HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK

Abstract

Background: HER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood.

Methods: Using Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively.

Results: All three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively.

Conclusion: We have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.

Fig 1. Schematic representation of HER-dependent signal transduction pathways associated with cell proliferation and apoptosis.

Fig 2. Dose-dependent growth inhibition in response to 72h exposure to lapatinib (a), canertinib (b), or afatinib (c) on malignant human cell lines with no HER2 expression (Jurkat), with moderate HER2 expression (T47D) and with HER2 overexpression (SKBR3).

Means ± SD, n = 3.

Fig 3. Effect of canertinib (white bar), lapatinib (light grey bar) and afatinib (dark grey bar) on activation (phosphorylation) of ERK/MAPK1/2, STAT3, CREB, STAT5A/B, p70 S6 kinase, IkBa, JNK, and p38 in SKBR3, T47D and Jurkat cells.

Serum-depleted cells were exposed for 6 h to vehicle (0.1% DMSO) or to 1 μM of the TKIs followed by a 3-min challenge with 100 ng/ml EGF and 1 nM HRGb1 and subjected to Milliplex assays. Results are shown relative to vehicle control (100%). Means ± SD.

Fig 4. Dose-dependent growth inhibition in response to 72h exposure to the JNK inhibitor SP600125 (a) or the STAT5 Inhibitor II IQDMA (b) on SKBR3 and T47D human breast cancer cells.

Means ± SD.

Fig 5. Dose-dependent effect of SP600125 when given for 72h (a) and time-dependent effect of 5μM canertinib after 2 and 6 hours (b) on apoptosis as examined by Milliplex immunoassay for detection of active (cleaved) caspase 3 in SKBR3 and T47D cells.

Results are shown relative to vehicle (0.1% DMSO) control. Means ± SD.

Fig 6. Effect of the HER-TKI canertinib on the expression of apoptosis-related PARP-1 cleavage products as demonstrated by Western blot analysis of SKBR3 cells grown for 48h (lanes 1–3) and 72h (lanes 4–6) in the absence (lanes 1 and 4), or in the presence of 1μM (lanes 2 and 5) and 10 μM (lanes 3 and 6) canertinib.

Actin was probed on a separate membrane to roughly estimate protein loading.

Fig 7. Effect of the HER-TKI canertinib on the expression of apoptosis-related PARP-1 cleavage products as demonstrated by Western blot analysis of T47D cells grown for 48h (lanes 1–3) and 72h (lanes 4–6) in the absence (lanes 1 and 4), or in the presence of 1μM (lanes 2 and 5) and 10 μM (lanes 3 and 6) canertinib.

Actin was probed on a separate membrane to roughly estimate protein loading.