoing clinical trials. Immunohistochemical analysis of human tumor tissue arrays with antibodies specific for Rgnef and FAK pY397 reveal a positive correlation between Rgnef expression and FAK activation and increased stage/grade of serous-type ovarian cancer. Stable knockdown of Rgnef in human ovarian carcinoma cells grown as subcutaneous or intraperitoneal xenografts in mice suggest that Rgnef may play roles in both primary tumor growth and ascites-associated cell survival and spread. Here, we use a transgenic model of spontaneous ovarian cancer (MISIIR-T-Antigen) to test the role of Rgnef in ovarian cancer progression. Female Rgnef+/+;TAg+ and Rgnef-/-;TAg+ mice were monitored by ultrasound imaging from age 12 to 17 weeks before euthanasia. Rgnef-/-;TAg+ tumors were significantly smaller (p=0.0006) and contained fewer Ki67 positive cells than Rgnef+/+;TAg+ controls at 17 weeks. Tumors and ovarian carcinoma cells isolated from Rgnef-/-;TAg+ mouse ascites have reduced FAK pY397 and increased E-cadherin expression compared to controls, suggesting that loss of Rgnef results in a less aggressive tumor phenotype. These findings identify Rgnef as a component in signaling pathways promoting FAK activation and regulating ovarian tumor progression.

Citation Format: Nichol L. G. Miller, Isabelle Tancioni, Sean Uryu, Elizabeth G. Kleinschmidt, Denise C. Connolly, David D. Schlaepfer. An Rgnef (p190RhoGEF/Arhgef28) signaling axis regulates ovarian cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2014-3157
Abstract 3158: Identifying novel genes critical to invasion in head and neck squamous cell carcinoma
Authors: Jayakar Sangeeta K. , Loudig Olivier D. , Brandwein-Gensler Margaret , Kim Ryung S. , Prystowsky Michael B. , Segall Jeffrey E. , Belbin Thomas J.
Published: May 2015

Oral squamous cell carcinomas (OSCC) have a poor patient prognosis, which is attributed to their invasive nature. Our goal was to identify specific genes that are important in OSCC invasion utilizing genome-wide expression data generated from HNSCC patient tumor samples of varying degrees of invasiveness. Invasion was assessed using the histological parameter pattern of invasion (POI), which describes the manner in which tumor cells infiltrate the normal tissue at the tumor-host interface. POI was used to categorize tumor resections into five types (1-5), type 5 being the most invasive with a “widely dispersed pattern of tumor infiltrate” and correlated significantly with the appearance of local recurrence and decreased overall survival of the patients after surgery. Global gene expression data was acquired from analysis of the surgical resections from 9 OSCC patient samples collected at Montefiore Medical Center between 2001 and 2008. Tumor cell-enriched cores from FFPE samples were used to generate global gene expression data using the Illumina® WG-DASL array to compare gene expression in four tumors that were WPOI type 5 and five tumors that were WPOI type 3. RNA extracted from flash-frozen tumor samples following surgical resection from the same patients was used to obtain global gene expression data using the Illumina® HumanHT-12 v4 Expression BeadChip. Candidate genes were assessed using in vitro invasion assays following siRNA knockdown of specific gene expression using siGENOME smart pools from Dharmacon. When both gene expression datasets were analyzed, we identified 104 genes that were overexpressed at least 1.5-fold in the WPOI type 5 tumors compared to the less invasive WPOI type 3 tumors. Fifty genes were initially tested based on functional criteria. In our initial screen, 16 of these genes showed a significant reduction in tumor cell invasion when expression was knocked down by siRNA in an OSCC cell line. The top gene from the screen, APOE, is now being further examined to determine a mechanistic explanation for its effect on invasion. Downstream signaling of APOE receptors and how this may interact with invasion are currently being assessed. This initial screen of global gene expression data in combination with pattern of invasion has revealed APOE as a novel gene that may play a critical role in OSCC invasion.

Citation Format: Sangeeta K. Jayakar, Olivier D. Loudig, Margaret Brandwein-Gensler, Ryung S. Kim, Michael B. Prystowsky, Jeffrey E. Segall, Thomas J. Belbin. Identifying novel genes critical to invasion in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3158. doi:10.1158/1538-7445.AM2014-3158
Abstract 3159: ZAR2 transcriptionally represses the ATPase ATP6V0A4 to negatively regulate invasiveness of breast cancer cells
Authors: Misra Smita , Chaudhuri Gautam
Published: May 2015

ZAR2 is a recently characterized RNA-dependent transcriptional repressor protein that is implicated in the cell cycle-dependent regulation of BRCA2 gene expression. We have shown previously that ZAR2 mRNA is transcribed from the overlapping bi-directional promoter of the BRCA2 gene, binds to the overlapping promoter and prevents the expression of BRCA2 through chromatin remodeling. ZAR2 level is significantly low in the invasive breast cancer cells and tissues as compared to the non-invasive cells. Knockdown of ZAR2 in the non-invasive breast cancer cells increased the in vitro invasiveness of these cells whereas forced expression of ZAR2 in the invasive breast cancer cells prevented their invasiveness. To understand the possible mechanism of ZAR2-mediated regulation of invasiveness of the breast cancer cells we studied differential gene expression in the ZAR2 knocked down non-invasive breast cancer cells by RNA-seq analysis. One of the genes that are significantly elevated in the ZAR2 knocked down cells is the invasion determining enzyme ATP6V0A4. ATP6V0A4 is transcribed from an overlapping bi-directional promoter along with its partner TMEM213 in the breast cancer cells. We found that both ATP6V0A4 and TMEM213 levels are increased in the ZAR2 knocked down cells whereas cells with forced expression of ZAR2 have a significant decrease in the levels of these proteins. ZAR2 cell cycle dependently binds to the ATP6V0A4/TMEM213 gene promoter to repress the activity of this bi-directional promoter. This study thus reports a new pathway for the regulation of the invasiveness of breast cancer cells. Supported in part by DOD grants BC990678 and BC050641 to GC and NIH grant 1U54RR026140 to SM.

Citation Format: Smita Misra, Gautam Chaudhuri. ZAR2 transcriptionally represses the ATPase ATP6V0A4 to negatively regulate invasiveness of breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2014-3159
Abstract 3167: Myosin-Va involvement in malignant properties of melanoma cells
Authors: Ramao Anelisa , Pontes Carmen Lucia S. , Espreafico Enilza M.
Published: May 2015

Evidence has emerged on the role of myosin-Va in cancer progression. Recently a role for myosin-Va in tumor cell migration, invasion and metastasis has been described for cancer cell lines from lung, colon, breast and prostate. One of the objectives of this work was to assess a possible role to myosin-Va in melanoma cells. We knocked down the expression of endogenous myosin-Va by using a lentiviral vector expressing a shRNA (shMYO5A) designed to target MYO5A mRNA in 1205Lu - a human melanoma cell line and functional studies were conductedonce efficient knockdown was attained. Myosin-Va depletion was showed to have no effect in cell proliferation as determined by crystal violet staining at a time course interval. However, cell viability was significantly reduced in MYO5A depleted cells compared to control ones. Also, a higher apoptotic index was observed in those cells suggesting that myosin-Va is involved in tumor growth. Subsequentlywe performed a cell migrationanalysis and found rates 60% lower for MYO5A depleted cells. This effect could have been caused by impairment in cell adhesion to the substrate as we could verify by a dynamic adhesion assay in different types of matrix. Adhesion ability to collagen, fibronectin, lamminin and matrigel was reduced in MYO5A depleted cells. Also, cell surface levels of integrins were lower for shMYO5A transfected cells compared to control as measured by flow cytometry approach and imply a defect in cell adhesion and migration caused by MYO5A knock-down.Next we evaluate cell survival under detachment conditions by plating cells on non-adherent surfaces and measuring cell death after 24h by annexin-V staining at cell surface. Results suggested that myosin-Va protects cells from anoikis (apoptosis caused by detachment) since MYO5A depleted cells presented higher rates of apoptosis compared to wild-type. Colony formation in soft agar showed that myosin-Va is required for anchorage-independent growth since depleted cells presented fewer and smaller colonies 30 days after culturing under those conditions. These findings indicate that myosin-Va is involved in several steps of tumor growth and progression including cell survival after detachment of the initial tumor mass and colonization of new microenvironments, suggesting that its upregulation in melanoma cells contributes to the rise of malignancy in this type of cancer.

Citation Format: Anelisa Ramao, Carmen Lucia S. Pontes, Enilza M. Espreafico. Myosin-Va involvement in malignant properties of melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3167. doi:10.1158/1538-7445.AM2014-3167
Abstract 3172: Detection of viral HPV DNA in sporadic colorectal cancers in relation to CpG island methylator phenotype (CIMP)
Authors: Bernabe-Dones Raul D. , Villavicencio-Torres Wesley , Munoz-Masso Cristina , Reyes -Medina Yaritza , Marcano-Bonilla Lorena , Perez-Cantalapiedra Hector , Fonseca-Williams Sharon , Lacourt-Ventura Mercedes Y. , Yamamura Yasuhiro , Rodriguez Nayra , Cruz-Correa Marcia R.
Published: May 2015

Background: There is evidence that insertion of viral DNA into a mammalian genome can lead to alterations of methylation patterns. To test the possible correlation between epigenetic modulations in colorectal cancer (CRC) and HPV infection, we conducted a MSP- PCR methylation analysis. The aim of the present study was to examine the presence of DNA sequences of human papillomavirus (HPV) in CRC and correlate this data with their CIMP status.

Methods: Thirty-seven colorectal adenocarcinomas were screened for HPV infection using nested-PCR (PGMY09/11-GP5+/6+). HPV typing was performed by type-specific PCR for HPV 16 and HPV 18 DNA. Eight CpG island methylator phenotype (CIMP) genes specific to sporadic colorectal cancer (MLH1, CACNA1G, NEUROG1, IGF2, SOCS1, RUNX3, CDKN2A and CRABP1) were assessed by methylation-specific PCR.

Results: Thirty-seven CRC cases (mean age at diagnosis 63.2 ± 11.7 years; 17 males) were evaluated. Tumors were mostly located in the left colon (73.0%), and moderate differentiated (61.1%). HPV's DNA was identified in 23 of 37 (62.2%) CRC studied cases. CRABP1 (59.5%), CDKN2A (37.8%), MLH1 (13.5%), and NEUROG1 (10.8%) were the most commonly methylated genes with a discrete clinic-pathological phenotype and gene methylation pattern. No statistical associations between HPV status and methylation patterns were found, after correlation with tumor staging, cell differentiation, tumor location, and life style.

Discussion: We reported that the CIMP-hypermethylation may be less frequent among Puerto Ricans Hispanics than previously reported in other racial and/or ethnic groups. However, a correlation could not be established (p>0.5) between HPV infection and the aberrant methylation pattern in the genes panel. We observed the trend that HPV is more prevalent in CRC without aberrant methylation patterns. This might suggest that hypermethylation could be a protective factor for HPV infection. This inference may mean that HPV positive colorectal cancer could represent a distinctive clinical and epidemiological condition compared with HPV-negative CRC.

Citation Format: Raul D. Bernabe-Dones, Wesley Villavicencio-Torres, Cristina Munoz-Masso, Yaritza Reyes -Medina, Lorena Marcano-Bonilla, Hector Perez-Cantalapiedra, Sharon Fonseca-Williams, Mercedes Y. Lacourt-Ventura, Yasuhiro Yamamura, Nayra Rodriguez, Marcia R. Cruz-Correa. Detection of viral HPV DNA in sporadic colorectal cancers in relation to CpG island methylator phenotype (CIMP). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2014-3172
Abstract 3174: The host genomic environment of the provirus determines the abundance of BLV-infected clones
Authors: Gillet Nicolas , Brogniez Alix de , Renotte Nathalie , Gutierrez Geronimo , Alvarez Irene , Trono Karina , Willems Luc
Published: May 2015

Human T-lymphotropic Virus 1 (HTLV-1) induces a persistent infection that remains generally asymptomatic. Nevertheless, in a small proportion of individuals and after a long latency, HTLV-1 infection leads to leukemia or lymphoma. Onset of clinical manifestations correlates with a persistently elevated number of infected cells. Because the vast majority of cells are infected at early stages, primary infection is a crucial period for HTLV-1 persistence and pathogenesis. Since HTLV-1 is transmitted through breast feeding and because systematic population screenings are rare, there is a lack of available samples at early infection. Therefore, we addressed this question in a closely related animal model by inoculating cows with Bovine Leukemia Virus (BLV). Initial BLV provirus insertion is not random but occurs nearby transcribed genomic regions (genes and CpG islands). This pattern of integration is highly similar to the observed pattern in HTLV-1 in vitro experiments. Later on, the proportion of clone carrying a provirus in transcribed regions decreases to reach levels comparable to those observed in asymptomatic HTLV-1 carriers. The negative selection against BLV clones carrying a provirus nearby transcribed regions is more efficient in animals that will subsequently present low proviral load set point. Finally, like for HTLV-1 infected people, the abundance of the BLV infected clones correlates with the integration of the provirus in transcribed regions. These observations highlight the high similarities between BLV and HTLV-1 clonality and suggest a key role exerted by the host immune system during primary infection.

Citation Format: Nicolas Gillet, Alix de Brogniez, Nathalie Renotte, Geronimo Gutierrez, Irene Alvarez, Karina Trono, Luc Willems. The host genomic environment of the provirus determines the abundance of BLV-infected clones. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2014-3174
Abstract 3175: The splice variant of the human papillomavirus 16 E6 protein, E6*, displays anti-tumor properties in vivo
Authors: Evans Whitney , Filippova Maria , Aragon Robert , Filippov Valeri , Reeves Mark , Duerksen-Hughes Penelope
Published: May 2015

High-risk types of the human papillomavirus (HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar, and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from high-risk types of HPV during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process (if any) has not yet been established. In the present study, we examined the behavior of the E6* protein during tumor growth in an in vivo nude mouse xenograft model. We created E6*-expressing SiHa (HPV+) and C33A (HPV-) cells, then examined the ability of both the parental and E6*-expressing cells to form tumors in nude mice. The difference in tumor size observed in the presence and absence of E6*, when expressed in both HPV+ (SiHa) and HPV- (C33A) cells, was dramatic and consistent, strongly indicating an anti-oncogenic role for E6* in this context. Interestingly, we found that tumor growth inhibition by E6* was greater in tumors derived from SiHa cells, which are HPV16-positive, than in tumors produced by C33A, which are HPV-negative. This difference implies that E6* acts by interfering with the oncogenic activity of the full-length protein as well as through one or more HPV-independent mechanisms. Consistent with this idea, we found that E6* does indeed bind to the full-sized isoform and inhibits its ability to accelerate degradation of p53 and procaspase 8. These data represent the first demonstration of biologically-relevant E6* activities distinct from those of the full-length isoform in vivo. The significance of these findings in the context of human cancers is in the possibility of mimicking or replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit.

Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark Reeves, Penelope Duerksen-Hughes. The splice variant of the human papillomavirus 16 E6 protein, E6*, displays anti-tumor properties in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2014-3175
Abstract 3178: Rack1 protects N-terminal phosphorylated c-Jun from Fbw7-mediated degradation
Authors: Zhang Jinhui , Zhu Feng , Li Xiang , Xu Yanming , Peng Cong , Li Shengqing , Cho Yong-Yeon , Yao Ke , Zykova Tatyana A. , Bode Ann M. , Dong Zigang
Published: May 2015

The c-Jun transcription factor is a highly unstable oncoprotein. Several ubiquitin ligases mediate c-Jun degradation. However, c-Jun can be stabilized once it is phosphorylated at the N-terminus by c-Jun N-terminal kinases (JNKs) or other protein kinases. This phosphorylation decreases c-Jun ubiquitination and degradation. The underlying mechanism for this phenomenon is still unknown. We show that receptor for activated C-kinase 1 (Rack1) can bind with c-Jun and ubiquitin ligase Fbw7 to form a complex. When c-Jun is phosphorylated at the N-terminus, c-Jun is released from the complex and cannot be ubiquitinated by Fbw7, which leads to increased stabilization and accumulation of c-Jun. These results reveal that Rack1 plays a very important role in tumorigenesis by maintaining the stability of c-Jun that has been phosphorylated at its N- terminus by JNKs or other kinases.

Citation Format: Jinhui Zhang, Feng Zhu, Xiang Li, Yanming Xu, Cong Peng, Shengqing Li, Yong-Yeon Cho, Ke Yao, Tatyana A. Zykova, Ann M. Bode, Zigang Dong. Rack1 protects N-terminal phosphorylated c-Jun from Fbw7-mediated degradation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2014-3178
Abstract 3179: Redox signaling to nuclear respiratory factor-1 proteins by reactive oxygen species contributes to the estrogen-induced breast tumor development
Authors: Roy Deodutta , Felty Quentin , Okoh Victor
Published: May 2015

We have investigated the molecular mechanisms by which reactive oxygen species (ROS)-mediated redox signaling regulates estrogen-dependent growth of breast tumorigenesis and prospects for targeting these signaling pathways for therapeutic prevention and control of tumor growth. We present here for the first time that ROS generated by E2 exposure were responsible for in vitro MCF-7 cell tumor formation. This is evident from the inhibition of E2-induced ROS formation by ROS scavengers. H2O2 and O2•− seem to play important role in estrogen-mediated tumor growth redox signaling by inactivation of PTPs, such as, CDC25A, PTEN to prolong signaling through kinases, such as ERK and AKT. We observed a novel link between ROS-generated by estrogen, redox regulation of ERK and AKT-mediated signaling to nuclear regulatory protein, nuclear respiratory factor-1 (NRF-1) and ERα responsive gene expression in breast cancer cells and their involvement in the growth of estrogen-dependent breast tumor. NRF-1 serves as a nucleating factor to recruit signaling proteins involved in regulating estrogen-responsive cell cycle genes, and oxidative stress regulatable proteins. Findings of this study not only identify the new redox signaling pathway critical to the estrogen-dependent growth of breast tumors; but also provides important information for the design of redox signaling based therapeutic agents for the prevention and treatment of breast cancer.

Citation Format: Deodutta Roy, Quentin Felty, Victor Okoh. Redox signaling to nuclear respiratory factor-1 proteins by reactive oxygen species contributes to the estrogen-induced breast tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2014-3179
Abstract 318: Specific role of Mieap α in Mieap-induced vacuole generation through an interaction with UVRAG
Authors: Nakamura Yasuyuki , Kamino Hiroki , Saito Yuri , Kitamura Noriaki , Sano Hitoya , Arakawa Hirofumi
Published: May 2015

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria via Mieap-induced accumulation of lysosome-like organelles within mitochondria (MALM) or Mieap-induced vacuole (MIV), respectively (1-4). BNIP3 and NIX are essential mediators of MALM. MIV generation is closely related to the endocytic pathway. However, the mechanism of MALM and MIV function and of their selection and regulation remains unclear. UVRAG was identified as a Mieap-interacting protein. UVRAG plays a role in canonical autophagy through the endocytic pathway. Interestingly, Mieap α, but not Mieap β, specifically interacts with UVRAG. Knockdown of UVRAG severely inhibited MIV generation induced by Mieap α. On the other hand, endogenous Mieap β, but not Mieap α, accumulated at and within mitochondria during the MALM process. These results, taken together, suggest that Mieap α has a specific role in MIV generation through an interaction with UVRAG by regulating the endocytic pathway. In addition, UVRAG and Mieap are inactivated in human cancers including colorectal cancer patients, suggesting that the MIV regulated by the interaction of Mieap and UVRAG may play a role in tumor suppression. In this paper, we discuss several possible mechanisms for the selection of MALM or MIV and the role of Mieap/UVRAG in tumorigenesis on the basis of our recent data.

1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011

2. Kitamura N et al. PLoS ONE 6: e16060, 2011

3. Nakamura Y et al. PLoS ONE 7: e30767, 2012

4. Miyamoto T et al. Sci Rep 2: 379, 2012

Citation Format: Yasuyuki Nakamura, Hiroki Kamino, Yuri Saito, Noriaki Kitamura, Hitoya Sano, Hirofumi Arakawa. Specific role of Mieap α in Mieap-induced vacuole generation through an interaction with UVRAG. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2014-318
Abstract 3180: Gastrokine 1 inhibits Helicobacter pylori CagA-induced gastric carcinogenesis
Authors: Yoon Junghwan , Park WonSang , Choi Won Seok
Published: May 2015

Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell mortality, and stimulates gastric carcinogenesis. We investigated the effects of CagA and GKN1 on cell proliferation, apoptosis, ROS production, epithelial-mesenchymal transition (EMT) and cell migration in CagA or GKN1 transfected gastric epithelial cells and tissues from humans and mice infected with H. pylori. We studied whether H. pylori CagA regulates gene copy number and expression of GKN1 in gastric cells and tissues of both humans and mice. On a molecular level, H. pylori CagA induced increased cell proliferation, ROS production, anti-apoptotic activity, and cell migration and invasion, and activation of NF-κB and PI3K/Akt signaling pathways and epithelial mesenchymal transition-related proteins. In addition, H. pylori CagA reduced GKN1 gene copy number and expression in gastric cells and tissues of humans and mice. However, GKN1 overexpression successfully suppressed these carcinogenic effects through directly binding to CagA. These study identifies GKN1 as pharmacological target that inhibits the development and progression of gastric cancers via suppression of H. pylori CagA-induced carcinogenic potential. Additionally, we propose that GKN1 is a potential diagnostic biomarker for H. pylori CagA status, gastritis and gastric cancers.

Citation Format: Junghwan Yoon, WonSang Park, Won Seok Choi. Gastrokine 1 inhibits Helicobacter pylori CagA-induced gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2014-3180
Abstract 3182: Tobacco smoke induces pulmonary neuroendocrine alterations in vivo
Authors: Xie Wei , Liang Zhaofeng , Yin Ying , Xie Chunfeng , Geng Hao , Zhao Li , Wu Rui , Li Xiaoting , Deng Feifei , Wu Jieshu , Geng Shanshan , Zhu Mingming , Zhu Jianyun , Zhu Weiwei , Huang Cong , Zhong Caiyun
Published: May 2015

Tobacco smoke is the leading cause of lung cancer. High-grade malignant pulmonary neuroendocrine tumors, including small cell lung cancers (SCLCs) and large cell neuroendocrine carcinomas (LCNECs), are almost exclusively associated with tobacco smoking. Unanalogous to most of other lung tumors such as squamous cell carcinomas, adenocarcinoma, adenosquamous carcinoma or carcinoid, no precursor lesions for high-grade lung neuroendocrine tumors have so far been identified. The purpose of the present study was to investigate the effects of tobacco smoke on pulmonary neuroendocrine alterations in phenotypically normal cells in vivo. Male BALB/c mice were exposed to tobacco smoke for 6h/day, 7days/week for 12 weeks. Pulmonary histology, neuroendocrine differentiation as well as MAPK/AP-1 activation were examined in lung tissues. Exposure to tobacco smoke significantly induced expression of neuroendocrine differentiation markers such as chromogranin A, neural cell adhesion molecule/(CD56), synaptophysin, and neuron specific enolase, as demonstrated by immunohistochemistry, Western blotting and real-time PCR. The expression levels of epithelial markers, including E-cadherin, zona ocludens-1, cytokeratin 5 and involucrin, were downregulated by tobacco smoke. Moreover, tobacco smoke significantly increased levels of p-ERK1/2, p-JNK and p-p38, while it suppressed p-ERK5 level. Expression of Jun and Fos proteins were differentially regulated by tobacco smoke. Taken together, the present study provides experimental evidence for the first time that tobacco smoke induces pulmonary neuroendocrine differentiation, shedding new light on the carcinogenic process of pulmonary neuroendocrine tumors.

Citation Format: Wei Xie, Zhaofeng Liang, Ying Yin, Chunfeng Xie, Hao Geng, Li Zhao, Rui Wu, Xiaoting Li, Feifei Deng, Jieshu Wu, Shanshan Geng, Mingming Zhu, Jianyun Zhu, Weiwei Zhu, Cong Huang, Caiyun Zhong. Tobacco smoke induces pulmonary neuroendocrine alterations in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3182. doi:10.1158/1538-7445.AM2014-3182
Abstract 3183: Modulation of NLRP3 by chemotherapeutics: Potential application in mesothelioma treatment
Authors: Shukla Arti , Westbom Catherine , Thompson Joyce , MacPherson Maximilian , Beuschel Stacie
Published: May 2015

Malignant mesothelioma (MM) is a deadly cancer and inflammation plays an important role in this carcinogenesis. In about 80% of cases, asbestos is the cause of MM development. We have previously shown that asbestos-induced NLRP3 priming and activation plays a significant role in MM development. We have also demonstrated that MM cells and tumor tissues show decreased levels of NLRP3 and caspase-1 activity as compared to normal mesothelial cells or tissue. Here using various human MM lines and two chemotherapeutic agents, doxorubicin and cisplatin, we show that chemotherapeutic agents can prime and activate NLRP3 in various MM cell lines, resulting in caspase-1 activation which may help in reducing drug resistance and increase tumor toxicity. The process of NLRP3 priming/activation was in part based on reactive oxygen species generated by these chemotherapeutic agents, as N-acetyl cysteine (NAC) was able to block these responses partly but significantly. The process of NLRP3 activation by these drugs also resulted in the release of pro-inflammatory cytokine IL-1β and IL-18. Our current studies are focused on blocking IL-1 receptor by an antagonist to find out if this can attenuate the release of IL-1-related cytokines like IL-6, IL-8 and VEGF. If successful, the combination of chemotherapeutic drugs and IL-1 receptor antagonists may have potential in MM treatment. This work is supported by NIH RO1 grant and a fellowship from the Department of Pathology, UVM.

Citation Format: Arti Shukla, Catherine Westbom, Joyce Thompson, Maximilian MacPherson, Stacie Beuschel. Modulation of NLRP3 by chemotherapeutics: Potential application in mesothelioma treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2014-3183
Abstract 3186: Transplacental arsenic exposure modifies the number of hair follicle keratinocytes stem cells and alters their cell-cycle control
Authors: Marval Paula L. Miliani de , Kim Sun Hye , Rodriguez-Puebla Marcelo L.
Published: May 2015

Drinking water contamination with arsenic is a worldwide problem and a concern in some areas of the United States, especially in the West (California, Nevada, Alaska and Utah) and small areas of New England. Inorganic arsenic is considered a human carcinogen with many target tissues such as the skin, urinary bladder and lung; and characterizing its mechanisms of action is key to designing methods of intervention. Arsenic has transplacental carcinogenic activity, and since their fetal abundance, stem cells (SCs) seem to be a main target of arsenic exposure during gestation. We have established that fetal arsenic exposure via the maternal drinking water, leads to a decrease number of keratinocyte's stem cells (KSC) in mouse epidermis of the offspring. Biochemical analysis of these KSCs shows increased mRNA levels of the cell-cycle regulators cyclin D1 and CDK4. Supporting this observation, overexpression of CDK4 in KSCs mimics arsenic exposure as demonstrated by the reduced number of KSCs, and the decreased number of benign skin tumors, and severe rise in the rate of malignant conversion to squamus cell carcinomas.

We expect that these studies will enable to determine the consequence of in utero arsenic exposure on the proliferative potential of KSCs and the impact on the selection of KSCs that give origin to skin tumors with high risk of malignant conversion. Therefore, these studies will contribute to understanding how early alterations of somatic stem cells results in functional changes that impact a late stage of tumorigenesis such as malignant progression.

Citation Format: Paula L. Miliani de Marval, Sun Hye Kim, Marcelo L. Rodriguez-Puebla. Transplacental arsenic exposure modifies the number of hair follicle keratinocytes stem cells and alters their cell-cycle control. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3186. doi:10.1158/1538-7445.AM2014-3186