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

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 4biological 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.

The stable transformation of the cell lines was assessed by PCR (testing the transgenes integration in the genome), RT-qPCR (testing the expression levels of ELK3 at the RNA level) and WesternBlot (testing the expression levels of ELK3 al the protein level) (Fig. 3).

Fig. 3 Validation of the stable transformation of MDA231 and MDA468 cell lines at the genome level by PCR with specific primers for the constructs used in the transformation (A), for the coding sequence of the ELK3 gene without introns (B) and for the shRNA specific sequence found in the ELK-KD1 construct (C) and at the transcriptome level by RT-qPCR , with primers specific for the ELK3 gene (D).

In the third phase of the project, we aimed to identify the modifications in the gene expression patterns triggered by ELK3 overexpression and knockdown, in a genomic approach, in order to determine the main molecular mechanisms by which this transcription factor affects TNBC metastasis. The rationale behind this objective is to group the genes that are modulated by ELK3 into signaling pathways associated with BC metastasis, by overlapping the results obtained for ELK3 overexpression with the ones obtained for ELK3 knock down.

We evaluated the expression level for all the genes in the human genome by a holistic functional genomic approach, using the microarray technology, starting with the total RNA extracted from one of the cell lines in which the ELK3 transcription factor is down-regulated. The expression levels of some of the genes from the microarray data were also validated by RT-qPCR in both transformed (ELK-OE and ELK-KD) and parental cell lines.

In the microarray analysis of MDA231_ELK-KD1 vs. MDA231_CTR-KD, we identified 740 genes modulated by the inhibition of the ELK3 transcription factor (FR > ±1.3, p<0.05), of which 322 were overexpressed and 418 were downregulated. The top 50 upregulated and downregulated genes based on the microarray data, plotted for each biological replicate, are shown in Figure 1.
Fig. 1 Top 50 upregulated and dowregulated genes following ELK3 inhibition (MDA231_ELK-KD1 vs. MDA231_CTR-KD), based on microarray data, represented for each biological replica (images created in GSEA_4.3.0 software).

The whole set of differentially expressed genes (n=740) obtained in the microarray analysis were used for the functional analysis in Ingenuity Pathway Analysis Software (IPA). Functional analysis in the IPA software revealed 43 cellular processes affected by ELK3 transcription factor inhibition, 22 of which are related to cancer. The main cellular processes triggered by the inhibition of the ELK3 transcription factor were the inhibition of cell invasion and migration and the inhibition of the self-renewal capacity of cells, doubled by the induction of apoptosis, with the impairment of cell viability, demonstrating, on the molecular level, the cellular processes induced by ELK3 in our cell lines. Therefore, we are now able to present the main molecular mechanisms by which ELK3 impacts TNBC metastasis in terms of cell motility and and stemness potential.