NEW ANTI-ERBB-2 THERAPIES AND RETINOIC ACID EFFECT ON HUMAN BREAST CANCER CELL MOTILITY

VANDERHOEVEN F.; FLAMINI MI.

Becas de formación inicial en investigación en cáncer 2016-2017

Instituto Nacional del Cancer

Año: 2018; p. 338 - 341

Breast cancer is classified in molecular subtypes according to the presence/absence of estrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 (ErbB-2, also known as HER-2). Tumors overexpressing ErbB-2 are more aggressive and metastatic, due to the signaling through this receptor contributes to the oncogenic transformation; therefore patients have a poor prognosis. Currently, new anti-HER-2 strategies that are used in clinic have been developed. One of these strategies is a monoclonal antibody that binds to the extracellular domain of HER-2 known as trastuzumab (Tz) or Herceptin®. Despite this progress, between 40-60% of patients do not respond to or develop resistance to the treatment. This fact leads to the requirement of new therapeutic approaches based on the combination of different drugs. Retinoids, mainly retinoic acid (RA), have been suggested in the adjuvant treatment of breast carcinoma because of their ability to inhibit cell growth and induce morphological or phenotypic differentiation. Its antitumor activity is mediated primarily by retinoic acid receptors (RAR), which belong to the nuclear receptor superfamily. Cell adhesion and migration are critical for the cell to spread, invade, and metastasize elsewhere. Moesin induces actin depolymerization and its translocation towards the edge of the cell membrane, being responsible of cortical actin complexes formation. Another key protein is focal adhesion kinase (FAK), which participates in the assembly and disassembly of focal adhesion complexes, reorganizing them in the migration direction; its overexpression is correlated with more aggressive tumors. The hypothesis of this project is that the combination of trastuzumab (Tz) and retinoic acid (RA) has a synergistic effect in decreasing the viability, adhesion and migration of HER-2 + /RAR+ human breast cancer cells by modifications in the expression/localization of proteins related to the cellular movement as moesin and FAK. The human breast cancer cell lines SKBR-3 (ER- /HER-2 + /RAR+ ) and BT-474 (ER+ /HER-2 + /RAR+ ) were used for a comparative analysis, since it has been shown that treatment with Tz has a lower effect in patients presenting ER+ tumors compared to those with ER- . Treatments for 72 hours were performed with Tz 1 and 10 μg/ml, RA 10-6 M and the combination of both drugs and then MTT assays, pharmacological interaction analysis, immunofluorescence, adhesion, migration and cell invasion assays, silencing with interfering RNA, FAK inhibitor and western blot were performed. 339 The 72 hours treatment with different doses of Tz (0.1 ? 1 ? 10 ? 100 μg/ml) and RA (10- 8 ? 10-7 ? 10-6 ? 10-5 M) decreases both cell lines proliferation in a dose-dependent manner (Fig. A). In SKBR-3 cells, treatment with Tz (1 ? 10 ? 100 μg/ml) in combination with RA 10-6 M has a synergistic effect (Fig. B). On the other hand, in the BT-474 cell line treatment with Tz 10 - 100 μg/ml in combination with RA 10-6 M exerts a synergistic effect and treatment with Tz 1 μg/ml combined with RA 10-6 M generates an additive effect in the diminution of the cellular proliferation. The dose reduction index (DRI) obtained for the SKBR-3 cell line indicates that within the synergistic combinations Tz 1 ? 10 μg/ml + RA 10-6 M, the Tz dose is reduced 10 times, while for the combination Tz 100 μg/ml + RA 10-6 M the reduction of Tz dose is 100 times (Fig. C). In the BT-474 line, in the combination Tz 10 μg ml + RA 10-6 M, the Tz dose employed is 10 times lower when it is used within this synergic group and for the combination Tz 100 μg/ml + RA 10-6 M, the DRI indicates that within this synergistic scenario the effect is 100 times more potent compared to Tz alone. Simultaneous administration of the two drugs significantly decreased adhesion, migration and invasion in both cell lines (Fig. D). Migration was also found to be decreased following treatment with FAK inhibitor or transfected with the interfering RNA for FAK in combination with the treatments. By immunofluorescence it was observed that Tz 10 μg/ml, RA 10-6 M and the combination of both drugs induce FAK translocation from the cytosol to the nucleus in both cell lines (Fig. E). In addition, a granular distribution of HER-2 receptor was observed after the combined treatments (Fig. F). By confocal microscopy, we confirmed that in control cells, HER-2 is found in the cell membrane and the co-administration of both drugs induces the internalization of this receptor, to a greater extent in the SKBR-3 cell line. RA and Tz combined treatments strongly decrease FAK, Moesin and HER-2 expression (Fig. G). In conclusion, in HER-2 + /RAR+ breast cancer cell, the coadministration of Tz and RA, exert a synergistic effect in cellular proliferation decrease, which allows to reduce the doses used, decreasing the toxicity while maintaining effectiveness. Also, this coadministration induces FAK nuclear relocation and cell adhesion/migration/invasion decrease to a greater degree in the ERcell line. Internalization and subsequent degradation of HER-2 receptor would have a beneficial effect upon the downregulation of activation cascade of this highly oncogenic pathways stimulated by this receptor. The Tz + RA combination resulted in a potent decrease in the expression of the essential proteins for migration, FAK/Moesin and HER-2 receptor. These findings suggest that the coadministration of both drugs in patients with this type of cancer could contribute to improve their prognosis and reduce the adverse effects of therapy because the Tz doses applied would be lower due to the adjuvant effect of RA.