The dark-side of the outside: how extracellular heat shock proteins promote cancer.

In addition to exerting several essential house-keeping activities in the cell, heat shock proteins (HSPs) are crucial players in a well-structured molecular program activated in response to stressful challenges. Among the different activities carried out by HSPs during emergency, they reach the extracellular milieu, from where they scout the surroundings, regulate extracellular protein activity and send autocrine and paracrine signals. Cancer cells permanently experience stress conditions due to their altered equilibrium and behaviour, and constantly secrete heat shock proteins as a result. Other than supporting anti-tumour immunity, extracellular heat shock proteins (eHSPs), can also exacerbate cancer cell growth and malignancy by sustaining different cancer hallmarks. eHSPs are implicated in extracellular matrix remodelling, resistance to apoptosis, promotion of cell migration and invasion, induction of epithelial to mesenchymal transition, angiogenesis and activation of stromal cells, supporting ultimately, metastasis dissemination. A broader understanding of eHSP activity and contribution to tumour development and progression is leading to new opportunities in the diagnosis and treatment of cancer.

The one thousand and one chaperones of the NF-κB pathway.

The NF-κB pathway represents a crucial signaling mechanism in sensing and integrating a multitude of environmental and intracellular stimuli and directing a coordinated response that from the cellular level may impact on the entire organism. A plethora of chaperone proteins work at multiple steps of the pathway, from membrane receptor activation to transcription factor binding to DNA. Indeed, chaperones are required to assist protein conformational changes, to assemble supramolecular complexes and to regulate protein ubiquitination, required for pathway activation. Some chaperones acquired a role as integral components of the signaling complexes, needed for signal progression. Here we describe the chaperones involved in the NF-κB pathway and their specific roles in the different contexts.

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.

miR-15a targets the HSP90 co-chaperone Morgana in chronic myeloid leukemia.

Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients' bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients.

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/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.

IκBα targeting promotes oxidative stress-dependent cell death.

BACKGROUND: Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy. METHODS: We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions. RESULTS: Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis. CONCLUSIONS: NFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.

Targeting the Extracellular HSP90 Co-Chaperone Morgana Inhibits Cancer Cell Migration and Promotes Anticancer Immunity.

HSP90 is secreted by cancer cells into the extracellular milieu, where it exerts protumoral activities by activating extracellular substrate proteins and triggering autocrine signals through cancer cell surface receptors. Emerging evidence indicates that HSP90 co-chaperones are also secreted and may direct HSP90 extracellular activities. In this study, we found that the HSP90 co-chaperone Morgana is released by cancer cells and, in association with HSP90, induces cancer cell migration through TLR2, TLR4, and LRP1. In syngeneic cancer mouse models, a mAb targeting Morgana extracellular activity reduced primary tumor growth via macrophage-dependent recruitment of CD8+ T lymphocytes, blocked cancer cell migration, and inhibited metastatic spreading. Overall, these data define Morgana as a new player in the HSP90 extracellular interactome and suggest that Morgana may regulate HSP90 activity to promote cancer cell migration and suppress antitumor immunity. SIGNIFICANCE: This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaperone Morgana to inhibit metastasis formation and enhance the CD8+ T-cell-mediated antitumor immune response.

The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma.

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Correction to: The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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.

The IKK/NF-κB signaling pathway requires Morgana to drive breast cancer metastasis.

NF-κB is a transcription factor involved in the regulation of multiple physiological and pathological cellular processes, including inflammation, cell survival, proliferation, and cancer cell metastasis. NF-κB is frequently hyperactivated in several cancers, including triple-negative breast cancer. Here we show that NF-κB activation in breast cancer cells depends on the presence of the CHORDC1 gene product Morgana, a previously unknown component of the IKK complex and essential for IκBα substrate recognition. Morgana silencing blocks metastasis formation in breast cancer mouse models and this phenotype is reverted by IκBα downregulation. High Morgana expression levels in cancer cells decrease recruitment of natural killer cells in the first phases of tumor growth and induce the expression of cytokines able to attract neutrophils in the primary tumor, as well as in the pre-metastatic lungs, fueling cancer metastasis. In accordance, high Morgana levels positively correlate with NF-κB target gene expression and poor prognosis in human patients.

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.

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.