Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 126.96.36.199 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: The role of “mitotic slippage” in the origin of amoeboid metastatic cell in therapy resistant cancers
Project No.: 188.8.131.52/VIAA/3/19/463
Period: 36 months (1 January 2020 – 31 December 2022)
Project costs: 133 806.00 EUR
Project implementer: Dr. biol. Kristīne Salmiņa
The mortality of cancer patients still remains high, mostly due to metastatic disease, where tumor cell migration is a critical mechanism in development of resistance to anticancer therapy. The amoeboid motility is a very efficient mode of migration and metastatic growth of single tumor cells. The working hypothesis of this project suggests the role of “mitotic slippage” – reverse of aborted mitosis enhanced by therapy in formation of polyploid tumour cells undergoing the reprogramming to amoeboid state. Beside motility, amoeboid cancer cells display various activities cooperatively enabling survival and drug-resistant metastatic growth. Impact of the key amoebal cdc42-kinase on these activities will be characterised in mechanistic experiments. The work will be performed on breast cancer and melanoma cell lines, and include the patient material. Genotoxic treatments, cdc42 inhibition will be examined by immunofluorescence, cytometry, western blot, qRT-PCR, live-imaging and confocal microscopy. In addition, the clonogenicity, migration, invasion, and tumorigenicity tests will be applied.
Information published 02.01.2020.
1 January 2020 – 31 March 2020
At this stage of the project, the breast cancer MDA-MB 231 cell line was treated with the anticancer agent doxorubicin, and mitotic slippage, which is the source of giant cell formation, was observed. The cell cycle, polyploidization, growth curve and microscopic counts of mitotic slippage were determined. Activation of the meiotic genes MOS, DMC1, REC8 and SPO11 after cell treatment was observed. The signs of amoeboidisation in giant cells resulting from the application of anti-cancer therapy in comparison to control cells have been investigated. Work has been done to prepare and submit the publication.
Information published 31.03.2020.
1 April 2020 – 30 June 2020
At this stage of the project, the human melanoma SK-MEL 28 cell line was treated with the anticancer agent Vemurafenib and the cell cycle, growth curve and expression of meiotic genes after optimization of cell treatment were determined. However, increased cell polyploidisation and mitotic slippage were not observed after treatment. A scientific article on the role of mitotic slippage and extranuclear DNA in cancer cell resistance has been published: doi: 10.3390/ijms21082779.
Information published 30.06.2020.
1 July 2020 – 30 September 2020
At this stage of the project, the human melanoma SK-MEL 28 cell line was treated with the anticancer agent doxorubicine. Optimization of cell treatment and characterization of resistance model were performed. Cell cycle analysis, polyploidisation, growth curve and microscopic detection of mitotic slippage were carried out, as well as the expression of meiotic kinase MOS and CyclinB1 using immunofluorescence was studied. It was found that doxorubicine in this cell line, as in MDA-MB 231 cells, induces mitotic slippage and subsequent polyploidisation.
Information published 30.09.2020.
1 October 2020 – 31 December 2020
At this stage of the project, samples were obtained for the analysis of the transcription profile at different time points after treatment of MDA-MB 231 and SK-MEL 28 cell lines with doxorubicin. Work has been done on the preparation and submission of the publication, as a result of which a scientific review article has been published https://doi.org/10.1016/j.semcancer.2020.12.009
Information published 30.12.2020.
1 January 2021 – 31 March 2021
At this stage of the project, the clonogenicity, the expression of meiotic genes and senescence markers of SK-MEL28 cells after treatment with doxorubicin and vemurafenib were studied. The results of the project were also presented at the conference (MDPI: The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response) as a poster presentation 10.3390 / IECC2021-09214.
Information published 30.03.2021.
1 April 2021 – 30 June 2021
At this stage of the project, RNA was isolated and purified from samples of MDA-MB 231 and SK-MEL 28 cell lines obtained before and at different time points after genotoxic treatment. Work has been done on the preparation and submission of the publication, as a result of which a scientific review article has been published https://doi.org/10.3390/cells10071582.
Information published 30.06.2021.
1 July 2021 – 30 September 2021
At this stage of the project, purification of RNA obtained from MDA-MB 231 and SK-MEL 28 cell lines at different time points after genotoxic treatment and creation of cDNA libraries for transcriptome sequencing were performed. Experiments to detect the expression of the amoeboidation marker Cdc42 before and after genotoxic treatment have also been initiated in these tumor cell lines.
Information published 30.09.2021.
1 October 2021 – 31 December 2021
At this stage of the project, cDNA libraries were created and transcriptome sequencing was performed on samples of MDA-MB 231 and SK-MEL 28 cell lines, which were obtained at different time points after genotoxic treatment. The expression of amoeboidation markers cdc42, Rac1 and Actin in giant cells, resulting from genotoxic treatment induced mitotic slippage was also determined. Experiments have been started to detect MAGEA10 in these cells. Transcription of YAP and TEAD in cell nuclei after doxorubicin treatment has been shown, indicating inactivation of the Hippo pathway in giant cells.
Information published 30.12.2021.
1 January 2022 – 31 March 2022
At this stage of the project, to investigate the role of Cdc42 in tumour cell resistance, Cdc42 inhibitor ML141 in combination with genotoxic treatment was applied.
The quality of the transcriptome samples obtained from the sequencing was checked with the FastQC program. Quality-filtered reads were mapped against Human reference genome using STAR software. In order to identify differentially expressed genes (DEGs) after treatments, differential gene expression analysis was performed (edgeR).
The results of the project were also presented at the conference (Salmina K, Rumnieks F, Pjanova D, Erenpreisa J. Role of mitotic slippage and amoeboid giant cell in tumor microenvironment and cancer resistance. IECC 2022: Tumor Microenvironment Heterogeneity in Cancer Progression: Challenge or Opportunity. 14–16 Feb 2022) as oral presentation.
Information published 31.02.2022.
1 April 2022 – 30 June 2022
At this stage of the project, the role of cdc42 in the resistance of tumor cells was investigated using cdc42 inhibitor ML141 in combination with genotoxic treatment. Clonogenicity of breast cancer MDA-MB 231 cells was found to be reduced after this combined treatment, however no changes in filopodia formation were observed when examining the actin cytoskeleton. Protein samples were obtained for testing the effectiveness of the cdc42 inhibitor, and the extracellular vesicle fraction was also separated for MAGEA protein expression analysis. The expression of CTA genes MAGEA10 and MAGEA4 was also determined before and after doxorubicin treatment. Expression of these proteins in tumor cells was found using immunofluorescence and western blot methods.
Information published 30.06.2022.
1 July 2022 – 30 September 2022
At this stage of the project, the expression of CTA genes MAGEA10 and MAGEA4, markers of extracellular vesicles (CD9, CD63), as well as cdc42 was investigated before and after doxorubicin treatment in tumor cells and the fraction of extracellular vesicles using immunofluorescence and westernblot methods. Double immunofluorescent staining of MAGEA and Actin was performed. Further investigation of the role of the amoeboidization marker cdc42 in tumor cell resistance using the cdc42 inhibitor ML141 in combination with genotoxic treatment also in the murine melanoma B16-F10 cell line was performed. Proliferation ability of tumor cells was studied using Ki67 protein as a marker. Immunohistochemical staining of cdc42 was performed in paraffin-embedded tumor sections of melanoma patients.
Information published 30.09.2022.
1 October 2022 – 31 December 2022
At this stage of the project, Differential gene (DEGs) expression analysis for samples of NT control cells and several time points after doxorubicin treatment was performed. Obtained data were evaluated from biological context using gene ontology analyser GOseq pathway analysis. It was found that after doxorubicine treatment cells show upregulation of immune response, reproductive process and also the locomotory behavior and migration compatible with their amoeboid conversion. The down-regulated genes were mostly indicating to suppression of cell division. Phylo-stratification analysis of the age index using full transcriptome data of all compared populations revealed upregulation of the Phylostratum 8 comprising gene modules of inflammation, viral and bacterial response, innate and adaptive immune response and also female pregnancy. The results of the project were also presented at the conference (Salmina K, Vainshelbaum NM, Inashkina I, Pjanova D, Erenpreisa J. Mitotic slippage promotes amoeboid transition in breast cancer cells after genotoxic treatment. 4th Danube Conference on Epigenetics. 18–21 October 2022) as a poster presentation.
Information published 30.12.2022.