(A) Western blot of SNAP25, LC3B, and SQSTM1 in the indicated cell lines with ACTB as a loading control. also acts as a putative tumor suppressor in prostate cancer, ovarian cancer and synovial sarcoma [15-17]. Recent studies have also demonstrated that this multifunctional protein influences cell fate determination, 2-Aminoethyl-mono-amide-DOTA-tris(tBu ester) which implicates it as a potential therapeutic target [18,19] Although substantial information exists regarding NUPR1 in the setting of gene regulation, the role of NUPR1 in the autolysosomal process is uncharacterized. We hypothesized that NUPR1 may facilitate the ability of cancer cells to survive in a stressful state. Here, we investigate the molecular and clinical consequences of NUPR1 activity as a critical transcriptional regulator controlling autolysosomal dynamics in lung cancers. Results NUPR1 expression is correlated with low overall survival rates in human NSCLC Using immunohistochemistry (IHC), we studied NUPR1 expression in 118 clinical non-small cell lung cancer (NSCLC) specimens and their adjacent tissues. Variable expressions of NUPR1 were found in lung tumor tissues, whereas cancer-adjacent lung tissues did not express significant levels of NUPR1 (Figure?1A). Quantification of staining on a scale of 0 to 10 showed that high NUPR1 expression correlated significantly with poor overall survival rates (= 0.00025) (Figure?1B). Subjects whose tumors had low NUPR1 expression had strikingly longer survival time than those whose tumors had high NUPR1 expression levels, with median survivals of 28 mo (high NUPR1) versus more than 80 mo (low NUPR1) (Figure?1B). NUPR1 staining intensity did not correlate with TNM status, smoking history, age, or gender (Table S1). Consistent with this observation, lung cancer cell lines also showed different expression of NUPR1 both at the mRNA and protein levels (Figure?1C and D, respectively). Normal human bronchial epithelial cells expressed undetectable levels of NUPR1 (Figure?1C and Figure 1.D, respectively). These differential expression levels of NUPR1 may correlate with its context-specific induction, as previously reported [8]. Open in a separate window Figure 1. depletion induces autolysosomal vacuolization. (A) IHC staining with anti-NUPR1 was performed on 118 NSCLC samples and their adjacent tissues. Representative images show moderate (case #1) and strong (case #2) NUPR1 staining. Scale bars: 10 m. (B) Kaplan-Meier overall survival rates for 118 NSCLC subjects with low (0 to 5.0 staining scores, blue lines; n = 68) versus high (5.1 to 10.0 staining scores, green lines; n = 50) NUPR1 expression. Median survival was more than 80 mo for the low NUPR1 expression group versus 28 mo for the high NUPR1 expression group (= 0.00025). (C and D) Relative transcript levels determined by quantitative RT-PCR shown as fold differences relative to in a normal lung epithelial cell line (NHBE) and cancer cell lines as indicated in (C), and the NUPR1 level determined by western blotting is shown with ACTB as a loading control in (D). (E) Western blot confirming the knockdown efficiency of 3 shRNAs against human shRNA in A549 cells. Large and small vacuoles can be seen scattered throughout the cytoplasm in shRNA cells at the indicated magnifications. depletion leads to accumulation of dilated autolysosomes (arrows). The right image is a higher magnification of the indicated portion, showing electron-dense material within autolysosomes. (G) Light micrographs and electron micrographs of cell morphology following depletion in H1299, H460 and H1155 cells. Arrows show the vacuole membrane location. NUPR1 depletion induces autolysosomal vacuolization To assess the role of in lung cancer Spn cells, we stably transduced lung adenocarcinoma A549 cells with lentiviral particles encoding 3 independent small hairpin RNAs (shRNAs) targeting or an irrelevant firefly luciferase shRNA (hereafter referred to as control, con, Table S2). The efficiency of these shRNAs in repressing this protein was assessed by western blotting (Figure?1E). Intriguingly, extensive perinuclear accumulation of phase-lucent vacuoles after depletion, but not in the shRNA control, was observed in A549 cells (Figure?1F) as well as in H460 and H1155 lung cancer cells (Figure?1G). These changes were confirmed by transmission 2-Aminoethyl-mono-amide-DOTA-tris(tBu ester) electron microscopy, which revealed that depletion originate from autolysosome dysfunction, we stably transfected GFP-LC3B (hereafter referred to as GFP-LC3) or tandem-tagged mCherry-GFP-LC3 plasmid into A549 cells to monitor the subcellular localization of LC3. In GFP-LC3 A549 cells, depletion led to the formation of massive vacuoles as well as increased LC3 puncta inside a time-dependent manner (Number?2A, lower panels 2-Aminoethyl-mono-amide-DOTA-tris(tBu ester) and 2B), indicating that autophagic circulation is impaired. In mCherry-GFP-LC3 A549 cells, depletion dramatically increased the transition of mCherry-GFP-LC3-positive autophagosomes (yellow puncta) to mCherry-positive, GFP.
(A) Western blot of SNAP25, LC3B, and SQSTM1 in the indicated cell lines with ACTB as a loading control