Department of transcription and cell signaling, IMBLD
Department of transcription and cell signaling, IMBLD, in generally engaged in the regulation of transcription by several transcription factors in tumor cells with the emphasis on melanoma and lung cancer. We explore also the influence of epigenetic changes, namely chromatin remodeling, which play a pivotal role in transcription of some genes. Currently, work is focused 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”, genes which are important for the embryonic development of the specific lineage and also maintain the survival normal cell lineage and a tumor arising from this lineage in an 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 the 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 have found that the chromatin-remodeling complex SWI/SNF is absolutely required for MITF gene transcription.
Recently, we found that transcription of the antiapoptotic protein survivin is activated (in many tumor types) by the Hedgehog/GLI signaling pathway, which is deregulated in most cancers. The most potent transcription factor of the Hedgehog route responsible for survivin gene activation is GLI2. Similarly, we found that the gene Slug (which is important for EMT and metastasis and is a target gene for MITF) is also positively regulated by the Hedgehog/GLI pathway, in melanomas. 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 “oncogene”, as it is capable of cooperating with several known oncogenes in cell transformation.
Combined targeted therapy of tumors, which utilize LMW inhibitors 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. These agents are often, logically, inhibitors of the oncogenes harboring the “driver” mutation (e.g. BRAF, RAS, EGFR) important for cancer initiation and progression. Unfortunately, resistant cells that appear usually after months of monotherapy are paradoxically very often dependent on the presence of the drug (so called “drug addiction”), so immediate cessation of administration of this drug is required (“drug holiday”). Another agent is then used, but a monotherapy can repeatedly lead to a relapse. The combination of more 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 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 malignant melanoma in vitro (cell cultures). The cell killing (through apoptosis) appeared even after 4 days of treatment at drug doses normally used for a single agent experiment. With the exception of about two very sensitive cell lines, no drug was efficient as a single agent, event after at least 10 days. Moreover, the cell killing was entirely independent of “driver” mutations that the tumors were carrying. The results are now patent pending, as both national and international applications which progress well. Similarly, we develop promising combination of 4 agents for the possible treatment of pancreatic cancer cells.
We further study signaling pathways of mTOR and MAPK that control many essential physiological processes in cells. Deregulation of the mTOR-dependent pathway occurs in many human diseases and may be a selective target for their therapy. We have found highly upregulated mTOR signaling in melanoma cells and this hyperactivity could be reduced by suppression of the activity of nonreceptor tyrosine kinase, c-Src. Such inhibition is comparable with the effects of the mTOR specific inhibitor, rapamycin, and moreover, it does not induce negative feedback mechanisms activating the Akt /MAPK-dependent signaling that may be responsible for attenuating therapeutic effects of rapamycin and its analogues.
In our laboratory, we use a number of methods of molecular biology, including analysis of gene expression by several methods, DNA cloning, preparation of recombinant proteins, gene transfer to cells in culture by several methods, silencing of genes in cultured cells by shRNAs, CRISPR/Cas9, study of DNA-protein interactions, immunofluorescence, study of the changes in cell cycle and apoptosis, and reporter studies, among others. DNA sequencing, microarrays, antibody preparation, and tumorigenicity testing in nude mice are performed in collaboration with other laboratories, or as a service in commercial companies. In the cell culture laboratory, we cultivate a large number of human tumor cell lines, some normal diploid human cells and several mouse tumor cell types. The laboratory is a workplace for work with radioisotopes and GMO used in molecular biology. In all the current projects (with one exception) is the principal investigator the head of our laboratory.