Investigation of the Role of NFIB and NR4A2 in Resistance to standard Chemotherapy in Triple Negative Breast Cancer

The patient tumour carrying an amplification associated with overexpression of NFIB had received a combination of anthracyclines and taxanes, and did not respond to both drug classes. To determine whether inhibition of NFIB improves sensitivity to chemotherapy, we decided to test the sensitivity of SUM149 cells to the anthracycline Doxorubicin.

We did not choose to test a taxane because of the already high sensitivity of SUM149 to Paclitaxel, as determined previously by a Ph.D. student from our laboratory (SUM149 cell line was the most sensitive of the cell lines in our possession to paclitaxel with an IC50 of 0.00002 μM). This would have rendered it difficult to observe an eventual NFIB-dependant increase in the already high sensitivity.

The cell line sensitivity to doxorubicin was determined using Alamar Blue cytotoxicity assay. The cells were plated at a concentration of 7,5x10 cells per well in a 6-well plate and transfected 24h later with 10uM of NFIB#2-siRNA, NFIB#5siRNA or ctl-siRNA. Three days after, cells were collected and counted with Tripan Blue to plate 2500 living cells per well in a 96-well plate.

The day after, the transfected cells were treated with increasing doses of DOXO (from 0,1pM to 100uM) or with drug-free medium (untreated cells) for 72 hours and then subsequently evaluated using the Alamar Blue cell viability assay to compare the viable cell fraction in relation to the control of non-treated cells. The concentration of DOXO required to inhibit the growth of 50% of the cells (the IC50) was determined using Graph Pad Prism software. All the experiments were performed in quadruplicate wells for each condition and were repeated at least three times. The untreated cells at 72h were compared also with the time zero cells (value of fluorescence 24h after plating).

As shown in the Figure 4.3A, the increase in the fluorescence intensity of the untreated cells at 72h relative to time zero was approximately the same for ctl-siRNA and NFIB-siRNA transfected cells. From this first result we ruled out an effect of NFIB expression on the proliferation of SUM149 cells since NFIB knockdown did not result in a lower cellular proliferation rate compared to the ctl cells in untreated cells. This outcome confirmed what was already visible 72h after siRNA transfection, when we did not notice a reduction in the cell number for the NFIB-siRNA transfected cells as compared with the cells transfected with the control siRNA (data not shown); in other words, cell counting after siRNA transfection confirmed this Alamar blue result.

As evident from the Alamar Blue assay (Fig. 4.3B), NFIB knockdown did not increase SUM149 cells sensitivity to DOXO: ctl-siRNA and NFIB-siRNAs-treated cells show a similar dose-response curve and a similar IC50 value, around 0,05 uM, to doxorubicin.
For each repetition of the assay we evaluated NFIB knockdown efficacy by Western blot analysis (Fig. 4.3C).
Taken together, our data suggest that increased NFIB expression does not affect drug resistance level to doxorubicin and proliferation in the SUM149 TNBC cell line.