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Understanding the effects of therapeutic HER2 antibodies trastuzumab and pertuzumab on HER2-mediated cell signaling
- Author / Creator
- Hamid Maadi
The ErbB family of receptor tyrosine kinases, also called the HER receptors, consists of four members including epithelial growth factor receptor (EGFR), erbB2 (HER2), erbB3 (HER3), and erbB4 (HER4). HER2 overexpression has been reported in 20-30% of breast cancer cells and is responsible for lower overall survival rates in HER2-positive breast cancer cells. Trastuzumab as the first HER2-targeted therapy for the treatment of HER2-positive breast cancer patients was introduced in 1998. Although several mechanisms have been suggested for the mode of action of trastuzumab, the findings regarding these mechanisms are unclear and controversial. In addition, a considerable number of patients with HER2-positive breast cancer do not respond, or become resistant, to trastuzumab. In recent years, several new chemotherapeutic and targeted-therapeutic drugs have been introduced to increase the efficacy of targeted therapy with trastuzumab. Pertuzumab was specifically designed as a humanized recombinant monoclonal antibody to block both homodimerization and heterodimerization of HER2. Therefore, the lack of a clear understanding of the mechanisms of both trastuzumab and pertuzumab action may limit their application and efficacy.
Significant proportions of HER2-positive breast cancer cells co-express multiple types of HER receptors. Therefore, it is hard to investigate the possible effects of trastuzumab and pertuzumab on HER2 homodimer formation and HER2 homodimer-mediated HER2 phosphorylation. To overcome this limitation, we studied the effects of these antibodies in Chinese hamster ovary (CHO) cells transfected with genes encoding different HER receptors. Our time course and dose response results revealed that trastuzumab and pertuzumab only bind to HER2 with high affinity and specificity. Moreover, we showed that when used as single agents trastuzumab and pertuzumab have no inhibitory effects on HER2 homodimerization and phosphorylation in CHO cells overexpressing HER2 (CHO-K6). However, the combination of trastuzumab and pertuzumab can inhibit the phosphorylation of HER2 at specific sites suggesting the possible inhibitory effects of combination therapy on HER2-mediated signaling.
I further studied the mechanism of action of trastuzumab in HER2-positive breast cancer cells including SKBR3 and BT474 cells. I determined the effects of trastuzumab on HER2 signaling under various conditions including treatment with DMEM, EGF, HRG, and 10% FBS. The HER2 signaling was assessed by its heterodimerization, phosphorylation, and activation of the major downstream signaling proteins including Erk and Akt. Results demonstrated two different mechanism of action for trastuzumab in these two different HER2-positive breast cancer cell lines. In SKBR3 cells, we found that trastuzumab reduced cell viability in all treated conditions; however, it only arrested cell cycle at the G1 phase in the absence of EGF and HRGα ligands. It is possible that trastuzumab can reduce the viability of the SKBR3 cells through inducing both cell cycle arrest and apoptosis and ligands suppress the inhibitory effects of trastuzumab on cell cycle progression. Indeed, our results showed that trastuzumab has inhibitory effects on PI3K/Akt and MAPK signaling pathways, which may act through inhibition of HER receptors phosphorylation. On the other hand, in BT474 cells we showed that trastuzumab reduced viability and arrest the cell cycle at G1 in the presence or absence of EGF but not in the presence of HRGα ligands. However, trastuzumab did not show any detectable inhibitory effects on HER receptors’ phosphorylation, heterodimerization, and MAPK activation under these conditions. Interestingly, we showed that trastuzumab inhibited Akt phosphorylation at both threonine and serine phosphorylation sites. Previous studies have shown that lipid raft localization of HER receptors in breast cancer cell lines can promote HER kinase-independent activation of Akt. In addition, it has been shown that trastuzumab can affect HER2 localization in the breast cancer cell membrane. Therefore, we hypothesis that trastuzumab inhibits the lipid raft localization of HER2, which leads to the inhibition of Akt phosphorylation. Our results showed that trastuzumab blocked HER2 and EGFR localization to lipid rafts. Moreover, we found that trastuzumab significantly reduced the phosphorylation of c-Src which plays an important role in HER-mediated Akt phosphorylation in lipid rafts.
Overall, my results showed that trastuzumab can exert its anti-proliferative effects through different mechanisms which is cell line dependent. Most importantly, we identified a novel mechanism underlying the action of trastuzumab. By this novel mechanism, trastuzumab targets a HER2 kinase-independent signaling pathway by hindering the movement of HER2 homodimers and HER2-EGFR heterodimers into the lipid rafts.
- Graduation date
- Fall 2020
- Type of Item
- Doctor of Philosophy
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