Fig 2: Carfilzomib and bortezomib increase the levels of the PEST type protein-tyrosine phosphatase BDP1 (PTPN18) but not PTPN12 and PTPN13(A) BT474 and (B) MDA-MB-361 cells were cultured in the presence of the indicated carfilzomib (right panels) and bortezomib (left panels) concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies.

Fig 3: Schematic model of how proteasome inhibitors disrupt bi-directional HER2/ERa cross-talk signaling pathways in lapatinib-resistant ER+/HER2+ breast cancer cellsSchematic overview of how PIs might counteract mechanisms that lead to lapatinib resistance in ERa+/HER2+ breast cancer cells, based on the results of this report and the literature. Continued activation of HER2 has a causal role in tumorigenesis, lead to tumor progression [1]. Aberrant signaling through HER2 and other members of the HER-family mediates endocrine resistance in ERa positive breast cancer [18, 19], while preclinical and clinical studies revealed that ERa co-expression attenuates the efficiency of anti-HER2 therapies [8, 9, 12, 13, 14]. Previously, published reports and our own observations demonstrate that in ERa and HER2-positive breast cancer cells that are initially sensitive to lapatinib, increased expression of ERa and enhanced ERa signaling is evident upon acquisition of resistance to lapatinib [15]. Thus, up-regulation of ERa expression and/or activity can function as an escape mechanism that leads to resistance to anti-HER2 therapy in HER2+/ER+ breast cancer patients. Thus, in HER2+/ER+ breast cancer cells, either ERa or HER2 can promote proliferation and survival. These studies and our observations demonstrate that concomitant inhibition of both HER2 and ERa is needed to achieve the best antitumor activity in ER+/HER2+ breast cancer. Our experiments demonstrate that carfilzomib and bortezomib strongly reduce phosphorylation and activation of HER2 most likely through protecting HER2-specific PEST protein tyrosine phosphatases such as BDP1 from proteasomal destruction. Additionally, both PIs strongly reduce expression of ERa protein expression. Thus, PIs might be effective in the treatment of HER2+/ERa+ breast cancer by disrupting bi-directional HER2/ERa cross-talk signaling pathways.

Fig 4: Carfilzomib and bortezomib-mediated inhibition of HER2 autophosphorylation, blockade of ERa expression and increase in BDP1 levels correlates temporally with induction of apoptotic cell death(A) Carfilzomib and bortezomib induce cell death in a time-dependent manner. BT474 cells were cultured in the presence or absence of the indicated carfilzomib or bortezomib concentrations. The percentage of SubG1 cells was evaluated at different time points using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented. (B) BT474 cells were cultured in the presence of 250 nM carfilzomib or 25 nM bortezomib. Cells were harvested at the indicated time points. Western blots of protein lysates were probed with the indicated antibodies. (C) Schematic model of a possible molecular network regulated by both proteasome inhibitors, based on the results of the Western blots and the literature. Continued activation of HER2 has a causal role in tumorigenesis and leads to tumor progression [1, 2]. Activation of HER2 triggers autophosphorylation of specific tyrosine residues within its cytoplasmic domain, consequently activating different intracellular signaling pathways such as Ras-MAPK and the PI3K/Akt axis [34] which determine cell growth, cell survival and therapy resistance to endocrine therapies. Different protein tyrosine phosphatases regulate phosphorylation of the HER2 signaling domain and are therefore important key regulators of HER2 activity [35, 36]. The PEST-type protein-tyrosine phosphatase BDP1 inhibits ligand-induced activation of HER2 [36, 37]. PEST sequences cause accelerated degradation by the proteasome/ubiquitin system [38], and PIs therefore increase the amount of BDP1 through blocking its proteasomal destruction. This in turn leads to accelarated dephosporylation and thereby inactivation of HER2 and its downstream target signaling pathways. Aberrant signaling through HER2 and other members of the HER family mediates endocrine resistance in ER+ positive breast cancer. On the other hand ERa co-expression with HER2 attenuates the efficiency of anti-HER2-targeted therapies. These findings indicate that HER2 and ERa act in concert to allow breast cancer cells to escape from both anti-ERa and anti-HER2-targeted therapies. Besides inhibiting HER2, PIs also suppress ERa protein expression.