Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone.

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.

Integrative Genomic Characterization Identifies Molecular Subtypes of Lung Carcinoids.

Lung carcinoids (LC) are rare and slow growing primary lung neuroendocrine tumors. We performed targeted exome sequencing, mRNA sequencing, and DNA methylation array analysis on macro-dissected LCs. Recurrent mutations were enriched for genes involved in covalent histone modification/chromatin remodeling (34.5%; MEN1, ARID1A, KMT2C, and KMT2A) as well as DNA repair (17.2%) pathways. Unsupervised clustering and principle component analysis on gene expression and DNA methylation profiles showed three robust molecular subtypes (LC1, LC2, LC3) with distinct clinical features. MEN1 gene mutations were found to be exclusively enriched in the LC2 subtype. LC1 and LC3 subtypes were predominately found at peripheral and endobronchial lung, respectively. The LC3 subtype was diagnosed at a younger age than LC1 and LC2 subtypes. IHC staining of two biomarkers, ASCL1 and S100, sufficiently stratified the three subtypes. This molecular classification of LCs into three subtypes may facilitate understanding of their molecular mechanisms and improve diagnosis and clinical management. SIGNIFICANCE: Integrative genomic analysis of lung carcinoids identifies three novel molecular subtypes with distinct clinical features and provides insight into their distinctive molecular signatures of tumorigenesis, diagnosis, and prognosis.

Function and clinical relevance of RHAMM isoforms in pancreatic tumor progression.

The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers. We previously screened genes upregulated in human hepatocellular carcinomas for their metastatic function in a mouse model of pancreatic neuroendocrine tumor (PNET) and identified that human RHAMMB promoted liver metastasis. It was unknown whether RHAMMB is upregulated in pancreatic cancer or contributes to its progression. In this study, we found that RHAMM protein was frequently upregulated in human PNETs. We investigated alternative splicing isoforms, RHAMMA and RHAMMB, by RNA-Seq analysis of primary PNETs and liver metastases. RHAMMB, but not RHAMMA, was significantly upregulated in liver metastases. RHAMMB was crucial for in vivo metastatic capacity of mouse and human PNETs. RHAMMA, carrying an extra 15-amino acid-stretch, did not promote metastasis in spontaneous and experimental metastasis mouse models. Moreover, RHAMMB was substantially higher than RHAMMA in pancreatic ductal adenocarcinoma (PDAC). RHAMMB, but not RHAMMA, correlated with both higher EGFR expression and poorer survival of PDAC patients. Knockdown of EGFR abolished RHAMMB-driven PNET metastasis. Altogether, our findings suggest a clinically relevant function of RHAMMB, but not RHAMMA, in promoting PNET metastasis in part through EGFR signaling. RHAMMB can thus serve as a prognostic factor for pancreatic cancer.

ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Uncoupling of hormone-dependence from chaperone-dependence in the L701H mutation of the androgen receptor.

The mechanisms underlying androgen receptor (AR)-mediated progression of prostate cancer following androgen ablation have yet to be fully determined. On this basis we screened naturally occurring mutants of human AR for hormone-independent activity using a yeast model system. An initial screen of 43 different mutants revealed that ARs having a Leu701His mutation (AR(L701H)) exhibited hormone-independent activation of a lacZ reporter gene. The AR(L701H) mutant bound dihydrotestosterone to a similar extent as did wild type AR, although its ability to be induced by hormone for transactivation was reduced substantially. Subsequent studies focused on the dependence of AR(L701H) on molecular chaperones for folding to the active state. We found that AR(L701H) was highly dependent on Hsp90 for its hormone-independent activation, suggesting that this chaperone functions in AR(L701H) folding. However, the mutant did not respond specifically to increased levels of FKBP52, suggesting that this chaperone functions at the hormone-dependent activation stage in the folding process. Further studies of AR(L701H) in PC3 cells suggested that this mutant is prohibited from hormone-independent transactivation in mammalian cells. However, basal expression of a reporter gene by AR(L701H) was not impaired by the presence of 17-allylamino-17-demethoxygeldanamycin as was wild type AR, suggesting differential interactions of these receptors with molecular chaperones in animal cells.

An acetylation site in the middle domain of Hsp90 regulates chaperone function.

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.