The Zinc-Finger AN1-Type Domain 2a Gene Acts as a Regulator of Cell Survival in Human Melanoma: Role of E3-Ligase cIAP2.

The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment. IMPLICATIONS: The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.

Human NF-κB repressing factor acts as a stress-regulated switch for ribosomal RNA processing and nucleolar homeostasis surveillance.

The nucleolus, a dynamic nuclear compartment long regarded as the cell ribosome factory, is emerging as an important player in the regulation of cell survival and recovery from stress. In larger eukaryotes, the stress-induced transcriptional response is mediated by a family of heat-shock transcription factors. Among these, HSF1, considered the master regulator of stress-induced transcriptional responses, controls the expression of cytoprotective heat shock proteins (HSPs), molecular chaperones/cochaperones constituting a major component of the cell protein quality control machinery essential to circumvent stress-induced degradation and aggregation of misfolded proteins. Herein we identify human NF-κB repressing factor (NKRF) as a nucleolar HSP essential for nucleolus homeostasis and cell survival under proteotoxic stress. NKRF acts as a thermosensor translocating from the nucleolus to the nucleoplasm during heat stress; nucleolar pools are replenished during recovery upon HSF1-mediated NKRF resynthesis. Silencing experiments demonstrate that NKRF is an unconventional HSP crucial for correct ribosomal RNA (rRNA) processing and preventing aberrant rRNA precursors and discarded fragment accumulation. These effects are mediated by NKRF interaction with the 5'-to-3' exoribonuclease XRN2, a key coordinator of multiple pre-rRNA cleavages, driving mature rRNA formation and discarded rRNA decay. Under stress conditions, NKRF directs XRN2 nucleolus/nucleoplasm trafficking, controlling 5'-to-3' exoribonuclease nucleolar levels and regulating rRNA processing. Our study reveals a different aspect of rRNA biogenesis control in human cells and sheds light on a sophisticated mechanism of nucleolar homeostasis surveillance during stress.

The proteasome inhibitor bortezomib is a potent inducer of zinc finger AN1-type domain 2a gene expression: role of heat shock factor 1 (HSF1)-heat shock factor 2 (HSF2) heterocomplexes.

The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment.

Regulation of cyclooxygenase-2 expression by heat: a novel aspect of heat shock factor 1 function in human cells.

The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E(2) in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position -2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.

AIRAP, a new human heat shock gene regulated by heat shock factor 1.

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones that protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27, and other proteins of the network. Herein we report that the zinc finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1 binding HSE sequence in the AIRAP promoter critical for heat-induced transcription was identified. Because its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.