Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan;19(1):73-88.
doi: 10.15252/embr.201744523. Epub 2017 Dec 7.

ROCK-dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

Masaharu Hazawa  1   2   3 , De-Chen Lin  4   5 , Akiko Kobayashi  2 , Yan-Yi Jiang  4 , Liang Xu  4 , Firli Rahmah Primula Dewi  2 , Mahmoud Shaaban Mohamed  2 , Hartono  2 , Mitsutoshi Nakada  6   7 , Makiko Meguro-Horike  8 , Shin-Ichi Horike  6   8 , H Phillip Koeffler  4   5 , Richard W Wong  1   2   3
Affiliations

ROCK-dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

Masaharu Hazawa et al. EMBO Rep. 2018 Jan.

Abstract

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Identification of NUP62 as epidermal differentiation‐preventing factor in SCC

  1. Heat map representing NUPs transcript levels across healthy tissues including skin, esophagus, bone morrow, brain, muscle, salivary grand, pancreas and liver from The Human Protein Atlas (see URLs).

  2. Human H1 embryonic stem cells were differentiated to keratinocyte progenitors in vitro, and their global RNA expression profiles during differentiation were analyzed using RNA‐seq. Rectangle highlights NUP62.

  3. Expression profiles of NUP62 and IVL in normal skin tissue. Bar = 30 μm.

  4. Heat map showing the expression of NUP62 in non‐tumor tissue, and primary head and neck squamous cell carcinoma (HNSCC) samples from TCGA. SI, SII, SIII, SIV denote stages I, II, III and IV.

  5. NUPs expression levels in cervical squamous cell carcinoma (CSCC) samples. NUP62 mRNA levels were retrieved from GEO (accession number is shown above the figure). Five data in healthy human samples (N) and 40 data in tumor patients (T). P values are based on unpaired two‐tailed t‐test with **indicating P < 0.01. n.s. indicates not significant.

  6. Western blot analysis of each NUP in SCC cells at 72 h after siRNA‐mediated knockdown.

  7. Plot showing the correlation between average effect of NUP depletion on SAS cell growth and differential gene expression (normal tissue versus HNSCC from TCGA). NUP62 was highlighted in orange. TP63 was marked in purple as positive control for oncogene. Data show mean from two independent experiments (n = 2).

  8. Summary of correlation coefficient between each NUPs mRNA and epidermal differentiation genes (SPRR1B and IVL) in head and neck SCC from TCGA.

  9. qRT–PCR analysis of genes associated with epidermal differentiation after individual depletion of each NUPs. Transcripts of each gene with scrambled siRNAs is considered 1.0. Data show mean ± SD (n = 3; NUP62i) or mean (n = 2; others). P values are based on one sample t‐test with *indicating P < 0.05.

Source data are available online for this figure.
Figure EV1
Figure EV1. Knockdown of NUP62 decreased proliferation of SCC cells
  1. A

    Levels of transcripts in cells depleted of the indicated NUPs were measured by qRT–PCR. Transcript levels of each NUPs with scrambled siRNAs are considered 100%. Data show mean from two independent experiments (n = 2).

  2. B

    Relative viable number of UMSCC1 and SAS SCC cells depleted of each NUP after siRNA treatment compared to transfection with scrambled siRNA (Control). Value of cells with scrambled siRNAs is considered 100%. Data show mean from two independent experiments (n = 2).

  3. C

    Western blot analysis of NUP62 in various SCC cell lines expressing shRNA NUP62.

  4. D, E

    SCC cells expressing shRNA NUP62 were examined by MTT assay (D) and foci formation assay (E). Number of colonies from cells expressing shRNA control is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P‐values are based on unpaired two‐tailed t‐test (D) or one sample t‐test (E) with *P < 0.05, **P < 0.01.

Source data are available online for this figure.
Figure 2
Figure 2. NUP62 is required for proliferation and undifferentiated status of SCC cells

  1. Western blot analysis of NUP62 in SCC cells after siRNA‐mediated NUP62 depletion.

  2. Proliferation of NUP62‐silenced SCC cells examined by MTT assay. Data show mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05 and **P < 0.01.

  3. qRT–PCR analysis of differentiation related genes in SCC cell line treated with scrambled siRNAs (CTLi) or NUP62 siRNAs (NUP62i). Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05.

  4. Heat map showing mutual exclusivity between NUP62 expression and differentiation related genes. Samples were divided according to mRNA expression levels [mRNA expression z‐Scores (RNA‐Seq V2 RSEM) > mean + 0.1 SD] from the TCGA cohorts. P values are based on fisher exact test.

Source data are available online for this figure.
Figure 3
Figure 3. Transcriptome analysis of NUP62‐regulated genes and processes

  1. Heat map from microarray data showing up‐ and down‐regulated genes after 3 days of knockdown of NUP62.

  2. GO analysis of the up‐regulated genes upon NUP62 depletion.

  3. GSEA of NUP62 knockdown microarray datasets with down‐regulated genes in HNSCC cell upon p63 knockdown (GSE88833).

  4. qRT–PCR analysis of p63 and NUP62 mRNA in various SCC cell lines depleted of NUP62. Expression levels of cells treated with scrambled siRNAs is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

  5. Western blot analysis of ΔNp63α in SCC cells after siRNA‐mediated NUP62 depletion.

Source data are available online for this figure.
Figure 4
Figure 4. NUP62 regulates ΔNp63α nuclear transport in SCCs
  1. A, B

    Immunofluorescence confocal microscopic analysis of ΔNp63α in SCC cells after transient depletion of NUP62 by siRNA (72 h post transfection), (A) and shRNA (B). Representative pictures [bar: 10 μm (A) and 20 μm (B)] and the quantifications as ratio of nuclear/cytoplasm signals were presented as scatter plot with mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05 and **P < 0.01.

  2. C

    Western blot analysis of ΔNp63α protein levels in cytosol and nuclear fractions of SCC cells depleted NUP62.

  3. D

    Transcriptional targets of ΔNp63α, grouped according to their known functions. Red; activated genes, Blue; suppressed genes.

  4. E

    qRT–PCR analysis of ΔNp63α target genes mRNA in SCC cells depleted NUP62. Expression levels of cells treated with scrambled siRNAs is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

Source data are available online for this figure.
Figure EV2
Figure EV2. Analysis of TAp63 targets gene levels in NUP62‐depleted SCC cells
Transcript amounts of TAp63 targets were measured by qRT–PCR. Transcripts of each gene with scrambled siRNAs are considered 1.0. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test. n.s. indicates not significant.
Figure 5
Figure 5. ROCK kinase inhibits ΔNp63α nuclear transport in SCCs

  1. Pathway analysis of the up‐regulated genes upon NUP62 depletion.

  2. GSEA after NUP62 knockdown with up‐regulated genes by ROCK inhibition microarray datasets (GSE61226).

  3. Immunofluorescence confocal microscopic analysis of ΔNp63α in SCC cells treated with ROCK1 inhibitor (Y27632, 10 μM, 24 h). Representative pictures (bar: 10 μm) and quantification as a ratio of nuclear/cytoplasm signals presented as a scatter plot with mean ± SD from three independent experiments (n = 3). P value are based on unpaired two‐tailed t‐test with *indicating P < 0.05.

  4. qRT–PCR analysis of ΔNp63α target genes mRNA in SCC cells after Y27632 treatment (24 h, 10 μM). Expression levels of cells treated with scrambled siRNAs is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05.

  5. Western blot analysis of exogenous constitutively active form of ROCK1 (Δ3‐ROCK1).

  6. Immunofluorescence confocal microscopic analysis of ΔNp63α in SCC cells expressing Δ3‐ROCK1 and the effect of ROCK inhibitor (Y27632, 10 μM, 24 h). Representative pictures (bar: 10 μm) and quantification as a ratio of nuclear/cytoplasm signals presented as a scatter plot with mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with **indicating P < 0.01.

  7. Proliferation of SCC cells expressing Δ3‐ROCK1 and effect of Y27632 (10 μM, analyzed by MTT assay). Data show mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05. and **P < 0.01.

  8. qRT–PCR analysis of IVL and SPRR1B mRNA in SCC cells treated with Y27632. Expression levels of cells with DMSO is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

Source data are available online for this figure.
Figure EV3
Figure EV3. ROCK suppresses ΔNp63α nuclear transport in SCC cells
  1. A

    Immunofluorescence confocal microscopic analysis of ΔNp63α in SCC cells expressing Δ3‐ROCK1, and the effect of CRM1 inhibitor (Leptomycin B, 10 μM, 24 h) on the cells. Representative pictures (bar: 10 μm) and quantification as a ratio of nuclear/cytoplasm signals presented as a scatter plot with mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with **indicating P < 0.01. n.s. indicates not significant.

  2. B, C

    SCC cell lines under Y27632 treatment (10 μM) were subjected to MTT assay (B) and foci formation assay (C). Number of colonies of control cells is considered 100%. Data show mean ± SD from three independents (n = 3). P values are based on two‐tailed t‐test (B) or one sample t‐test (C) with *indicating P < 0.05.

  3. D

    Proliferation of SCC cells expressing Δ3‐ROCK1 and the effect of Y27632 (10 μM) were analyzed by MTT assay. Data show mean ± SD from three independents (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05.

Figure 6
Figure 6. ROCK1‐dependent phosphorylation on the FG domain of NUP62 decreases the ΔNp63α–NUP62 interaction
  1. A

    Schematic representation of the structural and functional domains of ΔNp63α. DBD, DNA binding domain; OD, Oligomerization; TA, Transactivation; SAM, Steric a‐motif; TID, transactivation inhibitory domain (upper panel). Predicted NLSs in ΔNp63α by NLS mapper (middle panel). Diagram of mutations conducted in ΔNp63α (bottom panel).

  2. B, C

    Immunofluorescence confocal microscopic analysis of ΔNp63αWT, ΔNp63α∆NLS1 and ΔNp63α∆NLS2 in 293T. Representative pictures are shown (B). Bar indicates 10 mm. Phenotypes are quantified by counting [n = 2, (C)].

  3. D

    Comparison of NLS score p53, p73 and p63.

  4. E

    Western blot analysis of KPNB1 in SCC cells after siRNA knockdown.

  5. F

    Immunofluorescence confocal microscopic analysis of ΔNp63α in KPNB1 silenced SCC cells. Representative pictures (bar: 10 mm) and quantification as a ratio of nuclear/cytoplasm signals presented as scatter plot with mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05.

  6. G

    Schematic representation of the structural and functional domains of NUP62. FG., FG repeat, CC., coiled‐coil domain.

  7. H

    Schematic representation of NUP62 GFP fusion constructs overexpressed in HEK293T cells. HEK293T cells were co‐transfected with Flag‐tagged ΔNp63α and GFP‐fused NUP62 fragments (FG, Middle, and CC as shown in right panel). Seventy‐two hours after transfection, cells were harvested, lysed, followed by immunoprecipitation (IP) assay using an anti‐Flag antibody. Potential phosphorylation sites on FG is indicated.

  8. I, J

    HEK293T cells were co‐transfected with FG, Δ3‐ROCK1 (I), together with Flag‐tagged ΔNp63α (J). Twenty‐four hours after transfection, cells were treated with Y27632 (24 h, 10 μM). At 72 h after transfection, cells were harvested, lysed, and proceeded for IP assay using indicated antibody.

  9. K

    Interaction between ectopic NUP62 and ΔNp63α is shown either with wild‐type NUP62 (WT) or with a point mutation in the S2 and T20 of FG (Amt).

Source data are available online for this figure.
Figure EV4
Figure EV4. Expression profile of ΔNp63 isoforms and KPNB1 in SCC cells, and potential ROCK targeting region on FG of NUP62

  1. Western blot analysis of ΔNp63α and KPNB1.

  2. Evolutionarily conserved phosphorylation sites in FG domain of NUP62.

  3. Quantification of Western blots results showing ROCK1 activation resulted in elevated phosphorylation of FG of NUP62, while Y27632 reduced this phosphorylation. The relative amount of phosphorylation was quantified by ImageJ using pull‐downed FG amounts as an internal control and normalized further to this level in control (CTL). Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05. n.s. indicates not significant.

  4. Diagram of mutations made in FG domain.

Source data are available online for this figure.
Figure 7
Figure 7. ∆Np63α nuclear transport is mediated by the phosphorylation of FG domain of NUP62
  1. A, B

    Western blot analysis of ectopic WT and Amt NUP62 in SCC cells (A), and exogenous Δ3‐ROCK1 in those cells (B).

  2. C

    Immunofluorescence confocal microscopic analysis of ΔNp63α in SCC cells expressing either WT or Amt NUP62 after Δ3‐ROCK1 overexpression (72 h post transfection). Representative pictures (bar: 10 μm) and quantification as a ratio of nuclear/cytoplasm signals presented as a scatter plot with mean ± SD from three independent experiments (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05 and **P < 0.01. n.s. indicate not significant.

  3. D

    SCC cell lines expressing either WT or Amt NUP62 were subjected either to MTT assay. Data show mean ± SD from three independent experiments (n = 3). P‐values are based on unpaired two‐tailed t‐test with *indicating P < 0.05.

  4. E

    Cross‐section image of dermis and epidermis is modified from previous report 11. Hypothetical model of NUP62 action in regulating cell fate.

Source data are available online for this figure.
Figure EV5
Figure EV5. ∆Np63α nuclear transport is mediated by the phosphorylation of FG domain of NUP62
  1. A

    qRT–PCR analysis of ΔNp63α target genes mRNA in SCC cells expressing either WT or Amt NUP62. Expression levels of cells treated with scrambled siRNAs are considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

  2. B

    Co‐localization analysis of RanBP2 and exogenous NUP62 in SCC cells by immunofluorescence confocal microscopy. Representative pictures are shown (bar: 10 μm).

  3. C, D

    Various SCC cell lines expressing either WT or Amt NUP62 were subjected to foci formation assay (C). Number of colonies from cells expressing WT NUP62 is considered 100% (D). Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05.

See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cancer Genome Atlas Network (2015) Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 517: 576–582 - PMC - PubMed
    1. Lin DC, Hao JJ, Nagata Y, Xu L, Shang L, Meng X, Sato Y, Okuno Y, Varela AM, Ding LW et al (2014) Genomic and molecular characterization of esophageal squamous cell carcinoma. Nat Genet 46: 467–473 - PMC - PubMed
    1. Lee CS, Bhaduri A, Mah A, Johnson WL, Ungewickell A, Aros CJ, Nguyen CB, Rios EJ, Siprashvili Z, Straight A et al (2014) Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma. Nat Genet 46: 1060–1062 - PMC - PubMed
    1. Cancer Genome Atlas Research Network, Analysis Working Group: Asan University, BC Cancer Agency, Brigham and Women's Hospital, Broad Institute, Brown University, Case Western Reserve University, Dana‐Farber Cancer Institute, Duke University, Greater Poland Cancer Centre et al (2017) Integrated genomic characterization of oesophageal carcinoma. Nature 541: 169–175 - PMC - PubMed
    1. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713 - PubMed

Publication types

MeSH terms