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

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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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Essential cell-autonomous role of CPEB4 in melanoma growth.(a) Representative examples of animals subcutaneously implanted with SK-Mel-28, SK-Mel-103 or UACC-62 cells expressing control shRNA (shC) or CPEB4 shRNA (sh1). Key melanoma-associated mutations in the indicated cell lines are listed in parenthesis. Panels on the left correspond to immunoblots for visualization of the efficiency of the CPEB4 shRNA-depleting constructs in the different cell lines. (b) Differential tumour growth upon subcutaneous implantation of cells in immunosuppressed mice in a. N=5 mice per group. (c, upper) Depletion of CPEB4 in the indicated cell lines upon lentiviral-driven transduction of two validated shRNA for CPEB4 (sh1 and sh2) shown by immunoblotting using cells expressing a shRNA control (shC) as a reference. (lower) Melanoma cell proliferation after transduction of control or CPEB4 shRNAs. Graphs depict relative cell numbers at the indicated time points obtained from three independent experiments in triplicate. (d) Representative images of SA-β-Gal staining (blue) of the indicated melanoma cell lines expressing control or CPEB4 shRNAs. Scale bars, 20 μm. (e) Percentage of SA-β-Gal-positive cells from d. Error bars represent s.e.m. from three independent experiments. Student's t-test and ANOVA P values are indicated or represented as ***P<0.001, **P<0.01. ANOVA, analysis of variance.
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f2: Essential cell-autonomous role of CPEB4 in melanoma growth.(a) Representative examples of animals subcutaneously implanted with SK-Mel-28, SK-Mel-103 or UACC-62 cells expressing control shRNA (shC) or CPEB4 shRNA (sh1). Key melanoma-associated mutations in the indicated cell lines are listed in parenthesis. Panels on the left correspond to immunoblots for visualization of the efficiency of the CPEB4 shRNA-depleting constructs in the different cell lines. (b) Differential tumour growth upon subcutaneous implantation of cells in immunosuppressed mice in a. N=5 mice per group. (c, upper) Depletion of CPEB4 in the indicated cell lines upon lentiviral-driven transduction of two validated shRNA for CPEB4 (sh1 and sh2) shown by immunoblotting using cells expressing a shRNA control (shC) as a reference. (lower) Melanoma cell proliferation after transduction of control or CPEB4 shRNAs. Graphs depict relative cell numbers at the indicated time points obtained from three independent experiments in triplicate. (d) Representative images of SA-β-Gal staining (blue) of the indicated melanoma cell lines expressing control or CPEB4 shRNAs. Scale bars, 20 μm. (e) Percentage of SA-β-Gal-positive cells from d. Error bars represent s.e.m. from three independent experiments. Student's t-test and ANOVA P values are indicated or represented as ***P<0.001, **P<0.01. ANOVA, analysis of variance.

Mentions: Next, we took advantage of two previously validated short hairpin RNAs (shRNAs)35 to deplete CPEB4 in a panel of melanoma cell lines that recapitulate frequent characteristic oncogenic mutations in BRAF, NRAS or p53 of this disease (see depletion efficacies in Fig. 2a). Control and shRNA-expressing cells were injected subcutaneously in immunosuppressed mice. Curiously, 90% of implants of shCPEB4-cells failed to grow (Fig. 2a,b), halting progression even before sizable lesions could be generated that needed additional roles on vascularization. This was unexpected considering the reported situation in pancreatic cancer cells, where CPEB4 depletion delayed, but did not abrogate xenograft formation35.


Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers
Essential cell-autonomous role of CPEB4 in melanoma growth.(a) Representative examples of animals subcutaneously implanted with SK-Mel-28, SK-Mel-103 or UACC-62 cells expressing control shRNA (shC) or CPEB4 shRNA (sh1). Key melanoma-associated mutations in the indicated cell lines are listed in parenthesis. Panels on the left correspond to immunoblots for visualization of the efficiency of the CPEB4 shRNA-depleting constructs in the different cell lines. (b) Differential tumour growth upon subcutaneous implantation of cells in immunosuppressed mice in a. N=5 mice per group. (c, upper) Depletion of CPEB4 in the indicated cell lines upon lentiviral-driven transduction of two validated shRNA for CPEB4 (sh1 and sh2) shown by immunoblotting using cells expressing a shRNA control (shC) as a reference. (lower) Melanoma cell proliferation after transduction of control or CPEB4 shRNAs. Graphs depict relative cell numbers at the indicated time points obtained from three independent experiments in triplicate. (d) Representative images of SA-β-Gal staining (blue) of the indicated melanoma cell lines expressing control or CPEB4 shRNAs. Scale bars, 20 μm. (e) Percentage of SA-β-Gal-positive cells from d. Error bars represent s.e.m. from three independent experiments. Student's t-test and ANOVA P values are indicated or represented as ***P<0.001, **P<0.01. ANOVA, analysis of variance.
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f2: Essential cell-autonomous role of CPEB4 in melanoma growth.(a) Representative examples of animals subcutaneously implanted with SK-Mel-28, SK-Mel-103 or UACC-62 cells expressing control shRNA (shC) or CPEB4 shRNA (sh1). Key melanoma-associated mutations in the indicated cell lines are listed in parenthesis. Panels on the left correspond to immunoblots for visualization of the efficiency of the CPEB4 shRNA-depleting constructs in the different cell lines. (b) Differential tumour growth upon subcutaneous implantation of cells in immunosuppressed mice in a. N=5 mice per group. (c, upper) Depletion of CPEB4 in the indicated cell lines upon lentiviral-driven transduction of two validated shRNA for CPEB4 (sh1 and sh2) shown by immunoblotting using cells expressing a shRNA control (shC) as a reference. (lower) Melanoma cell proliferation after transduction of control or CPEB4 shRNAs. Graphs depict relative cell numbers at the indicated time points obtained from three independent experiments in triplicate. (d) Representative images of SA-β-Gal staining (blue) of the indicated melanoma cell lines expressing control or CPEB4 shRNAs. Scale bars, 20 μm. (e) Percentage of SA-β-Gal-positive cells from d. Error bars represent s.e.m. from three independent experiments. Student's t-test and ANOVA P values are indicated or represented as ***P<0.001, **P<0.01. ANOVA, analysis of variance.
Mentions: Next, we took advantage of two previously validated short hairpin RNAs (shRNAs)35 to deplete CPEB4 in a panel of melanoma cell lines that recapitulate frequent characteristic oncogenic mutations in BRAF, NRAS or p53 of this disease (see depletion efficacies in Fig. 2a). Control and shRNA-expressing cells were injected subcutaneously in immunosuppressed mice. Curiously, 90% of implants of shCPEB4-cells failed to grow (Fig. 2a,b), halting progression even before sizable lesions could be generated that needed additional roles on vascularization. This was unexpected considering the reported situation in pancreatic cancer cells, where CPEB4 depletion delayed, but did not abrogate xenograft formation35.

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