bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1938: Raf/MEK/ERK-mediated differential regulation of androgen receptor levels in prostate cancer cells
Authors: Hong Seung-Keun
Published: August 2014

Androgen receptor (AR), a member of the nuclear receptor superfamily, controls the growth regulatory and differentiation pathways in prostate epithelial cells, and its altered signaling is a pivotal event in the carcinogenesis of prostate epithelium. Previous studies show that basal Raf/MEK/extracellular signal-regulated kinase (ERK) activity is required for androgen-induced AR expression or for maintaining its levels in cells. However, loss of AR expression is correlated with upregulated Raf/MEK/ERK activity in certain contexts of PCa biology, including androgen withdrawal-induced PCa differentiation. Indeed, we found a strong correlation between AR downregulation and ERK1/2 phosphorylation in LNCaP cells cultured under a hormone-depleted condition and in the neuroendocrine derivates of LNCaP that were generated by prolonged culture under such condition. In this in vitro model, we investigated the role of Raf/MEK/ERK pathway in regulating AR expression. Sustained activation of the Raf/MEK/ERK pathway, mediated by an inducible Raf, was sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Interestingly, when the intensity of Raf induction was serially decreased to levels under a certain threshold, activation of the Raf/MEK/ERK pathway no longer downregulated but rather upregulated AR levels in cells. Our study therefore suggests that the Raf/MEK/ERK pathway can regulate AR levels in PCa cells in opposing contexts depending upon its different signaling intensity. We also found that sustained Raf/MEK/ERK activation can induce downregulation of full length AR as well as hormone binding domain-deficient AR isoforms in CWR22Rv1 cells, which are known to more potently stimulate PCa cell growth. Further investigation of the mechanisms underlying Raf/MEK/ERK-mediated AR downregulation may lead to a novel means to control AR levels in PCa 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 1938. doi:10.1158/1538-7445.AM2011-1938
Abstract 1939: Association between LRRK2 and 4E-BP1 protein levels in normal and malignant cells
Authors: Pons Berta , Armengol Gemma , Ramón y Cajal Santiago J.
Published: August 2014

Translational control is a crucial component of cancer development and progression. Eukaryotic initiation factor 4E (eIF4E) mediates eIF4F association with the mRNA 5’ cap structure to stimulate cap-dependent translation initiation. The eIF4E-binding protein, 4E-BP1, regulates cap-dependent translation through its phosphorylation at multiple sites. The search for new kinases that could be implicated in 4E-BP1 phosphorylation, and determination of the mechanisms that affect 4E-BP1 stability are important to understand the role of eIF4E in cell transformation.

In this study, we investigate 48 kinases that may be implicated in 4E-BP1 phosphorylation and stability. The screening study was based on analysis of 4E-BP1 status after inhibition of these kinases in a breast carcinoma cell line (MDA-MB 231 cells).

With the use of this method, several kinases that affect 4E-BP1 stability (LRRK2, RAF-1, p38γ, GSK3β, AMPKα, PRKACA and PRKACB) and 4E-BP1 phosphorylation (CDK1, PDK1, SRC, PRKCB1, PAK2, p38β PRKCA and CaMKKB) were identified. These novel findings provide evidence that 4E-BP1 can be regulated and stabilized by multiple kinases implicated in several cell signaling pathways. We also found that LRRK2 downregulation affects 4E-BP1 stability and causes degradation of this factor in various cell types. Interestingly, inhibition of proteasome activity by MG132 restored total 4E-BP1 protein levels in cells where LRRK2 was downregulated. In addition, these stabilization events appear to be functionally important for cell growth control in normal 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 1939. doi:10.1158/1538-7445.AM2011-1939
Abstract 194: The Golgi/ER protein p23/Tmp21 regulates PKCΔ-mediated apoptosis in prostate cancer cells
Authors: Wang Hongbin , Xiao Liqing , Kazanietz Marcelo G.
Published: August 2014

Protein kinase C isozymes have prominent and specific roles in the control of apoptosis and survival of cancer cells. Activation of PKCΔ in androgen-dependent prostate cancer cell models triggers an apoptotic response that is mediated by the release of death factors and the activation of the extrinsic apoptotic cascade. We have previously established that p23/Tmp21, a member of the p24 family ER/Golgi cargo protein, physically interacts with the C1 domains in PKC isozymes and regulates the their activation. Here we found that depletion of p23/Tmp21 significantly potentiates apoptosis induced by phorbol 12-myristate 13-acetate (PMA) in LNCaP prostate cancer cells and the DNA damaging agent doxorubicin. The potentiation of the PMA effect was prevented by the pan-PKC inhibitor GF 109203X or PKCΔ RNAi depletion. Yeast two-hybrid assay revealed that the PKCΔC1a-b domain interacts with p23/Tmp21. In addition, co-localization analysis by confocal microscopy demonstrated that GFP-PKCΔ C1b domain co-localizes with p23/Tmp21 in the perinuclear region. The interaction of PKCΔ and p23/Tmp21 in LNCaP cells was also confirmed by co-immunoprecipitation. Interestingly, disruption of PKCΔ-p23/Tmp21 association by depletion of p23/Tmp21 in LNCaP cells significantly accelerated PMA-induced PKCΔ translocation to the plasma membrane and potentiates the activation of PKCΔ downstream effectors ROCK and JNK. Moreover, a mutated PKCΔ that is unable to interact with p23/Tmp21 triggered significant apoptotic effect when expressed in LNCaP cells. In summary, our data provide evidence that p23/Tmp21 acts as an anchoring protein that retains PKCΔ at the perinuclear region, thus limiting its availability for signaling.

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 194. doi:10.1158/1538-7445.AM2011-194
Abstract 1942: Lactoferrin interferes with the Akt-GSK3b-cyclin D1 growth signaling of human prostate cancer cells
Authors: Banerjee Partha , Jagadeesh Shankar , Dobi Albert , Srivastava Shiv
Published: August 2014

The etiology of human prostatic carcinoma remains largely undefined. However, it is becoming clear that epigenetic inactivation of various tumor suppressor genes could play pivotal role in the development of various cancers including prostate cancer. One of such tumor suppressor is lactoferrin (LTF) gene.

Epigenetic inactivation of LTF may contribute to the development of human prostate cancer. Ectopic expression of LTF or supplementation of bovine LTF in human prostate cancer cells that lack endogenous LTF expression resulted in reduced growth in vitro supporting a role for LTF as a putative tumor suppressor gene. Therefore, it is important to delineate the molecular mechanisms by which LTF regulates prostate cancer cell growth.

In this investigation, we demonstrated that, purified bovine LTF treatment of LNCaP and PC3 cells blocked cell cycle progression and up-regulated cell cycle inhibitors, p15 (INK4b), p16 (INK4a), p21 (Cip1) and p27 (Kip1) and p57 (Kip2) in a dose dependent manner. As a consequence, Cdk4, Cdk6, cyclin D1 and cyclin E levels decreased significantly with bovine LTF treatment. Subsequently, LTF treatment decreased nuclear cyclin D1 and possibly facilitated its degradation. In order to examine the degradation of cyclin D1, we analyzed the levels of phospho-Akt-Ser473- and phospho-Akt-Thr308 after LTF treatment of LNCaP and PC3 cells. We observed that LTF dose dependently decreased phospho-Akt-Ser-473 and phospho-Akt-Thr-308 without altering the total Akt levels. As a consequence, activated GSK3-beta level increased, which is a known to degrade cyclin D1.

In summary, this study shows that bovine LTF treatment can inhibit human prostate cancer cell growth by disrupting Akt-GSK3b-cyclin D1 signaling pathway. Lactoferrin, therefore, promises an encouraging therapeutic potential for prostate 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 1942. doi:10.1158/1538-7445.AM2011-1942
Abstract 1944: Phospholipid scramblase 1 and 3 as novel effectors of cripto-1 signaling
Authors: Karasawa Hideaki , Nagaoka Tadahiro , Rangel Maria Cristina , Castro Nadia P. , Held Natalie , Gonzales Monica , Bianco Caterina , Salomon David S.
Published: August 2014

In the present study we performed a yeast two-hybrid (Y2H) screen to search for novel Cripto-1 (CR-1) binding proteins that might modulate CR-1-dependent signaling pathways involving Nodal/Gdf3/Smad 2/3 or GRP78/glypican-1/c-src. Screening a mouse embryo or human colon cDNA prey library with a peptide sequence of CR-1, we isolated six candidate genes, including mNotch3, mFibulin-4, mCHD4, hAHNAK, hHUWE1 and mouse phospholipid scramblase 3 (PLSCR3). PLSCRs consist of a family of four related genes that encode for endofacial transmembrane proteins, and have been proposed to play a role in the redistribution of plasma membrane phospholipids. Recent work on the PLSCRs has revealed their involvment in signal transduction and lipid metabolism. The most studied member of this family, PLSCR1, has been shown to partition into lipid rafts implicated in the regulation and endocytic trafficking of receptor-signaling complexes. We confirmed the results of the Y2H screen since we found that full-length GFP-tagged PLSCR1 and PLSCR3 were able to bind to 3xFLAG-CR-1 in transiently transfected 293T cells, as detected by coimmunoprecipitation and Western blot analysis. In addition, we were able to detect co-localization of these proteins in 293T cells that were transiently expressing GFP-PLSCR1/3 and 3xFLAG-CR-1 by immunocytochemistry. We then evaluated the effect of interaction between CR-1 and PLSCR-1/3 on Nodal signaling. We therefore analyzed the activity of the Nodal/Activin-responsive (n2)7-luciferase reporter ((n2)7-Luc), which contains mouse Fast-1 binding sites from the Nodal left side-specific enhancer, in 293T cells transiently expressing CR1-FLAG, PLSCR1-GFP, PLSCR3-GFP, (n2)7-Luc, TK-renilla, mouse Fast-1, mouse Nodal and human ALK4 expression vectors. Overexpression of PLSCR1 induced a very strong dose-dependent inhibition of CR-1 activation of Nodal-responsive luciferase reporter activity in 293T cells. In contrast, overexpression of PLSCR3 did not have a significant inhibitory effect on CR-1activation of Nodal-responsive luciferase reporter activity in 293T cells. The effect of knockdown of PLSCR-1/3 with specific siRNA on CR-1 ability to induce activation of a Nodal-responsive luciferase reporter assay and to modulate CR-1 Smad-dependent and Smad-independent signaling pathways is currently under investigation.

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 1944. doi:10.1158/1538-7445.AM2011-1944
Abstract 1947: FAM129B regulates cell proliferation and activates NF-κB and Nrf2 by inactivating KEAP1 in cancer cells
Authors: Lin You-Chin , Cheng Kai-Chung , Lai Alan Chuan-Ying , Chen Yu-Ju , Yu Alice L.
Published: August 2014

The Family with sequence similarity 129 (FAM129) contains three membrane proteins, FAM129A, B and C. FAM129A (also called Niban) and FAM129C (also called BCNP1) have been found to be up-regulated in thyroid tumors and chronic lymphocytic leukemia, respectively. Although phosphorylation of FAM129B has been suggested to play a role in controlling melanoma cell invasion, the molecular mechanism of FAM129B in tumorigenesis remains largely unknown. In this study, we first identified FAM129B as one of proteins with altered phosphorylation status in breast cancer stem cells by the phosphor-mass spectrum technique. Silencing of FAM129B expression in breast cancer cell line, MDA-MB-231, led to decreased proliferation, along with flattened and rounded cell morphology. By mammalian two-hybrid system and FRET (fluorescence resonance energy transfer) assay, KEAP1 (kelch-like ECH-associated protein 1) was detected to interact with FAM129B. KEAP1 has been reported to function as a substrate adaptor protein for Cul3-dependent E3 ligase complex to repress IKKβ and transcription factor Nrf2. We indeed observed that the expression of NF-κB and Nrf2 downstream genes, FN1 and NQO1, was down-regulated in FAM129B-silenced cells by qPCR analysis. On the other hand, FAM129B together with either IKKβ or Nrf2 synergistically activated NF-κB and Nrf2 promoter activity by reporter assays. Taken together, our results suggest that FAM129B might play an important role in the regulation of cell proliferation in cancers by inactivating KEAP1 which in turn activates Nrf2 and IKKβ activities.

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 1947. doi:10.1158/1538-7445.AM2011-1947
Abstract 1948: Functional profiling of signal transduction pathway proteins in gastric cancer (GC) patients
Authors: Lee Tani , Lee Jeeyun , Magonova Katya , Kang Won Ki , Harvie Gulia , Barham Robert , Leesman Glen , Kim Phillip , Singh Sharat , Kim Sung
Published: August 2014

Although gastrectomy is the only curative treatment in gastric cancer (GCA) patients, a high recurrence rate ranging from 40 ∼ 60% following curative surgery still accounts for poor overall survival. In order to further improve survival, there is an urgent need to identify reliable molecular prognostic markers for survival or recurrence following adjuvant chemoradiation therapy. With the advent of molecularly targeted therapy in cancer care, profiling of pathway proteins for targeted drug is of utmost importance. Hence, we have developed a multiplexed immunoassay platform for functional profiling of the signal transduction pathway proteins using a multiplexed immuno-microarray platform.

A multiplexed protein microarray platform, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies (d-abs) has been utilized to profile targetable biomarkers in 447 fresh frozen tumor tissues procured during surgical procedure from GCA patients with localized disease. The collaboration between two channeling-enzymes conjugated on two d-abs in proximity enables the profiling of the target proteins with extreme sensitivity and specificity in multiplexed fashion. As CEER can also be configured for each specific target protein, it allows differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). Here we report the profiling of HER2, p95HER2, HER1, HER3, PI3K, cMET, Shc and IGF1R in GCA for their levels of expression and phosphorylation. Approximately 12% of patients in this cohort expressed high levels of HER2. Varying levels of truncated HER2 were detected in 7.1% (27/447) of GCA pts. HER3 were found in approximately 40% of pts. The prevalence of biomarker expression/activation along with the outcome of unsupervised clustering analysis of signaling molecules revealing diverse and distinct disease subtypes will be presented.

The treatment of recurrent or metastatic GC may be optimized based on patient-specific functional profiling of biomarker leading to patient-tailored treatment strategies. In addition, the incidence of alteration in transduction pathway proteins may guide oncologists for further clinical development of targeted therapies or combined / sequenced targeted therapies.

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 1948. doi:10.1158/1538-7445.AM2011-1948
Abstract 1954: In vitro effects on gene expression of Casp9, GRB10, and CDKN1B following rG3 treatment of human breast cancer cells
Authors: Schalkoff Katie , Walker Heather , Scott Tom
Published: August 2014

Introduction: Integrins can stimulate cellular growth and proliferation, as well as apoptosis, and are important factors in AKT cell signaling. To assess their binding, a recombinant protein of the G3 domain of laminin-5 (rG3) that specifically binds the alpha subunit expressed on metastatic MDA-MB-231 cells in vitro was produced. Studying changes in expression of genes involved in cell signaling and the apoptosis cascade, specifically CDKN1B, GRB10, and Caspase 9, can help improve understanding of signaling pathways of metastatic cells and the AKT pathway.

Methods: rG3 was produced using ArcticExpress Competent Cells (Stratagene). Plasmid DNA was transformed into competent cells and transferred to ampicillin plates for colony production of successful transformation. A single colony was used to inoculate LB broth containing gentamycin and ampicillin for an overnight incubation at 37°C. LB without antibiotics was inoculated with the overnight culture and incubated at 30°C for 3 hours. The temperature was lowered to 11.5°C before the expression was induced using IPTG at a final concentration of 1mM. SDS-PAGE/immunoblotting confirmed successful soluble rG3 protein expression. MDA-MB-231 human breast cancer cells from ATCC were grown in tissue culture flasks and maintained until confluent. Cells were plated at 3 × 106 cells/well in a 6-well tissue culture plate in a 2 ml volume of DMEM without pen/strep in duplicate. rG3 was added to wells at a final concentration of 175 µg/ml for treated cells and corresponding control buffer for untreated cells. After incubation overnight, cells were collected at time points within 0-60 hours and RNA was isolated using Qiagen RNeasy. An eppendorf Realplex 96-well plate reader was used for PCR analysis using a β-actin standard curve. Differences in gene expression were shown in fold change using Pfaffl's Method.

Results: Our preliminary data show there are changes in gene expression after treatment with rG3.We are further investigating these changes in expression. Preliminary results suggest that changes in gene expression after treatment may be informative in understanding the AKT signaling pathway.

Conclusion: The study of these changes in gene expression after altering the AKT pathway from treatment with rG3 could prove to be a helpful tool in understanding the signaling mechanisms of metastatic cells and the details of the signaling pathways involved in apoptosis.

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 1954. doi:10.1158/1538-7445.AM2011-1954
Abstract 1956: Enhanced colony-forming ability, and motility and migratory properties of Cisplatin-resistant ovarian cancer cells
Authors: Yue Peibin , Zhang Xiaolei , Turkson James
Published: August 2014

Ovarian cancer causes the highest gynecologic neoplasm-related deaths in the United States. Although the majority of ovarian cancer patients respond to Cisplatin therapy, most develop recurrent and resistant disease, raising the need to understand the molecular mechanisms that support the resistant phenotype and to identify novel effective therapeutic and chemo-sensitizing modalities. In this study, we developed Cisplatin resistance in vitro to address the molecular and biological mechanisms that may be presented by the recurrent disease. The resistant lines, A2780S/CP3 and A2780S/CP5, which were derived from the Cisplatin-sensitive line, A2780S and are resistant to 3 and 5 μM Cisplatin, respectively, showed marked changes in morphology, compared to the parental cells. Notably, both resistant lines showed enhanced colony-forming ability and significantly enhanced cell motility and migratory properties. Molecular mechanistic analysis indicated the Cisplatin resistance was associated with hyperactive epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (Erk)1/2MAPK and EGFR-Signal Transducer and Activator of Transcription (Stat)3 pathways, the overexpression of Survivin and vascular endothelial growth factor receptor (VEGF), and F-actin and Cortactin, with F-actin localization that is predominantly at the cellular extensions. Treatment of the Cisplatin-resistant A2780S/CP5 line with the EGFR inhibitor, Iressa or the Stat3 inhibitor, S3I-201 inhibited the colony-forming ability of these cells, without suppressing their viability, suggesting EGFR-Erk1/2 and EGFR-Stat3 activation are required for the proliferation of the resistant cells. Furthermore, inhibition of EGFR or Stat3 activation significantly suppresses the migration of A2780S/CP3 cells, while the motility of both A2780S/CP3 and A2780S/CP5 lines are sensitive to EGFR inhibition. These results together suggest that Cisplatin resistance in ovarian cancer is mediated in part through the activation of the EGF receptor-mediated Erk and Stat3 pathways that promote increased cell proliferation, motility and migration. These findings raise the possibility that EGFR or Stat3 inhibitors combined with Cisplatin may be an effective therapeutic approach for Cisplatin-resistant ovarian 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 1956. doi:10.1158/1538-7445.AM2011-1956
Abstract 1957: PMEPA1 promotes androgen receptor negative prostate cell proliferation through suppressing the Smad3/4-c-Myc-p21Cip1 signaling pathway
Authors: Liu Rong , Zhou Zhongmei , Huang Jian , Chen Ceshi
Published: August 2014

The PMEPA1 gene has been shown to suppress the androgen receptor (AR) and TGFβ signaling pathways and is abnormally expressed in prostate tumors. However, the role and mechanism action of PMEPA1 in AR-negative prostate cancer are unclear. Here, we demonstrate that inhibition of PMEPA1 suppresses AR-negative RWPE1 and PC-3 prostate cell proliferation through upregulating the p21 transcription. Additionally, PMEPA1 overexpression suppresses the p21 expression and promotes cell proliferation. PMEPA1 is induced by TGFβ as a negative feedback loop to suppress Smad3 phosphorylation and nuclear translocation, upregulates c-Myc, downregulates p21, and promotes PC-3 cell proliferation. The PMEPA1 functions depend on its Smad2/3 binding motif. Consistently, depletion of Smad3/4, but not Smad2, blocks the PMEPA1's functions of regulating c-Myc and p21. Importantly, stable depletion of PMEPA1 in PC-3 inhibits xenograft growth. Finally, we found that PMEPA1 is overexpressed in a subset of prostate cancer cell lines and tumors. These findings suggest that PMEPA1 may promote AR-negative prostate cancer cell proliferation through p21. (This work was supported by a grant from the Department of Defense (W81XWH-07-1-0191) and a grant from the American Cancer Society (RSG-08-199-01). Dr. Liu is supported by a postdoctoral fellowship from American Urological Association Foundation.)

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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 1957. doi:10.1158/1538-7445.AM2011-1957
Abstract 1967: Ligand-dependent TGFβ signalling potential
Authors: McLean Sarah E. , Guglielmo John Di
Published: August 2014

Transforming growth factor beta (TGFβ) is a cytokine which regulates many normal biological processes such as organogenesis and cell cycle control. Its signaling pathway is under intense study as it is commonly dysregulated in pathophysiological conditions such as cancer and fibrosis. The TGFβ signalling cascade is initiated by the binding of ligand to cell-surface serine-threonine kinase receptors. Binding of ligand to the TGFβ receptors initiates phosphorylation of intracellular signalling proteins called Smads, which can then enter the nucleus and initiate specific transcriptional programs. There are three TGFβ ligands which can activate canonical Smad signalling: TGFβ1, TGFβ2 and TGFβ3. These ligands share significant sequence homology (70-80%) but have different spatial and temporal patterns of expression in development. Although TGFβ1 and TGFβ3 are highly expressed in the tumour microenvironment, the role of TGFβ3 has been largely over-looked and thought to be the same as TGFβ1. The objective of the present study is to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Using dose response studies, we show that TGFβ1 ligand induces Smad2 phosphorylation to a greater extent than TGFβ3. We also show that TGFβ3 induces a lower reduction in steady-state levels of E-cadherin, a protein involved in cell-cell adhesion, compared to TGFβ1. To evaluate the transcriptional programs of both ligands, we used microarray technology to assess gene transcription in non-small cell lung cancer cells treated with TGFβ1 or TGFβ3. Consistent with our signalling data, we found that TGFβ3 induces far fewer genes than TGFβ1. Future studies will evaluate the differential roles of TGFβ1 and TGFβ3 in cancer cell migration and epithelial-to-mesenchymal transition.

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 1967. doi:10.1158/1538-7445.AM2011-1967
Abstract 1970: SOCS-1,-3 gene delivery in gastric cancer cells induces a potent anti-proliferative effect via the suppression of JAK/STAT and P38 MAPK signaling pathways
Authors: Urase Maiko , Serada Satoshi , Souma Yoshihito , Iwahori Kouta , Shimada Kazuki , Nishida Toshirou , Fujimoto Minoru , Naka Tetsuji
Published: August 2014

BACKGROUND:Enhanced activation of inflammatory cytokines signaling transductions have been suggested as a driving force in the development of gastric tumors. The suppressor of cytokine signaling (SOCS) family proteins are important negative regulator of proinflammatory cytokine signals and have been reported to be frequently silenced by DNA methylation of CpG islands in gastric cancer (GC). The aim of this in vitro assessment was to evaluate the role of SOCS-1 and SOCS-3 in the proliferation of GC cell through regulating cytokine signaling pathways. MATERIAL AND METHODS:Six GC cell lines (NUGC3, AGS, MKN45, NUGC4, MKN7, and MKN74) were used in this study. Methylation status of SOCS-1/-3 was assessed by methylation specific PCR. The concentrations of IL-6 in the cell culture supernatant was measured by ELISA. Adenovirus vectors encoding either SOCS-1 or SOCS-3 were constructed for gene delivery to GC cells. The status of activated kinases and downstream proteins were determined by Western blot analysis. Anti-proliferative effects in GC cells were assessed by WST-8 assay.

RESULTS: In six GC cell lines used in our study, SOCS-1 and SOCS-3 genes were methylated except one cell line, and two cell lines (NUGC3, AGS) showed elevated endogenous IL-6 production with constitutively phosphorylated STAT3. Unexpectedly, anti-IL-6R antibody did not affect both of the cell proliferation and the status of phosphorylated STAT3 in these two cell lines. In contrast, adenovirus-mediated SOCS-1 /-3 gene delivery markedly reduced cell proliferation of these cells. The anti-proliferative effect of SOCS-1/-3 correlated with decreased levels of the activation of STAT3 and p38 MAPK. Cell proliferation assay confirmed that the blockade of JAK/STAT and p38 MAPK signaling pathways also reduced cell proliferation of these cell lines. CONCLUSIONS: Our results indicated that the DNA methylation of SOCS-1/-3 may be associated with GC proliferation, and highlighted an anti-proliferative potential of SOCS-1/-3 through the inhibition of JAK/STAT and p38 MAPK signaling pathways independently IL-6 signaling. Thus, DNA methylation of SOCS-1/-3 can be a molecular marker of GC progression, and their forced expression may represent a novel therapeutic application as a multi-growth signal inhibitor for GC treatment.

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 1970. doi:10.1158/1538-7445.AM2011-1970
Abstract 1972: Nicotine regulates DNA-binding protein inhibitor (Id1) through a Src-dependent pathway promoting tumorigenic properties and chemoresistance in pancreatic adenocarcinoma
Authors: Treviño José G. , Pillai Smitha R. , Kunigal Sateesh S. , Chellappan Srikumar P.
Published: August 2014

Nicotine, an addicting component of an established risk factor (tobacco) for pancreatic cancer is identified as a tumor promoter potentially acting through activation of oncogenes such as Src, a non-receptor protein tyrosine kinase. Recent studies from our laboratory have shown that the Id1 transcription factor (inhibitor of DNA binding/Differentiation) is induced by nicotine in NSCLC cells. Id1 has been shown to play a role in tumor progression by promoting angiogenesis, invasion, and chemoresistance. Further, Src and its family of non-receptor protein tyrosine kinases (SFKs) play a role in angiogenesis, tumor progression, and chemoresistance. Based on our observations on NSCLC cells, we hypothesize nicotine-induces Id1 expression in a Src-dependent fashion and promotes tumorigenesis and chemoresistance. Protein analysis, mRNA expression, and further experiments were determined in a panel of pancreatic cancer cells, PANC-1, Mia-Paca-2, L3.6pl, and L3.6plGemRes by western blot analysis and RT-PCR respectively. Our results demonstrate that stimulation with nicotine (1 µM), and activation of nAChR, resulted in phosphorylation of Src, indicative of activation, and its family of kinases. Nicotine also induced Id1 expression at similar concentrations. Inhibition of SFK activity with Dasatinib (500nM) did not allow serum or nicotine-induced SFK activity and subsequent Id1 expression. Specific inhibition of c-Src expression with short-interfering RNA (siRNA) decreased constitutive and nicotine-induced Id1 expression and depletion of Id1 expression by siRNA or short-hairpin RNA (shRNA) significantly reduced the proliferative (80%) and invasive property (75%) of pancreatic cancer cells, a property often mediated by Src. Additionally, inhibition of Src and Id1 expression in innate or established gemcitabine resistant cancer cells resulted in gemcitabine sensitization at IC50, 250 ng/ml. To determine if nicotine contributes to gemcitabine chemoresistance potentially through a Src-dependent Id1 signaling axis, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine. Nicotine promoted a 2-fold increase in cell proliferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key Src-dependent signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. We demonstrate that Id1, by a nicotine promoting Src-dependent pathway, contributes to signaling pathways involved in tumor progression and establishment of a chemoresistant phenotype in pancreatic 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 1972. doi:10.1158/1538-7445.AM2011-1972
Abstract 1973: Caveolin-1 promotes cell proliferation, EGFR activation and tumor growth in kidney cancer
Authors: Xu Chao Ying (Sarah) , Wang Yi , Roche Olga , Moriyama Eduardo , Ohh Michael
Published: August 2014

Tumor hypoxia is associated with disease progression, drug resistance and poor prognosis. It has been shown previously that hypoxia prolongs the activation of epidermal growth factor receptor (EGFR) by delaying clathrin-dependent endocytosis-mediated deactivation of receptors. Recently, caveolin (CAV) mediated signaling and trafficking have been reported to be involved in the oncogenesis of many tumours, including clear cell renal cell carcinoma (CCRCC). However, the role of CAV-1 in the tumor progression is unclear. Here, we show that the loss of von Hippel-Lindau (VHL) protein – the principal negative regulator of hypoxia-inducible factor (HIF) – increases the auto-phosphorylation of epidermal growth factor receptor (EGFR), thus attributing to an enhanced ligand-independent cell signaling pathway, majorly Ras-Raf-MEK-ERK pathway, and leading to cell proliferation. The auto-phosphorylation of EGFR is due to the HIF-dependent overexpression of caveolin-1 (CAV-1) at the level of transcription, which enhances the association between EGFR and CAV-1 and the auto-dimerization of EGFR. Primary CCRCC tumors and numerous hypoxia treated tumor cell lines exhibit significantly higher expression of CAV-1 protein. Using a dorsal skin-fold window chamber on SCID mice, shCAV-1 knockdown 786-O tumor cell line exhibits lower growth than that of shscramble counterpart in xenografts. These findings support a model in which tumour hypoxia or oncogenic activation of HIF prolongs ligand-independent RTK signaling through the over-expression of CAV-1. Given the oncogenic role of CAV-1, it should be considered as a future therapeutic target in cancer treatment.

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 1973. doi:10.1158/1538-7445.AM2011-1973
Abstract 1977: N-glycosylation level of the type II TGF-β receptor is important for the stability and cell surface localization and affects TGF-β resistance in cancer cells
Authors: Kim Young-Woong , Park Jinah , Lee Hyun-Ju , Kim Seong-Jin
Published: August 2014

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability, localization, ligand-receptor binding and intracellular signaling. The type II TGF-β receptor (TβRII) plays a crucial role in the TGF-β signaling pathway. Although N-linked glycosylation of TβRII was first demonstrated over a decade ago, it is unclear how this modification affects TβRII biology. By generating deficient N-linked glycosylation (dNG) mutants, in which each asparagine was converted to glutamine, we show here that N-linked glycosylation occurs on two conserved asparagine residues in the extracellular domain of TβRII. dNG mutant TbRII lines indeed exhibited defective N-linked glycosylation. Notably, non-glycosylated TβRII was more sensitive to proteasome-mediated degradation than fully N-glycosylated TβRII, suggesting that the N-linked glycosylation protects TβRII from proteasome-mediated degradation. Defective N-linked glycosylation also induced TβRII to accumulate in the endoplasmic reticulum (ER), resulting in decreased expression on the cell surface compared to N-glycosylated TβRII. Ultimately, upon TGF-β stimulation, cells expressing dNG mutant TβRII lines were not efficiently able to activate downstream signaling due to the decreased number of cell surface-expressed receptor molecules. These findings demonstrate that N-linked glycosylation is essentially required for the stability and cell surface localization of TβRII. Moreover, by comparing the N-linked glycosylation levels of TβRII in various cancer cell lines, we suggest that the degree of N-linked glycosylation of TβRII might be one of molecular mechanisms involved in the different cellular responses of cancer cell lines upon TGF-β treatment, shedding light on the N-linked glycosylation that fosters TGF-β resistance in many cancers.

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 1977. doi:10.1158/1538-7445.AM2011-1977
Abstract 1979: Role of Akt1 on the viability, migration and cytoskeleton organization of murine mammary tumor cells reexpresing glypican-3 (GPC3)
Authors: Romero Silvina A. , Lago Huvelle María A. , Peluffo Guillermo , de Kier Joffé Elisa Bal , Peters María Giselle
Published: August 2014

Previously, we have demonstrated that the restatement of GPC3 in the murine mammary adenocarcinoma LM3 cell line induces the inhibition of its metastatic ability in vivo, event associated with a partial reversion of the epithelial-mesenchymal transition. We also found that LM3-GPC3 cells present a constitutive lower activity of the Akt pathway.

The Akt kinases 1, 2 and 3 have a critical role in regulating survival, metabolism and other cellular activities. Their role on migration and metastasis is less clear. The aim of this study was to analyze the function of Akt1 on the viability, migration and cytoskeleton organization of LM3-GPC3 cells. Experiments were done employing two clones reexpressing GPC3 (LM3-GPC3) and two control clones transfected with the empty vector (LM3-vector).

By means of WB we determined that all the clones express similar levels of total Akt (1, 2 and 3). However, while LM3-vector cells presented a higher proportion of Akt2 (Akt1/Akt2=0.75), GPC3 reexpression reversed this relationship (Akt1/Akt2=1.45).

To study the role of Akt1, we introduced by viral infection a constitutively active variant of this isoform (CA Akt1) into LM3-GPC3 cells. We corroborated previous findings that GPC3 reexpression increases LM3 cell line susceptibility to death under serum deprivation (ANOVA-Bonferroni p<0.001). However, CA Akt1 did not affect the viability of LM3-GPC3 cells (ANOVA, ns). While no changes were detected in the actin cytoskeleton organization, we found by wound healing assays that LM3-GPC3 cells expressing CA Akt1 were more motile than their controls (Wound coverage: LM3-GPC3 control=45% vs LM3-GPC3 CA Akt1=90%, ANOVA-Bonferroni p<0.001).

These results suggest that the Akt family would have isoform-specific mechanisms to regulate the studied cellular parameters. Since the migration of LM3-GPC3 cells expressing CA Akt1 reached similar levels to those of LM3-vector clones, we conclude that GPC3 reduces the migratory ability of LM3 cells by inhibiting Akt1. On the other hand, CA Akt1 did not affect either viability or the organization of the actin cytoskeleton of LM3-GPC3 cells. Thus, we deduce that other isoforms of Akt and/or other signaling pathways are involved in the modulation of these cellular properties.

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 1979. doi:10.1158/1538-7445.AM2011-1979
Abstract 1980: The SIAH2-SMAD2/3/4 antagonism downstream of the K-RAS and TGFβ signaling pathways during tumor initiation, progression and metastasis of human cancer
Authors: Liao Yang , Edens Maryanne , Janknecht Ralf G. , Leof Edward B. , Tang Amy H.
Published: August 2014

The pivotal roles of the K-RAS and TGFβ signaling pathways have been well characterized in a number of normal and pathological conditions including human cancer. Hyperactivation of the ERBB/KRAS pathway is known to drive neoplastic transformation, tumorigenesis and metastasis in a large percentage of human cancers while the growth inhibitory actions of TGFβ are critical in preventing cancer. A better understanding of the antagonistic interaction and cross-talk between the K-RAS and TGFβ signaling pathways that control tumorigenesis and metastasis may give rise to novel therapeutic interventions. Our previous work has highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. In this study, we showed that SMAD2/3/4 – the essential downstream mediators of TGFβ signaling, are new SIAH2 substrates. SIAH2 physically interacts with, ubiquitinates and degrades SMAD2/3/4 (but not SMAD1 – the downstream mediator of the BMP signaling pathway). The identification of the SIAH2-mediated SMAD2/3/4 modulation/degradation may provide an additional mechanism to explain the well documented, but less understood ERBB/K-RAS and TGFβ antagonism described in normal development, tumorigenesis and metastasis. We propose that the tumor promoting activity of RAS/SIAH2 may be mediated through the degradation of SMAD2/3/4 proteins and thus the relief of TGFβ-dependent growth inhibition in response to ERBB/K-RAS activation. The discovery of the SIAH2-SMAD2/3/4 antagonism downstream of K-RAS and TGFβ signaling pathways may provide a novel mode of antagonistic cross-talk between these two important signaling pathways to explain the growth inhibitory action of the TGFβ pathway and the growth-promoting activity of the K-RAS pathway in the genesis, progression and metastasis of human cancer. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligase, our results provide new insights into the role of altered proteolysis in mediating the context-dependent ERBB/RAS-TGFβ antagonism and cross-talk in human cancers.

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 1980. doi:10.1158/1538-7445.AM2011-1980
Abstract 1981: Tumor suppressor functions of the Src-like adaptor protein (SLAP) in colorectal cancer cells
Authors: Naudin Cécile , Leroy Cédric , Sirvent Audrey , Simon Valérie , Bourgaux Jean-François , Robert Bruno , Pannequin Julie , Hollande Frédéric , Roche Serge
Published: August 2014

Background : The cytoplasmic tyrosine kinase Src is frequently deregulated in colorectal cancer (CRC) and this deregulation is implicated in tumour growth and in the induction of metastasis. How Src is activated in this cancer has not been clearly established. In contrast to Ras, Src activating mutations are rare events, suggesting the existence of alternative mechanisms for the induction of Src oncogenic activity. Here, we addressed whether Src deregulation involves inhibition of its negative regulators, including the Src-Like Adaptor Protein (SLAP).

Experimental procedures : SLAP expression was analyzed in colon cancer cells and in microdissected tissue samples of patients with CRC. The effect of SLAP was analyzed on the capacity of CRC cells to grow in soft agar and to induce tumours once engrafted in nude mice. The effect of SLAP on the invasive properties of CRC cells was investigated in vitro using Matrigel Boyden chamber assays, as well as in vivo following injection in the spleen of Balb-c mice.

Results : We found that SLAP is expressed in the healthy colon and that its expression is reduced in 60% of CRC biopsies tested. Moreover, we showed that SLAP overexpression dramatically reduced the capacity of CRC cells to induce primary tumours and liver metastases in vivo. Conversely, SLAP knock-down in early-stage CRC cells led to a remarkable induction of liver metastasis in mice. This novel function of SLAP required its myristoylation site as well as intact SH3 and SH2 domains. A proteomic approach is under way to identify SLAP partners involved in this tumor suppressor activity.

Conclusions: Our results suggest that, in addition to lymphocytes, SLAP also regulates transforming properties of CRC cells. We hypothesize that SLAP negatively regulates oncogenic signalling initiated by tyrosine kinases including Src.

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 1981. doi:10.1158/1538-7445.AM2011-1981
Abstract 1983: A cell-permeable Stat3 SH2 domain mimetic inhibitor of Stat3 activation inhibits growth of human breast tumor xenografts
Authors: Zhao Wei , Jaganathan Soumya , Zhang Xiaolei , Turkson James
Published: August 2014

Human tumors harbor multiple molecular aberrations that have become the driving force for uncontrolled cell proliferation and survival, and other tumor-associated processes. Constitutive activation of the Signal Transducer and Activator of Transcription (Stat) 3 protein is a frequent occurrence in many human cancers, including breast cancer. Given the role of aberrant Stat3 activity in human tumors, small-molecule Stat3 inhibitors would be useful novel therapeutics and tools for probing Stat3-mediated tumor processes. We herein report that a 28-mer peptide, SPI, composed of the amino acid residues 588-615 of the Stat3 SH2 domain, replicates Stat3 biochemical properties and functions as a Stat3 inhibitor. Studies show SPI and Stat3 (or Stat3 SH2 domain) both bind with similar affinities to known Stat3-binding phosphotyrosine (pY) peptide motifs, including the epidermal growth factor receptor (EGFR) peptides, pY1068EGFR and pY1086EGFR, and the high-affinity, interleukin-6 receptor (IL-6R)/gp130-derived peptide, GpYLPQTV-NH2. Consequently, SPI potently and selectively inhibits Stat3 SH2 domain:pY interactions and disrupts the binding of Stat3 to the IL-6R/gp130 peptide, GpYLPQTV-NH2. Fluorescence imaging studies show SPI is cell membrane-permeable. Treatment with SPI of malignant cells that harbor aberrant Stat3 activity resulted in the selective inhibition of constitutive Stat3 phosphorylation, and of Stat3 DNA-binding and transcriptional activities. By contrast, similar treatments have little or no effects on the induction of the Stat1, Stat5, and Erk1/2MAPK pathways in malignant cells or in EGF-stimulated fibroblasts. In contrast to normal cells, human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring constitutively-active Stat3 are highly sensitive to SPI and undergo extensive morphology changes, concomitant with the loss of viability and apoptosis. Significantly, treatment with SPI or its inverse sequence version strongly inhibited the growth of human breast tumor xenografts in mice, which is associated with the down-regulation of the expression of the known Stat3-regulated genes, Cyclin D1, Bcl-xL and Survivin. Our study identifies SPI as a novel molecular probe for interrogating Stat3 signaling and that functions as a selective Stat3 inhibitor, which induces antitumor response in vivo.

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 1983. doi:10.1158/1538-7445.AM2011-1983