Transform NIH 3T3 Research Articles

Abstract PO4-28-11: Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells

Abstract Several studies in the past have demonstrated the role of cell-to-cell interaction (crosstalk) between tumor cells and normal (nontumorigenic) cells in cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have a potential to transform the phenotypes and the behaviors of normal cells. Application of the 2D in-vitro cultures has been limited due to its inability to replicate the complex in-vivo TME. Conditioned Medium (CM) obtained from cultured cancer cells contains secreted growth factors that potentially regulate the phenotype and the functionality of normal cells. In this study, a culture of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells with conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in an altered phenotype with increased cell viability. 4T1CM treated NIH3T3 and RAW 264.7, in comparison with the respective control cells, resulted in an upregulation of the genes including- α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF). In addition, 4T1.CM treated NIH3T3 showed an EMT phenotype as indicated by the regulation of EMT markers such as, E-cadherin, β-catenin, N-cadherin, and Vimentin. Interestingly, an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1) was observed in 4T1CM treated RAW 264.7, a tendency towards exhibiting an inhibitory immune response. Intriguingly, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers including- sex determining region Y-box 2, and Aldehyde dehydrogenase. Collectively, our study suggested a role for 4T1CM in transforming the normal NIH3T3 and RAW 264.7 into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). RNAseq experiments are underway to map differentially expressed genes (DEGs) that potentially regulate the 4T1CM induced transformation of NIH3T3 and RAW 264.7 cells. Furthermore, invitro drug testing in 3D model of these transformed cells to target the pathway intermediates may provide novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Joseph Francis. Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-11.

Herceptin: A First Assault on Oncogenes that Launched a Revolution

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.

PO-314 ALK inhibitor, ceritinib, abrogates activation of the novel ALK-I1171T mutation in neuroblastoma

IntroductionMutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) are undoubtedly implicated in the pathogenesis of the childhood cancer, neuroblastoma. However, clinical response of ALK positive neuroblastoma patients to the first generation ALK inhibitor has been rather disappointing.Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression.Material and methodsGenomic tumour DNA from biopsy samples were extracted and exome sequencing was performed through paired-end sequencing on Illumina plateforms.The novel ALK-I1171T mutant was biochemically analysed by western blot and neurite-outgrowth assay in PC12 cells, and foci formation assay in NIH3T3 cells.Results and discussionsAnalyses of genomic tumour DNA from biopsy samples initially showed an 11q deletion, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALK-I1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signalling activation. Secondly, ALK-I1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALK-I1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALK-I1171T compared with crizotinib.ConclusionThis study suggests that ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Evolutionary acquired robustness and vulnerability in cancer genome: negligible negative selection in carcinogenesis

Cancer is a disease involving the accumulation of somatic genetic alterations accompanied by clonal evolution (1,2). A wide range of oncogenes and tumor suppressor genes are known to be mutated, deleted, rearranged, and/or amplified in most cancers. These genes have been identified by functional analyses (transformation of NIH3T3), genetic linkage analyses, and structural genomic analyses such as cytogenetics.

Abstract B40: A novel activating I1171T ALK mutation in neuroblastoma responds better to ceritinib compare to the first generation inhibitor crizotinib

Abstract Mutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) have been suggested to be important players in the genetics and progression of the childhood tumor neuroblastoma. Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression. Analyses of genomic tumor DNA from biopsy samples initially showed an 11q minus, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALKI1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signaling activation. Secondly, ALKI1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALKI1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALKI1171T compared with crizotinib. Citation Format: Joachim T. Siaw, Jikui Guan, Tommy Martinsson, Magnus Hansson, Per Kogner, Ruth Palmer. A novel activating I1171T ALK mutation in neuroblastoma responds better to ceritinib compare to the first generation inhibitor crizotinib [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B40.

Genotoxicity of a Low-Dose Nitrosamine Mixture as Drinking Water Disinfection Byproducts in NIH3T3 Cells.

N-nitrosamines (NAms), which can arise as byproducts of disinfection agents, are reportedly found in drinking water, and their potential carcinogenicity is a concern; however, little research exists regarding the genotoxicity or carcinogenicity of NAms exposure as a low-dose mixture. The three most common NAms components in China's drinking water are N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosomethylethylamine (NMEA). Thus, we measured the genotoxic and carcinogenic potential of these compounds and measured the cell cycle and gene expression. The data show that exposure to the NAms-mixture doubled the revertants in the TA98 and TA100 S. typhimurium strains and increased the DNA double-strand breaks and the micronuclear frequency in the NIH3T3 cells compared to a single exposure. After long-term NAms mixture exposure, a malignant transformation of NIH3T3 and a significantly increased G2/M distribution were observed. Furthermore, P53, CDK1, P38, CDC25A and CyclinB expressions were down-regulated in the NAms-mixture exposure group; however, P21 and GADD45A genes were up-regulated. Interestingly, the CHK1/CHK2 and CDC25A genes had two responses, depending on the NAms concentrations. Thus, we observed mutagenic, genotoxic and carcinogenic effects after a low-dose NAms-mixture exposure in drinking water, and DNA repair and apoptosis pathways may contribute to these adverse effects.

Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer.

BackgroundThe 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers.MethodsDNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression.ResultsHere we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells.ConclusionTo our knowledge, this is the first study to identify the 14-3-3 eta gene as a tumor suppressor and that its expression is suppressed in colon tumors by DNA hypermethylation. These data suggest a link between 14-3-3 expression levels and the development of colon cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1856-y) contains supplementary material, which is available to authorized users.

Abstract 271: Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy.

Abstract Cancer stem cells (CSCs) or side-population (SP) constitute a sub-population of cells in a tumor which are responsible for the maintenance of several cancers, including pancreatic cancer (PC). In the process of identification of a CSC maintenance marker for PC, we have focused on the human polymerase association factor 1 (hPaf1) or pancreatic differentiation 2 (PD2) due to its critical role in self-renewal of mouse embryonic stem cells (mESCs) through the interaction with Oct3/4, a gatekeeper of pluripotency and self-renewal for ESCs. Furthermore, PD2 has been found to be highly overexpressed in poorly differentiated PC compared to well-differentiated PC due to gene amplification in the 19q13 locus. Additionally, ectopic overexpression of PD2 led to the oncogenic transformation of NIH3T3 immortalized cells. An essential property of stem cells is its ability to self-renew, which is also an important property of CSCs. Based on the aforementioned work, we hypothesize that PD2 is involved in the maintenance of self-renewal and drug resistance of pancreatic CSCs. To test our hypothesis, SP and Non-SP were isolated from pancreatic tumors as well as from cell lines using Hoechst 33342 exclusion assay followed by FACS analysis. SP and NSP were characterized using both in vitro and in vivo tumorigenicity assays. In vitro assays such as tumorsphere assay revealed that SP maintained its colony formation and formed large tumorspheres whereas the NSP cells were dispersed and formed smaller spheres. Both confocal and western bolt analysis revealed that SP overexpressed PD2 as well as CSC markers (CD133, ALDH1) and self-renewal markers (Shh and β-catenin). In vivo assays such as subcutaneous and orthotopic injection of 5000 SP and NSP cells into athymic nude mice were performed. SP injected mice produced a larger tumor as compared to NSP injected mice. Gemcitabine treatment enriched the SP cells thereby increasing PD2 expression compared to NSP cells. Furthermore, we carried out transient knockdown of PD2 by specific siRNA against PD2 in pancreatic CSCs and subsequently treated with gemcitabine which resulted in reduced viability thereby indicating a novel role of PD2 in CSC mediated drug resistance. Also, it was further corroborated by the downregulation of CD133 and multidrug resistance gene 2 expressions in SP. Further, analysis of PD2 expression along with CSC markers in PC progression mouse models revealed co-expression of PD2 and CSC markers in specific cells. Altogether, our results propose a novel role of PD2 in CSC maintenance as well as in drug resistance, which could be anticipated to be developed as a novel targeted therapy against pancreatic CSCs. Citation Format: Arokia Priyanka Vaz, Moorthy P. Ponnusamy, Satyanarayana Rachagani, Parama Dey, Surinder Batra. Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 271. doi:10.1158/1538-7445.AM2013-271

In vitro transforming potential, intracellular signaling properties, and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu, and Arg886Trp

Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.

Role and Relevance of TrkB Mutations and Expression in Non–Small Cell Lung Cancer

TrkB has been involved in poor cancer outcome. TrkB mutations have been reported in non-small cell lung cancer. In this study, we aimed at characterizing the role of three potentially sensitizing TrkB mutations previously reported in lung cancer. We characterized three activation loop mutants of TrkB (M713I, R715G, and R734C) in terms of pathway activation/phosphorylation, migration, anchorage-independent growth, and sensitivity to a Trk inhibitor, using NIH3T3 cells and Baf3 cells. We also sequenced the tyrosine kinase domain of TrkB in a large number of lung cancer samples of East-Asian origin and cell lines. None of the mutants were constitutively active in NIH3T3 transformation and migration assays. M713I and R734C mutants showed low levels of autophosphorylation in comparison with wild-type TrkB. Although R715G showed similar level of autophosphorylation to wild-type TrkB on brain-derived neurotrophic factor stimulation, the mutant was not as competent as wild-type TrkB in supporting interleukin-3-independent growth of Baf3 cells. In addition, the Trk inhibitor AZD6918 inhibited wild-type TrkB-induced cell migration and cell growth, whereas the mutants were relatively resistant to the Trk inhibitor compared with wild-type TrkB. We could not confirm the presence of nonsynonymous mutation in 78 lung cancer samples and 29 cell lines. Wild-type, but not mutant, TrkB enhances cell migration and transformation. Our study suggests that TrkB mutations should not be used for selection of patients with lung cancer treated with Trk inhibitors. High expression of wild-type TrkB might be beneficial for studies of Trk inhibitors.

Abstract 1120: Role and relevance of TrkB mutations and expression in non-small cell lung cancer

Abstract Purpose: TrkB oncogene has been involved in poor cancer outcome. TrkB mutations have been reported in non-small cell lung cancer. In this study, we aimed at characterizing the role of 3 potentially sensitizing mutations previously reported in TrkB in lung cancer. Experimental design: We characterized three activation loop mutants of TrkB (M713I, R715G and R734C) in terms of pathway activation/phosphorylation, migration, anchorage independent growth and sensitivity to a Trk inhibitor, using NIH3T3 cells and Baf3 cells. We also sequenced the tyrosine kinase domain of TrkB in a large number of lung cancer samples and cell lines. Results: None of the mutants were constitutively active in NIH3T3 transformation and migration assays. M713I and R734C mutants showed low levels of autophosphorylation in comparison with wild-type TrkB. Although R715G showed similar level of autophosphorylation to wild-type TrkB upon BDNF stimulation, the mutant was not as competent as wild-type TrkB in supporting IL-3 independent growth of Baf3 cells. In addition, the Trk inhibitor inhibited wild-type TrkB induced cell migration and cell growth, whereas the mutants were relatively resistant to the Trk inhibitor compared to wild-type TrkB. We could not confirm the presence of non-synonymous mutation in 78 lung cancer samples and 29 cell lines. Conclusions: Wild-type TrkB but not its activation loop mutations enhances cell migration and transformation. Our study suggests that TrkB mutations should not be used for selection of patients with lung cancer treated with Trk inhibitors. High expression of wild-type TrkB might be beneficial for studies of Trk inhibitors. 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 1120. doi:10.1158/1538-7445.AM2011-1120

The Hippo Tumor Pathway Promotes TAZ Degradation by Phosphorylating a Phosphodegron and Recruiting the SCFβ-TrCP E3 Ligase

The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1(β-TrCP) E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ε and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.

14-3-3γ Induces Oncogenic Transformation by Stimulating MAP Kinase and PI3K Signaling

The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3γ was shown by focus formation and tumor formation in SCID mice using 14-3-3γ transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3σ, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3γ. In addition, we found that 14-3-3γ interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3γ as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3γ induced transformation.

Abstract 3108: Functional delineation of structural domains in Yorkie/Yes-associated protein

Abstract The Salvador/Warts/Hippo (SWH) pathway plays an important role in organ size control, and deregulation of the pathway can lead to organ overgrowth and human cancer as well. The SWH pathway regulates tissue growth through inhibiting the activity of the downstream regulator, Yorkie (Yki) in D. melanogaster and Yes-associated protein (YAP) in mammals, which act as transcriptional co-activators. Yki and YAP have a conserved N-terminal domain, through which they associate and cooperate with the transcription factors, Scalloped (Sd)/TEAD, to promote tissue growth. In addition, Yki and YAP possess two conserved WW domains that mediate interaction between several proteins that either promote or inhibit YAP's ability to drive transcription. Furthermore, the C-terminus of YAP includes three further motifs: a SH3 domain-binding motif, transcriptional activation domain and a PDZ domain-binding motif, none of which are conserved in Yki. To study the role of the Yki/YAP WW domains and the YAP C-terminus, several Yki/YAP mutants were constructed and their function was analyzed with assays for tissue growth in Drosophila, or with assays for colony growth, cell transformation, migration and invasion in MCF10A or NIH-3T3 cells. Results showed that: 1. WW domain mutant YAP promoted transformation, migration and invasive ability of MCF10A cells, suggesting that WW domains impart inhibitory regulation of YAP in these cells. In contrast, the YAP WW domains were essential to promote NIH-3T3 cell growth and transformation, and for the capability of Yki to drive tissue growth in D. melanogaster. These results also suggested that YAP WW domains are required for tissue growth but inhibit invasion. 2. Deletion of YAP's transcriptional activation domain abolished its ability to enhance transcriptional activity of TEAD2, as well as YAP's ability to stimulate invasion of MCF10A cells. However, this mutant promoted growth and transformation of both MCF10A and NIH-3T3 cells. These results suggest that the transcriptional activation domain is required for YAP-mediated invasion but inhibits its ability to stimulate colony growth and cell transformation. 3. Mutation of YAP's PDZ domain-binding motif lowered its ability to activate TEAD2 and promote invasive behavior of MCF10A cells. In contrast, PDZ binding motif-deleted YAP promoted 3D growth of MCF10A cells and transformation in both MCF10A and NIH-3T3 cells. These results suggest that YAP's PDZ domain-binding motif is required for YAP-mediated invasion but has an inhibitory effect on growth and transformation. Taken together, these results suggest that YAP is able to mediate tissue growth, transformation and invasion through different domains and the total effect of YAP on growth and invasion is dependent on the coordination of protein interactions via its WW and transcriptional activation domains, as well as its PDZ domain-binding motif. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3108.

Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth.

High Expression of the PAX3-FKHR Oncoprotein Is Required to Promote Tumorigenesis of Human Myoblasts

PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis.

Antigen(s)-specific tumour-infiltrating lymphocytes from tumour induced by human herpes virus-6 (HHV-6) DNA transfected NIH 3T3 transformants

Tumour infiltrating lymphocytes (TIL) have recently been shown to mediate potent therapeutic effects in certain malignancies in mice and in humans. To understand the mechanism of TIL immunotherapy it would be advantageous to generate tumour-specific TIL and to study a defined system of TIL and target cells in which the tumour epitope(s) recognized by TIL might be identified. We have established tumourigenic cell lines by transfection of NIH 3T3 cells with the entire genome of human herpesvirus-6 (HHV-6) and its small fragment (about 5% of the viral DNA sequence). Injection of these cells into nude mice produced tumours termed G-2T and 14-2T, respectively. Cell lines derived from these tumours when injected in NIH Swiss mice produced tumours, G-2TS and 14-2TS, respectively. We have generated TIL from G-2TS tumour that can kill G-2TS tumour cells in vitro but not other related tumours (14-2TS or MCA-106). These TIL can be expanded between 2-6.5 every 3-5 days. The TIL proliferated in tissue culture in response to recombinant interleukin-2 and interleukin-4 and maintained their tumor specificity for up to 6 months in vitro. Their phenotype was Thy 1.2+, Lyt-2+ and L3T4-. The availability of such tumour-specific stable TIL lines and specific viral-transformed targets will provide an opportunity to characterize the tumour-associated antigen critical for the specific cytotoxicity in this system and thereby to clarify the mechanism of this promising immunological approach to cancer therapy.

Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon

The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.

A transforming gene, hst, found in NIH 3T3 cells transformed with DNA from three stomach cancers and a colon cancer.

DNA samples from 5 out of 15 stomach cancers and from 5 out of 15 colon cancers showed transforming activity in NIH 3T3 cells upon transfection. A transforming gene from an NIH 3T3 transformant induced by transfection of DNA from a stomach cancer was cloned, and showed transforming activity amounting to 120 focus-forming units/micrograms. The transforming gene was identified as hst. The hst gene was also responsible for acquisition of the transforming activity in DNA samples from 2 other stomach cancers and one colon cancer.