Abstract A5: Can activation of the UPR lead to Trastuzumab resistance?
Authors: Kumandan Sreekanth , Mahadevan Navin R. , DeLaney Alexandra , Zanetti Maurizio 
Published: March 2013

Abstract
HER2+ breast cancer accounts for 25–30% of patients with breast cancer. HER2+ breast cancer responds poorly to traditional hormone and chemotherapy but responds to treatment with the humanized IgG antibody, trastuzumab, which targets HER2 and disables homodimer formation and consequent downstream growth signaling. When used in conjunction with other forms of therapy, trastuzumab induces initial clinical remission and reduces the probability of recurrence. However, trastuzumab becomes ineffective over time in a significant number of cases (trastuzumab resistance), leaving patients with few viable treatment options. Several mechanisms have been proposed to explain trastuzumab resistance. These include (1) the upregulation and signaling through other members of the HER family, such HER3 and HER4; (2) steric hindrance of trastuzumab binding by overexpressed MUC-4; (3) compensatory signaling through other growth signaling receptors, e.g., IGF-R; and (4) alternative downstream signaling that reactivates the PI3K/AKT pathway. For example, it has been shown that the loss of PTEN, a potent negative regulator of PI3K/AKT signaling, results in the loss of trastuzumab sensitivity in breast cancer cells. Another yet unappreciated source of PI3K/AKT activation is the unfolded protein response (UPR), a cellular signaling mechanism that adapts to disruption of endoplasmic reticulum homeostasis evoked by accumulation of un/misfolded proteins. The UPR has been identified as a key cell-intrinsic regulator of tumor survival, promoting tumorigenesis during hypoxia and nutrient starvation as occurs in the tumor microenvironment. A downstream target of the UPR is lipocalin 2 (LCN2), a small chaperone which functions as an iron transporter. As a novel oncoprotein, LCN2 has been shown to be crucially required in models of mammary cancers, including Her2+ cancer. LCN2 is able to promote EMT in breast cancer cells in vitro, and LCN2 levels in bodily fluids correlate positively with the progression of breast cancer in humans.
We thus hypothesized that, in HER2+ cancer cells, the UPR could lead to the re-activation of downstream targets such as PI3K/AKT and LCN2, possibly leading to trastuzumab resistance. Treatment of HER2+ SKBr3 human breast cancer cells with trastuzumab significantly inhibited the basal expression of LCN2. The induction of a UPR with thapsigargin completely abrogated trastuzumab-mediated LCN2 downregulation, and the inhibition of the UPR with phenyl-4 butyric acid (PBA) resulted in the near complete restoration of trastuzumab sensitivity. The addition of LY294002, a PI3K/AKT inhibitor, to trastuzumab-treated/UPR-induced SKBr3 cells, only partially reduced the upregulation of LCN2, indicating that UPR-mediated activation of LCN2 as a downstream target occurs in part via PI3K/AKT-independent mechanisms. Taken together, these results suggest that the upregulation of the UPR in the tumor microenvironment may constitute an alternative trigger of trastuzumab resistance in HER2+ cancer cells. Thus, targeting the UPR may represent a new form of adjuvant therapy for HER2+ breast cancer.