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. 2014 Feb 18:3:e01460.
doi: 10.7554/eLife.01460.

miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

Matteo Forloni  1 , Shaillay Kumar Dogra, Yuying Dong, Darryl Conte Jr, Jianhong Ou, Lihua Julie Zhu, April Deng, Meera Mahalingam, Michael R Green, Narendra Wajapeyee
Affiliations

miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

Matteo Forloni et al. Elife. .

Abstract

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001.

Keywords: BRAF; NRAS; melanoma; miRNA.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. BRAFV600E and NRASQ61K upregulate miR-146a expression.
(A) Schematic summary of study design. (B) Volcano plot showing miR-146a (red arrow) as the most upregulated miRNA. (C) qRT-PCR analysis of miR-146a expression in primary melanocytes transduced with BRAFV600E (gray) or Vector control (black) retrovirus particles. Quiescent cells were used as a control (light gray). (D) qRT-PCR analysis (top) measuring miR-146a expression in MEL-ST cells expressing BRAFV600E in the presence (+) or absence (−) of the MEK inhibitor U0216 (10 µM) relative to Vector control. Immunoblots (bottom) of phosphorylated (p-) ERK (upper) and total (t-) ERK (lower) show that BRAFV600E activates MEK-dependent phosphorylation of ERK. (E) qRT-PCR analysis (top) measuring miR-146a expression in MEL-ST cells expressing NRASQ61K relative to Vector control. Immunoblots (bottom) of phosphorylated (p-) ERK (upper) and total (t-) ERK (lower) show that NRASQ61K activates phosphorylation of ERK. (F) qRT-PCR analysis (top) measuring miR-146a expression in MEL-ST cells expressing constitutively active MEK (DD), relative to the Vector control. Immunoblots (bottom) of p-ERK (upper) and t-ERK (lower) show that MEK (DD) activates phosphorylation of ERK. (G) qRT-PCR analysis monitoring miR-146a expression in melanocyte and melanoma cell lines. BRAF (blue) and NRAS (orange) mutant cell lines are indicated. DOI: http://dx.doi.org/10.7554/eLife.01460.003
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Ectopic expression of BRAFV600E in WI-38 and IMR-90 cells stimulates miR-146a expression.
qRT-PCR analysis of miR-146a expression in WI-38 or IMR-90 cells transduced with BRAFV600E (gray) or Vector control (black) retrovirus particles. Quiescent cells were used as a control (light gray). DOI: http://dx.doi.org/10.7554/eLife.01460.004
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Activation of MAP Kinase pathway target genes upon ectopic expression of BRAFV600E in MEL-ST cells.
qRT-PCR analysis of MEK-ERK transcriptional targets FOS, EGR1 and FOSL1 under indicated conditions in MEL-ST cells expressing either an empty vector or BRAFV600E. DOI: http://dx.doi.org/10.7554/eLife.01460.005
Figure 1—figure supplement 3.
Figure 1—figure supplement 3.. Ectopic expression of HRAS v12 or HRAS v12 S35 in MEL-ST cells stimulates miR-146 expression.
qRT-PCR analysis of miR-146a (left) and immunoblot analysis (right) of phosphorylated (p-) ERK, total (t-) ERK in MEL-ST cells transduced with empty vector, HRAS v12, or HRAS v12s35. DOI: http://dx.doi.org/10.7554/eLife.01460.006
Figure 1—figure supplement 4.
Figure 1—figure supplement 4.. Inhibition of MAP kinase signaling by MEK inhibitor blocks HRAS v12-mediated miR-146a upregulation.
qRT-PCR analysis (left) to monitor miR-146a expression and immunoblot analysis (right) of phosphorylated (p-) ERK and total (t-) ERK in MEL-ST cells transduced with HRAS v12 in the presence (+) or absence (−) of the MEK inhibitor U0216. DOI: http://dx.doi.org/10.7554/eLife.01460.007
Figure 1—figure supplement 5.
Figure 1—figure supplement 5.. Ectopic expression of MEK DD stimulates the transcription of MAP kinase target genes.
qRT-PCR analysis of MEK-ERK transcriptional targets FOS, EGR1 and FOSL1 in MEL-ST cells expressing either a empty vector or constitutively active MEK (MEK DD). DOI: http://dx.doi.org/10.7554/eLife.01460.008
Figure 2.
Figure 2.. BRAFV600E upregulates miR-146a through MYC oncogene.
(A) Schematic representation of MYC promoter and the miR-146a binding site that has been identified by PROMO analysis software. ‘+1’ indicates the transcription start site. (B) Immunoblot analysis of p-MYC and total MYC in whole cell lysates of MEL-ST cells transduced with empty vector or BRAFV600E. Actin was used as a loading control. (C) Chromatin immunoprecipitation (ChIP) assay measuring MYC binding to the miR-146a promoter in MEL-ST cells stably expressing BRAFV600E in the presence (+) or absence (−) of the MEK inhibitor U0216 relative to cells transduced with the empty vector. A non-specific control region served as a negative control for MYC recruitment. (D) qRT-PCR analysis of MYC mRNA (left) and miR-146a (right) expression in SKMEL-28 cells infected with MYC shRNAs. (E) qPCR analysis of MYC ChIP of miR-146a promoter and negative control in SKMEL-28 cells infected with MYC shRNAs. DOI: http://dx.doi.org/10.7554/eLife.01460.009
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Analysis of MYC and miR-146a expression in M14 cells expressing shRNAs against MYC.
qRT-PCR analysis of MYC and miR-146a expression in M14 cells transduced with MYC shRNA expression vectors relative to cells transduced with a non-specific (NS) shRNA vector. DOI: http://dx.doi.org/10.7554/eLife.01460.010
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. shRNA-mediated downrgulation of MYC in M14 cells inhibits the expression of MYC transcriptional target genes.
qRT-PCR analysis of MYC targets CCDN1 and CDC25C in M14 cells transduced with MYC shRNA expression vectors relative to cells transduced with a non-specific (NS) shRNA vector. DOI: http://dx.doi.org/10.7554/eLife.01460.011
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. shRNA-mediated downrgulation of MYC in SKMEL-28 cells inhibits the expression of MYC transcriptional target genes.
qRT-PCR analysis of MYC targets CCDN1 and CDC25C in SKMEL-28 cells transduced with MYC shRNA expression vectors relative to cells transduced with a non-specific (NS) shRNA vector. DOI: http://dx.doi.org/10.7554/eLife.01460.012
Figure 2—figure supplement 4.
Figure 2—figure supplement 4.. Transcriptional regulation of miR-146a.
qRT-PCR analysis of ETS1, ELK1, NF-κB, c/EBPβ and miR-146a in SKMEL-28 expressing indicated shRNA. DOI: http://dx.doi.org/10.7554/eLife.01460.013
Figure 3.
Figure 3.. Oncogenic activity of pre-miR-146a/C and pre-miR-146a/G.
(A) Schematic representation of pre-miR-146a/C and pre-miR-146a/G sequences. (B and C) Number of colonies formed in liquid (B) or soft-agar (C) by M14, SKMEL-28 or A375 melanoma cells expressing pre-miR-146a harboring a C or G at position 40, as compared to the Vector (−) control. Colonies were counted after 2 weeks (B) or 4 weeks (C) of growth. (D) qRT-PCR analysis of miR-146a expression in SKMEL-28 cells infected with miRZip-146a (+) or an empty vector (−). (E and F) Number of colonies formed in liquid (E) or soft-agar (F) by SKMEL-28 expressing miRZip-146a (+) or the vector (−) control. Colonies were counted after 2 weeks (E) or 4 weeks (F) of growth. (G) Average tumor volumes at 1 month time points for mice injected with SKMEL-28 expressing a control miRZip or miRZip-146a. (H) Average volume of tumors formed by SKMEL-28 cells (2.5 × 106) expressing pre-miR-146a/C or pre-miR-146a/G, as compared to the Vector control, injected subcutaneously into the flanks of nude mice (n = 5). (I) Representative images (left) and colony number in soft-agar (right) for the NRASQ61K transformed MEL-ST cells that express an empty vector or miRZip-146a. (J) Average tumor volumes at the indicated time points for mice injected with NRASQ61K transformed MEL-ST cells expressing a control miRZip or miRZip-146a. DOI: http://dx.doi.org/10.7554/eLife.01460.014
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Analysis of miR-146a expression in SKMEL-28, A375 and M14 cells expressing either pre-miR-146a/G or pre-miR-146a/C construct.
SKMEL-28, M14 and A375 cells stably expressing pre-miR-146a/C (C) or pre-miR-146a/G (G) or an empty vector (−) were analyzed for miR-146a expression by qRT-PCR. DOI: http://dx.doi.org/10.7554/eLife.01460.015
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. pre-miR-146a/G promotes proliferation of melanoma cells more effective than pre-miR-146a/C.
M14, SKMEL-28 and A375 cells stably expressing pre-miR-146a/C (blue) or pre-miR-146a/G (red) or an empty vector (black) were analyzed for proliferation at indicated days. Relative proliferation is plotted. *, ** and *** represents p values <0.01, <0.001 and <0.0001 respectively. DOI: http://dx.doi.org/10.7554/eLife.01460.016
Figure 3—figure supplement 3.
Figure 3—figure supplement 3.. Inhibition of miR-146a expression blocks the proliferation and anchorage-independent growth of M14 cells.
M14 cells either expressing a control miRZip vector or miRZip-146a were analyzed for miR-146a expression, colony formation, or growth in soft-agar or tumor formation in mice. Tumor volume at 1 month timepoint is plotted. DOI: http://dx.doi.org/10.7554/eLife.01460.017
Figure 3—figure supplement 4.
Figure 3—figure supplement 4.. Inhibition of miR-146a expression blocks the proliferation and anchorage-independent growth of SKMEL-28 cells.
SKMEL-28 cells either expressing a scrambled LNA-antagomiR or LNA-based miR146a antagomiR were analyzed for miR-146a expression (left), colony formation (middle) or growth in soft-agar (right). DOI: http://dx.doi.org/10.7554/eLife.01460.018
Figure 3—figure supplement 5.
Figure 3—figure supplement 5.. Inhibition of miR-146a expression blocks the proliferation and anchorage-independent growth of M14 cells.
M14 cells either expressing a scrambled LNA-antagomiR or LNA-based miR146a antagomiR were analyzed for miR-146a expression (left), colony formation (middle) or growth in soft-agar (right). DOI: http://dx.doi.org/10.7554/eLife.01460.019
Figure 3—figure supplement 6.
Figure 3—figure supplement 6.. Inhibition of miR-146a expression does not block the proliferation and anchorage-independent growth of YUSIV cells.
(Top panel) YUSIV cells either expressing a control miR-ZIP vector or miR-Zip-miR146a were analyzed for miR-146a expression (left), colony formation (middle) or growth in soft-agar (right). (Bottom Panel) YUSIV cells either expressing a control LNA-based control antagomiR or LNA-based miR-146a antagomiR were analyzed for miR-146a expression (left), colony formation (middle) or growth in soft-agar (right). DOI: http://dx.doi.org/10.7554/eLife.01460.020
Figure 3—figure supplement 7.
Figure 3—figure supplement 7.. Analysis of miR-146a expression in indicated melanoma cell lines transfected with increasing concentration of synthetic miR-146a.
qRT-PCR analysis of miR-146a expression in indicated melanoma cell lines. DOI: http://dx.doi.org/10.7554/eLife.01460.021
Figure 3—figure supplement 8.
Figure 3—figure supplement 8.. miR-146a enhances the growth of melanoma cell lines in a concentration dependent manner.
Relative proliferation of indicated melanoma cell lines 5 days after the transfection of synthetic mature miR-146a at indicated concentrations. DOI: http://dx.doi.org/10.7554/eLife.01460.022
Figure 3—figure supplement 9.
Figure 3—figure supplement 9.. NRASQ61K is sufficient to transform MEL-ST cells.
Representative images of soft-agar colonies formed by MEL-ST cells expressing BRAFV600E. NRASQ61K was used as a positive control. DOI: http://dx.doi.org/10.7554/eLife.01460.023
Figure 4.
Figure 4.. Downregulation of NUMB and activation of NOTCH signaling by pre-miR-146a/C and pre-miR-146a/G.
(A) Schematic representation of the NUMB mRNA and potential miR-146a target sites in the coding region (blue arrow) and 3′-UTR. (B) qRT-PCR analysis of NUMB mRNA levels in SKMEL-28 cells expressing the indicated pre-miR-146a allele relative to the Vector control. (C) Western blot of NUMB expression in SKMEL-28 cells transfected with either wild-type NUMB (NUMB-WT) or miR-146a-resistant NUMB (NUMB-MUT) and increasing amount of synthetic miR-146a. (D) Dual luciferase assay using a CSL-Luciferase reporter to measure NOTCH activity in SKMEL-28 cells expressing the indicated pre-miR-146a alleles relative to the Vector control. (E) qRT-PCR analysis of the NOTCH targets HES1, HEY2 and CCDN1 mRNA in indicated samples. Actin mRNA was used as an internal control. (F) Dual luciferase assay using a CSL-Luciferase reporter to measure NOTCH activity in SKMEL-28 cells expressing miRZip-146a relative to the Vector control. (G) qRT-PCR analysis of HES1, HEY2 and CCDN1 mRNA in indicated samples. Actin mRNA was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.01460.024
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Ectopic expression of miR-146a downregulates NUMB protein expression.
Immunoblot analysis of NUMB protein in SKMEL-28 cells stably transduced with empty vector, pre-miR-146a/C or pre-miR-146a/G. DOI: http://dx.doi.org/10.7554/eLife.01460.025
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Relative luciferase activity of NUMB 3′UTR luciferase construct in SKMEL-28 cells expressing an empty vector (−), pre-miR-146a/C (C) or pre-miR-146a/G (G).
DOI: http://dx.doi.org/10.7554/eLife.01460.026
Figure 5.
Figure 5.. miR-146 oncogenic activity depends on the activation of the NOTCH signaling through downregulation of the tumor suppressor NUMB.
(A) qRT-PCR analysis (top) and immunoblot (bottom) of NUMB expression levels in SKMEL-28 cells infected with two different shRNAs against NUMB relative to the control non-silencing shRNA (NS). (B) Number of colonies in soft-agar of SKMEL-28 cells expressing NUMB shRNAs relative to the control non-silencing shRNA (NS). (C) Average volume of tumors formed by SKMEL-28 cells (2.5 × 106) expressing NUMB shRNAs, relative to the non-silencing shRNA (NS shRNA) control, injected subcutaneously into the flanks of nude mice (n = 5). (D and E) Immunoblot of NOTCH (D) and colony formation assay (E) of SKMEL-28 cells stably transduced with the activated intracellular NOTCH domain (ICN) or empty vector. (F) Average volume of tumors formed by SKMEL-28 cells expressing the activated intracellular NOTCH domain (ICN) relative to vector control. 2.5 × 106 cells were injected subcutaneously into the flank of nude mice (n = 5). (G) Colony formation in soft-agar of SKMEL-28 cells expressing pre-miR-146a/G and transfected with either an empty vector, NUMB wild-type (WT) or an miR-146a-resistant NUMB (MUT). (H) qRT-PCR analysis of HES1, HEY2 and CCDN1 mRNA in indicated samples. Actin was used as an internal control. (I) Colony formation in soft-agar of MEL-ST/NRASQ61K transfected with either an empty vector, NUMB wild-type (WT) or miR-146a-resistant NUMB (MUT). (J and K) Number of colonies formed in liquid (J) or soft-agar (K) by SKMEL-28 expressing miRZip-146a (+) or the control miRZip (−) that either express a non-silencing shRNA or an shRNA against NUMB. Colonies were counted after 2 weeks (J) or 4 weeks (K) of growth. (L and M) Number of colonies formed in liquid (L) or soft-agar (M) by M14 expressing miRZip-146a (+) or the control miRZip (−) that either express a non-silencing shRNA or an shRNA against NUMB. Colonies were counted after 2 weeks (L) or 4 weeks (M) of growth. DOI: http://dx.doi.org/10.7554/eLife.01460.027
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Inhibition of NUMB expression promotes growth of SKMEL-28 cells.
Colony formation assay of SKMEL-28 cells expressing two different NUMB shRNAs relative to a non-silencing (NS) shRNA. DOI: http://dx.doi.org/10.7554/eLife.01460.028
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. Inhibition of NUMB promotes proliferation of melanoma cells.
Proliferation assay of SKMEL-28 cells expressing two different NUMB shRNAs relative to a non-silencing (NS) shRNA. DOI: http://dx.doi.org/10.7554/eLife.01460.029
Figure 5—figure supplement 3.
Figure 5—figure supplement 3.. Ectopic expression of activated Notch promotes the growth of melanoma cells.
Colony formation assay of SKMEL-28 cells stably transduced with the activated intracellular NOTCH domain (ICN) or empty vector. DOI: http://dx.doi.org/10.7554/eLife.01460.030
Figure 5—figure supplement 4.
Figure 5—figure supplement 4.. Ectopic expression of intracellular Notch promotes proliferation of melanoma cells.
Proliferation rate of SKMEL-28 cells stably expressing Intracellular NOTCH (ICN) relative to cells with empty vector. Proliferation rate was measured after 72 hr of growth using a colorimetric MTT assay. DOI: http://dx.doi.org/10.7554/eLife.01460.031
Figure 6.
Figure 6.. Synergistic melanoma growth inhibition by simultaneous blockage of Notch and BRAF signaling.
(A) MTT proliferation assay. (B) Immunoblot analysis of phosphorylated (p-) ERK and total (t-) ERK and (C) qRT-PCR analysis of NOTCH target HES1 in SKMEL-28 and SKMEL-19 cells left untreated or treated with U0216, DAPT or combination of both drugs. (D) Model. DOI: http://dx.doi.org/10.7554/eLife.01460.032
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