Phosphorylated ribosomal S6 (p-rpS6) as a post-treatment indicator of HER2 signalling targeted drug resistance

Abstract

Objective: To identify clinically relevant predictive biomarkers of trastuzumab resistance.

Material and methods: MTT, FACS assays, immunoblotting and immunocytochemistry were used to phenotypically characterize drug responses of two cell models BT474R and SKBR3R. Student's t-test and Spearman's correlation were applied for statistic analysis.

Results: The activity of a downstream effector of the HER2 pathway phosphorylated ribosomal protein S6 (p-rpS6), was suppressed by trastuzumab in the parental cell lines yet remained unchanged in the resistant cells following treatment. The level of p-rpS6 was inversely correlated to the drug induced growth inhibition of trastuzumab-resistant cells when they are treated with selected HER2 targeting drugs.

Conclusion: p-rpS6 is a robust post-treatment indicator of HER2 pathway-targeted therapy resistance.

Keywords: Biomarker; breast cancer; cell model; drug response; trastuzumab resistance.

Figure 1.

Trastuzumab sensitivities of BT474R, SKBR3R and their parental cell lines BT474 and SKBR3. (a) Dose response curves of the four cell lines after 7 days trastuzumab treatments. Cell viability is the percentage of viable cells with respect to cells untreated with trastuzumab. (b) GraphPad Prism 5 modelled IC₅₀ values of the resistant sublines BT474R and SKBR3R and their parental cells BT474 and SKBR3 to trastuzumab (μg/ml). Bars are means of three independent experiments and standard deviations.

Figure 2.

Trastuzumab-resistant cells BT474R and SKBR3R expressed more aggressive phenotypes than parental BT474 and SKBR3. (a) Cell proliferation measured by EdU incorporation. EdU incorporated cells are shown in green. Cell nuclei were stained blue (DAPI). A: BT474, B: BT474+T, C: BT474R, D: BT474R+T, E: SKBR3, F: SKBR3+T, G: SKBR3R, H: SKBR3R+T. (b) Quantification of the data from a Student’s t-test were used to process the data. [* significant (t<0.05); ** most significant (t<0.01)]. Experiments were repeated at least three times. Bars represent mean and standard deviation values. Ctl: control untreated; T: trastuzumab treated. (c) FACS data demonstrates the G0/G1 fraction of the cell cycle was increased and S-phase was decreased after exposing to trastuzumab in BT474 and SKBR3 cells; in the trastuzumab-resistant BT474R and SKBR3R cells, neither G0/G1 or S-phase was affected by trastuzumab exposure.

Figure 3.

p-rpS6 expression was not suppressed by trastuzumab treatment in trastuzumab-resistant BT474R and SKBR3R cells, but it was decreased in parental BT474 and SKBR3 cells. (a) Immunoblotting of whole cell lysate of key effectors in HER2 signalling pathway demonstrating altered expression of some markers in response to trastuzumab treatment. (b) Immunocytochemistry staining of p-rpS6 in BT474, SKBR3 and trastuzumab-resistant BT474R and SKBR3R cells. P-rpS6 was stained green in the cytoplasm, cell nuclei were stained with DAPI (blue lighter).

Figure 4.

Higher intensity of p-rpS6 stain is correlated with a higher percentage of Ki67 positive cell numbers. (a) Dual staining of p-rpS6 (green in the cytoplasm) and Ki67 (red in the nuclei) of BT474R, SKBR3R treated or untreated with trastuzumab in comparison with their parental cells BT474 and SKBR3. (b) Quantified fluorescence intensity data from a fluorescence intensity of p-rpS6 was measured with imageJ, Ki67 positive cells were manually counted.

Figure 5.

Decreased p-rpS6 expression correlates with the sensitivity of cells to HER2 signalling targeted drugs in BT474R and SKBR3R cells (r=0.8). (a) Immunoblotting of key proteins of HER2 signalling pathway in BT474R cells. (b) Growth inhibition of BT474R by HER2 signalling targeted drugs. Ctl: untreated; T: trastuzumab; R: rapamycin; A: AZD2014; B: BEZ235; E: erotinib; L: lapatinib; M: MK2206; O: OSI-906 (linsitinib). (c) Immunoblotting of key proteins of HER2 signalling pathway in SKBR3R cells. (d) Growth inhibition of SKBR3R by HER2 signalling targeted drugs.

Figure 6.

Synergistic effect of trastuzumab and lapatinib on growth inhibition (cell viability shown with bars) as well as p-rpS6 expression (shown with western blot bands on the top) of BT474R (a) and SKBR3R (b). Synergism was determined by Colby methods (Colby, 1967). T: trastuzumab; L: lapatinib; TL: trastuzumab +lapatinib.