Genetic heterogeneity of pseudoxanthoma elasticum: the Chinese signature profile of ABCC6 and ENPP1 mutations.

Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder characterized by ectopic mineralization, is caused by mutations in the ABCC6 gene. We examined clinically 29 Chinese PXE patients from unrelated families, so far the largest cohort of Asian PXE patients. In a subset of 22 patients, we sequenced ABCC6 and another candidate gene, ENPP1, and conducted pathogenicity analyses for each variant. We identified a total of 17 distinct mutations in ABCC6, 15 of them being, to our knowledge, previously unreported, including 5 frameshift and 10 missense variants. In addition, a missense mutation in combination with a recurrent nonsense mutation in ENPP1 was discovered in a pediatric PXE case. No cases with p.R1141X or del23-29 mutations, common in Caucasian patient populations, were identified. The 10 missense mutations in ABCC6 were expressed in the mouse liver via hydrodynamic tail-vein injections. One mutant protein showed cytoplasmic accumulation indicating abnormal subcellular trafficking, while the other nine mutants showed correct plasma membrane location. These nine mutations were further investigated for their pathogenicity using a recently developed zebrafish mRNA rescue assay. Minimal rescue of the morpholino-induced phenotype was achieved with eight of the nine mutant human ABCC6 mRNAs tested, implying pathogenicity. This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations. These genetic data have implications for allele-specific therapy currently being developed for PXE

Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1

Cytotoxic effects of DOX and LMB on A549 cells.

<p>A, Cytotoxic effects of DOX alone and DOX+LMB on cell viability of A549 cells as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for DOX and LMB (0.5 nM) for DOX+LMB. Values are represented as means ± SD, n = 6. B, Cytotoxic effects of LMB alone and LMB+DOX on cell viability of A549 cells as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for LMB and DOX (0.5 µM) for LMB+DOX. Values are means ± SD, n = 6. C, Cytotoxic effects of DOX alone and pre-LMB+DOX on cell viability of A549 cells at 48 h as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for DOX and pre-LMB for pre-LMB+DOX. Values are means ± SD, n = 6. D, Cytotoxic effects of LMB alone and pre-DOX+LMB on cell viability of A549 cells at 48 h as determined by the MTT assay. Data are expressed as the percentage by comparing to vehicle control for LMB and pre-DOX for pre-DOX+LMB. Values are means ± SD, n = 6. Experiments performed in triplicate yielded similar results.</p

Western blot analyses of protein expression in A549 cells after DOX and LMB treatment.

<p>A, Effects of pre-DOX+LMB treatment on the protein expression of p53, phospho-p53 (Ser15), phospho-p53 (Thr55), p21, and survivin in A549. Cells were treated with 0.5 µM DOX 24 h before treatment with LMB (1 nM or 5 nM). After 48 h LMB treatment, cells were harvested for Western blot analysis to determine protein levels. Blots were also probed for α-tubulin to confirm equal protein loading. B, The relative protein intensities of p53, phospho-p53 (Ser15), phospho-p53 (Thr55), p21, and survivin as compared with the intensity of α-tubulin. The intensity of each band was quantified using Quantity One software. Data are means ± SD, n = 3. The experiments were conducted in triplicate. LMB1: 1 nM LMB; LMB5: 5 nM LMB; *, <i>P</i><0.05 compared to control; **, <i>P</i><0.01 compared to control; #, <i>P</i><0.05, compared to LMB5; ##, <i>P</i><0.01, compared to LMB5.</p

Effects of DOX and LMB on cell cycle and apoptosis of A549 cells.

*<p><i>P</i><0.05 in comparison to control;</p>**<p><i>P</i><0.01 in comparison to control.</p>#<p><i>P</i><0.05 in comparison to LMB alone;</p>##<p><i>P</i><0.01 in comparison to LMB alone.</p

Prevalence and prognosis of hypoxia‐inducible factor‐2α (HIF‐2α) pathway gene mutations across advanced solid tumors

Abstract Introduction Hypoxia‐inducible factor‐2α (HIF‐2α) modulates the hypoxic response pathway in tumors; however, mutations in pathways (including SDHA, SDHB, SDHC, SDHD, FH, and VHL genes) that are suspected to activate HIF‐2α are poorly understood, with limited understanding of the prevalence and clinical prognosis. Methods This retrospective observational study used a de‐identified nationwide (US‐based) clinico‐genomic database (CGDB) across 15 available tumor types. Results Among the 9467 adult patients with advanced/metastatic solid tumors included in the analysis, any mutation at the above‐mentioned six genes was observed in 1.8% (95% CI: 1.5–2.1) of patients. The mutation prevalence ranged from 0.05% of SDHD to 0.93% of VHL. When further stratified by tumor type, the prevalence of gene mutation in each tumor type was well below 1%, except for VHL with 44% in renal cell carcinomas (RCC). Excluding RCC, the prevalence of any HIF‐2α gene mutations in the study population was 0.9% (95% CI: 0.8–1.2). The median overall survival (OS) from 1 and 2 L therapy among patients with any HIF‐2α gene mutation was 14.5 (95% CI: 11.5–24.2) and 9.3 (95% CI: 6.0–18.1) months, respectively, compared with 13.4 (95% CI: 12.9–13.9) and 9.8 (95% CI: 9.3–10.4) months among patients without HIF‐2α gene mutations. Discussion and Conclusions The prevalence of HIF‐2α related gene mutations was generally low (<1%) across the 15 solid tumor types, except for VHL in RCC. No significant association between HIF‐2α gene mutation status and OS was identified among patients evaluated in this study

Nuclear proteome profiling in A549 cells after DOX and/or LMB treatment.

<p>A, Western blot of nuclear and cytoplasmic protein extractions from A549; α-tubulin served as an internal control for cytoplasmic proteins, and histone 3 served as a control for nuclear proteins. B, 2D-DIGE analyses of nuclear proteins in A549 cells and 3D views of SQSTM1 in A549 cell with vehicle control or LMB treatment. Nuclear proteins treated with LMB or vehicle control were labeled with Cy3 (green channel) and Cy5 (red channel), respectively. Nuclear proteins were separated based on isoelectric point (PI, horizontal axis) and molecular weight (MW, vertical axis). Approximately 1,000 protein spots were detected in nuclear extractions of A549 cells. Spots labeled with red color indicate decreased expression after LMB treatment, while spots labeled with green color indicate increased expression after LMB treatment (left panel). Magnification of 5 protein spots (right upper panel) and 3D view of vehicle control and LMB treated (right bottom panel) (identified by LC/MS/MS as sequestosome 1 (SQSTM1/p62)). C, Protein sequence and tandem mass spectrometry identification of SQSTM1. The MS/MS fragmentation spectrum (obtained after trypsin digestion) of AYLLGKEDAAR for SQSTM1 is shown. The resultant MS/MS data were processed using Mascot. D, Western blot analysis of SQSTM1 in cytoplasm and nucleus of A549 cells after LMB treatment. E, Effects of LMB alone or pre-DOX+LMB treatment on protein expression of SQSTM1 in A549 cells. The relative protein intensity of SQSTM1 was compared with the intensity of corresponding α-tubulin. The intensity of each band was quantified using Quantity One software. Data are means ± SD. Experiments were conducted in triplicate. LMB1: 1 nM LMB; LMB5: 5 nM LMB; **, <i>P</i><0.001 compared to control.</p