Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial–mesenchymal transition (EMT) and its inverse process, mesenchymal–epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future.

https://www.mdpi.com/2073-4409/8/12/1644/pdf

Genetically Engineered Lung Cancer Cells for Analyzing Epithelial-Mesenchymal Transition.

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.

/pdf/genetical-engineered-lung-cancer-cell-for-analyzing-40piefn69f.pdf

Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Breast cancer is the global leading cause of cancer-related death in women and it represents a major health burden worldwide. One of the promising breast cancer therapeutic avenues is through small molecule inhibitors (SMIs) which have undergone rapid progress with successful clinical trials. Recently, three emerging and vital groups of proteins are targeted by SMIs for breast cancer treatment, namely cyclin-dependent kinase 4 and 6 (CDK4/6), poly (adenosine diphosphate-ribose) polymerase (PARP) and phosphoinositide 3-kinase (PI3K). Several of these inhibitors have been approved for the treatment of breast cancer patients or progressed into late-stage clinical trials. Thus, modeling from these successful clinical trials, as well as their limitations, is pivotal for future development and trials of other inhibitors or therapeutic regimens targeting breast cancer patients. In this review, we discuss eight recently approved or novel SMIs against CDK4/6 (palbociclib, ribociclib and abemaciclib), PARP (olaparib, veliparib and talazoparib), and PI3K (buparlisib and alpelisib). The mechanisms of action, series of clinical trials and limitations are described for each inhibitor.

https://journals.sagepub.com/doi/pdf/10.1177/1758835918808509

Inhibitors targeting CDK4/6, PARP and PI3K in breast cancer: a review.

Cancer metastasis continues to be the leading cause of mortality in cancer patients around the world. One of the prominent biological processes implicated in cancer metastasis is epithelial-to-mesenchymal transition (EMT). EMT has also been shown to have roles in many aspects of cancer initiation and progression, including tumorigenesis and drug resistance. Despite the accumulation of a large body of data on the association of EMT with cancer, to date, EMT has not been an active target for therapeutic development partly due to the lack of appropriate in vitro models. Utilizing some of the basic biology of EMT, we have created a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs. In lung cancer, vimentin (VIM) intermediate filament (IF) proteins are associated with EMT, and the metastatic spread of cancer. Vimentin expression is generally upregulated when epithelial cells transition to the mesenchymal phenotype. We capitalized on this biological phenomenon, using CRISPR/Cas9 gene editing to generate a VIM RFP (red fluorescent protein) reporter cell line in the A549 non-small cell lung cancer (NSCLC) cell line; one of the most widely used and recognized lung cancer cell lines. The A549 VIM RFP cell line harbors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene which enables end-point or real-time tracking of the EMT status as cells transition from epithelial to mesenchymal phenotype under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the A549 VIM RFP cell line shows sensitivity to metastatic NSCLC drugs PP1 (SRC inhibitor) and A83-01 (ALK5 inhibitor). These results provide the foundation for the use of this cell line in high throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic NSCLC. Furthermore, the A549 Vim RFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. Citation Format: Metewo S. Enuameh, Sangeeta Kumari, Chauzhong Zou, John Foulke, Elizabeth Turner, Weiguo Shu, Robert Newman. The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development [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 2012.

Abstract 2012: The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development

Development of the next generation of anti-cancer drugs, biologics, and immunotherapies is currently hampered by extremely poor success rates of seemingly promising experimental therapies in human clinical trials. This poor success rate is partially due to a lack of biologically relevant cancer model systems. The 2D culture system, while relatively inexpensive and easy to use, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. Combining 3D culture with CRISPR/Cas9 genome editing technologies can generate powerful cell-based model systems with both physiological relevance and well-controlled genetic and drug-susceptibility profiles. These new models will enable the investigation of specific molecular mechanisms, biofunctional outcomes of newly identified genetic alternations, and targeted therapeutic drug responses within a more biologically intricate context. In this study, we use CRISPR/Cas9 to generate isogenic drug-resistant melanoma models that can be used as either 2D or 3D cancer models, as well as for studies of the acquired drug resistance. Two different models were generated starting with the BRAF-inhibitor sensitive A375 melanoma cell line. We introduced either the NRAS Q61K or the KRAS G13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF-resistant tumor samples. We then assessed the susceptibility of these new isogenic lines to BRAF inhibitors in both 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of Programmed Death-Ligand 1 (PD-L1), which recent advances in cancer immunology have directly linked to cancer immune evasion and poor clinical outcomes. Our results show that A375 melanoma isogenic cells carrying KRAS G13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRAS Q61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRAS G13D , but not in NRAS Q61K A375 cells, indicating that expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in RAS-RAF-MAPK signaling. These results have direct implications for cancer immune checkpoint molecule studies, and highlight the utility of these isogenic melanoma models in both 2D and 3D applications in research and development of novel anti-cancer drugs and combination therapies. Citation Format: Elizabeth Turner, Luping Chen, Johnathan Haag, Alexei Miagkov, Lysa-Anne Volpe, Metewo Selase Enuameh, Robert Newman, Fang Tian. Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma [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 5021.

Abstract 5021: Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma

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Abstract 2012: The generation of a NSCLC EMT reporter cell line for metastatic lung cancer drug discovery and development

Abstract 5021: Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma