Toumy Guettouche

The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.

BACKGROUND: Heat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser303, Ser307 and Ser363, which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser230 contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. RESULTS: An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444. Phosphorylation of Ser326 but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser326 increased rapidly during heat stress as shown by experiments using a pSer326 phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor. CONCLUSION: Twelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser326 plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer326 also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues.

In the absence of stress, human heat shock factor 1 (hHSF1) is in its unactivated form. hHSF1 polypeptide is in a dynamic heterocomplex with Hsp90 and is incapable of specifically binding DNA. When cells are stressed, heterocomplex assembly is disrupted. Unbound hHSF1 homotrimerizes, acquires DNA binding activity, and concentrates in the nucleus, but remains transcriptionally inactive. A subsequent reaction converts this inactive, trimeric form into the active, hyperphosphorylated transcription factor. Subsequent to the stressful event, hHSF1 is deactivated and eventually returned to its unactivated form. Evidence is presented herein that trimeric hHSF1 has the propensity to dynamically associate with an Hsp90-immunophilin-p23 complex through its regulatory domain. Formation of this heterocomplex results in repression of the transcriptional activity of trimeric hHSF1. Stress-denatured proteins effectively compete with trimeric hHSF1 for Hsp90-immunophilin-p23 complex, counteracting assembly of the heterocomplex and repression of hHSF1 transcriptional activity. This repression mechanism may be required for a proportional transcriptional response to stress. Formation of the heterocomplex may also represent the first step toward returning the hHSF1 to its unactivated form.