To further study the mechanisms of resistance to HER2-targeted therapies, we developed a panel of over 10 different HER2-positive human breast cancer cell lines de novo or acquired resistant to T, L, or L + T. We find that while de novo and acquired resistance to T is associated with reactivation of the HER2 pathway, resistance to L or L + T is due to alternative signaling through the ER pathway, providing clues to strategies to improve HER2-targeted therapies in the clinic. Here, ESR1 is linked to breast cancer.