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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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CPEB4 is induced at early stages of melanoma development.(a) CPEB4 mRNA expression in 27 solid tumours including melanoma extracted from the CCLE data set (CCLE_Expression_Entrez_2012-10-18.res). mRNA levels are normalized by inter-sample variability across the cell lines included in the data set (RMA-Log2). The number of cell lines from each cancer type is indicated in parenthesis. Box colours represent the P-values from pairwise comparisons between melanoma and each tumour type. (b) Representative micrographs of sections from the indicated lesions showing CPEB4 staining in pink. Nuclei are counterstained with hematoxylin. Scale bars, 200 μm (upper images); and 50 μm (lower images). (c) Quantification of CPEB4 staining intensity in the indicated lesion type scored from 0 (negative) to 3 (highest). The number of lesions per category is indicated in Supplementary Methods. (d) Percentage of CPEB4-positive cells in melanoma lesions (four independent areas were analysed by stained specimen, using hematoxylin as a reference to estimate total cell number per section; epidermal and stromal cells were excluded from the analysis). χ2-test P values (P) are indicated. NS, non-significant.
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f1: CPEB4 is induced at early stages of melanoma development.(a) CPEB4 mRNA expression in 27 solid tumours including melanoma extracted from the CCLE data set (CCLE_Expression_Entrez_2012-10-18.res). mRNA levels are normalized by inter-sample variability across the cell lines included in the data set (RMA-Log2). The number of cell lines from each cancer type is indicated in parenthesis. Box colours represent the P-values from pairwise comparisons between melanoma and each tumour type. (b) Representative micrographs of sections from the indicated lesions showing CPEB4 staining in pink. Nuclei are counterstained with hematoxylin. Scale bars, 200 μm (upper images); and 50 μm (lower images). (c) Quantification of CPEB4 staining intensity in the indicated lesion type scored from 0 (negative) to 3 (highest). The number of lesions per category is indicated in Supplementary Methods. (d) Percentage of CPEB4-positive cells in melanoma lesions (four independent areas were analysed by stained specimen, using hematoxylin as a reference to estimate total cell number per section; epidermal and stromal cells were excluded from the analysis). χ2-test P values (P) are indicated. NS, non-significant.

Mentions: One of the defining features of melanomas is their inherent metastatic potential36. Pro-angiogenic roles of CPEB4 identified in pancreatic adenocarcinomas and other pathologies1135 were of interest as possible contributors to the aggressive behaviour of advanced melanomas. CPEB4 was also attractive as in cancer, this is the only CPEB for which antibodies have been validated for genome-wide RNA immunoprecipitation and target identification35. Publicly accessible transcriptomic profiles were mined for a global evaluation of CPEB4 mRNA expression in melanoma and to identify possible differences with other malignancies. The Cancer Cell Line Encyclopedia (CCLE) offered an ideal platform for comparative analyses as it covers over 1,000 cell lines of 37 different cancer types collected and processed in a uniform manner37. Focusing on solid tumours, melanoma cells were found among the highest expressors of CPEB4 (Fig. 1a). Intriguingly, while CPEB4 was not exclusive of melanoma, pairwise analyses identified statistically significant differences with 18 tumour types, including glioblastoma and pancreatic cancer (Fig. 1a), where CPEB4 functions and targets have been addressed in more detail. High CPEB4 mRNA in melanoma cells was supported by two independent data sets3839 corresponding to human clinical biopsies (N=198 and 119 cases, respectively) that span across various cancer types (see Supplementary Fig. 1a). These results were further validated by quantitative PCR after reverse transcription (qRT-PCR) in a panel of cell lines from melanoma and other tumour types (Supplementary Fig. 1b). Histological analyses were then performed on tissue microarrays containing representative examples of 15 tumour types (N=108 specimens; see Supplementary Information for detail). This analysis confirmed a high CPEB4 expression in melanoma, although again, not restricted to this disease (see Supplementary Fig. 1c for comparative analyses with other malignancies with strong, medium and low CPEB4 staining).


Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers
CPEB4 is induced at early stages of melanoma development.(a) CPEB4 mRNA expression in 27 solid tumours including melanoma extracted from the CCLE data set (CCLE_Expression_Entrez_2012-10-18.res). mRNA levels are normalized by inter-sample variability across the cell lines included in the data set (RMA-Log2). The number of cell lines from each cancer type is indicated in parenthesis. Box colours represent the P-values from pairwise comparisons between melanoma and each tumour type. (b) Representative micrographs of sections from the indicated lesions showing CPEB4 staining in pink. Nuclei are counterstained with hematoxylin. Scale bars, 200 μm (upper images); and 50 μm (lower images). (c) Quantification of CPEB4 staining intensity in the indicated lesion type scored from 0 (negative) to 3 (highest). The number of lesions per category is indicated in Supplementary Methods. (d) Percentage of CPEB4-positive cells in melanoma lesions (four independent areas were analysed by stained specimen, using hematoxylin as a reference to estimate total cell number per section; epidermal and stromal cells were excluded from the analysis). χ2-test P values (P) are indicated. NS, non-significant.
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f1: CPEB4 is induced at early stages of melanoma development.(a) CPEB4 mRNA expression in 27 solid tumours including melanoma extracted from the CCLE data set (CCLE_Expression_Entrez_2012-10-18.res). mRNA levels are normalized by inter-sample variability across the cell lines included in the data set (RMA-Log2). The number of cell lines from each cancer type is indicated in parenthesis. Box colours represent the P-values from pairwise comparisons between melanoma and each tumour type. (b) Representative micrographs of sections from the indicated lesions showing CPEB4 staining in pink. Nuclei are counterstained with hematoxylin. Scale bars, 200 μm (upper images); and 50 μm (lower images). (c) Quantification of CPEB4 staining intensity in the indicated lesion type scored from 0 (negative) to 3 (highest). The number of lesions per category is indicated in Supplementary Methods. (d) Percentage of CPEB4-positive cells in melanoma lesions (four independent areas were analysed by stained specimen, using hematoxylin as a reference to estimate total cell number per section; epidermal and stromal cells were excluded from the analysis). χ2-test P values (P) are indicated. NS, non-significant.
Mentions: One of the defining features of melanomas is their inherent metastatic potential36. Pro-angiogenic roles of CPEB4 identified in pancreatic adenocarcinomas and other pathologies1135 were of interest as possible contributors to the aggressive behaviour of advanced melanomas. CPEB4 was also attractive as in cancer, this is the only CPEB for which antibodies have been validated for genome-wide RNA immunoprecipitation and target identification35. Publicly accessible transcriptomic profiles were mined for a global evaluation of CPEB4 mRNA expression in melanoma and to identify possible differences with other malignancies. The Cancer Cell Line Encyclopedia (CCLE) offered an ideal platform for comparative analyses as it covers over 1,000 cell lines of 37 different cancer types collected and processed in a uniform manner37. Focusing on solid tumours, melanoma cells were found among the highest expressors of CPEB4 (Fig. 1a). Intriguingly, while CPEB4 was not exclusive of melanoma, pairwise analyses identified statistically significant differences with 18 tumour types, including glioblastoma and pancreatic cancer (Fig. 1a), where CPEB4 functions and targets have been addressed in more detail. High CPEB4 mRNA in melanoma cells was supported by two independent data sets3839 corresponding to human clinical biopsies (N=198 and 119 cases, respectively) that span across various cancer types (see Supplementary Fig. 1a). These results were further validated by quantitative PCR after reverse transcription (qRT-PCR) in a panel of cell lines from melanoma and other tumour types (Supplementary Fig. 1b). Histological analyses were then performed on tissue microarrays containing representative examples of 15 tumour types (N=108 specimens; see Supplementary Information for detail). This analysis confirmed a high CPEB4 expression in melanoma, although again, not restricted to this disease (see Supplementary Fig. 1c for comparative analyses with other malignancies with strong, medium and low CPEB4 staining).

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