An Experimental Analysis of the Molecular Effects of Trastuzumab (Herceptin) and Fulvestrant (Falsodex), as Single Agents or in Combination, on Human HR+/HER2+ Breast Cancer Cell Lines and Mouse Tumor Xenografts

Abstract

Purpose: To investigate the effects of trastuzumab (herceptin) and fulvestrant (falsodex) either in combination or alone, on downstream cell signaling pathways in lab-cultured human HR+/HER2+ breast cancer cell lines ZR-75-1 and BT-474, as well as on protein expression levels in mouse xenograft tissue.

Methods: Cells were cultivated in the presence of trastuzumab or fulvestrant or both. Molecular events that resulted in an inhibition of cell proliferation and cell cycle progression or in an increased rate of apoptosis were studied. The distribution and abundance of the proteins p-Akt and p-Erk expressed in these cells in response to single agents or combinatorial treatment were also investigated. In addition, the effects of trastuzumab and fulvestrant, either as single agents or in combination on tumor growth as well as on expression of the protein p-MED1 expressed in in vivo mouse xenograft models was also examined.

Results: Cell proliferation was increasingly inhibited by trastuzumab or fulvestrant or both, with a CI<1 and DRI>1 in both human cell lines. The rate of apoptosis increased only in the BT-474 cell line and not in the ZR-75-1 cell line upon treatment with fulvestrant and not trastuzumab as a single agent (P<0.05). Interestingly, fulvestrant, in combination with trastuzumab, did not significantly alter the rate of apoptosis (in comparison with fulvestrant alone), in the BT-474 cell line (P>0.05). Cell accumulation in the G1 phase of cell cycle was investigated in all treatment groups (P<0.05), and the combination of trastuzumab and fulvestrant reversed the effects of fulvestrant alone on p-Akt and p-Erk protein expression levels. Using ZR-75-1 or BT-474 to generate in vivo tumor xenografts in BALB/c athymic mouse models, we showed that a combination of both drugs resulted in a stronger inhibition of tumor growth (P<0.05) and a greater decrease in the levels of activated MED1 (p-MED1) expressed in tumor issues compared with the use of either drug as a single agent.

Conclusions: We demonstrate that the administration of trastuzumab and fulvestrant in combination results in positive synergistic effects on both, ZR-75-1 and BT-474 cell lines. This combinatorial approach is likely to reduce physiological side effects of both drugs, thus providing a theoretical basis for the use of such combination treatment in order to resolve HR+/HER2+ triple positive breast cancer that has previously been shown to be resistant to endocrine treatment alone.

Fig 1. Drug dose versus ZR-75-1 cell proliferation inhibition rate curve (A) and Drug dose versus BT-474 cell proliferation inhibition rate curve (B) using trastuzumab or fulvestrant treatment or both.

After 72h of continuous exposure to drugs (either trastuzumab or fulvestrant as single agents) higher cell proliferation inhibition rates were observed with increasing drug concentrations (P<0.05). The combination of both drugs led to much stronger effect compared with individual drug treatments in both cell lines (P<0.05); ED₅₀ values have been indicated in Table 1.

Fig 2. Effects on BT-474 cell apoptosis in either untreated or cultured in the presence of absolute ethanol (vehicle) or trastuzumab at ED₅₀ or fulvestrant at ED₅₀ or both for 72h.

Data are representative of at least three independent experiments with similar results. Fulvestrant as a single agent or combined with trastuzumab induced BT-474 cell apoptosis. However, although significant differences were observed between the trastazumab-treated and combination groups, no statistical differences were observed between the fulvestrant-treated and combination groups. * P<0.05 compared with control group. ** P<0.05 compared with combination group.

Fig 3. Effects on ZR-75-1 cell apoptosis either untreated or cultured in the presence of the vehicle or trastuzumab at ED₅₀ or fulvestrant at ED₅₀ or both for 72h.

Data are representative of at least three independent experiments with similar results. Statistically significant differences were not observed in the rate of cell apoptosis between treatment groups and control groups in the ZR-75-1 cell line (P>0.05).

Fig 4. Effects on the cell cycle stage distribution of ZR-75-1 cells after continuous exposure to trastuzumab and fulvestrant as single agents or in combination.

Trastuzumab and fulvestrant used as single agents promoted a statistically significant accumulation of ZR-75-1 cells in the G1 phase of the cell cycle when compared to the control groups. The combination of trastuzumab and fulvestrant caused a statistically significant further increase in the percentage of ZR-75-1 cells in the G₁ phase compared with the effect of fulvestrant alone (P = 0.030). * P<0.05 compared to the control group. ** P<0.05 compared to the combination group. The cell cycle stage distribution of ZR-75-1 cells in the five different groups are shown in S3 Fig of the supplementary information section.

Fig 5. Effects on the cell cycle progression of BT-474 cells after continuous exposure to trastuzumab and fulvestrant as single agents or in combination for 72h.

Trastuzumab and fulvestrant as single agents or as a combination promoted the accumulation of BT-474 cells in the G1 phase of the cell cycle. However, no statistically significant differences were detected between the combination treatment group and individual drug-treated groups (P>0.05). The cell cycle stage distribution of BT-474 cells in the five different groups are shown in S4 Fig of the supplementary information section. * P<0.05 compared with control group.

Fig 6. Effects on the levels of the proteins p-Akt and p-Erk extracted from ZR-75-1 cells either untreated or cultured in the presence of vehicle or trastuzumab at ED₅₀ or fulvestrant at ED₅₀ or with a combination of both for 72h.

p-Akt and p-Erk proteins from two samples in the control group and vehicle group and from three samples in each treatment group were detected. It was found that trastuzumab decreased p-Akt levels and that fulvestrant increased both, p-Akt and p-Erk levels. In addition, the combination of both drugs significantly reduced levels of p-Akt and notably inhibited the effect that fulvestrant had on the levels of both proteins. * P<0.05 compared with control group. ** P<0.05 compared with combination group.

Fig 7. Effects on the levels of the proteins p-Akt and p-Erk extracted from BT-474 cells either untreated or cultured in the presence of vehicle or trastuzumab at ED₅₀ or fulvestrant at ED₅₀ or with a combination of both for 72h.

The figure shows that trastuzumab when used as a single agent decreased the levels of both p-Akt and p-Erk, while fulvestrant raised the levels of both proteins. The combination of both drugs significantly prevented fulvestrant from raising the levels of both proteins. * P<0.05 compared with control group. ** P<0.05 compared with combination group.

Fig 8. Nude Mouse Xenograft Model Studies.

ZR-75-1 (a) and BT-474 (b) tumor xenografts excised from female BALB/c (nu/nu) nude mice after four-week treatments with trastuzumab and fulvestrant as single agents or in combination. Days versus tumor volume curves for ZR-75-1 (c) and BT-474 (d) tumor xenografts show that trastuzumab and fulvestrant as single agents or in combination significantly inhibited tumor growth (P<0.05) and that tumor growth in the combination group was notably much more inhibited compared with that in individually treated groups (P<0.05).

Fig 9. Immunoblot analysis of the levels of the proteins p-MED1 and MED1 expressed in ZR-75-1 tumor xenografts.

Three samples from each group were randomly selected. Similar results were obtained after two experimental repeats. Thus, representative bands from a single experiment are shown here. Trastuzumab and fulvestrant either as single agents or in combination decreased levels of the protein p-MED1 (P<0.05) and increased levels of MED1 total protein (P<0.05). However, no significant differences were observed between combination groups and individually treated groups (P>0.05).

Fig 10. Immunoblot analysis of the levels of the proteins p-MED1 and MED1 expressed in BT-474 tumor xenografts.

Three samples from each group were randomly selected. Similar results were obtained after two experimental repeats. Thus, representative bands from a single experiment are shown here. Trastuzumab and fulvestrant as single agents decreased levels of the protein p-MED1 (P<0.05) and the combination resulted in lower expression levels when compared with fulvestrant alone (P = 0.039). In addition, total protein levels of MED1 increased in the combination group compared with the vehicle-treated group (P = 0.017).