Department of transcription and cell signaling, IMBLD

The work of the department is focused on the transcriptional regulation by several transcription factors in tumor cells with the emphasis on melanoma and lung cancer. We explore also the influence of epigenetic changes such as chromatin remodeling, which play a pivotal role in transcription of some genes. Currently, work is concentrated mainly on transcription factors MITF and GLI1-3, among others. MITF is a transcriptional activator of dozens of genes in normal and malignant melanocytes and is a critical survival factor for melanoma cells. It is therefore regarded as a paradigm of so called “lineage addiction oncogenes”, i.e. genes which are important for the embryonic development of the specific lineage and also maintaining the survival of normal cell lineage and tumors arising from this lineage in the adult. The approaches used for melanoma cells could be potentially applicable also for some other tumors expressing “lineage addiction oncogenes”, such as prostate cancer, gliomas, breast cancer and some others. These genes are promising targets for their downregulation or gene therapy, as inhibition of their expression or function kills tumor cells. This topic includes also studying the epigenetic mechanisms required for MITF expression. We found that the chromatin-remodeling complex SWI/SNF is absolutely required for MITF gene transcription.

In our laboratory, we found that transcription of the gene for the antiapoptotic protein survivin is activated (in more tumor types) by the Hedgehog/GLI (HH) signaling pathway, which is upregulated in cancer cells. The most potent transcription factor of the HH responsible for survivin gene activation is GLI2. In melanomas, we revealed that the gene SLUG (which is important for EMT and metastasis) is also positively regulated by the HH/GLI2. Furthermore, we often use adenoviral gene E1A 12S and its mutated forms as helpers in the transcriptional studies. E1A 12S protein is a transcriptional repressor and a specific viral “oncogene”, as it is capable of cooperating with several known oncogenes in cell transformation.

Combined targeted therapy of tumors, which utilize low molecular weight inhibitors (LMWi) of various molecular targets important for the growth of tumor cells, is considered as a very perspective future therapy of cancer. Utilizing only one drug in this type of therapy is almost always accompanied with the development of resistence to the drug. Resistant cells arise almost invariantly after months of monotherapy and are paradoxically dependent on the presence of the drug (so called “drug addiction”), so immediate cessation of the drug administration is required (“drug holiday”). Another substance is then used, but a monotherapy can repeatedly lead to a relapse. The combination of more LMWi agents that inhibit independent but crucial pathways in the tumor cell, when given at the beginning of treatment, efficiently prevents the development of resistence and gives much better chance to kill tumor cells earlier. In line with this, we have developed five different combinations, each composed of three inhibitors, that all have appeared very efficient against several tumor types in vitro (cell cultures). The cell killing (through apoptosis) appeared even after 3-4 days of treatment at drug doses normally used for a single agent experiment. No drug was so much efficient as a single agent, with the exception of very sensitive cell lines. Moreover, the cell killing was entirely independent of “driver” mutations that the tumors were carrying. Some of the results appeared in a Czech patent. Currently, we test event better drug combinations consisting only from substances which are already used in the clinical treatment or in the last phases of clinical trials. About 50 human tumor cell lines tested (in 2D and 3D cell cultures) were extremely sensitive to drug cocktails and only one cell line was resistant. At present, the in vivo experiments with these combinations of LMWi on nude mice are underway.

We further study signaling pathways mTOR and MAPK that control many essential physiological processes in cells. Deregulation of the AKT/mTOR-dependent pathway occurs in many human cancers and may be a selective target for their therapy. We have found highly upregulated AKT/mTOR signaling in melanoma cells and this hyperactivity could be reduced by suppression the activity of nonreceptor tyrosine kinase c-Src through the application of dasatinib on cells. Such inhibition is comparable with effects of the mTOR specific inhibitor rapamycin. Our further study is an exploration of possible effect of dedifferentiation as a primary cancer-forming stimulus. These long-term experiments aim at the possible identification of (pre)incipient cancer cells and tumor stimuli changing the normal diploid cell into a cancer cell.

We also study how the transcription of genes coding the known immune checkpoint inhibitors (eg. PD-1, CTLA-4 and others, that function as inhibitory receptors on the surface of CD8+ T-lymphocytes) is regulated and what transcription factors are involved. These T-cells infiltrate the tumor stroma and hinder the effective antitumor immune response. Blocking their repressive action by using antibodies (which prevent the ligand binding to the receptor, eg. Nivolumab is anti-PD-1) is now widely used as a kind of immunotherapy for increasing the immune antitumor therapeutic response.

In our laboratory, we use a number of molecular and cellular biology methods, such as analysis of gene expression by several methods (incl. realtime PCR, Western blots), DNA cloning, mutagenesis, preparation of recombinant proteins, gene transfer to cells in culture by several methods (including transfer by using lentiviruses), silencing of genes in cultured cells by shRNAs, CRISPR/Cas9, study of DNA-protein and protein-protein interactions, immunofluorescence and confocal microscopy, apoptosis, promoter-reporter studies, among others. DNA sequencing, RNA-seq, microarrays, and other omic studies are performed as a servis in companies. Tumorigenicity testing in nude mice is performed in collaboration with The Center of Experimental Biomodels of our faculty (including imaging). Immunohistochemistry is performed in collaboration with the Second Medical Faculty, Charles University Prague. In our cell culture laboratory, we cultivate a large number of human tumor cell lines, several types of normal diploid human cells and selected mouse tumor cell types.

The department has permissions to work with several radioisotopes and genetically modified organisms (GMOI and GMOII) used in molecular biology.