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Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers

View Article: PubMed Central - PubMed

ABSTRACT

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

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RAB27A as a novel CPEB4-controlled melanoma driver.(a) Immunoblots showing the downregulation of RAB27A protein expression in MITF-negative (SK-Mel-103) and MITF-positive (UACC-62) cell lines at the indicated times upon lentiviral-driven expression of control or CPEB4 shRNA. (b) RAB27A mRNA Poly(A) tail shortening visualized by PAT assays in CPEB4-depleted melanoma cells. RNase H was used as a reference control for poly(A) removal. nt, nucleotides. (c) RAB27A mRNA levels from RIP experiments performed with CPEB4 antibody or IgG control antibody in the indicated melanoma cells. Inputs were used to normalize mRNA expression in the immunoprecipitated fraction and data are presented as means±s.e.m. from triplicates. (d) BrdU incorporation in the indicated melanoma cell lines visualized by flow cytometry 4 days after lentiviral-driven expression of control or RAB27A shRNA. The corresponding cell cycle distribution is shown in e. (f) Micrographs of paraffin-embedded sections of xenografts generated with the indicated cell lines expressing shC or shRNA against CPEB4, and processed for the visualization of RAB27A (pink staining). Nuclei are counterstained with hematoxylin. Scale bars, 50 μm. (g) Quantification of RAB27A expression represented as a function of positive cells. (h) RAB27A mRNA downregulation determined by quantitative qRT-PCR in xenografts generated as in f. (i) Mosaic image corresponding to dual immunohistochemistry performed on human melanoma tumours (whole-lesion analysis) and visualized by confocal microscopy for single-cell quantification of CPEB4 (red) and RAB27A (green). Scale bars, 1,000 μm. Images in the right correspond to higher magnification of three selected areas of the lesion (labelled as 1, 2 and 3; scale bars, 100 μm) demonstrating the correlation between these two proteins. (j) Relative expression of CPEB4 and RAB27A quantified at a single-cell level by an intelligent matrix screening remote control tool (iMSRC) from images processed by the Definiens XD software. Data points were pseudo-coloured to separate cells with dual expression of CPEB4 and RAB27A (yellow) from those with dominance of one of the two proteins (green for RAB27A and red for CPEB4). Student's t-test P values (P) are indicated in the corresponding panels. iMSRC, intelligent matrix screening remote control.
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f9: RAB27A as a novel CPEB4-controlled melanoma driver.(a) Immunoblots showing the downregulation of RAB27A protein expression in MITF-negative (SK-Mel-103) and MITF-positive (UACC-62) cell lines at the indicated times upon lentiviral-driven expression of control or CPEB4 shRNA. (b) RAB27A mRNA Poly(A) tail shortening visualized by PAT assays in CPEB4-depleted melanoma cells. RNase H was used as a reference control for poly(A) removal. nt, nucleotides. (c) RAB27A mRNA levels from RIP experiments performed with CPEB4 antibody or IgG control antibody in the indicated melanoma cells. Inputs were used to normalize mRNA expression in the immunoprecipitated fraction and data are presented as means±s.e.m. from triplicates. (d) BrdU incorporation in the indicated melanoma cell lines visualized by flow cytometry 4 days after lentiviral-driven expression of control or RAB27A shRNA. The corresponding cell cycle distribution is shown in e. (f) Micrographs of paraffin-embedded sections of xenografts generated with the indicated cell lines expressing shC or shRNA against CPEB4, and processed for the visualization of RAB27A (pink staining). Nuclei are counterstained with hematoxylin. Scale bars, 50 μm. (g) Quantification of RAB27A expression represented as a function of positive cells. (h) RAB27A mRNA downregulation determined by quantitative qRT-PCR in xenografts generated as in f. (i) Mosaic image corresponding to dual immunohistochemistry performed on human melanoma tumours (whole-lesion analysis) and visualized by confocal microscopy for single-cell quantification of CPEB4 (red) and RAB27A (green). Scale bars, 1,000 μm. Images in the right correspond to higher magnification of three selected areas of the lesion (labelled as 1, 2 and 3; scale bars, 100 μm) demonstrating the correlation between these two proteins. (j) Relative expression of CPEB4 and RAB27A quantified at a single-cell level by an intelligent matrix screening remote control tool (iMSRC) from images processed by the Definiens XD software. Data points were pseudo-coloured to separate cells with dual expression of CPEB4 and RAB27A (yellow) from those with dominance of one of the two proteins (green for RAB27A and red for CPEB4). Student's t-test P values (P) are indicated in the corresponding panels. iMSRC, intelligent matrix screening remote control.

Mentions: Downregulation of BUB1B, CDK1 and DEK provides a mechanistic explanation for the aberrant spindles and defects in cytokinesis observed after sustained depletion of CPEB4 in melanoma cells (Fig. 4a–d). However, these genes cannot account for the acute and melanoma-enriched G1 arrest after CPEB4 downregulation, and could not explain why melanomas would be more dependent on this polyadenylation factor than other tumour types. The CPEB4 RIP-seq networks mentioned above in SK-Mel-103 (Fig. 6b,c) were then further screened for genes with unique functions in melanoma. This identified RAB27A (Fig. 6c), a vesicle trafficking modulator known for its essential roles in melanosome maturation (a defining trait of this cell lineage)46. Moreover, RAB27A is upregulated during melanoma progression and it is required for cell division, although by still incompletely defined mechanisms22. Although not expressed in SK-Mel-103, we questioned whether CPEB4 could also control an upstream modulator of RAB27A, the transcription factor MITF. MITF was relevant as the prototypical lineage-specific oncogene in melanocytic tumours47. Interestingly, we found the MITF 3′-UTR to contain the largest amount of CPEs, HEX and PBS of all the genes analysed, which as the case for RAB27A were predicted by our computational models to be optimally organized for the control by cytoplasmic polyadenylation (Fig. 7). Therefore, we set to test the functional impact of CPEB4 in the levels and polyadenylation status of both, MITF and RAB72A in additional melanoma cell lines and in tissue specimens (Figs 8 and 9, respectively).


Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers
RAB27A as a novel CPEB4-controlled melanoma driver.(a) Immunoblots showing the downregulation of RAB27A protein expression in MITF-negative (SK-Mel-103) and MITF-positive (UACC-62) cell lines at the indicated times upon lentiviral-driven expression of control or CPEB4 shRNA. (b) RAB27A mRNA Poly(A) tail shortening visualized by PAT assays in CPEB4-depleted melanoma cells. RNase H was used as a reference control for poly(A) removal. nt, nucleotides. (c) RAB27A mRNA levels from RIP experiments performed with CPEB4 antibody or IgG control antibody in the indicated melanoma cells. Inputs were used to normalize mRNA expression in the immunoprecipitated fraction and data are presented as means±s.e.m. from triplicates. (d) BrdU incorporation in the indicated melanoma cell lines visualized by flow cytometry 4 days after lentiviral-driven expression of control or RAB27A shRNA. The corresponding cell cycle distribution is shown in e. (f) Micrographs of paraffin-embedded sections of xenografts generated with the indicated cell lines expressing shC or shRNA against CPEB4, and processed for the visualization of RAB27A (pink staining). Nuclei are counterstained with hematoxylin. Scale bars, 50 μm. (g) Quantification of RAB27A expression represented as a function of positive cells. (h) RAB27A mRNA downregulation determined by quantitative qRT-PCR in xenografts generated as in f. (i) Mosaic image corresponding to dual immunohistochemistry performed on human melanoma tumours (whole-lesion analysis) and visualized by confocal microscopy for single-cell quantification of CPEB4 (red) and RAB27A (green). Scale bars, 1,000 μm. Images in the right correspond to higher magnification of three selected areas of the lesion (labelled as 1, 2 and 3; scale bars, 100 μm) demonstrating the correlation between these two proteins. (j) Relative expression of CPEB4 and RAB27A quantified at a single-cell level by an intelligent matrix screening remote control tool (iMSRC) from images processed by the Definiens XD software. Data points were pseudo-coloured to separate cells with dual expression of CPEB4 and RAB27A (yellow) from those with dominance of one of the two proteins (green for RAB27A and red for CPEB4). Student's t-test P values (P) are indicated in the corresponding panels. iMSRC, intelligent matrix screening remote control.
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f9: RAB27A as a novel CPEB4-controlled melanoma driver.(a) Immunoblots showing the downregulation of RAB27A protein expression in MITF-negative (SK-Mel-103) and MITF-positive (UACC-62) cell lines at the indicated times upon lentiviral-driven expression of control or CPEB4 shRNA. (b) RAB27A mRNA Poly(A) tail shortening visualized by PAT assays in CPEB4-depleted melanoma cells. RNase H was used as a reference control for poly(A) removal. nt, nucleotides. (c) RAB27A mRNA levels from RIP experiments performed with CPEB4 antibody or IgG control antibody in the indicated melanoma cells. Inputs were used to normalize mRNA expression in the immunoprecipitated fraction and data are presented as means±s.e.m. from triplicates. (d) BrdU incorporation in the indicated melanoma cell lines visualized by flow cytometry 4 days after lentiviral-driven expression of control or RAB27A shRNA. The corresponding cell cycle distribution is shown in e. (f) Micrographs of paraffin-embedded sections of xenografts generated with the indicated cell lines expressing shC or shRNA against CPEB4, and processed for the visualization of RAB27A (pink staining). Nuclei are counterstained with hematoxylin. Scale bars, 50 μm. (g) Quantification of RAB27A expression represented as a function of positive cells. (h) RAB27A mRNA downregulation determined by quantitative qRT-PCR in xenografts generated as in f. (i) Mosaic image corresponding to dual immunohistochemistry performed on human melanoma tumours (whole-lesion analysis) and visualized by confocal microscopy for single-cell quantification of CPEB4 (red) and RAB27A (green). Scale bars, 1,000 μm. Images in the right correspond to higher magnification of three selected areas of the lesion (labelled as 1, 2 and 3; scale bars, 100 μm) demonstrating the correlation between these two proteins. (j) Relative expression of CPEB4 and RAB27A quantified at a single-cell level by an intelligent matrix screening remote control tool (iMSRC) from images processed by the Definiens XD software. Data points were pseudo-coloured to separate cells with dual expression of CPEB4 and RAB27A (yellow) from those with dominance of one of the two proteins (green for RAB27A and red for CPEB4). Student's t-test P values (P) are indicated in the corresponding panels. iMSRC, intelligent matrix screening remote control.
Mentions: Downregulation of BUB1B, CDK1 and DEK provides a mechanistic explanation for the aberrant spindles and defects in cytokinesis observed after sustained depletion of CPEB4 in melanoma cells (Fig. 4a–d). However, these genes cannot account for the acute and melanoma-enriched G1 arrest after CPEB4 downregulation, and could not explain why melanomas would be more dependent on this polyadenylation factor than other tumour types. The CPEB4 RIP-seq networks mentioned above in SK-Mel-103 (Fig. 6b,c) were then further screened for genes with unique functions in melanoma. This identified RAB27A (Fig. 6c), a vesicle trafficking modulator known for its essential roles in melanosome maturation (a defining trait of this cell lineage)46. Moreover, RAB27A is upregulated during melanoma progression and it is required for cell division, although by still incompletely defined mechanisms22. Although not expressed in SK-Mel-103, we questioned whether CPEB4 could also control an upstream modulator of RAB27A, the transcription factor MITF. MITF was relevant as the prototypical lineage-specific oncogene in melanocytic tumours47. Interestingly, we found the MITF 3′-UTR to contain the largest amount of CPEs, HEX and PBS of all the genes analysed, which as the case for RAB27A were predicted by our computational models to be optimally organized for the control by cytoplasmic polyadenylation (Fig. 7). Therefore, we set to test the functional impact of CPEB4 in the levels and polyadenylation status of both, MITF and RAB72A in additional melanoma cell lines and in tissue specimens (Figs 8 and 9, respectively).

View Article: PubMed Central - PubMed

ABSTRACT

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

No MeSH data available.


Related in: MedlinePlus