p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi

Abstract

Cancer cells exhibit hyperactive secretory states that maintain cancer cell viability and remodel the tumor microenvironment. However, the oncogenic signals that heighten secretion remain unclear. Here, we show that p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi. p53 loss up-regulates the expression of a Golgi scaffolding protein, progestin and adipoQ receptor 11 (PAQR11), which recruits an adenosine diphosphate ribosylation factor 1-containing protein complex that loads cargos into secretory vesicles. PAQR11-dependent secretion of a protease, PLAU, prevents anoikis and initiates autocrine activation of a PLAU receptor/signal transducer and activator of transcription-3-dependent pathway that up-regulates PAQR11 expression, thereby completing a feedforward loop that amplifies prometastatic effector protein secretion. Pharmacologic inhibition of PLAU receptor impairs the growth and metastasis of p53-deficient cancers. Blockade of PAQR11-dependent secretion inhibits immunosuppressive processes in the tumor microenvironment. Thus, Golgi reprogramming by p53 loss is a key driver of hypersecretion in cancer.

Fig. 1. p53-regulated expression of Golgi genes.

(A and B) Volcano plots comparing the mRNA levels of genes in the Gene Ontology term “Golgi apparatus” (n = 453) in TP53–wild-type (WT) and KO A549 cells (A) and TP53–wild-type and -mutant (MT) LUADs in TCGA (n = 230 tumors) (B). (C to F) PAQR11 mRNA levels in paired primary and metastatic tumor biopsies (www.oncomine.org). (G) Correlation between PAQR11 mRNA levels and somatic driver mutations (rows) in the TCGA LUAD and pan-cancer cohorts (columns). t value, t statistic. t > 0, positive correlation; t < 0, negative correlation (heatmap). (H and I) PAQR11 mRNA levels in TP53–wild-type and -mutant LUADs (H) and pan-cancers (I) (dots) (TCGA). Del, deletion; LOF, loss-of-function mutations including nonsense and frameshift mutations; MIS, missense mutations. (J and K) Quantitative reverse transcription polymerase chain reaction (qPCR) analysis of PAQR11 mRNAs in H1299 cells (I) and wild-type and KO A549 cells (J) transfected with empty (Vec) or p53-expressing (p53) vectors. (L) Luciferase reporter assays in H1299 cells transfected with PAQR11 3′ untranslated region (3′UTR) reporters (wild-type or miR-182–binding site mutant) and miR-182 or control (miR-NC) mimics. n = 4. (M) qPCR analysis of PAQR11 mRNA levels in parental and p53 KO A549 cells transfected with miR-182 or miR-NC. Results expressed relative to “P + miR-NC.” (N) qPCR analysis of PAQR11 mRNA levels in control (Vec) and p53-reconstituted (p53) H1299 cells transfected with miR-182 antagomirs (anti-182) or anti-NC. Results expressed relative to “Vec + anti-NC.” Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way analysis of variance (ANOVA) test for multiple comparisons.

Fig. 2. PAQR11 promotes lung colonization by suppressing anoikis of metastatic cells.

(A) Lung metastases in nu/nu mice injected by tail vein with green fluorescent protein (GFP)–tagged PAQR11 short hairpin RNA (shRNA; shPAQR11)– or control shRNA (shCTL)-transfected H1299 cells. (B) Lung metastases in syngeneic, immunocompetent mice injected by tail vein with red fluorescent protein (RFP)–tagged shPAQR11- or shCTL-transfected 344SQ murine LUAD cells. (C) Kaplan-Meier analysis of the mice in (B). (D) Colonies formed in soft agar. Values expressed relative to shCTL. (E and F) Western blot (WB) analysis of cleaved PARP1 (C-PARP1) (E) and annexin V/propidium iodide flow cytometry (F) to detect apoptotic cells on adherent or nonadherent (suspension) plates. α-Tubulin loading control. (G) Orthotopic lung tumor size (left dot plot) and numbers of metastases to contralateral lung and mediastinal lymph nodes (right dot plot) in syngeneic, immunocompetent mice. (H and I) qPCR analysis of PAQR11 mRNA levels in lung tumors (dots) generated in Kras^LSL-G12D mice subjected to aerosolized delivery of lentiviruses that coexpress Cre and PAQR11 or GFP. (J) Hematoxylin and eosin–stained lung tumors (arrows) in GFP- and PAQR11-expressing cohorts. Scale bars, 8 mm. (K) Lung tumor diameters (color-coded) in each cohort. (L) Lung tumor numbers per mouse (dots). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig. 3. PAQR11-dependent secretion promotes anoikis resistance.

(A and B) Colonies formed by Vec- and p53-transfected H1299 cells (A) or shCTL- and shPAQR11-transfected H1299 cells (B). Cells were treated for 14 days with CM samples from indicated H1299 transfectants. Fresh medium included as a control (−). (C and D) Western blot analysis of cleaved PARP1 (C) and annexin V/propidium iodide flow cytometry (D) to detect apoptotic cells following treatment of shPAQR11-transfected cells with CM or fresh medium (−). α-Tubulin loading control. (E) qPCR confirmation of PAQR11 depletion by siPAQR11. (F) CM samples (triplicate) from siCTL- and siPAQR11-transfected H1299 cells (schema) were subjected to liquid chromatography–mass spectrometry (LC-MS) analysis to identify PAQR11-regulated proteins (volcano plot). The identities of proteins that are significantly down-regulated (P < 0.05) by siPAQR11 are indicated. The vertical dashed line indicates a 1.4-fold change threshold. (G) Spectrum reads of down-regulated proteins. (H) Kaplan-Meier analysis of the TCGA LUAD cohort (n = 504) based on average FAM3C, PLAU, and PLOD3 mRNA levels (three-gene signature). High and low expression levels were defined using the autoselect best cutoff method. (I) Western blot analysis of CM samples. Relative protein levels quantified densitometrically (bar graph). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig. 4. Secreted PLAU inhibits anoikis and promotes metastatic colonization.

(A) Western blot confirmation of target gene depletion in siRNA-transfected 344SQ cells. (B to D) Lung metastases in tail vein–injected syngeneic, immunocompetent mice were imaged (B) and quantified on the basis of mean number per mouse (C) and mean size (D). (E) Colonies formed in soft agar by siRNA-transfected H1299 cells and CALU-1 cells. Values expressed relative to siCTL. (F and G) Western blot analysis of cleaved PARP1 (F) and annexin V/propidium iodide flow cytometry (G) to detect apoptotic cells under adherent (Adh.) and suspension (Susp.) conditions. (H) Western blot analysis of C-PARP1 in siRNA-transfected H1299 cells treated for 2 days with (+) or without (−) recombinant PLAU (10 ng/ml). (I) Annexin V/propidium iodide flow cytometry of H1299 cells in (H). (J and K) Western blot analysis of cleaved PARP1 (J) and annexin V/propidium iodide flow cytometry (K) to detect apoptotic cells following treatment with CM samples or fresh medium (−). (L) Annexin V/propidium iodide flow cytometry of 393P murine LUAD cells cotransfected with PAQR11 or empty (Vec) expression vectors and siPLAU or siCTL. (M) Lung metastases generated in syngeneic, immunocompetent mice by tail vein injection of shRNA-transfected 344SQ cells alone or in combination with 10 μg of recombinant PLAU (+PLAU; in 100-μl cell suspension). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig. 5. Secreted PLAU inhibits anoikis through autocrine PLAUR activation.

(A) qPCR confirmation of PLAUR depletion in siRNA-transfected H1299 and CALU-1 cells. (B and C) Western blot analysis of cleaved PARP1 (B) and annexin V/propidium iodide flow cytometry (C) to detect apoptotic cells under adherent and suspension conditions. (D) Colonies formed in soft agar by siRNA-transfected cells. (E and F) Western blot analysis of cleaved PARP1 (E) and annexin V/propidium iodide flow cytometry (F) of siRNA-transfected cells treated with (+) or without recombinant PLAU (10 ng/ml) under adherent or suspension conditions. (G and H) Western blot analysis of cleaved PARP1 (G) and annexin V/propidium iodide flow cytometry (H) following treatment for 2 days with indicated concentrations of PLAUR inhibitor (PLAUR IN). (I) Colonies formed in soft agar following 14 days of PLAUR inhibitor treatment. (J) Lung metastases generated in syngeneic, immunocompetent mice by tail vein–injected RFP-tagged 344SQ cells that had been pretreated for 2 days with 10 μM PLAUR inhibitor or vehicle [dimethyl sulfoxide (DMSO)]. Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig. 6. Autocrine PLAUR activation regulates PAQR11 expression.

(A and B) qPCR analysis of cells treated for 2 days with 1 μM JAK inhibitor (P6) (A) and in shSTAT3-transfected cells (B). (C) Pearson correlation between PLAU and PAQR11 mRNAs in TCGA LUAD cohort. (D and E) qPCR analysis of siPLAU-transfected cells treated for 2 days with recombinant PLAU (10 ng/ml) (D) and in cells cotransfected with siPLAU and empty vector (Vec) or constitutively active mutant STAT3 (STAT3-CA) (E). (F) MMD gene promoter (schema). Luciferase assays on H1299 cells cotransfected with MMD promoter constructs and STAT3-CA or Vec. Empty reporter (pGL3). (G) Luciferase assay on H1299 cells cotransfected with wild-type (−500) or E-box–mutated MMD promoter and indicated expression vectors. (H) Chromatin immunoprecipitation assays on the MMD promoter (−500) using immunoglobulin G (IgG) or anti-ZEB1 antibodies. PAQR11 3′UTR (negative control). (I and J) qPCR analysis of H1299 cells treated for 2 days with 1 μM P6 (I) or cotransfected with STAT3-CA or Vec and siZEB1 or siCTL (J). (K) Heatmap depiction of ZEB1 and PAQR11 mRNA levels; pSTAT3/total STAT3 ratio; and secreted PLAU levels (sPLAU) in murine LUAD cell line panel. (L) Heatmap depiction of Pearson correlation (R values) of results in (K). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments. P values, two-tailed Student’s t test (two-group comparisons) and one-way ANOVA test (multiple comparisons).

Fig. 7. N-terminal amino acids of PAQR11 are required to rescue PAQR11-deficient cells.

(A) A topological model of PAQR11. N5: The first five N-terminal amino acids. (B) Colonies formed in soft agar by shCTL- and shPAQR11-transfected H1299 cells. The latter were rescued with full-length (PAQR11) or N-terminally truncated (∆N5) PAQR11. (C and D) Western blot analysis of cleaved PARP1 (C) and annexin V/propidium iodide flow cytometry (D) of cells under suspension conditions. (E) Western blot analysis of FAM3C and PLAU in CM samples (CM) and cell lysates (lysates). (F) Western blot analysis of pSTAT3 and total STAT3. (G) A competing peptide that includes the first 28 N-terminal amino acids of PAQR11 (PAQR11-N) (schema) was detected by anti-GFP Western blot analysis in H1299 cells. (H and I) Western blot analysis of cleaved PARP1 (H) and annexin V/propidium iodide flow cytometry (I) of H1299 cells transfected with (PAQR11-N) or without (GFP) competing peptide under adherent or nonadherent conditions. (J) Colonies formed in soft agar by H1299 cells transfected with (PAQR11-N) or without (GFP) competing peptide. (K) Lung metastases generated in syngeneic, immunocompetent mice by 344SQ cells transfected with (PAQR11-N) or without (GFP) competing peptide. Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig. 8. PAQR11 recruits ARF1 to the trans-Golgi network.

(A) PAQR11-interacting proteins were identified by using a proximity-dependent biotin identification (BioID) assay. (B) A confocal micrograph demonstrates that a Golgi marker (GM130) colocalizes with a hemagglutinin (HA)–tagged BioID/PAQR11 fusion protein but not control vector (HA-BioID). (C) Venn diagram illustration of proteins identified by both methodologies (BioID assay on H1299 cells and N-terminal PAQR11 peptide pull-down assay on 344SQ cells). (D) Validation of hits identified by Western blot analysis of streptavidin beads isolated from H1299 cells transfected with HA-BioID or HA-BioID-PAQR11 and treated with (+) or without (−) 100 μM biotin. (E) Confocal micrographs of EGFP-tagged PAQR11 and mCherry-tagged ARF1 in 344SQ cells. Blue indicates DAPI. Scatter plot shows the percentage of mCherry-ARF1 that colocalizes with EGFP-PAQR11 in each cell (dot). Scale bar, 10 μm. (F) Confocal micrographs of ARF1-EGFP in PAQR11-deficient and -replete 344SQ cells. Red indicates Golgin-97, and blue indicates DAPI. Scale bars, 10 μm. Right: Scatter plot shows the percentage of Golgin-97 that colocalizes with ARF1 in each cell (dot). (G) Intensity–pseudo-colored confocal micrographs of Golgi-associated and cytosolic ARF1-EGFP. Scale bars, 10 μm. The scatter plot shows the Golgi/cytosolic ARF1-EGFP ratio in each cell (dots). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test.

Fig. 9. ARF1-dependent cargo loading into secretory vesicles is PAQR11 dependent.

(A) Western blot analysis of whole cell lysate (WCL) and vesicle- and Golgi-fractionated H1299 cell lysates to confirm enrichment of fractionated lysates in organelle-specific markers. ER, endoplasmic reticulum. (B and C) Western blot analysis of FAM3C and PLAU in WCL and Golgi- and vesicle-enriched fractions of siRNA-transfected H1299 cells (B). Protein levels relative to siCTL controls quantified densitometrically (C). (D) FAM3C and PLAU expression constructs. (E) Confocal micrographs of H1299 cells transfected with expression constructs. Vesicles containing ectopic FAM3C and PLAU were detected using fluorescent tags (EGFP-FAM3C) or antibodies against Flag (PLAU). Golgi localization determined on the basis of colocalization with the trans-Golgi marker Golgin-97. Nuclei were counterstained with DAPI (blue). Scale bar, 8 μm. (F) Contrast-adjusted confocal micrographs of H1299 cells cotransfected with Flag-PLAU and indicated siRNAs and stained with anti-Flag antibodies. Arrows indicate Flag-PLAU⁺ vesicles. Dotted lines indicate cell boundary. Scale bar, 10 μm. Vesicle numbers per cell (dots) were quantified. (G) Schematic illustration of a working model in which p53 loss leads to PAQR11 up-regulation, initiating an ARF1-dependent secretory process that is amplified through an autocrine PLAUR-dependent signaling pathway. Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.