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During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Ribonucleoprotein (RNP) granules transport, store, or degrade messenger RNAs, thereby indirectly regulating protein synthesis. Normally, RNP granules are highly dynamic compartments. However, because of aging or severe environmental stress, RNP granules, in particular stress granules (SGs), convert into solid, aggregate-like inclusions. There is increasing evidence that such RNA-protein inclusions are associated with several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD) and Alzheimer’s disease (AD). Thus, understanding what triggers the conversion of RNP granules into aggregates and identifying the cellular players that control RNP granules will be critical to develop treatments for these diseases. In this review article, we discuss recent insight into RNP and SG formation. More specifically, we examine the evidence for liquid-liquid phase separation (LLPS) as an organizing principle of RNP granules and the role of aggregation-prone RNA-binding proteins (RBPs) in this process. We further discuss recent findings that liquid-like SGs can sequester misfolded proteins, which promote an aberrant conversion of liquid SGs into solid aggregates. Importantly, very recent studies show that a specific protein quality control (PQC) process prevents the accumulation of misfolding-prone proteins in SGs and, by doing so, maintains the dynamic state of SGs. This quality control process has been referred to as granulostasis and it relies on the specific action of the HSPB8-BAG3-HSP70 complex. Additional players such as p97/valosin containing protein (VCP) and other molecular chaperones (e.g., HSPB1) participate, directly or indirectly, in granulostasis, and ensure the timely elimination of defective ribosomal products and other misfolded proteins from SGs. Finally, we discuss recent findings that, in the stress recovery phase, SGs are preferentially disassembled with the assistance of chaperones, and we discuss evidence for a back-up system that targets aberrant SGs to the aggresome for autophagy-mediated clearance. Altogether the findings discussed here provide evidence for an intricate network of interactions between RNP granules and various components of the PQC machinery. Molecular chaperones in particular are emerging as key players that control the composition and dynamics of RNP granules, which may be important to protect against age-related diseases.

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

Summary Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy., Graphical Abstract, Highlights • HSPB2 undergoes concentration-dependent liquid-liquid phase separation in cells • HSPB2 phase separation requires its intrinsically disordered C-terminal tail • Aberrant HSPB2 phase separation mislocalizes lamin A • HSPB3, but not two HSPB3 myopathy mutants, inhibits HSPB2 phase separation, Morelli et al. show that, in mammalian cells, HSPB2 forms liquid-like nuclear compartments that affect lamin A localization and mobility, with detrimental consequences for chromatin organization and nuclear integrity. Aberrant compartment formation by HSPB2 is regulated by HSPB3, but not by two identified HSPB3 mutants linked to myopathy.