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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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Cell cycle defects in CPEB4-depleted melanoma.(a) Time-lapse analysis of aberrant mitosis in cell line SK-Mel-103 labelled with RFP-Cdt1 and GFP-Geminin fusion proteins (FUCCI system)41 and transduced with control or CPEB4 shRNAs (see Supplementary Movies 1 and 2 for real time imaging of this process in shC- and shCPEB4-transduced cells, respectively). (b) Quantification of cells undergoing normal or aberrant mitosis or blocked in G1 from experiments shown in a. (c) Confocal imaging of mitotic alterations of cultured UACC-62 melanoma cells expressing control or CPEB4 shRNA. DNA and spindles were visualized by DAPI (blue) and α-Tubulin (green) immunofluorescence, respectively. (d) Aberrant mitosis detected by confocal immunomicroscopy in xenografts generated with UACC-62 or SK-Mel-103 cells expressing control or CPEB4 shRNAs. Proliferating cells were identified by phospho-Histone 3 (red immunofluorescence). DAPI (blue) and α-Tubulin (green) were used to label DNA and spindles, respectively. (e) Cell cycle profiles of UACC-62 and HeLa cells infected with lentiviruses coding for control or CPEB4 shRNA (see depletion in the immunoblots of the upper panels). Shown are flow cytometry plots of the indicated cells processed to visualize BrdU incorporation. (f) Distribution of the indicated cell populations at the different phases of the cell cycle. The percentages of cells at the G0/G1, S or G2/M phases were determined by BrdU and PI staining.
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f4: Cell cycle defects in CPEB4-depleted melanoma.(a) Time-lapse analysis of aberrant mitosis in cell line SK-Mel-103 labelled with RFP-Cdt1 and GFP-Geminin fusion proteins (FUCCI system)41 and transduced with control or CPEB4 shRNAs (see Supplementary Movies 1 and 2 for real time imaging of this process in shC- and shCPEB4-transduced cells, respectively). (b) Quantification of cells undergoing normal or aberrant mitosis or blocked in G1 from experiments shown in a. (c) Confocal imaging of mitotic alterations of cultured UACC-62 melanoma cells expressing control or CPEB4 shRNA. DNA and spindles were visualized by DAPI (blue) and α-Tubulin (green) immunofluorescence, respectively. (d) Aberrant mitosis detected by confocal immunomicroscopy in xenografts generated with UACC-62 or SK-Mel-103 cells expressing control or CPEB4 shRNAs. Proliferating cells were identified by phospho-Histone 3 (red immunofluorescence). DAPI (blue) and α-Tubulin (green) were used to label DNA and spindles, respectively. (e) Cell cycle profiles of UACC-62 and HeLa cells infected with lentiviruses coding for control or CPEB4 shRNA (see depletion in the immunoblots of the upper panels). Shown are flow cytometry plots of the indicated cells processed to visualize BrdU incorporation. (f) Distribution of the indicated cell populations at the different phases of the cell cycle. The percentages of cells at the G0/G1, S or G2/M phases were determined by BrdU and PI staining.

Mentions: Main known roles of CPEB4 are related to the control of the second meiotic division in oocytes40, as well as the exit from mitosis in HeLa17 and HEK-293 cells16. Therefore, the FUCCI system41 was exploited for real-time imaging of cell cycle progression in living melanoma cells expressing or made deficient for CPEB4. This strategy is based on fluorescence-dependent visualization of cells in the G1 phase by means of a RFP-Cdt1 fusion protein (red emission), and cells in S/G2/M by GFP-Geminin (green emission). Real-time videomicroscopy revealed aberrant mitosis visualized by chromosome misalignment in metaphase and aberrant cytokinesis in telophase, after sustained depletion of CPEB4 in melanoma cells (see Fig. 4a–c, as well as Supplementary Movies 1 and 2 for comparative analyses of control- or CPEB4-shRNA cells, respectively). In vivo, defects in centrosome number, spindle disorganization, improper alignment of chromosomes and aberrant segregation were also evident by histological evaluation of CPEB4-depleted mouse xenografts (see Fig. 4d for examples of paraffin-embedded lesions stained with α-Tubulin and phosphorylated histone H3, as readouts for spindle formation and the detection of proliferative cells). Single-cell quantifications in cultured cells and subcutaneous melanoma lesions in mice demonstrated that although consistent, mitotic defects in CPEB4-downregulated melanoma cells were infrequent, with <10% increase over basal levels detected in the shControl (shC) cells (Fig. 4b). These results therefore suggested roles of CPEB4 acting at an earlier stage (that is, before mitosis) in the cell division cycle. To evaluate this possibility, cell cycle progression was analysed in greater detail by flow cytometry in melanoma versus non-melanoma lines (UACC-62 and HeLa) transduced with control or CPEB4 shRNAs. As shown in Fig. 4e, BrdU incorporation revealed a distinct decrease in S-phase population of CPEB4-depleted melanoma cells (see quantification in Fig. 4f). Analysis of cell cycle entry after thymidine block confirmed a marked inability to progress through G1-S in melanoma cells, with virtually no changes in the cell cycle profile of the HeLa counterparts (Supplementary Fig. 4). Together, these results demonstrate a new role of CPEB4 in G1/S transition in melanoma superseding a secondary function in mitotic control.


Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers
Cell cycle defects in CPEB4-depleted melanoma.(a) Time-lapse analysis of aberrant mitosis in cell line SK-Mel-103 labelled with RFP-Cdt1 and GFP-Geminin fusion proteins (FUCCI system)41 and transduced with control or CPEB4 shRNAs (see Supplementary Movies 1 and 2 for real time imaging of this process in shC- and shCPEB4-transduced cells, respectively). (b) Quantification of cells undergoing normal or aberrant mitosis or blocked in G1 from experiments shown in a. (c) Confocal imaging of mitotic alterations of cultured UACC-62 melanoma cells expressing control or CPEB4 shRNA. DNA and spindles were visualized by DAPI (blue) and α-Tubulin (green) immunofluorescence, respectively. (d) Aberrant mitosis detected by confocal immunomicroscopy in xenografts generated with UACC-62 or SK-Mel-103 cells expressing control or CPEB4 shRNAs. Proliferating cells were identified by phospho-Histone 3 (red immunofluorescence). DAPI (blue) and α-Tubulin (green) were used to label DNA and spindles, respectively. (e) Cell cycle profiles of UACC-62 and HeLa cells infected with lentiviruses coding for control or CPEB4 shRNA (see depletion in the immunoblots of the upper panels). Shown are flow cytometry plots of the indicated cells processed to visualize BrdU incorporation. (f) Distribution of the indicated cell populations at the different phases of the cell cycle. The percentages of cells at the G0/G1, S or G2/M phases were determined by BrdU and PI staining.
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f4: Cell cycle defects in CPEB4-depleted melanoma.(a) Time-lapse analysis of aberrant mitosis in cell line SK-Mel-103 labelled with RFP-Cdt1 and GFP-Geminin fusion proteins (FUCCI system)41 and transduced with control or CPEB4 shRNAs (see Supplementary Movies 1 and 2 for real time imaging of this process in shC- and shCPEB4-transduced cells, respectively). (b) Quantification of cells undergoing normal or aberrant mitosis or blocked in G1 from experiments shown in a. (c) Confocal imaging of mitotic alterations of cultured UACC-62 melanoma cells expressing control or CPEB4 shRNA. DNA and spindles were visualized by DAPI (blue) and α-Tubulin (green) immunofluorescence, respectively. (d) Aberrant mitosis detected by confocal immunomicroscopy in xenografts generated with UACC-62 or SK-Mel-103 cells expressing control or CPEB4 shRNAs. Proliferating cells were identified by phospho-Histone 3 (red immunofluorescence). DAPI (blue) and α-Tubulin (green) were used to label DNA and spindles, respectively. (e) Cell cycle profiles of UACC-62 and HeLa cells infected with lentiviruses coding for control or CPEB4 shRNA (see depletion in the immunoblots of the upper panels). Shown are flow cytometry plots of the indicated cells processed to visualize BrdU incorporation. (f) Distribution of the indicated cell populations at the different phases of the cell cycle. The percentages of cells at the G0/G1, S or G2/M phases were determined by BrdU and PI staining.
Mentions: Main known roles of CPEB4 are related to the control of the second meiotic division in oocytes40, as well as the exit from mitosis in HeLa17 and HEK-293 cells16. Therefore, the FUCCI system41 was exploited for real-time imaging of cell cycle progression in living melanoma cells expressing or made deficient for CPEB4. This strategy is based on fluorescence-dependent visualization of cells in the G1 phase by means of a RFP-Cdt1 fusion protein (red emission), and cells in S/G2/M by GFP-Geminin (green emission). Real-time videomicroscopy revealed aberrant mitosis visualized by chromosome misalignment in metaphase and aberrant cytokinesis in telophase, after sustained depletion of CPEB4 in melanoma cells (see Fig. 4a–c, as well as Supplementary Movies 1 and 2 for comparative analyses of control- or CPEB4-shRNA cells, respectively). In vivo, defects in centrosome number, spindle disorganization, improper alignment of chromosomes and aberrant segregation were also evident by histological evaluation of CPEB4-depleted mouse xenografts (see Fig. 4d for examples of paraffin-embedded lesions stained with α-Tubulin and phosphorylated histone H3, as readouts for spindle formation and the detection of proliferative cells). Single-cell quantifications in cultured cells and subcutaneous melanoma lesions in mice demonstrated that although consistent, mitotic defects in CPEB4-downregulated melanoma cells were infrequent, with <10% increase over basal levels detected in the shControl (shC) cells (Fig. 4b). These results therefore suggested roles of CPEB4 acting at an earlier stage (that is, before mitosis) in the cell division cycle. To evaluate this possibility, cell cycle progression was analysed in greater detail by flow cytometry in melanoma versus non-melanoma lines (UACC-62 and HeLa) transduced with control or CPEB4 shRNAs. As shown in Fig. 4e, BrdU incorporation revealed a distinct decrease in S-phase population of CPEB4-depleted melanoma cells (see quantification in Fig. 4f). Analysis of cell cycle entry after thymidine block confirmed a marked inability to progress through G1-S in melanoma cells, with virtually no changes in the cell cycle profile of the HeLa counterparts (Supplementary Fig. 4). Together, these results demonstrate a new role of CPEB4 in G1/S transition in melanoma superseding a secondary function in mitotic control.

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