BCL-xL and MCL-1 are pivotal anti-apoptotic proteins responsible for the survival of MDA-MB-468 and MDA-MB-231 cell lines.
At first, we assessed the impact of various anti-apoptotic proteins from the BCL-2 family on the survival of two TNBC cell lines, MDA-MB-468 and MDA-MB-231. To achieve this, we generated silenced cells for MCL-1, BCL-xL, or BCL-2, designated as sg MCL-1, sg BCL-xL, and sg BCL-2, respectively, using a CRISPR-Cas9 based approach. Our findings reveal that MDA-MB-468 and MDA-MB-231 control lines express all three anti-apoptotic proteins, albeit with lower levels of BCL-2 in MDA-MB-468 (Fig. 1A). Western blot analysis corroborated that MDA-MB-468 and MDA-MB-231 sg BCL-xL, sg MCL-1, and sg BCL-2 cells exhibit a deficiency in BCL-xL, MCL-1, and BCL-2, respectively (Fig. 1A).
Subsequently, we investigated the effects of both loss and pharmacological inhibition of BCL-xL, MCL-1, or BCL-2 on the survival of MDA-MB-468 and MDA-MB-231 (Fig. 1B). In control cell lines, only the pharmacological inhibition of BCL-xL using A1331852 resulted in cell death in MDA-MB-468 and MDA-MB-231 (20% and 60% respectively). This outcome underscores the critical role of BCL-xL in the fundamental survival of these cells, particularly in MDA-MB-231. To explore potential compensatory mechanisms between anti-apoptotic agents when inhibiting only one of them, we employed BH3 mimetics in the different anti-apoptotic-deficient cell lines. Inhibition of BCL-xL with A1331852 in MCL1-deficient cell lines led to a substantial 90% cell death, suggesting that simultaneous targeting of both BCL-xL and MCL-1 is necessary to induce massive cell death. Conversely, pharmacological inhibition of MCL-1 by S63845 in BCL-xL-deficient cell lines resulted in a less pronounced cell death of only 40%-50%. Interestingly, the use of A1331852 in MDA-MB-231 sg BCL-xL cells caused an unexpected 35% cell death, possibly due to the expression of an unidentified target for this inhibitor in these cells. Inhibition of BCL-2 by ABT-199 in BCL-xL or MCL-1-deficient MDA-MB-468 cells did not induce cell death compared to their respective controls. Similarly, pharmacological inhibition of BCL-xL or MCL-1 in MDA-MB-468 sg BCL-2 cells did not trigger cell death. Hence, BCL-2 does not play a significant role in the survival of MDA-MB-468 cells, consistent with their low expression of BCL-2. Likewise, BCL-2 does not appear to have a significant impact on the of MDA-MB-231 cells survival albeit pharmacological inhibition of BCL-2 in MDA-MB-231 sg MCL-1 induced a moderate 30% cell death.
In conclusion, our results highlight that BCL-xL and MCL-1 play significant roles in promoting the survival of MDA-MB-468 and MDA-MB-231 cell lines. However, the inhibition of either BCL-xL or MCL-1 alone is insufficient to initiate a massive apoptotic response in these studied cancer lines. The concomitant inhibition of BCL-xL and MCL-1 clearly increases cancer cells apoptosis, highlighting the presence of compensatory mechanisms between these two proteins.
BCL-xL significantly contributes to the resistance of the MDA-MB-231 and MDA-MB-468 cell lines to chemotherapy.
One standard of care for triple-negative breast cancers involves the combination of three chemotherapies: anthracycline (such as doxorubicin), alkylating agents (like cisplatin) and anti-metabolites (e.g., 5-fluorouracil). Combination of doxorubicin, cisplatin and 5-fluorouracil resulted in the death of only 25–30% of the cell lines as depicted in Fig. 2A. To identify the specific anti-apoptotic proteins responsible for impeding chemotherapy effectiveness in these cells, we employed CRISPR-Cas9 gene silencing and BH3 mimetics, as illustrated in Fig. 2B and 2C. Combining chemotherapy with the silencing of BCL-xL significantly increased cancer cell death from less than 20% to 60–70% in both cell lines, while silencing MCL-1 or BCL-2 had no substantial impact on sensitivity to chemotherapy (Fig. 2B). In accordance with this, combining chemotherapy with pharmacological inhibition of BCL-xL resulted in nearly 100% cell death in both cell lines, whereas MCL-1 inhibition had no effect (Fig. 2C). Contrary to BCL-2 silencing, the use of BH3 mimetics ABT-199 in combination with chemotherapy enhanced cell death to 60% in both cell lines, possibly due to an ABT-199 inhibitory effect on BCL-xL at the 1µM dose employed.
To confirm the apoptotic nature of cell death induced by chemotherapy and BCL-xL inhibitor, we employed a pan-caspase inhibitor (Q-VD-OPh), as shown in Fig. 2D. In line with expectations, massive cell death induced by the combination of BH3 mimetics S63845 and A1331852 targeting MCL-1 and BCL-xL respectively was entirely blocked by Q-VD-OPh. Cell death induced by chemotherapy alone or in combination with BCL-xL inhibitor was reversed by Q-VD-OPh. This observation corroborates that cell death induced by chemotherapy, either alone or in combination with BCL-xL inhibitor, is apoptotic in nature.
In conclusion, BCL-xL plays a critical role in limiting the effectiveness of chemotherapy in both the MDA-MB-231 and MDA-MB-468 cell lines.
CAFs protect MDA-MB-468 cells from apoptosis triggered by simultaneous chemotherapy and BCL-xL silencing.
Beyond their intrinsic resistance to chemotherapy, cancer cells survival may also be influenced by the surrounding tumor environment. Consequently, we explored how the presence of CAFs affects the susceptibility of MDA-MB-468 cells to apoptosis using a 2D coculture model. Our findings revealed that the presence of CAFs diminishes the apoptosis in MDA-MB-468 cells triggered by the combination of chemotherapeutic agents (Fig. 3A). Simultaneously, the response of CAFs to chemotherapy was variable, with CAF mortality ranging from 20–60% under chemotherapy conditions, and notably, the presence of MDA-MB-468 cells did not affect the sensitivity of CAFs to chemotherapy (Fig. 3B).
Since inhibiting BCL-xL makes cells more responsive to chemotherapy, we sought to determine whether CAFs could also counteract cell death induced by chemotherapy when BCL-xL was silenced. As illustrated in Fig. 3C, our results demonstrate that the presence of CAFs reverses the cell death triggered by the combination of chemotherapy and BCL-xL silencing. It is worth noting that the survival rate of BCL-xL deficient cells treated with chemotherapy under the influence of stromal pressure closely resembled that of control cancer cells treated with chemotherapy in monoculture (Fig. 3C). Concurrently, CAFs displayed variable responses to chemotherapy, and neither the presence of MDA-MB-468 control cells nor MDA-MB-468 cells with BCL-xL silencing influenced the sensitivity of CAFs to chemotherapy (Fig. 3D).
Hence, our findings emphasize that CAFs play a protective role against chemotherapy-induced apoptosis, even when the BCL-xL resistance factor is overcome in cancer cells.
BCL-xL expression is associated with patient-derived tumoroids responsiveness to chemotherapy.
To investigate the responsiveness of primary human breast cancer cells to chemotherapy we used 7 breast cancer organoid cultures (hereinafter called tumoroids). Tumoroids derived from treatment naive breast cancer samples were grown ex vivo using a stem cell culture method [13] that preserves mixtures of multiple tumor populations of flexible differentiation status [14,15] (see Fig. 4A for microscopic analysis). We assessed their response to chemotherapy via FACS analysis and identified variations in sensitivity among the tumoroids (Fig. 4B). In particular, tumoroids #2 and #5 showed greater sensitivity to chemotherapy than the other tumoroids, with high sensitivity as early as 0.5µM of chemotherapy. In contrast, tumoroids #4, #6 and #7 were the less sensitive tumoroids to high dose of chemotherapy. Subsequently, we investigated the expression of BCL-xL and MCL-1 in tumoroids using Western blot analysis of bulk lysates (Fig. 4C). Remarkably, while MCL-1 expression appears relatively uniform we observed heterogeneity in BCL-xL expression within the tumoroids. Tumoroid #2, #3 and #5 displayed low levels of BCL-xL, while tumoroids #1, #4, # 6 and #7 demonstrated high levels of BCL-xL. Tumoroids expressing the least BCL-xL are the most sensitive to chemotherapy (except tumoroid #3) and conversely the most responsive are those in which BCL-xL expression is high. There is in fact a negative correlation between sensitivity to 0.5 µM chemotherapy and the level of BCL-xL expression in tumoroids (Fig. 4D). We treated the most chemoresistant tumoroid (#4) with a combination of chemotherapy and BH3 mimetic antagonist of BCL-xL (A1331852, 100 nM). Consistent with our prior findings in resistant cancer cell lines, the combination effectively reversed the resistance of tumoroid #4 (Fig. 4E). These findings indicate that BCL-xL expression and activity limit the chemosensitivity of patient-derived breast tumoroids. Finally, we observed that the most sensitive tumors (#2 and #5) were rendered more resistant to chemotherapy when treated in the presence of CAFs-conditioned media (Fig. 4F). This result confirms the impact of CAFs on cancer cells in tumoroid model of human breast cancer.