Combined targeting of HER-2 and HER-3 represents a promising therapeutic strategy in colorectal cancer

Abstract

Background: Abrogation of growth factor-dependent signaling represents an effective therapeutic strategy for patients with colorectal cancer (CRC). Here we evaluated the effectiveness of targeting the epidermal growth factor (EGF) receptors HER-2 and HER-3 in the three cell lines LS513, LS1034 and SW837.

Methods: Treatment with HER-2-specific antibodies trastuzumab and pertuzumab resulted in a mild reduction of cellular viability. In contrast, the antibody-drug conjugate T-DM1 mediated a strong and dose-dependent decrease of viability and Akt phosphorylation.

Results: The most striking effects were observed with the dual tyrosine kinase inhibitor lapatinib, and the Pan-ErbB inhibitor afatinib. Selectively, the effect of EGF receptor inhibition was augmented by a combination with 5-fluorouracil and oxaliplatin. Finally, high expression of HER-3 was detected in 121 of 172 locally advanced rectal cancers (70.3%). In conclusion, inhibition of EGF receptors effectively blocks downstream signaling and significantly impairs viability of CRC cells. However, the effectiveness of receptor inhibition highly depends on the inhibitors' mode of action, as targeting HER-2 alone is not sufficient.

Conclusion: Since HER-2 and HER-3 are expressed in a relevant number of patients, targeting both receptors may represent a promising therapeutic strategy for CRC.

Keywords: Colorectal cancer; HER-2; HER-3; Inhibitors; Targeted therapy.

Fig. 1

Influence of HER-2/HER-3 inhibition on cellular viability. Cellular viability of LS513, LS1034, and SW837 cells was determined 24 h (black curve), 48 h (red curve), and 72 h (blue curve) after treatment with increasing concentrations of trastuzumab (a), pertuzumab (b), T-DM1 (c), lapatinib (d), and afatinib (e). To assess the inhibitory effect on downstream signaling, cells were treated with increasing concentrations of the respective inhibitors for 24 h, stimulated with 100 ng/ml neuregulin for 10 min, and referred to Western blot analysis. All experiments were performed in triplicate, independently repeated three times. The respective P-values and the (estimated) EC₅₀ for all drugs and time points are listed in Additional file 5: Table S1

Fig. 2

Cellular viability upon combined targeting of HER-2/HER-3. Cellular viability of LS513, LS1034, and SW837 cells was assessed 24 h (black curve), 48 h (red curve), and 72 h (blue curve) after treatment with various combinations of trastuzumab and pertuzumab. The inhibitory effect on downstream signaling was evaluated by Western blot analysis. All experiments were performed in triplicate, independently repeated three times. (a + b) Two different doses of pertuzumab were combined with increasing concentrations of trastuzumab. (c + d) Two different doses of trastuzumab were combined with increasing concentrations of pertuzumab. The respective P-values and the (estimated) EC₅₀ for all drugs and time points are listed in Additional file 5: Table S1

Fig. 3

Effectiveness of HER-2/HER-3 inhibition combined with 5-FU and oxaliplatin. Cellular viability of LS513, LS1034, and SW837 cells was assessed 24 h (black curve), 48 h (red curve), and 72 h (blue curve) after treatment with 5-FU and oxaliplatin combined with trastuzumab (a), pertuzumab (b), T-DM1 (c), lapatinib (d), and afatinib (e). All experiments were performed in triplicate, independently repeated three times. The respective P-values and the (estimated) EC₅₀ for all drugs and time points are listed in Additional file 5: Table S1

Fig. 4

Comparison of anti-HER-2/HER-3 monotherapy and a combination with 5-FU and oxaliplatin. Displayed are the respective EC₅₀ 24 h (black bar charts), 48 h (red bar charts), and 72 h (blue bar charts) after treatment. Compared to monotherapy, the combination of T-DM1 (a), lapatinib (b), and afatinib (c) with 5-FU and oxaliplatin mediated stronger effects only in LS513 and LS1034, but not in SW837. Not shown are the bar charts for trastuzumab and pertuzumab, as they were not time- and dose-dependent

Fig. 5

HER-3 protein expression of primary rectal cancer visualized by immunohistochemistry staining. a shows different intensities of HER-3 expression and the grading from no staining (IHC 0) to an intensive staining for HER-3 (IHC 3+). The positivity rate for HER-3 protein expression and the distribution of different staining grades within the analysed cohort of 172 CRC patients are depicted in the bar graph in b. Kaplan-Meier curves showing the time to recurrence (TTR) (c) and the cancer specific overall survival (d) in patients with tumors negative or with low expression of HER-3 (IHC 0–1+) versus a high HER-3 expression (IHC 2–3+)