Research ArticleCancer

Hypoxic cancer–associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling

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Science Signaling  05 Feb 2019:
Vol. 12, Issue 567, eaan8247
DOI: 10.1126/scisignal.aan8247
  • Fig. 1 The secretome of hypoxic CAFs induces endothelial sprouting angiogenesis.

    (A) Scheme of the 3D fibrin-based sprouting assay for ECs cocultured with CAFs or alone and treated with CAF-derived CM. (B) Representative images and quantification of the sprouting of ECs cocultured with CAFs under normoxia and hypoxia for 72 hours. Scale bar, 50 μm. N = beads assessed in three biological replicates; cCAF normoxia, n = 120; cCAF hypoxia, n = 135; pCAF normoxia, n = 128; pCAF hypoxia, n = 134. (C) Representative image and quantification of the sprouting of ECs treated with CM derived from CAFs cultured under normoxia or hypoxia for 72 hours. Scale bar, 50 μm. N = beads assessed in three biological replicates; cCAF normoxia, n = 201; cCAF hypoxia, n = 195; pCAF normoxia, n = 112; pCAF hypoxia, n = 135. (D) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showing VEGFA transcription in cCAFs and pCAFs under hypoxic condition (normalized to the VEGFA levels in normoxic condition. VEGFA normoxia = 1 = dashed line). VEGFA mRNA levels were normalized to TBP. N = three biological replicates, one-sample t test. (E) Enzyme-linked immunosorbent assay (ELISA) showing VEGFA levels in the CM from cCAFs and pCAFs. N = two biological replicates. (F) Quantification of the sprouting of ECs treated with the CM from normoxic and hypoxic CAFs in the presence of bevacizumab (1 μg/ml) or immunoglobulin K (IgGK) (control). N = beads assessed in three biological replicates; CM normoxia + IgGK, n = 178; CM hypoxia + IgGK, n = 161; CM normoxia + bevacizumab, n = 176; CM hypoxia + bevacizumab, n = 179. *P < 0.05, **P < 0.01, ****P < 0.0001, t test.

  • Fig. 2 Hypoxia-induced remodeling of the proteome and secretome of mammary CAFs.

    (A) SILAC-based workflow used to characterize the total proteome and secretome of hypoxic cCAFs and pCAFs. (B) Physical and functional relationship among the hypoxia-regulated proteins in the intracellular proteome and secretome, as determined by STRING analysis and visualized with Cytoscape. Gene ontology (GO) categories and KEGG pathways enriched in the subset of proteins up-regulated and down-regulated by hypoxia are indicated on the right. FASP, filter-aided sample preparation; SAX, strong anion exchange. Results from n = three biological replicates for both cCAF and pCAF.

  • Fig. 3 Hypoxic CAFs increase NCBP2-AS2 protein levels through enhanced translation.

    (A and B) Scatter plot of the averaged SILAC ratio, hypoxia/normoxia, measured by MS in the pCAF and cCAF proteome (A) and secretome (B) highlighting the induction of NCBP2-AS2 by hypoxia in both cell types. Results from n = 3 biological replicates. (C) RT-qPCR showing mRNA levels of NCBP2-AS2 and VEGFA normalized to TBP in CAFs upon exposure to hypoxia for 72 hours (normalized to the levels in CAFs in normoxia. Normoxia = 1 = dashed line). VEGFA was used as a positive control. N = three biological replicates. One-sample t test. (D) RT-qPCR showing the mRNA levels of NCBP2-AS2 and VEGFA upon treatment with 1 mM DMOG for 24 hours [normalized to the levels in dimethyl sulfoxide (DMSO) control-treated cells. DMSO = 1 = dashed line]. The mRNA levels were normalized to TBP. VEGFA was used as a positive control. N = three biological replicates. One-sample t test. (E) Polysomal profiling of cCAFs exposed to hypoxia and normoxia for 24 hours. Representative of three biological replicates. (F) RT-qPCR analysis for NCBP2-AS2 and actin mRNAs, which was used as control, on the nontranslating and polysomal ribosomal fractions. N = three biological replicates. *P < 0.05, **P < 0.01, t test. NS, not significant.

  • Fig. 4 The CM from hypoxic CAFs silenced for NCBP2-AS2 show a decreased pro-angiogenic activity in vitro and in vivo.

    (A and C) Sprouting quantification of ECs treated with CM from hypoxic and normoxic cCAFs (A) or pCAFs (C) transfected with siCTL or siNCBP2-AS2 [two different small interfering RNAs (siRNAs)]. N = beads assessed in three biological replicates; cCAF hypoxic CM: siCTL, n = 129; siNCBP2-AS2 #1, n = 124; and siNCBP2-AS2 #2, n = 116. Normoxic CM: siCTL, n = 121; siNCBP2-AS2 #1, n = 131; and siNCBP2-AS2 #2, n = 122. pCAF hypoxic CM: siCTL, n = 119; siNCBP2-AS2 #1, n = 107; and siNCBP2-AS2 #2, n = 97. Normoxic CM: siCTL, n = 111 beads; siNCBP2-AS2 #1, n = 127; and siNCBP2-AS2 #2, n = 104. (B and D) RT-qPCR showing the remaining mRNA levels of NCBP2-AS2 normalized to TBP in (B) cCAFs and (D) pCAFs used to produce CM. (E) Vascular density quantification, as measured by Meca32+ and Ng2+ staining of the blood vessels (y axis), of subcutaneous sponges, which were embedded with CM derived from hypoxic CAFs transfected with siCTL or siNCBP2-AS2. N = 1/2 sponges derived from six mice per group; t test. (F) RT-qPCR showing the remaining mRNA levels of NCBP2-AS2 normalized to TBP in CAFs producing the CM used in the sponge assay in (E). *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 ECs treated with the CM from hypoxic CAFs silenced for NCBP2-AS2 have a decreased migratory capacity.

    (A) Proliferation of ECs cultured in 2D and treated with the CM from normoxic and hypoxic CAFs transfected with siCTL or siNCBP2-AS2. N = 45 fields assessed per condition from three biological replicates. (B) Proliferation of ECs cultured in 3D fibrin gel and treated with the CM from normoxic and hypoxic CAFs transfected with siCTL or siNCBP2-AS2. N = beads assessed in three biological replicates. Normoxic CM: siCTL, n = 124; siNCBP2-AS2 #1, n = 132; and siNCBP2-AS2 #2, n = 136. Hypoxic CM: siCTL, n = 127; siNCBP2-AS2 #1, n = 120; and siNCBP2-AS2 #2, n = 134. (C) RT-qPCR showing remaining mRNA levels of NCBP2-AS2 normalized to TBP. (D) RT-qPCR showing remaining mRNA levels of NCBP2-AS2 normalized to TBP. (E and F) Scratch wound migration assay using ECs treated with the CM from hypoxic (E) or normoxic (F) cCAFs transfected with siCTL or siNCBP2-AS2. N = two biological replicates. (G) Relative wound density measured at 12 hours in the two independent replicate experiments [dashed lines in (E) and (F)]. (H) Representative images of lamellipodia formation in ECs treated with the CM from cCAFs silenced or not for NCBP2-AS2. (I) Quantification of cortactin at the cell membrane. (J) Quantification of the total cell perimeter. N = cells measured in three biological replicates. siCTL, n = 111; siNCBP2-AS2 #1, n = 126; and siNCBP2-AS2 #2, n = 125. Scale bar, 10 μm; scale bar in zoomed inset, 5 μm. P value based on Kruskal-Wallis test corrected for multiple testing using Dunn’s test; t test was used for the other panels. **P < 0.01, ****P < 0.0001.

  • Fig. 6 NCBP2-AS2 regulates the levels of CAF-derived VEGFA.

    (A and B) Volcano plot showing the SILAC ratio of the proteins quantified in the total proteome (A) and secretome (B) of hypoxic cCAFs transfected with siCTL and siNCBP2-AS2. The dashed lines show the regulation cutoff of at least onefold change and P < 0.05. Results from three biological replicates. (C) ELISA of VEGFA in the CM from hypoxic CAFs silenced or not for NCBP2-AS2 and normoxic CAFs siCTL. One-tailed t test. N = three or four biological replicates. (D) RT-qPCR showing remaining mRNA levels of NCBP2-AS2 normalized to TBP in cells used in (C). (E) RT-qPCR for VEGFA in hypoxic cCAFs and pCAFs transfected with siCTL or siNCBP2-AS2. The mRNA levels were normalized to TBP. One-sample t test. N = three biological replicates. (F) RT-qPCR for NCBP2-AS2 showing the silencing efficiency in cells used in (E). NCBP2-AS2 mRNA levels were normalized to TBP. N = three biological replicates. (G) Influence of NCBP2-AS2 silencing on the angiogenic secretome of hypoxic CAFs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 NCBP2-AS2 regulates EC sprouting through VEGFA signaling.

    (A) Sprouting quantification of ECs treated with the CM from hypoxic cCAFs transfected with siCTL or siNCBP2-AS2 (two different siRNAs) with or without VEGFA. N = beads assessed in three biological replicates. CM without VEGFA: siCTL, n = 149; siNCBP2-AS2 #1, n = 128; and siNCBP2-AS2 #2, n = 110. CM with VEGFA: siCTL, n = 132; siNCBP2-AS2 #1, n = 130; and siNCBP2-AS2 #2, n = 126. (B) Sprouting quantification of ECs treated with the CM from hypoxic pCAFs transfected with siCTL or siNCBP2-AS2 (two different siRNAs) with or without VEGFA. N = beads assessed in three biological replicates. CM without VEGFA: siCTL, n = 162 beads; siNCBP2-AS2 #1, n = 132; and siNCBP2-AS2 #2, n = 124. CM with VEGFA: siCTL, n = 127; siNCBP2-AS2 #1, n = 133; and siNCBP2-AS2 #2, n = 136. (C) RT-qPCR showing remaining mRNA levels of NCBP2-AS2 normalized to TBP. (D) Sprouting quantification of ECs treated with the CM from hypoxic cCAFs transfected with siCTL or siNCBP2-AS2 (two different siRNAs) in the presence of bevacizumab or IgGK (control). N = beads assessed in three biological replicates. CM with IgG: siCTL, n = 135; siNCBP2-AS2 #1, n = 129; and siNCBP2-AS2 #2, n = 107. CM with bevacizumab: siCTL, n = 127; siNCBP2-AS2 #1, n = 147; and siNCBP2-AS2 #2, n = 122. (E) Sprouting quantification of ECs treated with the CM from hypoxic pCAFs transfected with siCTL or siNCBP2-AS2 (two different siRNAs) in the presence of bevacizumab or IgGK (control). N = beads assessed in three biological replicates. CM with IgG: siCTL, n = 121; siNCBP2-AS2 #1, n = 108; and siNCBP2-AS2 #2, n = 107. CM with bevacizumab: siCTL, n = 126; siNCBP2-AS2 #1, n = 130; and siNCBP2-AS2 #2, n = 110. (F) RT-qPCR showing the remaining mRNA levels of NCBP2-AS2 normalized to TBP. (G) Representative Western blot for proteins downstream of VEGFR signaling in ECs treated with the CM from cCAFs silenced or not for NCBP2-AS2. N = three biological replicates. (H) RT-qPCR showing remaining mRNA levels of NCBP2-AS2 normalized to TBP. (I) Working model. *P < 0.05, **P < 0.01, ****P < 0.0001, t test.

  • Fig. 8 NCBP2-AS2 is expressed in the stroma of breast tumors.

    (A) Pie chart showing, for each type of breast cancer contained in a breast cancer TMA of 100 patient samples, how many showed a positive staining (as assessed by in situ hybridization with RNAscope) for NCBP2-AS2 in the tumor stroma. (B) Representative images of breast cancer tissues with positive or negative staining (as assessed by in situ hybridization with RNAscope) for NCBP2-AS2 in the tumor stroma. Scale bar, 100 μm (left) and 10 μm (right).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/567/eaan8247/DC1

    Fig. S1. Response of CAFs to hypoxia.

    Fig. S2. Hypoxia remodels the secretome of CAFs to promote angiogenesis.

    Fig. S3. Hypoxia-responsive proteins discovered in our study.

    Fig. S4. NCBP2-AS2 identification and quantification.

    Fig. S5. NCBP2-AS2 regulation by hypoxia.

    Fig. S6. PRM of NCBP2-AS2 in pulsed-SILAC and siRNA experiment.

    Fig. S7. Extracellular NCBP2-AS2 does not induce sprouting in ECs and VEGF expression in CAFs.

    Fig. S8. Western blot quantification of VEGFR signaling in HUVECs.

    Fig. S9. NCBP2-AS2 gene expression levels do not correlate with hypoxia in tumors.

    Data file S1. Proteome and secretome analysis of mammary CAFs.

    Data file S2. Hypoxia-regulated proteins in CAFs.

    Data file S3. Proteome of CAFs expressing siNCBP2-AS2.

    Data file S4. Secretome of CAFs expressing siNCBP2-AS2.

  • The PDF file includes:

    • Fig. S1. Response of CAFs to hypoxia.
    • Fig. S2. Hypoxia remodels the secretome of CAFs to promote angiogenesis.
    • Fig. S3. Hypoxia-responsive proteins discovered in our study.
    • Fig. S4. NCBP2-AS2 identification and quantification.
    • Fig. S5. NCBP2-AS2 regulation by hypoxia.
    • Fig. S6. PRM of NCBP2-AS2 in pulsed-SILAC and siRNA experiment.
    • Fig. S7. Extracellular NCBP2-AS2 does not induce sprouting in ECs and VEGF expression in CAFs.
    • Fig. S8. Western blot quantification of VEGFR signaling in HUVECs.
    • Fig. S9. NCBP2-AS2 gene expression levels do not correlate with hypoxia in tumors.
    • Legends for Data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Proteome and secretome analysis of mammary CAFs.
    • Data file S2 (Microsoft Excel format). Hypoxia-regulated proteins in CAFs.
    • Data file S3 (Microsoft Excel format). Proteome of CAFs expressing siNCBP2-AS2.
    • Data file S4 (Microsoft Excel format). Secretome of CAFs expressing siNCBP2-AS2.

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