| Approximately 20% of breast cancer tumors over-express the human epidermal growth factor receptor (HER2). Treatment with trastuzumab, a humanized HER2 monoclonal antibody, has improved patient survival and remains the standard of care for patients with HER2+ breast cancer in the adjuvant and metastatic settings. However, the majority of these patients relapse within one year of treatment, indicating a need to overcome acquired drug resistance. This body of work further examines the role of t-Darpp, a truncated form of the dopamine and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32), in acquired trastuzumab resistance. Although over-expression of t-Darpp is sufficient to confer trastuzumab resistance in HER2+ breast cancers, little is known about its regulation or mechanism of action. We examine the role of phosphorylation of T75, a key threonine residue of t-Darpp, and demonstrate that both cdk1 and cdk5 are able to phosphorylate this site. Further, we demonstrate that over-expression of t-Darpp in resistant cells may cause an increased sensitivity to EGFR tyrosine kinase inhibitors. These cells are also characterized by a more robust activation of EGFR signaling and greater EGFR stability than parental cells. To better understand the role that phosphorylation plays in resistance, we mutated the T75 residue of t-Darpp to an alanine. We found that T75 phosphorylation may be required for increased PKA activity conferred by t-Darpp and that the T75A-mutated form no longer causes sensitivity to EGFR inhibition or activation of EGFR signaling. Taken together, these results suggest that the over-expression t-Darpp may induce a compensatory shift to EGFR signaling as part of the trastuzumab resistance phenotype. The presence of t-Darpp might predict response to dual HER2/EGFR signaling and its involvement in the EGFR and PKA signaling networks should be explored further. |
