Fig. 5: Substrate receptor protein Prame is over-expressed in human cancer and mediates p14/ARF degradation.

A Downregulation of Prame induces the accumulation of p14/ARF. H1299 cells were transfected with Ctrl or Prame siRNA for 96 h and harvested for immunoblotting using antibodies against p14/ARF and Prame with Actin as a loading control. B Downregulation of Prame delays the degradation and extends the half-life of p14/ARF. H1299 cells were transfected with Ctrl or Prame siRNA for 96 h and then treated with 50 μg/mL CHX at indicated time before subjected to immunoblotting analysis using antybody against p14/ARF with Actin as a loading control. C P14/ARF interacts with Prame. Endogenous p14/ARF interacted with Prame. H1299 cells were pretreated with MG-132 for 2 h before harvesting and the lysates were subjected to immunoprecipitation with anti-p14/ARF Ab followed by immunoblotting with Prame Ab. D Exogenous p14/ARF interacted with Prame. Flag-tagged p14/ARF and HA-tagged Prame were constructed and transfected into HEK293T cells as indicated for 24 h. Total cell lysates were subjected to immunoprecipitation with anti-HA beads and immunoblotting with Flag Ab or subjected to immunoprecipitation with anti-Flag M2 beads and immunoblotting with HA Ab. E Prame mediates p14/ARF ubiquitination. Prame depletion suppressed ubiquitination of p14/ARF (left). H1299 cells were transfected with Ctrl or Prame siRNA for 96 h and followed with MG-132 treatment. Total cell lysates were harvested and subjected to immunoprecipitation with anti-p14/ARF Ab and immunoblotting with ub Ab. Prame overexpression promoted p14/ARF ubiquitination. HEK293T cells were transfected with indicated plasmids combinations of His-ub, Flag-p14/ARF and HA-Prame. Total cell lysates were subjected to immunoprecipitation assay with anti-Flag M2 beads and immunoblotting with ub Ab. F–H Prame is overexpressed in tumor tissues vs. adjacent tissues and indicates an unfavorable prognosis. (F) TCGA RNA-Seq database demonstrated Prame expression levels in breast cancer (left, n = 112) and lung cancer (right, n = 107). (G) Kaplan–Meier plots of OS and Prame expression in breast cancer (left, n = 1402, P < 0.0001) and lung cancer (right, n = 1926, P < 0.0001) using the program Kaplan–Meier Plotter. (H) Immunohistochemical staining of human lung adenocarcinoma tissue arrays using specific antibodies for Prame (n = 86, P < 0.0001). Scale bar for ×10 images: 500 μm; Scale bar for ×200 images: 25 μm. (I) Inverse relationship between Prame and p14/ARF. 10 paired breast cancer tissues with adjacent tissues and 4 paired lung cancer tissues with adjacent tissues were lysed and subjected to immunoblotting, the inverse relationship between Prame and p14/ARF was detected in 4/6 of paired breast cancer tissues (P1–P4, 66%) and 2/3 of lung cancer tissues (P5, P6, 66%). T: Tumor tissue, A: Adjacent tissue. All data were representative of three independent experiments. Data represented means, and error bars were standard deviation. Two‐sided t test.