AlphaB-crystallin and breast cancer: role and possible therapeutic strategies.

AlphaB-crystallin (HSPB5) is one of the most prominent and well-studied members of the small heat shock protein (sHsp) family. To date, it is known that this protein modulates significant cellular processes and therefore, it is not surprising that its deregulation is involved in various human pathologies, including cancer diseases. Despite the pathogenic significance of HSPB5 in cancer and its regulatory mechanism related to aggressiveness is poorly understood, several reports describe the association of breast carcinoma progression with HSPB5, whose expression is also considered an independent predictor of breast cancer metastasis to the brain. Indeed, numerous authors indicate HSPB5 as a new valuable biomarker for clinicopathological parameters and poor prognosis in breast cancer. Considering the cytoprotective, anti-apoptotic, pro-angiogenic, and pro-metastatic properties of the sHsps, it is not surprising that they are considered as promising targets for anticancer treatment, even though, at present, a deeper understanding of their mode of action is needed to allow the development of precise therapeutic interventions. Data on the direct inhibition of different sHsps demonstrate promising results in cancer pathologies; however, specific strategies against HSPB5 have not been considered. This review highlights the most relevant findings on HSPB5 and its role in breast cancer, as well as the possible strategies in using HSPB5 inhibition for therapeutic purposes.

MiRNA-671-5p Promotes prostate cancer development and metastasis by targeting NFIA/CRYAB axis.

Prostate cancer (PCa) is the second cause of death due to malignancy among men, and metastasis is the leading cause of mortality in patients with PCa. MicroRNAs (miRNAs) play important regulatory roles in tumor development and metastasis. Here, we identified 13 miRNAs related to PCa metastasis by bioinformatics analysis. Moreover, we found that miR-671-5p was increased in metastatic PCa tissues, and its high expression indicated poor prognosis of PCa. MiR-671-5p could facilitate PCa cells proliferation, migration, and invasion in vitro and vivo. We confirmed that miR-671-5p directly bound to the 3' untranslated regions of NFIA mRNA, and NFIA directly bound to the CRYAB promoter. High expression of NFIA and CRYAB negatively correlated with the advanced clinicopathological characteristics and metastasis status of PCa patients. Our study demonstrated that miR-671-5p promoted PCa development and metastasis by suppressing NFIA/ CRYAB axis.

A monoclonal antibody targeted to the functional peptide of αB-crystallin inhibits the chaperone and anti-apoptotic activities.

αB-Crystallin is a member of the small heat shock protein family. It is a molecular chaperone and an anti-apoptotic protein. Previous studies have shown that the peptide (73DRFSVNLDVKHFSPEELKVKV93, hereafter referred to as peptain-1) from the core domain of αB-crystallin exhibits both chaperone and anti-apoptotic properties similar to the parent protein. We developed a mouse monoclonal antibody against peptain-1 with the aim of blocking the functions of αB-crystallin. The antibody reacted with peptain-1, it did not react with the chaperone peptide of αA-crystallin. The antibody strongly reacted with human recombinant αB-crystallin but weakly with Hsp20; it did not react with αA-crystallin or Hsp27. The antibody specifically reacted with αB-crystallin in human and mouse lens proteins but not with αA-crystallin. The antibody reacted with αB-crystallin in human lens epithelial cells, human retinal endothelial cells, and with peptain-1 in peptain-1-transduced cells. Unlike the commercial antibodies against αB-crystallin, the antibody against peptain-1 inhibited the chaperone and anti-apoptotic activities of peptain-1. The antibody might find use in inhibiting αB-crystallin's chaperone and anti-apoptotic activities in diseases where αB-crystallin is a causative or contributing factor.

Discovery of structure-based small molecular inhibitor of αB-crystallin against basal-like/triple-negative breast cancer development in vitro and in vivo.

αB-crystallin (CRYAB) is present at a high frequency in poor prognosis basal-like breast tumours, which are largely absent of oestrogen, progesterone receptors and HER2 known as triple-negative breast cancer (TNBC). CRYAB functions as a molecular chaperone to bind to and correct intracellular misfolded/unfolded proteins such as vascular endothelial growth factor (VEGF), preventing non-specific protein aggregations under the influence of the tumour microenvironment stress and/or anti-cancer treatments including bevacizumab therapy. Directly targeting CRYAB can sensitize tumour cells to chemotherapeutic agents and decrease tumour aggressiveness. However, growing evidence shows that CRYAB is a critical adaptive response element after ischemic heart disease and stroke, implying that directly targeting CRYAB might cause serious unwanted side effects. Here, we used structure-based molecular docking of CRYAB and identified a potent small molecular inhibitor, NCI-41356, which can strongly block the interaction between CRYAB and VEGF165 without affecting CRYAB levels. The disruption of the interaction between CRYAB and VEGF165 elicits in vitro anti-tumour cell proliferation and invasive effects through the down-regulation of VEGF signalling in the breast cancer cells. The observed in vitro anti-tumour angiogenesis of endothelial cells might be attributed to the down-regulation of paracrine VEGF signalling in the breast cancer cells after treatment with NCI-41356. Intraperitoneal injection of NCI-41356 greatly inhibits the tumour growth and vasculature development in in vivo human breast cancer xenograft models. Our findings provide 'proof-of-concept' for the development of highly specific structure-based alternative targeted therapy for the prevention and/or treatment of TNBC.

Beneficial effects of αB-crystallin in spinal cord contusion injury.

αB-crystallin is a member of the heat shock protein family that exerts cell protection under several stress-related conditions. Recent studies have revealed that αB-crystallin plays a beneficial role in a mouse model of multiple sclerosis, brain ischemia, and Alexander disease. Whether αB-crystallin plays a role in modulating the secondary damage after CNS trauma is not known. We report here that αB-crystallin mediates protective effects after spinal cord injury. The levels of αB-crystallin are reduced in spinal cord tissue following contusion lesion. In addition, administration of recombinant human αB-crystallin for the first week after contusion injury leads to sustained improvement in locomotor skills and amelioration of secondary tissue damage. We also provide evidence that recombinant human αB-crystallin modulates the inflammatory response in the injured spinal cord, leading to increased infiltration of granulocytes and reduced recruitment of inflammatory macrophages. Furthermore, the delivery of recombinant human αB-crystallin promotes greater locomotor recovery even when the treatment is initiated 6 h after spinal cord injury. Our findings suggest that administration of recombinant human αB-crystallin may be a good therapeutic approach for treating acute spinal cord injury, for which there is currently no effective treatment.

αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects muscle myoblasts from TNF-α induced cytotoxicity.

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB. We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKß and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.

Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by alpha-crystallin-B.

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.

αB-crystallin is elevated in highly infiltrative apoptosis-resistant glioblastoma cells.

We have previously established two distinct glioma phenotypes by serial xenotransplantation of human glioblastoma (GBM) biopsies in nude rats. These tumors undergo a gradual transition from a highly invasive nonangiogenic to a less-invasive angiogenic phenotype. In a protein screen to identify molecular markers associated with the infiltrative phenotype, we identified α-basic-crystallin (αBc), a small heat-shock protein with cytoprotective properties. Its increased expression in the infiltrative phenotype was validated by immunohistochemistry and Western blots, confirming its identity to be tumor-derived and not from the host. Stereotactic human GBM biopsies taken from MRI-defined areas verified stronger αBc expression in the infiltrative edge compared to the tumor core. Cell migration assays and immunofluorescence staining showed αBc to be expressed by migrating cells in vitro. To determine αBc function, we altered its expression levels. αBc siRNA depletion caused a loss of migrating tumor cells from biopsy spheroids and delayed monolayer wound closure. In contrast, glioma cell migration in a Boyden chamber assay was unaffected by either αBc knockdown or overexpression, indicating that αBc is not functionally linked to the cell migration machinery. However, after siRNA αBc depletion, a significant sensitization of cells to various apoptotic inducers was observed (actinomycin, tumor necrosis factor α, and TNF-related apoptosis-inducing ligand [TRAIL]). In conclusion, αBc is overexpressed by highly migratory glioma cells where it plays a functional role in apoptosis resistance.

The effect of small molecules in modulating the chaperone activity of alphaB-crystallin against ordered and disordered protein aggregation.

Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as alphaB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of alphaB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of alphaB-crystallin against aggregation of the disease-related amyloid fibril forming protein alpha-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.

Hsp27 (HspB1) and alphaB-crystallin (HspB5) as therapeutic targets.

Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.