Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition

Abstract

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable whereas active PI3K-Akt and MAPK were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFK) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Direct Inhibition of PI3K in Combination with Dual HER2 Inhibitors is Required for Optimal Antitumor Activity in HER2+ Breast Cancer Cells

“…We previously showed that LR cells exhibit continued activation of PI3K signaling despite inhibition of the HER2 tyrosine kinase [25]. To identify mechanisms associated with maintenance of PI3K signaling in these drugresistant cells, we profiled them using the SNaPshot assay [27- Table S1).…”

“…SUM190 cells were purchased from Asterand (Detroit, MI, USA). Lapatinibresistant (LR) cell lines were generated as described previously [25] and cultured in the presence of 1 to 2 μM lapatinib. Lapatinib ditosylate and BIBW2992 were obtained from LC Laboratories (Woburn, MA, USA).…”

Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy

“…2). In particular, SFKs have been shown to be hyperactivated in breast cancer lines whose resistance is associated with the increased expression of certain RTKs, including ErbB2 itself, EGFR, and IGF1R (Rexer et al 2011;Zhang et al 2011), as well as cells that mediate an alternative survival pathway through b1 integrin ). Moreover, SFKs may help drive resistance in cells that overexpress RTKs by initiating a positive feedback loop.…”

Mining the proteome: the application of tandem mass spectrometry to endocrine cancer research

“…However, as observed with CA125 earlier, if high-molecular weight proteins are examined, the presence of PTMs may obscure the data; analyses must therefore be tailored to specific events, such as phosphorylation or glycosylation. It has been demonstrated that resistance to the TKI lapatinib in breast cancer cell lines is linked to increased phosphorylation of Src family kinases (Rexer et al 2011). Further, by treating these cells with Src inhibitors, the sensitivity to lapatinib is restored.…”