Federica Fusella

ERK1/2 activation in heart is controlled by melusin, focal adhesion kinase and the scaffold protein IQGAP1.

Extracellular signal-regulated kinase 1/2 (ERK1/2) signalling is a key pathway in cardiomyocyte hypertrophy and survival in response to many different stress stimuli. We have previously characterized melusin as a muscle-specific chaperone protein capable of ERK1/2 signalling activation in the heart. Here, we show that in the heart, melusin forms a supramolecular complex with the proto-oncogene c-Raf, MEK1/2 (also known as MAPKK1/2) and ERK1/2 and that melusin-bound mitogen-activated protein kinases (MAPKs) are activated by pressure overload. Moreover, we demonstrate that both focal adhesion kinase (FAK) and IQ motif-containing GTPase activating protein 1 (IQGAP1), a scaffold protein for the ERK1/2 signalling cascade, are part of the melusin complex and are required for ERK1/2 activation in response to pressure overload. Finally, analysis of isolated neonatal cardiomyocytes indicates that both FAK and IQGAP1 regulate melusin-dependent cardiomyocyte hypertrophy and survival through ERK1/2 activation.

Morgana and melusin: two fairies chaperoning signal transduction.

Chaperones and scaffold proteins are key elements involved in controlling the assembly of molecular complexes required for coordinated signal transduction. Here we describe morgana and melusin, two phylogenetically conserved chaperones that cooperate with Hsp90 and regulate signal transduction in important physiopathological processes. While morgana is ubiquitously expressed, melusin expression is restricted to striated muscles. Despite high sequence homology, the two chaperones have distinct functions. Morgana controls genomic stability by regulating the centrosome cycle via ROCKII kinase. Melusin, however, organizes ERK signal transduction in cardiomyocytes and regulates cardiac compensatory hypertrophy in response to different stress stimuli.

Morgana/CHP-1 is a novel chaperone able to protect cells from stress.

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

Morgana acts as an oncosuppressor in chronic myeloid leukemia

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

Morgana acts as a proto‐oncogene through inhibition of a ROCK–PTEN pathway

Morgana/CHP‐1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down‐regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH‐3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down‐regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy. Copyright

The double face of Morgana in tumorigenesis

Morgana is a chaperone protein able to bind to ROCK I and II and to inhibit their kinase activity. Rho kinases are multifunctional proteins involved in different cellular processes, including cytoskeleton organization, centrosome duplication, cell survival and proliferation. In human cancer samples Morgana appears to be either downregulated or overexpressed, and experimental evidence indicate that Morgana behaves both as an oncosuppressor and as a proto-oncogene. Our most recent findings demonstrated that if on the one hand low Morgana expression levels, by inducing ROCK II hyperactivation, cause centrosome overduplication and genomic instability, on the other hand, Morgana overexpression induces tumor cell survival and chemoresistance through the ROCK I-PTEN-AKT axis. Therefore, Morgana belongs to a new class of proteins, displaying both oncogenic and oncosuppressor features, depending on the specific cellular context.

Escaping NK cells and recruiting neutrophils: How Morgana/NF-κB signaling promotes metastasis

ABSTRACT Cancer cells escape immune surveillance and induce immune cell aberrant activation to support tumour growth and progression. We recently reported that Morgana/NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling in breast cancer cells is responsible for NK (Natural Killer) cell inactivation and neutrophil recruitment in the primary tumour and in the lung pre-metastatic niche.

Abstract P1-07-27: Neutrophil elastase modulates breast cancer progression by fostering collective cell detachment and tumor emboli dissemination

Proteases constitute a large family of enzymes involved in several processes, including degradation and remodeling of the extracellular matrix to drive dissemination of cancer cells into adjacent tissue. Within this large family, the serine protease neutrophil elastase (NE) has been proven clinically meaningful in breast cancer. Indeed, high levels of NE correlate with poor outcome and endocrine resistance in breast cancer patients. In an experimental model it has been demonstrated that estrogen receptor positive (ER+) MCF7 breast cancer cells grow in suspension as 3D-spheroids in NE-addicted medium, thus resembling the micropapillae of a human breast cancer characterized by high propensity to metastasize, i.e. the micropapillary carcinoma. We hypothesized that NE may produce disarrangement of tumor cell adhesion to the substrate fostering neoplastic lymphovascular invasion (LVI) and metastasis. ER+/E-Cadherin (E-CAD)+/HER2- (MCF7, T-47D, ZR-75-1), ER+/E-CAD+/HER2+ (BT-474), ER-/E-CAD-/HER2+ (SK-BR-3) and ER-/E-CAD-/HER2- (MDA-MB-231) cells were grown with serine proteases (NE, cathepsin-G), hyaluronidase and collagenase. NE and cathepsin-G led to 3D-spheroid formation of ER+/E-CAD+ cells only. In 3D-spheroids from MCF7 cells grown with NE the luminal Epithelial Membrane Antigen (EMA) lined the external border of cell clusters, which faced cancer associated fibroblasts in co-cultures experiments, thus recapitulating the inverted polarity of MPCs. MCF7 3D-spheroids were tamoxifen resistant. Injection of NE in tumors of MCF7 cells in SCID mice triggered neoplastic cluster detachment, micropapillae, vascular emboli similar to 3D-spheorids and metastases. In a cohort of human breast carcinomas with LVI the MCF7-3D-spheroid-alike pattern was the most prevalent in tumor emboli. Immunohistochemical NE expression was mainly detected in polymorphous neutrophilic granulocytes (PMNs) within vessels and in the stroma and PMNs were significantly higher in breast carcinomas with LVI. In fully developed metastases within lymph-nodes, which reverted the EMA expression as in the primary tumor, no NE+ PMNs were observed. Our results may explain why high levels of NE negatively act on patient prognosis by creating a favorable environment for breast cancer invasion and metastasis. Citation Format: Caterina Marchio, Laura Annaratone, Davide Balmativola, Maria Stella Scalzo, Stefania Bolla, Silvia Grasso, Isabella Russo, Federica Fusella, Luigia Macri, Mara Brancaccio, Paola Defilippi, Anna Sapino. Neutrophil elastase modulates breast cancer progression by fostering collective cell detachment and tumor emboli dissemination [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-27.

P11.46 Morgana overexpression confers resistance from apoptotic cell death

Resumen del poster presentado al 36th FEBS Congress celebrado en Torino (Italia) del 25 al 30 de Junio de 2011.-- et al.