Fig 1: Identification of Apt63 target as cell surface FoF1-ATP synthase ß-subunit ATP5B. a Apt63 associates with a 56kd protein in the LNCaP-LN3 plasma membrane. Representative SDS-PAGE gel of proteins immunoprecipitating with Apt63 from LN3 and Pro5 cell lines. Lane 1: LN3 whole cell lysate; Lane 2: LN3, membrane-enriched fraction; Lane 3: Pro5 total cell lysate; Lane 4: Pro5 membrane-enriched fraction. Equal amounts of protein were loaded in each lane. Bar at 56 kd indicates the predominant LN3 aptamer-associated band subjected to mass spectroscopy as described in “Materials and methods” and Table 1. b Confirmation of FoF1-ATP synthase ß-subunit as aptamer target. Membrane fractions of LN3 and Pro5 prostate cells were separated as in (a). Plasma membrane-bound Apt63/target complexes (lanes 1–3), and whole cell lysates (lanes 4–5) were obtained from indicated cell lines and immunoblotted with an anti-ATP5B monoclonal antibody. A single ~ 56 kd protein band co-migrating with recombinant ATP5B (lane 6) is present on the membranes of LN3 (lanes 1, 2) but not Pro5 cells (lane 3). Similar bands are present in whole cell lysates from both cell lines, reflecting the mitochondrial protein (lanes 4–5). c Apt63 co-localizes on plasma membrane with anti-ATP5B antibody. ATP5B antibody (green) and Apt63 (red) were bound to live LN3 cells, followed by fixation and imaging by confocal microscope. d The Apt63 target is extractable by detergent treatment of fixed cells. (left) Cy3-Apt63 (red) and plasma membrane marker WGA (green) were incubated with live cells followed by fixation and imaging as in (c). (right) Similarly treated cells, except that fixed slides were subjected to a short 0.05% Triton X-100 treatment. Note loss of Cy3 Apt63 signal from plasma membrane. e Apt63 stains mitochondria in permeabilized cells. Cells were fixed, then permeabilized with 0.05% Triton X-100 and incubated with Cy3-Apt63 or Cy3-AptScr and ATP5B antibody. In these permeabilized LN3 cells, Apt63 and ATP5B antibody staining are co-localized within mitochondria. (Color figure online)
Fig 2: Increased ATP5B expression is associated with greater risk of metastasis and reduced overall survival in prostate and breast cancer. a ATP5B expression is increased in primary prostate tumors that later metastasize. Primary prostate cancers with metastatic disease progression were compared to samples with no evidence of metastasis. Data were obtained from GSE46691 [27] (n = 545; p-value = 0.0063). b High expression of ATP5B mRNA correlates with decreased overall survival in prostate cancer patients of the TCGA-PRAD dataset (n = 551). Patients with high and low ATP5B expression are shown in red and blue, respectively (see “Materials and methods”). Patients with high expression (n = 250) had lower overall survival probability over time (log rank p-value = 0.050). c High expression of ATP5B mRNA correlates with reduced metastasis-free survival in patients of a compendium of 3661 breast cancer samples. Patients with high and low ATP5B expression are shown in red and blue, respectively (see “Materials and methods”). Patients with high expression (n = 1656) had higher probability to develop metastasis over time (log rank p-value = 0.0039). d High expression of ATP5B mRNA correlates with poor overall survival in breast cancer patients of the TCGA-BRCA dataset (n = 522). Patients with high and low ATP5B expression are shown in red and blue, respectively (see “Materials and methods”). Patients with high expression (n = 263) had lower overall survival probability over time (log rank p-value = 0.0198). (Color figure online)
Fig 3: Apt63 binds to a subset of aggressive breast tumors and co-localizes with ATP5B. a Primary tumor core biopsies (top row) and adjacent normal tissue (bottom row) with anti-ATP5B (Abcam #14730) using HRP (far left) or Alexa Fluor® 647 ATP5B antibodies (ab223436, ABCAM) (near left). ATP5B (assigned green color) and Cy3-Apt63 (red color) extensively co-localize both at cell membranes and in the cytosol. b Heterogeneous binding of Cy3-Apt63 to primary breast tumors and lymph node metastases. Brackets indicate normal adjacent tissues in the top row. c–f Representative high-resolution confocal images of normal tissue (c), ductal carcinoma in situ (DCIS) (d), invasive ductal carcinoma (CA) (e), and lymph node metastases (LN) (f), imaged with Cy3-Apt63. A membrane staining pattern, as well as cytosolic staining, can be seen in (e) and (f). Original magnification: x20. Results shown are representative of findings in a total of 417 breast biopsies. (Color figure online)
Fig 4: ATP5B expression is increased in prostate and breast cancer vs. normal tissue. Datasets were reviewed as described in “Materials and methods.” a ATP5B expression is increased in prostate adenocarcinoma compared with normal prostate tissue (p = 3.54e-4). GS = Gleason stage. Data from Vanaja et al. [74]. b ATP5B overexpression in GSE62872 prostate samples compared with normal prostate tissues (n = 424; p-value < 0.0001). c ATP5B overexpression in TCGA-PRAD samples compared with normal prostate tissues (n = 551; p-value = 0.0001). d ATP5B overexpression in GSE109169 breast cancers compared with their paired normal breast counterparts (n = 50; p-value = 0.0004). Gray lines link normal and tumor paired samples. e ATP5B overexpression in breast ductal cancers as compared to normal mammary tissues (n = 544; p-value = 0.0005). Data obtained from TCGA-BRCA database. f ATP5B copy number variation in breast cancer compared to normal blood cells. Copy number is significantly increased in ER + tumors. Data from TCGA Breast 2 dataset. (Color figure online)
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