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14 pages, 8 figures., 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., We acknowledge funding from the European Community, the Fundación Ramón Areces, and the Spanish Ministry of Education and Culture. A.E.-O. was supported by a postdoctoral contract from the Spanish Ministry of Education and Culture. E.D.-R. and L.Y. were supported by a predoctoral fellowship from the same ministry.

Under a Creative Commons license.-- et al., Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas., This work was supported by grants from Fundación Leticia Castillejo Castillo and Ministerio de Economía y Competitividad (SAF2009-07329 and SAF2012-30862) and grant JCCM PPII10-0141-040 to R.S.P., grant FIS PI080432 and a grant from Fundación Para la Investigación en Urología to A.S.S.-S., grant FISCAM PI2007-38 to J.M.G.-B., and grant FIS PS09/00868 to A.E.-O. R.S.P. and A.P. Research Institutes and the work carried out in their laboratories receive support from the European Community through the Regional Development Funding Program (FEDER).

Breast cancer (BC) is a heterogeneous disease in which several distinct genetic and histopathological types have been defined. Triple-negative breast cancer (TNBC) lacking ER/PR and HER-2 is the most aggressive and untreatable form. In the case of HER2+ tumors, and despite the emergence of trastuzumab and lapatinib, in the metastatic setting these tumors remain incurable due to the innate or acquired drug resistance. A number of kinases are implicated in the proliferation and survival of breast cancer cells. Here, we characterize the antitumor activity of the multikinase inhibitor TG02 in TNBC and HER2+ BC. TG02 is a novel drug with a unique kinase inhibitory spectrum, targeting both the cell cycle regulatory CDKs 1/2 and the transcriptional regulators CDKs 7/9, along with the emerging oncogenic MAPK ERK5 and the DNA damage response mediator CDK5, all within a narrow low nanomolar potency range. In panels of breast cancer cell lines, TG02 was potently and consistently antiproliferative at low (100nM) and proapoptotic at moderate (250-1000nM), clinically-achievable concentrations. In TNBC cells, the drug usually induced a G2/M arrest, whereas blockade of cell cycle progression at the G1/S boundary was more common in HER2+ lines. Simultaneous multikinase inhibition was demonstrated in several cellular models, showing that cell cycle arrest induced by inhibition of CDK1 and CDK2 progresses to apoptosis associated with profound depletion of the short-lived survival proteins of the Bcl-2 and IAP families downstream of CDK9-dependent loss of RNA polymerase II-driven transcription. TG02 was synergistic in combination with chemotherapy in TNBC models in vitro and in vivo and with trastuzumab and lapatinib in HER2-overexpressing cell lines. A majority of TNBC and HER2+ lines expressed constitutively active ERK5, which was efficiently inhibited by TG02 and may contribute to the antitumor activity of the drug. A robust DNA damage response including CHK1/2 activation and formation of H2AX foci was induced in TNBC cells, presumably due to inhibition of CDK5 by TG02, so we will also report on the mechanism-based combination of TG02 with PARP inhibitors in this setting. These data support clinical development of this compound for the treatment of TNBC and HER2+ BC, especially in combination with standard of care treatments used in these breast neoplasias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2830. doi:1538-7445.AM2012-2830

Breast cancer represents a heterogeneous disease in which several distinct genetic and histopathological types have been defined. Depending on the latter, treatment is established based on the presence of estrogen or progesterone receptors, the transmembrane tyrosine kinase HER2, or the absence of these markers. One of the most aggressive subtypes of breast cancer is offered by the HER2 positive tumors. Treatment of these tumors is based on agents, such as the monoclonal antibody trastuzumab, or the small molecule tyrosine kinase inhibitor lapatinib, which target HER2. The incorporation of these therapies to HER2 positive tumors has improved patient outcome. However, in the metastatic setting HER2 positive tumors still represent an important oncological problem. Moreover, some patients are either primary resistant, or acquire resistance to anti-HER2 therapies along time. Therefore, novel therapies are required to fight the disease once disseminated and under instances of anti-HER2 resistance. In addition to HER2, several studies have involved other kinases in the pathophysiology of breast cancer. Because of these facts, we have investigated the antitumoral action of TG02 in HER2 positive breast cancers. TG02 represents a novel molecule with a unique kinase inhibitory sprectrum. TG02 targets several receptor tyrosine kinases, such as FLT3 and various cytosolic serine/threonine kinases, such as ERK5, p38, CDK1, CDK2, CDK5, CDK9. We used HER2 overexpressing cell lines SKBR3 and BT474 to explore the action of TG02 on these models of HER2 positive breast cancer. TG02 caused a dose-dependent decrease in MTT metabolization, an assay used to explore cellular proliferation and viability. IC50 values were in the low nanomolar range. The drug also potentiated the action of trastuzumab or lapatinib, with whom TG02 exerted a synergistic action, as indicated by the Chou and Talalay algorithm. In BT474 cells, which bear constitutively active ERK5, treatment with TG02 caused inhibition of ERK5 phosphorylation, at concentrations of 100 nM and above, without affecting HER2 tyrosine phosphorylation. Cell cycle analyzes indicated that TG02 caused a decrease in G0/G1, and an increase in the subG0 region of the histogram. This result suggested that TG02 could trigger apoptotic cell death in BT474 cells. In fact, treatment with TG02 caused a dose-dependent increase in the processing of PARP, which was accompanied by increased annexin V staining. Therefore TG02 may indice cell cycle effects together with an induction of apoptotic cell death. These data support further development of this compound for the treatment of HER2 positive tumors, especially in combination with other standard of care treatments used in these breast neoplasias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A153.

21077 Background: Targeting ErbB2 with the monoclonal antibody trastuzumab (Herceptin) has shown to be active in breast cancer. However, a proportion of patients do not benefit from this treatment due to primary resistance. Mechanisms proposed for this resistance include activation of other receptors involved in proliferation as is the case of the insulin-like growth factor receptor (IGF-1R).Different strategies have been proposed for the inhibition of IGF-1R such as antibodies or small tyrosine-kinase inhibitors. In this study we analyzed the mechanism of action and the antiproliferative activity of an specific IGF-1R tyrosine-kinase inhibitor termed NVP-AEW541 alone and in combination with Herceptin. Methods: MCF7, SKBR3, BT474, and MDA-MB-231 breast cancer cells were used for this study. Proliferation and apoptosis were analyzed by MTT uptake assays or Annexin-V-FITC, respectively. The levels of different signalling proteins were analyzed by Western blotting. Microarray studies were performed using the HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA). Real time quantitative PCR was used to confirm the differentially regulated genes. Results: The IGF-1R was expressed in all the cell lines studied. Exogenous addition of IGF-1 increased the tyrosine phosphorylation of IGF-1R, and this was prevented by preincubation with NVP- AEW541. The latter also decreased MTT uptake, and provoked a slight increase in apoptosis of BT474 cells. Combination of NVP-AEW541 and Herceptin had a synergistic effect on the inhibition of BT474 proliferation. Cell cycle analyses indicated that the combined addition of NVP- AEW541 and Herceptin increased the number of cells in the G0/G1 phases. These data were complemented with an increase in p27 levels upon treatment with both compounds. Microarray analyses identified several genes whose expression was modified by each agent alone and by the combination. Conclusions: Our results indicate that the combined targeting of two receptor tyrosine kinases known to play important roles in tumor cell proliferation may be more efficient than individual targeting of these receptors. No significant financial relationships to disclose.

The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca2+. Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-α and pro-neuregulin-α2c, the receptor tyrosine kinase, TrkA, and the β-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca2+ increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction.