The ectodomain of certain transmembrane proteins can be released by the action of cell surface proteases, termed secretases. Here we have investigated how mitogen-activated protein kinases (MAPKs) control the shedding of membrane proteins. We show that extracellular signal-regulated kinase (Erk) acts as an intermediate in protein kinase C-regulated TrkA cleavage. We report that the cytosolic tail of the tumor necrosis factor α-converting enzyme (TACE) is phosphorylated by Erk at threonine 735. In addition, we show that Erk and TACE associate. This association is favored by Erk activation and by the presence of threonine 735. In contrast to the Erk route, the p38 MAPK was able to stimulate TrkA cleavage in cells devoid of TACE activity, indicating that other proteases are also involved in TrkA shedding. These results demonstrate that secretases are able to discriminate between the different stimuli that trigger membrane protein ectodomain cleavage and indicate that phosphorylation by MAPKs may regulate the proteolytic function of membrane secretases.

The four receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes and have also been implicated in tumor generation and/or progression. Activation of ErbB1/EGFR is mainly triggered by epidermal growth factor (EGF) and other related ligands, while activation of ErbB2, ErbB3, and ErbB4 receptors occurs by binding to another set of EGF-like ligands termed neuregulins (NRGs). Here we show that the Erk5 mitogen-activated protein kinase (MAPK) pathway participates in NRG signal transduction. In MCF7 cells, NRG activated Erk5 in a time- and dose-dependent fashion. The action of NRG on Erk5 was dependent on the kinase activity of ErbB receptors but was independent of Ras. Expression in MCF7 cells of a dominant negative form of Erk5 resulted in a significant decrease in NRG-induced proliferation of MCF7 cells. Analysis of Erk5 in several human tumor cell lines indicated that a constitutively active form of this kinase was present in the BT474 and SKBR3 cell lines, which also expressed activated forms of ErbB2, ErbB3, and ErbB4. Treatments aimed at decreasing the activity of these receptors caused Erk5 inactivation, indicating that the active form of Erk5 present in BT474 and SKBR3 cells was due to a persistent positive stimulus originating at the ErbB receptors. In BT474 cells expression of the dominant negative form of Erk5 resulted in reduced proliferation, indicating that in these cells Erk5 was also involved in the control of proliferation. Taken together, these results suggest that Erk5 may play a role in the regulation of cell proliferation by NRG receptors and indicate that constitutively active NRG receptors may induce proliferative responses in cancer cells through this MAPK pathway.

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVβ3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, β3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVβ3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.

The neuregulins represent the largest subclass of polypeptide factors of the epidermal growth factor family of ligands. These molecules are synthesized as membrane-bound, biologically active growth factors that act by binding to the HER/ErbB receptor tyrosine kinases. Preclinical data have indicated that increased expression and function of neuregulins may provoke cancer. Furthermore, neuregulin expression has been detected in several neoplasias, and their presence may correlate with response to treatments that target the HER receptors such as trastuzumab. In addition, the neuregulins have also been implicated in resistance to anti-HER therapies. Therefore, targeting of the neuregulins may be helpful in neoplastic diseases in which these polypeptide factors contribute to tumor generation and/or maintenance.

Triple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model. OVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells. The BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft model, reducing the expression levels of BRD4. Using both in vitro and in vivo approaches, we describe the profound activity of BET-PROTACs in parental and BETi-resistant TNBC models. This data provides options for further clinical development of these agents in TNBC.