BRAFV600E induces reversible mitotic arrest in human melanocytes via microRNA-mediated suppression of AURKB
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Evaluation Summary:
This interesting, timely and well-done study focuses on the mechanism underlying nevus growth arrest, which has received renewed attention as a result of recent studies that question the dogma that such arrest is mediated by oncogene-induced senescence. Through experiments involving both cultured primary human melanocytes and cells derived from clinical samples, the authors show that Braf-oncogene-induced nevus cell growth arrest results from microRNA-dependent suppression of the mitotic kinase Aurkb, which influences whether Braf activity is proliferative versus antiproliferative. While the conclusions made within the manuscript are justified, and there is sound acknowledgment of certain pitfalls that could be addressed in future research, more expansive sample sizes and further in vivo work would aid in providing more clinical relevance. This manuscript would appeal to researchers in the melanoma field, especially those studying the underlying mechanisms behind phenotypic plasticity and tumor heterogeneity.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Benign melanocytic nevi frequently emerge when an acquired BRAF V600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs—MIR211-5p and MIR328-3p—induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAF V600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAF V600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.
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Evaluation Summary:
This interesting, timely and well-done study focuses on the mechanism underlying nevus growth arrest, which has received renewed attention as a result of recent studies that question the dogma that such arrest is mediated by oncogene-induced senescence. Through experiments involving both cultured primary human melanocytes and cells derived from clinical samples, the authors show that Braf-oncogene-induced nevus cell growth arrest results from microRNA-dependent suppression of the mitotic kinase Aurkb, which influences whether Braf activity is proliferative versus antiproliferative. While the conclusions made within the manuscript are justified, and there is sound acknowledgment of certain pitfalls that could be addressed in future research, more expansive sample sizes and further in vivo work would aid in providing …
Evaluation Summary:
This interesting, timely and well-done study focuses on the mechanism underlying nevus growth arrest, which has received renewed attention as a result of recent studies that question the dogma that such arrest is mediated by oncogene-induced senescence. Through experiments involving both cultured primary human melanocytes and cells derived from clinical samples, the authors show that Braf-oncogene-induced nevus cell growth arrest results from microRNA-dependent suppression of the mitotic kinase Aurkb, which influences whether Braf activity is proliferative versus antiproliferative. While the conclusions made within the manuscript are justified, and there is sound acknowledgment of certain pitfalls that could be addressed in future research, more expansive sample sizes and further in vivo work would aid in providing more clinical relevance. This manuscript would appeal to researchers in the melanoma field, especially those studying the underlying mechanisms behind phenotypic plasticity and tumor heterogeneity.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Through a very substantial set of experiments, involving both cultured primary human melanocytes and cell lines, these authors show that nevus cell growth arrest is reversible, is primarily a G2-arrest, and depends on the actions of microRNAs on levels of Aurkb, which are themselves dependent on TPA-regulable aspects of cell state. The result is a phenomenon of reversible, Braf-dependent arrest that is strongly dependent on cell context. They continue on to make observations using clinical samples that support this view in vivo. The experiments are well done and generally well-explained, and the results should be of wide interest to those interest in melanocyte biology as well as those interested in cancer initiation more generally.
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Reviewer #2 (Public Review):
This manuscript puts forth new insight into the complicated nature of phenotypic plasticity often seen in melanoma progression, both in the clinic and on the bench. As many in the melanoma field have demonstrated time and time again, identifying clinically relevant molecular hallmarks that can be used to differentiate the stages and treat melanoma is made all the more difficult by the adaptive and plastic nature of the disease. The classical BRAF oncogene has been widely studied leading to many advancements in the mechanistic understanding of melanoma, and yet the details of its role in tumorigenesis and metastasis still remains largely elusive as the path from one stage to the next is not as straight forward as originally thought.
Utilizing publicly available RNA datasets and clinical samples, McNeil et al. …
Reviewer #2 (Public Review):
This manuscript puts forth new insight into the complicated nature of phenotypic plasticity often seen in melanoma progression, both in the clinic and on the bench. As many in the melanoma field have demonstrated time and time again, identifying clinically relevant molecular hallmarks that can be used to differentiate the stages and treat melanoma is made all the more difficult by the adaptive and plastic nature of the disease. The classical BRAF oncogene has been widely studied leading to many advancements in the mechanistic understanding of melanoma, and yet the details of its role in tumorigenesis and metastasis still remains largely elusive as the path from one stage to the next is not as straight forward as originally thought.
Utilizing publicly available RNA datasets and clinical samples, McNeil et al. were able to successfully demonstrate that proliferative arrest in melanocytes is induced by MIR211-5p/MIR328-3p-facilitated AURKB inhibition, limiting BRAF V600E's hyperproliferative tendencies. However, BRAF V600E proliferation is also dependent on environmental stimuli and melanocyte differentiation, which could provide new directions in diagnostics and therapeutic options. Overall, the authors' conclusions are well supported by the data, however in addition to the strengths of the manuscript, there remain a few weaknesses that lessen the scope of their findings in a clinical setting.
Strengths:
The ability to compare matched patient samples across multiple stages of disease remains a difficult hurdle that McNeil et al. was able to overcome by utilizing previously published datasets as well as by analyzing tissue obtained from partnering clinics. Throughout the manuscript, there are aspects of each experiment that demonstrate a well thought out analysis in order to properly justify and increase the impact of the authors' conclusions. In particular, the partial rescue of melanocytes from miRNA-induced proliferative arrest using the lentiviral-based AURKB and GPR3 mRNA expression constructs aided in solidifying the relationship, both indirect and direct, of these computationally predicted mRNA targets to the pseudo-senescence-associated miRNAs. Additionally, the in-depth analysis demonstrating how the role of the BRAF V600E oncogene in melanocytic proliferation arrest is highly dependent on environmental stimuli and melanocytic differentiation was clearly summarized in a single figure panel (4H). Finally, the QPI imaging was a unique addition, which visually drove home the claims of mitotic failure making it a hard conclusion to refute. Despite the quality of data, the authors took the time to recognize their own weaknesses within the manuscript by pointing out how the use of an artificial stimulant like TPA does not translate in identifying clinically relevant environmental stimuli that may be involved in miRNA expression regulation in skin. They do, however provide well thought out hypotheses to these unknowns they were not able to identify, which would provide a basis for future work.
Weaknesses:
While the authors present strong conclusions supported by their data, the limited sample sizes (n=3) in earlier experiments in addition to the lack of in vivo work lessens the potential clinical impact this manuscript could have on the melanoma community. They also make limited mention of future directions apart from investigating whether dysregulation of the spindle checkpoint may be a contributor to copy number variations in nevi.
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