Transformation Of NIH 3T3 Cells Research Articles

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation.

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3' untranslated region (3' UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3'UTR APA sites, leading to mRNA isoforms that differ substantially in 3'UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRAS G12V . Perturbations of isoform expression with long 3'UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3'UTR isoform contributes to the secreted Timp2 protein much more than the short 3'UTR isoform. The short and long 3'UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRAS G12V . Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3'UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

NUP98-BPTF promotes oncogenic transformation through PIM1 upregulation.

Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98-BPTF (NB) fusion in patients with T-cell acute lymphoblastic leukemia (T-ALL) using next-generation sequencing. The FG-repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown. To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline-inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T-ALL cells. NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto-oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB-induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T-ALL cells by inactivating the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion-positive leukemia.

The stem cell transcription factor ZFP296 transforms NIH3T3 cells and promotes anchorage-independent growth of cancer cells.

Cancer cells and embryonic stem (ES) cells share several biological properties, suggesting that some genes expressed in ES cells may play an important role in cancer cell growth. In this study, we investigated the possible role of zinc finger protein 296 (ZFP296), a transcription factor expressed in ES cells, in cancer development. First, we found that overexpression of Zfp296 in NIH3T3 mouse fibroblasts induced two phenomena indicative of cell transformation: enhanced proliferation under low-serum conditions and anchorage-independent growth. We also found that Zfp296 expression was upregulated in the tumor area of a mouse model of colon carcinogenesis. In addition, the expression levels of ZFP296 in various human cell lines were generally low in normal cells and relatively high in cancer cells. Finally, using a soft agar assay, we found that overexpression of ZFP296 promoted the anchorage-independent growth of cancer cells, while its knockdown had the opposite effect. Overall, these results suggest a possible role of the ES-specific transcription factor ZFP296 in cancer.

Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma.

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gαq/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gαq/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gαq/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

Phase transition and remodeling complex assembly are important for SS18-SSX oncogenic activity in synovial sarcomas

Oncoprotein SS18-SSX is a hallmark of synovial sarcomas. However, as a part of the SS18-SSX fusion protein, SS18’s function remains unclear. Here, we depict the structures of both human SS18/BRG1 and yeast SNF11/SNF2 subcomplexes. Both subcomplexes assemble into heterodimers that share a similar conformation, suggesting that SNF11 might be a homologue of SS18 in chromatin remodeling complexes. Importantly, our study shows that the self-association of the intrinsically disordered region, QPGY domain, leads to liquid-liquid phase separation (LLPS) of SS18 or SS18-SSX and the subsequent recruitment of BRG1 into phase-separated condensates. Moreover, our results show that the tyrosine residues in the QPGY domain play a decisive role in the LLPS of SS18 or SS18-SSX. Perturbations of either SS18-SSX LLPS or SS18-SSX’s binding to BRG1 impair NIH3T3 cell transformation by SS18-SSX. Our data demonstrate that both LLPS and assembling into chromatin remodelers contribute to the oncogenic activity of SS18-SSX in synovial sarcomas.

Proteomic analysis of chemically transformed NIH-3T3 cells reveals novel mechanisms of action of amaranth lunasin-like peptide

Food-derived biopeptides can interact with genes and proteins to preserve health and prevent the development of diseases. Lunasin is a soybean cancer-preventive peptide that has been well characterized; however, few studies have been carried out to characterize the function of amaranth lunasin-like peptide (AhLun). The aim of this work was to analyze the proteomic profile changes in NIH-3T3 cells when they are chemically transformed with the carcinogen 3-methylcholanthrene (3MC) in the absence or presence of AhLun. The addition of AhLun into the culture medium did not affect the cell morphology; however, as a chemopreventive agent, it significantly reduced anisokaryosis formation when cells were treated with 3MC. Changes in protein accumulation in NIH-3T3 cells were evaluated by gel-based proteomics analysis. Differentially accumulated protein spots that exhibited at least a twofold change in spot intensity (p < 0.05), when compared with control cells, were analyzed by LC-MS/MS. Successfully identified proteins were grouped into six main categories according to their localization and function (nuclear, ribosomal, mitochondrial, metabolism, cytoskeletal, and miscellaneous). The gel-based proteomic approach for the evaluation of the chemopreventive potential of AhLun reveals novel pathways of action and provides new clues about the possible mechanisms of action of this bioactive peptide present in amaranth seeds.

Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP.

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45kDa, much larger than expected (22.5kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

RBM15 condensates modulate m6A modification of STYK1 to promote tumorigenesis

RBM15 expression is recurrently upregulated in several types of malignant tissues, and its high expression level is typically associated with poor prognosis. However, whether and how RBM15 is involved in the tumor progression remains unclear. In this study, we found that overexpressing RBM15 in NIH3T3 cells was able to enhance proliferation rate in vitro and induced subcutaneous tumor formation in vivo. Moreover, we imaged the subcellular localization of RBM15 with our home-built structured illumination super-resolution microscopy, and revealed that RBM15 formed substantial condensates dispersed in the nucleus, undergoing dynamic fusion and fission activities. These condensates were partially colocalized with m6A-modified transcripts in the nucleus. In addition, we confirmed that RBM15 formed “liquid-like” droplets in a protein/salt concentration-dependent manner in vitro, and the addition of RNA further enhanced its phase-separation propensity. To identify downstream targets of RBM15, we performed meRIP-seq and RNA-seq, revealing that RBM15 preferentially bound to and promoted the m6A modification on the mRNA of Serine/threonine/tyrosine kinase 1 (STYK1), thereby enhancing its stability. The upregulated STYK1 expression caused MAPK hyperactivation, thereby leading to oncogenic transformation of NIH3T3 cells.

Morin inhibits the transformation of fibroblasts towards myofibroblasts through regulating “PPAR-γ-glutaminolysis-DEPTOR” pathway in pulmonary fibrosis

Morin, a natural flavonoid exists in many foods and dietary plants, owns good bioactivities. Herein, we investigated its effect on pulmonary fibrosis (PF), and further explored the mechanisms. Results showed that morin remarkably improved the pathologic alterations, and inhibited the transformation of fibroblasts towards myofibroblasts in lungs of mice with bleomycin-induced PF as well as TGF-β1 or hypoxia-stimulated NIH-3T3 cells. Mechanistic studies revealed that morin activated peroxisome proliferator activated receptor-gamma (PPAR-γ), and GW9662 or siPPAR-γ significantly weakened the inhibition of morin on the transformation of NIH-3T3 cells. Furthermore, morin restricted glutaminolysis by down-regulating the level of glutaminase 1 (GLS1), which was confirmed by glutamine deprivation, and GLS1 overexpression. Replenishment of metabolite α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG) inhibited morin-prevented transformation of fibroblasts, but neither TGF-β1 nor hypoxia could induce the transformation of IDH2-knockdown fibroblasts, suggesting 2-HG was directly involved in the action of morin. Then, ubiquitination of DEPTOR was demonstrated to be prevented by morin, which was attributed to KDM4A, an enzyme inactivated by 2-HG, and leucine as well as KDM4A inhibitor obstructed the effect of morin. Finally, the mechanisms of morin were further confirmed in vivo. Collectively, morin inhibited PF through intervening in “PPAR-γ-glutaminolysis-DEPTOR” signals, and subsequent restriction on the transformation of fibroblasts towards myofibroblasts.

The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

Expression and cell transformation activity of dynactin-associated protein isoforms.

Overexpression of human dynactin‐associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single‐pass transmembrane protein with a carboxy‐terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino‐terminal residues (NH2‐MVA in dynAPa and NH2‐MEYQLL in dynAPc). DynAPb has the same amino‐terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy‐terminal region, including the transmembrane domain. Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki‐1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy‐terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U‐bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.

Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization and Auto-phosphorylation.

Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1-6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930-34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1-6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR.

Abstract 2400: Strong biological bias for ALK intron 19 breakpoints in NSCLC

Abstract Introduction: EML4-ALK translocations are detected in 4-8 % of non-small cell lung cancer (NSCLC). While different EML4-ALK variants are defined by different breakpoints in the EML4 gene, most frequently located in intron 6 or 13, ALK breakpoint is almost invariably in intron 19. Rare reports describe EML4-ALK translocations with breakpoints in intron 17 or intron 18 of the ALK gene. Despite all these ALK breakpoints potentially generate oncogenic fusions, the reasons of this strong imbalance toward intron 19 (exon 20) breakpoints in ALK positive NSCLC are currently unknown. The aim of this study is to investigate the mechanisms that underlie ALK translocation in NSCLC. Methods: Eight different EML4-ALK fusions were either overexpressed with a retroviral system or generated with CRISPR/Cas9 lentivirus from the endogenous loci. EML4 exons 1-6 (E6) or exons 1-13 (E13) with different ALK exons: E6;A18, E6;A19, E6;A20 or E6;A21 and E13;A18, E13;A19, E13;A20, E13;A21. The fusions E6;A21 and E13;A21 contained an early stop codon not producing functional proteins. These EML4-ALK fusions were expressed in NIH3T3 and PC9 by retroviral vectors or engineered in PC9 (an EGFR-dependent lung cancer cell line that is sensitive to inhibition with the EGFR inhibitor osimertinib) and BEAS-2B cells by CRISPR/Cas9. HTGTS was performed on engineered PC9 cells. NSG immunodeficient mice were used for in vivo experiments with NIH3T3 and BEAS-2B cells. Results: The retroviral overexpression system showed that all EML4-ALK fusion proteins were expressed leading to an actively phosphorylated ALK in NIH3T3 and PC9 cells, except E6;A21 and E13;A21 fusions where no protein was detected, as expected. Accordingly, all active variants were able to induce NIH3T3 cell transformation and tumor formation in vivo and conferred resistance to osimertinib in PC9 cells. In contrast, when EML4-ALK translocations were induced by CRISPR/Cas9 in PC9 cells, only E6;A20 and E13;A20 variants rapidly overcame osimertinib inhibition while other variants rescued osimertinib-inihibited cells slowly and less efficiently. Surprisingly, the E6;A20 and E13;A20 fusions were invariably found in all resistant clones independently of the translocation induced by CRISPR/Cas9. Similarly, all tumors formed in vivo by BEAS-2B cells contained the E6;A20 or E13;A20 variants irrespective of the translocation induced originally. By cloning translocation junctions at large scale with HTGTS, we found that clones initially forced to break into ALK intron 17 eventually acquired a second breakpoint in ALK intron 19. Conclusions: Our data show that all EML4-ALK fusion variants were equally oncogenic when overexpressed. In contrast, when EML4-ALK variants were generated from the endogenous loci there was a strong selection bias toward ALK fusions originating in intron 19 suggesting that intron 19 variants have the strongest oncogenic potential in lung epithelial cells. Citation Format: Giulia C. Leonardi, Taek-Chin Cheong, Chiara Ambrogio, Tao Chen, Wei-Tien Tai, Elif Karaca, Ines Mota, Massimo Libra, Mark M. Awad, Rafael Blasco-Patino, Roberto Chiarle. Strong biological bias for ALK intron 19 breakpoints in NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2400.

Loss of function Cbl-c mutations in solid tumors.

Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression. Cbl proteins (Cbl, Cbl-b and Cbl-c) are a family of RING finger (RF) ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, and RET. Recent studies have identified Cbl mutations associated with human myeloid neoplasias in approximately 5% of the cases. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. We identified a novel cDNA that was isolated from a mouse mammary cancer from the C3(1) Large T Antigen transgenic model. This mutant cDNA encodes a protein that has a deletion in the RF domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely a somatic mutation. The mutant protein enhances transformation of NIH 3T3 cells when expressed in combination with SV40 Large T antigen. Together these data are consistent with a second hit mutation. In overexpression studies, this mutant Cbl-c protein fails to mediate ubiquitination of activated EGFR and acts in a dominant negative fashion to prevent ubiquitination and downregulation of the activated EGFR by wild type Cbl proteins. Mechanistically, the mutant Cbl-c binds to the EGFR and prevents recruitment of the wild type Cbl protein. Furthermore, data mining reveals Cbl-c mutations associated with solid tumors in humans. Subsequent cell-based analysis demonstrates a similar loss of E3 function and dominant negative effects for one of these human mutations. These data suggest that like Cbl mutations in myeloid neoplasms, loss of Cbl-c function may contribute to the pathogenesis of solid tumors in murine models and in humans.

Enzymatically Prepared Dual Functionalized Hydrogels with Gelatin and Heparin To Facilitate Cellular Attachment and Proliferation.

Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors.

The sensitivity to Hsp90 inhibitors of both normal and oncogenically transformed cells is determined by the equilibrium between cellular quiescence and activity.

The molecular chaperone Hsp90 is an essential and highly abundant central node in the interactome of eukaryotic cells. Many of its large number of client proteins are relevant to cancer. A hallmark of Hsp90-dependent proteins is that their accumulation is compromised by Hsp90 inhibitors. Combined with the anecdotal observation that cancer cells may be more sensitive to Hsp90 inhibitors, this has led to clinical trials aiming to develop Hsp90 inhibitors as anti-cancer agents. However, the sensitivity to Hsp90 inhibitors has not been studied in rigorously matched normal versus cancer cells, and despite the discovery of important regulators of Hsp90 activity and inhibitor sensitivity, it has remained unclear, why cancer cells might be more sensitive. To revisit this issue more systematically, we have generated an isogenic pair of normal and oncogenically transformed NIH-3T3 cell lines. Our proteomic analysis of the impact of three chemically different Hsp90 inhibitors shows that these affect a substantial portion of the oncogenic program and that indeed, transformed cells are hypersensitive. Targeting the oncogenic signaling pathway reverses the hypersensitivity, and so do inhibitors of DNA replication, cell growth, translation and energy metabolism. Conversely, stimulating normal cells with growth factors or challenging their proteostasis by overexpressing an aggregation-prone sensitizes them to Hsp90 inhibitors. Thus, the differential sensitivity to Hsp90 inhibitors may not stem from any particular intrinsic difference between normal and cancer cells, but rather from a shift in the balance between cellular quiescence and activity.

Roles of Cyclin A, Myc, Jun and Ppm1l in tumourigenic transformation of NIH3T3 cell

To analyse the mechanism of tumourigenic transformation of NIH3T3 cells at the transcriptional level, we used cancerogen 3-methylcholanthrene (3-MCA) and cancerogenic substance phorbol-12-myristate-13-acetate (PMA) to transform NIH3T3 cells and the assessment of transformation was performed using Giemsa staining and methylcellulose colony formation assay. Changes in gene expression profile were detected by Mouse Genome 430 2.0 microarray; and quantitative real-time polymerase chain reaction and Western blotting were used to verify the expression changes of mRNAs and proteins, respectively. With the aid of bioinformatics method, five signalling pathways were identified to participate in different stages of NIH3T3 cell transformation. Further, our study suggested that oncogenes Cyclin A, Myc, Jun and the tumour suppressor gene Ppm1l may play important roles in these pathways.

Aurora-A kinase and cancers: A biomarker to detect and a target to inhibit

In 1995, the Aurora-A kinase was discovered in Drosophila. In 1997, it was found to be overexpressed in breast cancer cell lines. In 1998, the function of the kinase in the assembly of the mitotic spindle was identified as well as its oncogenic activity. These results immediately triggered an intensive search by Big Biopharma for kinase inhibitors to be used in the treatment of cancer.Overexpression of the kinase is sufficient to transform NIH3T3 cells that induce tumor formation when implanted in nude mice. A more direct relationship between Aurora-A and cancer was discovered in 2006 by demonstrating that its overexpression in the mammary epithelium was sufficient to induce cancer. Aurora-A is then a key cell cycle kinase, whose overexpression induces cancer, making it indeed a good target for the treatment of cancers. A large number of inhibitors have therefore been manufactured and used in clinical trials for the treatment of cancers. However, after 20 years, none has yet managed to pass any phase III. So how can we improve the effectiveness of Aurora-A inhibitors in cancer treatments?

Abstract 555: Loss of function Cbl-c mutations in solid tumors

Abstract Receptor Tyrosine Kinase (RTK) signaling, which is essential for cellular growth and proliferation, can lead to malignant transformation and tumorigenesis when aberrantly activated. Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a conserved family of RING finger (RF) ubiquitin ligases (E3) that negatively regulate the activity of several tyrosine kinases such as the EGFR. Cbl mutations have recently been identified in 5% of human myeloid neoplasms, with a majority of mutations clustered within the RF and linker domains. Cbl-c is the most recently identified Cbl protein and is exclusively expressed in epithelial cells. We recently identified a novel cDNA isolated from a mouse mammary tumor in the C3(1) Large T Antigen transgenic mouse model. This mutant cDNA encodes a protein that has a deletion in the RF domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeloid neoplasias. Genomic analyses of both parental and transgenic lines show no evidence of germline mutation, suggesting that this mutation is most likely somatic. The mutant Cbl-c protein enhances transformation of NIH 3T3 cells when co-transfected with SV40 Large T Antigen. In overexpression studies, this mutant Cbl-c fails to mediate ubiquitination of activated EGFR and acts in a dominant negative manner to prevent ubiquitination of activated EGFR by wildtype Cbl proteins. Datamining also reveals Cbl-c mutations associated with human solid tumors. Cell-based analyses demonstrate a similar loss of E3 function in some of these human mutations. These data collectively suggest that, like Cbl mutations in myeloid neoplasms, loss of function Cbl-c mutants may also contribute to the pathogenesis of solid tumors in murine models and in humans. Citation Format: Silvano R. Daniels, Stephen C. Kales, Mariya Liyasova, Marion M. Nau, Phil E. Ryan, Jeffrey E. Green, Stanley Lipkowitz. Loss of function Cbl-c mutations in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 555.

Development of inhibitors for activated Cdc42‐associated kinase (ACK) to reverse v‐Ras‐induced cancerous phenotype of mammalian cells

The superfamily of Ras GTPase has been reported to control different aspects of mammalian cell growth. Our lab demonstrated a possible role of Ras‐Cdc42‐ACK signaling in the survival of v‐Ras‐transformed cells. We also found that ACK‐deficient v‐Ras transformed NIH 3T3 cells undergo apoptosis while the parental NIH 3T3 cells grow normally. We proposed a hypothesis that “ACK‐induced phosphorylation plays a critical role in the survival of Ras‐induced transformed cells”. Based on three‐dimensional structure of ACK, our lab in collaboration with other group screens chemicals to identify potential specific inhibitors for ACK. In this report, I will present computer aided screening protocol and the effect of these inhibitors on growth of v‐Ras‐induced transformed and parental NIH 3T3 cells. I have used software to screen and identify potential inhibitors for ACK. These inhibitors were then used to treat v‐Ras transformed and parental NIH 3T3 cells. The cell growth was monitored by MTT assay. I have further studied the effect of these inhibitors on gene expression using immunostaining, immunoblotting, and polymerase chain reaction. I have identified some compounds that have strong affinity for ACK and inhibit growth of v‐Ras transformed cells. Treatment of v‐Ras cells with these inhibitors was found to activate apoptosis. I have used several markers of apoptosis. These results will be described in this report. We are designing novel compounds with more specific binding to ACK. Development of specific ACK inhibitors will be useful in better understanding the role of ACK in the growth of normal and cancer cells.Support or Funding InformationNIH‐RISEThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.