Azucena Esparís-Ogando

Differential shedding of transmembrane neuregulin isoforms by the tumor necrosis factor-α-converting enzyme

The neuregulins (NRGs) are a family of EGF-like factors that activate receptor tyrosine kinases of the ErbB/HER type. Some NRGs are membrane anchored and are released upon cleavage of the ectodomain. Here we have investigated the characteristics of the cleavage of different transmembrane NRG isoforms (proNRG) that diverge in domains that have been implicated in the regulation of the cleavage of other membrane-anchored growth factors. We show that cleavage of proNRGs is complex and generates several cell-bound truncated fragments. Comparison of the resting generation of these truncated fragments between proNRG forms that diverge in the linker region that connects the EGF-like module to the transmembrane domain revealed that proNRG beta 2a was relatively resistant to processing compared to proNRG beta 4a which was processed more efficiently than proNRG alpha 2a. An important role for this linker in proNRG cleavage was supported by deletion analysis of this region that prevented NRG solubilization. Studies aimed at the identification of the proteolytic machinery responsible for proNRG processing indicated that metalloproteases were involved in proNRG processing. This was further supported by the fact that cleavage of proNRG alpha 2c was defective in fibroblasts derived from TACE(-/-) animals that express an inactive form of the metalloprotease TACE.

Activation of ErbB2 by Overexpression or by Transmembrane Neuregulin Results in Differential Signaling and Sensitivity to Herceptin

The ligands of the epidermal growth factor family and their receptors, the ErbB proteins, have been linked to the development of different types of cancer. Particular attention has focused on ErbB2, whose activation may occur by receptor overexpression or by ligand-induced oligomerization with other ErbB receptors. Whether these two modes of ErbB2 activation cause the same biological responses is unknown. Here, we uncovered important differences in the signaling, proliferation rates, and the response to anti-ErbB2 antibodies when comparing MCF7 cells expressing the ligand neuregulin, to MCF7 cells overexpressing ErbB2. Expression of neuregulin caused higher proliferation than ErbB2 overexpression. Transmembrane neuregulin expression was accompanied by constitutive activation of ErbB2, ErbB3, and ErbB4 receptors. ErbB2 overexpression caused tyrosine phosphorylation of ErbB2, whereas ErbB3 and ErbB4 were only slightly tyrosine phosphorylated. Autocrine transmembrane neuregulin also caused constitutive activation of several signaling pathways, such as the Erk1/2, Erk5, and Akt routes, which have been linked to breast cancer cell proliferation. Interestingly, expression of neuregulin increased p21 levels and this was required for the proliferation of MCF7 cells. Treatment with the anti-ErbB2 receptor antibody Herceptin had an inhibitory effect on proliferation only in cells expressing neuregulin but not on cells overexpressing ErbB2, and its inhibitory activity was accompanied by a decrease in p21. These results suggest that Herceptin may also be of help in the treatment of tumors in which neuregulin feeds the tumoral tissue.

Cleavage of the TrkA neurotrophin receptor by multiple metalloproteases generates signalling-competent truncated forms.

The ectodomain of the neurotrophin receptor TrkA has been recovered as a soluble fragment from the culture media of cells by a process that involves endoproteolytic cleavage. This cleavage may be upregulated by several treatments, including NGF treatment or protein kinase C activation. In this report we have investigated the cellular site and proteolytic activities involved in TrkA cleavage, and the effects of ectodomain truncation on signalling. Cleavage occurs when the receptor is at, or near, the cell surface, and it can be prevented by agents that affect protein sorting. Cleavage generates several cell‐bound fragments, and their generation can be differentially blocked by inhibitors, documenting the involvement of multiple plasma membrane metalloendoproteases. The major cell‐bound receptor fragment (i) is tyrosine‐phosphorylated inu2003vivo; (ii) does autophosphorylate inu2003vitro; and (iii) is able to associate with intracellular signalling substrates. Artificial deletion of the TrkA ectodomain results in an active receptor that induced neurite outgrowth in pheochromocytoma cells. Cleavage by this natural cellular mechanism appears thus to serve not only as an outlet of receptor binding fragments, but also to generate signalling‐competent cell‐bound receptor fragments. In the nervous system this ligand‐independent receptor activation could play important roles in the development and survival of neurons.

Mitogen-activated protein kinase-dependent and -independent routes control shedding of transmembrane growth factors through multiple secretases.

Solubilization of a number of membrane proteins occurs by the action of cell-surface proteases, termed secretases. Recently, the activity of these secretases has been reported to be controlled by the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and the p38 mitogen-activated protein kinase (MAPK) routes. In the present paper, we show that shedding of membrane-anchored growth factors (MAGFs) may also occur through MAPK-independent routes. In Chinese-hamster ovary cells, cleavage induced by protein kinase C (PKC) stimulation was largely insensitive to inhibitors of the ERK1/ERK2 and p38 routes. Other reagents such as sorbitol or UV light stimulated MAGF cleavage independent of PKC. The action of sorbitol on cleavage was only partially prevented by the combined action of inhibitors of the p38 and ERK1/ERK2 routes, indicating that sorbitol can also stimulate shedding by MAPK-dependent and -independent routes. Studies in cells devoid of activity of the secretase tumour necrosis factor-alpha-converting enzyme (TACE) indicated that this protease had an essential role in PKC- and ERK1/ERK2-mediated shedding. However, secretases other than TACE may also cleave MAGFs since sorbitol could still induce shedding in these cells. These observations suggest that cleavage of MAGFs is a complex process in which multiple secretases, activated through different MAPK-dependent and -independent routes, are involved.

ERK2, but Not ERK1, Mediates Acquired and “De novo” Resistance to Imatinib Mesylate: Implication for CML Therapy

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.

P3-16-14: Effect of TG02, a Multikinase Inhibitor, on Triple Negative Breast Cancer Cells.

Background Breast cancer is the most common neoplasia in women. Formerly, we reported that the MAPK Erk5 participates in the proliferation of breast cancer cells in vitro, it is overexpressed in the tumours of a number of breast cancer patients, and its overexpression is an independent prognostic marker for disease-free survival. In addition, inhibition of Erk5 sensitized cells to treatments commonly used in the breast cancer clinic. Therefore, Erk5 may represent a novel therapeutic target in breast cancer. Here we describe the preclinical activity of TG02, a novel multi-kinase inhibitor being developed by Tragara Pharmaceuticals, in triple negative breast cancer (TNBC). TG02 presents a unique kinase inhibitory spectrum, combining Erk5 inhibitory properties with inhibition of CDKs and certain receptor tyrosine kinases. Material and methods : The action of TG02 on cell proliferation of TNBC cell lines was carried out by the MTT-based assay, and its action on cell death and cell cycle progression was analyzed by flow cytometry. The expression of different kinases and other proteins implicated in cell cycle, apoptosis and DNA damage responses were analyzed by western blotting. Studies on the action of TG02 in combination with chemotherapy were performed in MDAMB231 and HBL100 cells, and the potency of the combination was quantitated with the Calcusyn software. In vivo studies on the action of TG02 were performed in mice xenografted with the TNBC cell line MDA-MB231. Results : The TNBC cell lines analyzed showed high levels of Erk5 expression, and Erk5 was active under resting conditions in some of them. TG02 inhibited the kinase activity of Erk5 even though TG02 did not affect the Erk5 upstream activating kinase Mek5. TG02 showed an inhibitory effect of phosphorylation of residue Thr732 in the C-terminal tail of Erk5 without affecting the phosphorylation of the activation loop TEY motif. Cell proliferation studies indicated that one group of TNBC cells were very sensitive to the action of this compound (IC50 ≤100 nM) and another group were more resistant. TG02 induced cell cycle arrest at the early G1 and G2 phases of cell cycle, and triggered cell death in MDAMB231 (representative cell line of the most resistant group), and induced a strong effect of apoptosis and a DNA damage response in HCC1187(representative of the most sensitive group). In vitro studies indicated that TG02 sensitizes TNBC cells to chemotherapy, showing additive or synergistic effects depending of the doses. In vivo studies indicated that TG02 exerted a strong antitumoral action in mice bearing MDA-MB231-derived tumours. Conclusions : TNBC cells are very sensitive to TG02, both in vitro and in vivo. The inhibits the kinase activity of Erk5, which, together with the targeting of other kinases, may contribute to the induction of cell cycle arrest or apoptosis in response to the compound in TNBC cells. TG02 synergized with chemotherapy, supporting the possibility of using this drug in combination therapy. Taken together, these preclinical studies establish the bases for the clinical development of this compound for the treatment of TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-14.