Investigating the role of ELK3 transcription factor in the migration of triple negative breast cancer cells, using genomic and microfluidic approaches

Postdoctoral Research Project

Project title: Investigating the role of ELK3 transcription factor in the migration of triple negative breast cancer cells, using genomic and microfluidic approaches

Acronym: GENOMICROELK
Project Type: Postdoctoral research project
Project ID: PN-III-P1-1.1-PD-2021-0525
Contract No: PD62/2022
Contracting unit: UEFISCDI
Total budget: 249.960 Lei (aprox. 51.200 €)
Project duration: 24 months (01/04/2022 - 31/03/2024)

Project summery
Metastasis is the main cause of breast cancer (BC)-related death, no therapies that target this process being available. Therefore, there is a need to elucidate the molecular mechanisms that govern metastasis in order to develop therapeutic strategies that are able to target the metastatic cells. The ELK3 transcription factor is a molecule whose expression we found to be correlated with the migration capacity of BC cells, and thus it might be a key regulator of cancer metastasis. In this context, the goal of this project is to evaluate the role of ELK3 in BC metastasis, by studying its involvement in cell migration, epithelial-to-mesenchymal transition and the cancer stem cell phenotype, and some of the underlying molecular mechanisms. BC cell lines in which ELK3 is either overexpressed (ELK3-OE) or knocked-down (ELK3-KD) will be generated by a lentiviral cell transduction procedure. The transformed cells will be evaluated in terms of their cell migration capacity in 3D microfluidic devices, EMT phenotype by flow-cytometry and stemness potential in mammosphere formation assays. On the other hand, in a functional genomic approach (microarray technology), the molecular effects triggered by both ELK3-OE and ELK3-KD will be assessed by identifying the gene expression patterns that are modulated in these transformed cells. The results regarding the cellular effects will be confronted with the molecular changes triggered by the overexpression/knockdown of ELK3, emphasizing the molecular mechanisms by which ELK3 might affect BC metastasis.
The overall goal of this project is to elucidate the impact of the transcription factor ELK3 in the metastasis of triple negative breast cancer, in an attempt to nominate this molecule as a molecular target for future therapeutic strategies in breast cancer.

Specific objective 1. Generation of genetically modified triple negative breast cancer cell lines with stable overexpression (ELK3-OE) or stable inhibition (ELK3-KD) of the ELK3 transcription factor, by lentiviral transduction.
Specific objective 2. Evaluation of the cellular effects determined by the overexpression and inhibition of the transcription factor ELK3 in triple negative breast cancer cells, in terms of the cells’ migration capacity in 3D microfluidic devices, their phenotype on the epithelial-mesenchymal continuum by flow cytometry and their stemness potential in mammosphere formation assays
Specific objective 3. Identification of the molecular signaling pathways modulated by the transcription factor ELK3 that could be involved in the metastatic process of triple negative breast cancer, by using microarray technology and RT-qPCR.
Stage 1 Generation of genetically modified triple negative breast cancer cell lines with stable overexpression (ELK3-OE) or stable inhibition (ELK3-KD) of the ELK3 transcription factor by lentiviral transduction
Activity 1.1 Design of the overall experimental procedure and genetic constructs to be used in the generation of the genetically modified triple negative breast cancer cell lines.
Activity 1.2 Lentiviral-mediated transduction of triple-negative breast cancer cell lines and generation of 4 genetically modified cell lines
Activity 1.3 Validation of the genetic modification for the 4 triple negative breast cancer cell lines, in terms of their phenotype (GFP expression) and at the level of genomic DNA, messenger RNA and ELK3 protein.

Stage 2_Phase 1 Evaluation of the cellular effects determined by the overexpression and inhibition of the transcription factor ELK3 in triple negative breast cancer cells, in terms of the cells’ migration capacity in 3D microfluidic devices, their phenotype on the epithelial-mesenchymal continuum by flow cytometry and their stemness potential in mammosphere formation assays
Activity 2.1 Assessment of the impact of the transcription factor ELK3 in the migration of triple-negative breast cancer cells in 3D microfluidic devices
Activity 2.2 Assessment of the impact of the transcription factor ELK3 in the epithelial-mesenchymal transition of triple-negative breast cancer cells by flow cytometry
Activity 2.3 Assessment of the impact of the transcription factor ELK3 on the stemness capacity of triple negative breast cancer cells in mammosphere formation assays

Stage 2_Phase 2 Identification of the molecular signaling pathways modulated by the transcription factor ELK3 that could be involved in the metastasis of triple negative breast cancer, by using microarray technology and RT-qPCR.
Activity 2.4 Identification of all gene expression changes triggered by the overexpression and inhibition of the transcription factor ELK3 in triple-negative breast cancer cells, by microarray technology
Activity 2.5 Validation of gene expression changes determined by the overexpression and inhibition of the transcription factor ELK3 in triple negative breast cancer cells that could be involved in metastasis, by RT-qPCR
Activity 2.6 Identification of the molecular mechanisms that could explain the impact of the ELK3 transcription factor in the metastasis of triple negative breast cancer, through the functional analysis of gene expression differences
 
Stage 3 Elucidation of the impact of the transcription factor ELK3 in the metastatic process of triple negative breast cancer
Activity 3.1 Elucidation of the impact of the ELK3 transcription factor in  triple negative breast cancer metastasis by corroborating previously obtained cell biology and molecular biology data.
In the first stage of this project, we aimed to obtain two genetically modified cell lines of triple negative breast cancer (TNBC) by lentiviral transduction, MDA231 and MDA468, in order to overexpress or inhibit the expression of the transcription factor ELK3, and thus, to elucidate the impact of this molecule in the process of breast cancer metastasis.

In order to achieve this objective, in a first phase, the general experimental design of lentiviral transduction (Fig. 1) and the design of genetic constructs inserted into plasmids (Activity 1.1) were created. Subsequently, lentiviral-mediated genetic modification (Activity 1.2) was implemented by: 1. cloning the plasmids (containing the gene constructs) into bacteria and further isolating them, 2. viral packaging with the plasmids of interest by cell transfection into the HEK293T cell line and harvesting the lentivirus, 3. transducing breast cancer target cells by their infection with the previously obtained lentivirus and 4. the selection of genetically modified cells based on the marker gene included in the construct. These cells, which were possibly genetically modified, were characterized in terms of their viability on the one hand (labeling with propidium iodide PI), and in term of GFP expression (a reporter gene inserted into the genetic constructs) on the other hand, by fluorescence microscopy and flow cytometry. The success of the genetic modification of these cell lines (Activity 1.3) is also to be investigated by PCR (testing for the presence of the sequences of interest in the target cells’ genome), RT-qPCR (evaluation of ELK3 expression at the mRNA level) and WesternBlot (evaluation of ELK3 expression at the protein level).

The viral packaging system used to obtain the virus was a third-generation one, the actual genetic modification being based, like all lentiviral transduction systems, on the ability of the human immunodeficiency virus (HIV-1) to insert foreign genetic material into the genome of cells. The third generation systems use only 4 genes from the HIV genome (in addition to the gene of interest): GAG, POL, REV and a gene for viral capsid proteins, which represent the minimum genetic information required for viral packaging and subsequent transduction. At the same time, the 4 genes are distributed in 3 different plasmids, and the expression of viral particles in the process of viral packaging is conditioned by molecular elements present only in virus-producing cells (HEK293T in this experimental design). All this increases the level of biosafety in experiments where the lentivirus is used.

Fig. 1 The general experimental design used to obtain the genetically modified MDA231 and MDA468 cell lines, in which the transcription factor ELK3 in up-/down-regulated. The efficiency of both infection and selection were assessed by fluorescence microscopy and flow cytometry. All experiments were implemented in 3 biological replicates. The details of the experimental protocol that were optimized during the implementation of this project are highlighted by red callouts next to the word "OPTIMIZED".

Therefore, following the implementation of all the activities within this stage, 9 genetically modified breast cancer cell lines were obtained (in 3 biological replicates each) in which over 90% of the cells expressed the GFP gene, the reporter gene included in the genetic constructs. The obtained cell lines are:
                                 1. MDA231_ELK-OE – cell line with ELK3 overexpression
                                2. MDA231_ELK-KD1 - cell line with ELK3 inhibition
                                3. MDA231_ELK-KD2 - cell line with ELK3 inhibition
                                4. MDA468_ELK-KD1 - cell line with ELK3 inhibition
                                5. MDA468_ELK-KD2 - cell line with ELK3 inhibition
                               +4 genetically modified control cell lines
Morphological aspects and GFP expression among all these cell lines at the end of stage 1 of the project are shown in Figure 2. The number of viable, genetically modified cells for each cell line, for each biological replicate, is shown in Table 1.

Fig. 2 Morphological aspects and GFP expression among the 10 genetically modified cell lines by lentiviral transduction (biological replica 1), two days after cessation of selection: cell cultures under phase contrast (PH) and UV light (GFP) (DMi8, Leica - 10X objective).

Table 2. Number of viable, genetically modified (GFP+) cells with each genetic construct of interest for each cell line and biological replica, available in liquid nitrogen-stored cryotubes.
Genetic constructReplica 1Replica 2Replica 3
MDA468MDA231MDA468MDA231MDA468MDA231
CTR OE171.733424.742993.200532.062570.14328.485
ELK OE061.357080.448076.820
CTR KD355.421982.096154.460744.59501.164.096
ELK KD1623.427371.655699.205526.671538.260517.005
ELK KD21.213.279474.198515.393684.875451.051430.711
Project director: Lect. Cruceriu Daniel, PhD
Mentor: Prof. Banciu Manuela, PhD
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