Knockdown of individual proteasome subunits results in NSCLC cytotoxicity.

<p>Diagram of the 26 S proteasome showing multiple whole genome shRNA screen hits with the following color code: top hit (red), strong hit (>1 shRNA sequence per gene in both cell lines, dark orange), minor hit (1 shRNA sequence per gene in both cell lines, light orange), chymotrypsin-like proteolytic catalytic site (not a hit but highlighted for illustrative purposes, green). Each hit is labeled using the last two alphanumeric characters of the gene’s HUGO nomenclature; e.g., A1 = PSMA1, B5 = PSMB5, M1 = SHFM1.</p

Proteasome inhibition delays DNA repair <i>in vivo</i>.

<p>(A) FANCD2 immunofluorescence in 10 Gy irradiated vs. unirradiated NCI-H460 xenografts recovered from mice with or without doxycycline-induced <i>PSMA1</i> shRNA expression in tumor cells. Bar = 10 µm. (B) γ-H2AX immunohistochemistry in NCI-H460 xenograft tumors with or without doxycycline-induced <i>PSMA1</i> shRNA knockdown, recovered from mice 1, 6, and 24 hours after 10 Gy irradiation. Bar = 10 µm. (C) Quantification of immunohistochemistry for γ-H2AX in (B). Cells with ≥5 foci were scored as positive (n>400 cells). All results are mean ± SEM. P values were calculated using a two-tailed Student’s t test.</p

Proteasome inhibition blocks expression of Fanconi Anemia/Homologous Recombination genes.

<p>(A) Diagram of NF-kB promoters on <i>FANCD2</i> and <i>BRCA1</i> genes. (B) Expression by qPCR of <i>FANCD2</i> following proteasome inhibition ±30 nM (A549) or 50 nM (NCI-H460) bortezomib or ± inducible <i>PSMA1</i> shRNA, and ±10 Gy IR. All values are normalized to ACTB and all results are mean ± SEM. (C) As in (B) but with BRCA1. (D) NSCLC cell survival following bortezomib and IR is partially rescued by <i>IkBα</i> siRNA knockdown when performed in advance. Cell viability was assayed using an ATP luminescence-based assay measuring relative luminescent units (RLU). All results are mean ± SEM.</p

Role of proteasome inhibition in modulating NF-κB pathway-mediated expression of Fanconi Anemia (FA)/homologous recombination (HR) genes.

<p>Proteasome inhibition by bortezomib or <i>PSMA1</i> knockdown results in an increase in IκBα, which in turn decreases NF-κB binding to the promoters of FA/HR genes including <i>FANCD2</i> and <i>BRCA1</i>. This reduces the availability of these DNA repair proteins for recruitment to DNA damage sites, resulting in decreased RAD51 focus formation and HR following induction of DNA double strand breaks by ionizing radiation.</p

Proteasome inhibition sensitizes NSCLC cells to radiation.

<p>(A) Western blot showing protein levels of PSMA1 and PSMB5 in A549 and NCI-H460 NSCLC cells after <i>PSMA1</i> shRNA knockdown compared to non-silencing shRNA control. (B) Chymotrypsin-like (CTL) proteasome activity assay in A549 (left) and NCI-H460 (right) NSCLC cells after treatment with bortezomib, or <i>PSMA1</i> siRNA knockdown. All results are mean ± SEM and normalized to DMSO vehicle control. (C) Clonogenic survival assay of A549 (left) and NCI-H460 (right) following IR and bortezomib. Marked bars show the percent kill of bortezomib-treated samples compared to DMSO vehicle control at each IR dose. All results are mean ± SEM and normalized to DMSO vehicle control. (D) Apoptosis detection assay of NCI-H460 following 2 and 4 Gy IR and 50 nM bortezomib. Bars show percentage of cells in early apoptosis (left) or late apoptosis (right) via Annexin V and propidum iodide staining, respectively. All results are mean ± SD and P values were calculated using a two-tailed Student’s <i>t</i> test.</p