Accepted Article
Received Date : 11-Oct-2014 Revised Date : 25-Nov-2014 Accepted Date : 01-Dec-2014 Article type
: Original Article
Manuscript Category: Signaling & Cell biology (SCB)
microRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis
*Varsha Tembe1,2, *Sarah-Jane Schramm1,2, *Mitchell S. Stark3,4, Ellis Patrick5, Vivek Jayaswal5, Yue Hang Tang6, Andrew Barbour6,7, Nicholas K. Hayward3, John F. Thompson2,8, Richard A. Scolyer2,9,10, Yee Hwa Yang5, Graham J. Mann1,2
1
Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
2
Melanoma Institute Australia, Sydney, NSW, Australia
3
Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Brisbane,
QLD, Australia 4
Institute of Health and Biomedical Innovation, Queensland University of
Technology, Brisbane, QLD, Australia 5
School of Mathematics and Statistics, The University of Sydney, Sydney, NSW,
Australia 6
Surgical Oncology Group, School of Medicine, The University of Queensland,
Princess Alexandra Hospital, Brisbane, QLD, Australia
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pcmr.12343 This article is protected by copyright. All rights reserved.
Accepted Article
7
Department of Surgery, School of Medicine, The University of Queensland,
Princess Alexandra Hospital, Brisbane, QLD, Australia Disciplines of 8Surgery and 9Pathology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia 10
Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney,
NSW, Australia
*Contributed equally to the work.
Correspondence Varsha Tembe Westmead Millennium Institute, Darcy Road, Westmead, NSW, 2145, Australia. T: +612 98459052, F: +61 2 9845 9102 E: [email protected]
SUMMARY Objectives/experimental design The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh-frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumors and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation.
Principal observations Associations with prognosis were replicated for miR-150-5p, miR-142-3p, and miR142-5p. Co-analysis of miRNA and mRNA uncovered a network associated with poor
This article is protected by copyright. All rights reserved.
Accepted Article
prognosis
that
paradoxically
favoured
expression
of
miRNAs
opposing
tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves.
Conclusions Robust association of miR-150-5p and the miR-142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with poor prognosis in an autoregulatory fashion will not be suitable therapeutic targets.
SIGNIFICANCE More work is needed to understand the behaviour and translational relevance of miRNAs in melanoma. We report the largest study (n=45) of miRNA expression profiling in fresh-frozen metastatic melanomas to date. Association of miR-150-5p, miR-142-3p, and miR-142-5p with poor prognosis across independent cohorts of tumors and sera highlighted potential prognostic biomarkers. Integration of miRNA and mRNA expression data identified a dominant network of miRNAs in the worst prognosis samples: most of which appeared to be reactive to tumor progression rather than driving it. This has implications for selection of miRNAs as therapeutic targets. KEYWORDS bioinformatics,
biomarker,
BRAF,
melanoma,
pathology, prognosis
This article is protected by copyright. All rights reserved.
microRNA,
miRNA,
network,
Accepted Article
RUNNING TITLE microRNA profiling in metastatic melanoma
INTRODUCTION Despite recent advances in targeted therapies, metastatic melanoma continues to be among the most aggressive and treatment-resistant tumor types (Flaherty et al., 2012). Moreover, incidence is rising in the U.S., Australia, and parts of Europe (de Vries and Willem Coebergh, 2004; Garbe and Leiter, 2009; Thompson et al., 2005), and in some cases, faster than for any other cancer (Guy and Ekwueme, 2011).
The molecular mechanisms that drive melanoma development and progression are complex and have not been fully elucidated. Critical progress has been made in identifying a number of causally implicated gene mutations e.g., BRAF, NRAS KIT, CDKN2A, MITF (Whiteman et al., 2011) and recent efforts to uncover the remaining driver mutations have been accelerated by high-throughput sequencing e.g., (Hodis et al., 2012). However, it is now evident from work in both melanoma and other cancers that such oncogenic mutations do not fully explain the development and progression of tumors (Richards and Medrano, 2009). Changes to the epigenetic landscape, including but not limited to microRNA(miRNA)-based gene regulation, are now firmly associated with human malignancies (Dawson and Kouzarides, 2012). There is still much to be learned about the role of miRNAs as dynamic regulators and/or associates of melanoma progression and prognosis (Crea et al., 2014). Since the earliest report on miRNA expression in melanoma was published (Lu et al., 2005), various studies have revealed deregulated expression of miRNAs in tumor cell lines (Levati et al., 2009; Mueller et al., 2009; Satzger et al., 2010), fresh-frozen
This article is protected by copyright. All rights reserved.
Accepted Article
or formalin-fixed paraffin-embedded (FFPE) tissues (Caramuta et al., 2010; Segura et al., 2010), and patient-derived serum or plasma (Friedman et al., 2012; Leidinger et al., 2010). miRNAs have been nominated as biomarkers that can differentiate between tumor and normal tissue (reviewed in (Bonazzi et al., 2012)), discriminate melanoma subtypes (Chan et al., 2011; Poliseno et al., 2012), and potentially aid in both diagnosis (Leidinger et al., 2010) and prognosis (Caramuta et al., 2010; Friedman et al., 2012; Nguyen et al., 2011; Satzger et al., 2010; Segura et al., 2010). However, in these studies, only a handful have involved microarray expression profiling of miRNA in clinical tumor samples (Caramuta et al., 2010; Chen et al., 2010; Glud et al., 2009; Segura et al., 2010), and since all but one of those studies involved few samples (n≤16 (Caramuta et al., 2010; Chen et al., 2010; Glud et al., 2009)) further analysis including validation of their findings is needed. Consistent with these prior related reports we focus our analysis on metastatic melanoma: a setting in which there is an urgent need for new biomarkers of melanoma progression and prognosis, as well as therapeutic interventions (Slipicevic and Herlyn, 2012).
The role of miRNAs in the effects of the common BRAFV600E activating mutation, which drives the mitogen activated protein kinase (MAPK) pathway, is also poorly understood. This mutation is not only a critical predictive marker of sensitivity to effective systemic therapy (Long et al., 2011) but is also negatively correlated (in a BRAF-inhibitor treatment naïve setting) with patient survival in metastatic melanoma (Barbour et al., 2014; Jayawardana et al., 2014; Long et al., 2011; Mann et al., 2013). Studies leveraging clinical samples to evaluate miRNA associations with BRAF mutation status are scarce (Caramuta et al., 2010). The present study, which This article is protected by copyright. All rights reserved.
Accepted Article
relates miRNA expression to both patient clinical outcome and BRAF mutation status, was therefore intended to point to new prognostic tools, and to inform new avenues for novel therapeutic strategies by characterising the miRNA networks affected by BRAF mutation (Völler et al., 2013).
We have also examined how miRNAs exert their regulatory effects on important melanoma target genes and pathways. miRNAs have the potential to regulate multiple inter-related pathways due to their binding efficiency (primarily in the 3’UTR) to target genes and, as such, have the predicted ability to bind to thousands of genes. Recent evidence suggests that complex diseases such as melanoma can be characterized by the cooperative effects of multiple dysfunctional miRNAs rather than a single miRNA per se (Couts et al., 2013). However, while there have been several candidate-driven studies i.e., hypothesis-driven work focussed on exploring relationships between one miRNA and one or a few target genes (reviewed in (Bonazzi et al., 2012)), there has been no large-scale analysis of such networks to date. We therefore performed the first global integration of miRNA and mRNA microarray expression data derived from the same clinical specimens in American Joint Committee on Cancer (AJCC) stage III melanomas. Here, we employed both an unsupervised approach (the identification of associations between computationally predicted groups of miRNA and mRNA transcripts informed by their relative expression values) and a pathway-supervised approach (in which putative miRNAmRNA relationships were identified based on an intersection of prior knowledge in miRBase (Griffiths-Jones et al., 2008) and KEGG (Kanehisa et al., 2012)).
This article is protected by copyright. All rights reserved.
Accepted Article
Surprisingly, the deregulated network of miRNAs identified in association with reduced survival was largely anti-proliferative in the tumors from patients with worse prognosis, as judged by the effects of their target mRNA products.
RESULTS miRNA microarray expression profiling in metastatic melanoma – discovery cohort Global miRNA expression profiles were evaluated in 45 metastatic melanomas from AJCC stage III patients surviving more than 4 years after resection of lymph node metastatic disease with no sign of relapse (good prognosis (GP), nGP=22) and from patients who died of melanoma within 12 months of same (poor prognosis (PP), nPP=23) (Figure 1). Gene (mRNA) expression profiling in this cohort has been previously described (Jayawardana et al., 2014; Mann et al., 2013) and correlated with detailed clinico-pathologic, and molecular data including BRAF mutation status. Discovery analysis was restricted to clinical stage III patients to reduce the potential for confounding effects due to sample heterogeneity, which would be expected with the inclusion of tissue from AJCC stage IV disease. It is also (arguably) the more clinically urgent research setting for the prognostic stratification of patients, based on the wide range of survival outcomes indicated by the current set of prognostic markers for that group (Balch et al., 2009). Tissue levels of 12 miRNAs were associated with patient clinical outcome in AJCC stage III metastatic melanoma We examined which miRNAs (nprobes=1347) were differentially expressed (DE) between patients with GP and PP. Twelve miRNAs were significantly (adjusted
This article is protected by copyright. All rights reserved.
Accepted Article
P1.5-fold, in good compared with poor prognosis patients (adjusted P2 years) compared with short (
Received Date : 11-Oct-2014 Revised Date : 25-Nov-2014 Accepted Date : 01-Dec-2014 Article type
: Original Article
Manuscript Category: Signaling & Cell biology (SCB)
microRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis
*Varsha Tembe1,2, *Sarah-Jane Schramm1,2, *Mitchell S. Stark3,4, Ellis Patrick5, Vivek Jayaswal5, Yue Hang Tang6, Andrew Barbour6,7, Nicholas K. Hayward3, John F. Thompson2,8, Richard A. Scolyer2,9,10, Yee Hwa Yang5, Graham J. Mann1,2
1
Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
2
Melanoma Institute Australia, Sydney, NSW, Australia
3
Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Brisbane,
QLD, Australia 4
Institute of Health and Biomedical Innovation, Queensland University of
Technology, Brisbane, QLD, Australia 5
School of Mathematics and Statistics, The University of Sydney, Sydney, NSW,
Australia 6
Surgical Oncology Group, School of Medicine, The University of Queensland,
Princess Alexandra Hospital, Brisbane, QLD, Australia
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pcmr.12343 This article is protected by copyright. All rights reserved.
Accepted Article
7
Department of Surgery, School of Medicine, The University of Queensland,
Princess Alexandra Hospital, Brisbane, QLD, Australia Disciplines of 8Surgery and 9Pathology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia 10
Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney,
NSW, Australia
*Contributed equally to the work.
Correspondence Varsha Tembe Westmead Millennium Institute, Darcy Road, Westmead, NSW, 2145, Australia. T: +612 98459052, F: +61 2 9845 9102 E: [email protected]
SUMMARY Objectives/experimental design The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh-frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumors and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation.
Principal observations Associations with prognosis were replicated for miR-150-5p, miR-142-3p, and miR142-5p. Co-analysis of miRNA and mRNA uncovered a network associated with poor
This article is protected by copyright. All rights reserved.
Accepted Article
prognosis
that
paradoxically
favoured
expression
of
miRNAs
opposing
tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves.
Conclusions Robust association of miR-150-5p and the miR-142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with poor prognosis in an autoregulatory fashion will not be suitable therapeutic targets.
SIGNIFICANCE More work is needed to understand the behaviour and translational relevance of miRNAs in melanoma. We report the largest study (n=45) of miRNA expression profiling in fresh-frozen metastatic melanomas to date. Association of miR-150-5p, miR-142-3p, and miR-142-5p with poor prognosis across independent cohorts of tumors and sera highlighted potential prognostic biomarkers. Integration of miRNA and mRNA expression data identified a dominant network of miRNAs in the worst prognosis samples: most of which appeared to be reactive to tumor progression rather than driving it. This has implications for selection of miRNAs as therapeutic targets. KEYWORDS bioinformatics,
biomarker,
BRAF,
melanoma,
pathology, prognosis
This article is protected by copyright. All rights reserved.
microRNA,
miRNA,
network,
Accepted Article
RUNNING TITLE microRNA profiling in metastatic melanoma
INTRODUCTION Despite recent advances in targeted therapies, metastatic melanoma continues to be among the most aggressive and treatment-resistant tumor types (Flaherty et al., 2012). Moreover, incidence is rising in the U.S., Australia, and parts of Europe (de Vries and Willem Coebergh, 2004; Garbe and Leiter, 2009; Thompson et al., 2005), and in some cases, faster than for any other cancer (Guy and Ekwueme, 2011).
The molecular mechanisms that drive melanoma development and progression are complex and have not been fully elucidated. Critical progress has been made in identifying a number of causally implicated gene mutations e.g., BRAF, NRAS KIT, CDKN2A, MITF (Whiteman et al., 2011) and recent efforts to uncover the remaining driver mutations have been accelerated by high-throughput sequencing e.g., (Hodis et al., 2012). However, it is now evident from work in both melanoma and other cancers that such oncogenic mutations do not fully explain the development and progression of tumors (Richards and Medrano, 2009). Changes to the epigenetic landscape, including but not limited to microRNA(miRNA)-based gene regulation, are now firmly associated with human malignancies (Dawson and Kouzarides, 2012). There is still much to be learned about the role of miRNAs as dynamic regulators and/or associates of melanoma progression and prognosis (Crea et al., 2014). Since the earliest report on miRNA expression in melanoma was published (Lu et al., 2005), various studies have revealed deregulated expression of miRNAs in tumor cell lines (Levati et al., 2009; Mueller et al., 2009; Satzger et al., 2010), fresh-frozen
This article is protected by copyright. All rights reserved.
Accepted Article
or formalin-fixed paraffin-embedded (FFPE) tissues (Caramuta et al., 2010; Segura et al., 2010), and patient-derived serum or plasma (Friedman et al., 2012; Leidinger et al., 2010). miRNAs have been nominated as biomarkers that can differentiate between tumor and normal tissue (reviewed in (Bonazzi et al., 2012)), discriminate melanoma subtypes (Chan et al., 2011; Poliseno et al., 2012), and potentially aid in both diagnosis (Leidinger et al., 2010) and prognosis (Caramuta et al., 2010; Friedman et al., 2012; Nguyen et al., 2011; Satzger et al., 2010; Segura et al., 2010). However, in these studies, only a handful have involved microarray expression profiling of miRNA in clinical tumor samples (Caramuta et al., 2010; Chen et al., 2010; Glud et al., 2009; Segura et al., 2010), and since all but one of those studies involved few samples (n≤16 (Caramuta et al., 2010; Chen et al., 2010; Glud et al., 2009)) further analysis including validation of their findings is needed. Consistent with these prior related reports we focus our analysis on metastatic melanoma: a setting in which there is an urgent need for new biomarkers of melanoma progression and prognosis, as well as therapeutic interventions (Slipicevic and Herlyn, 2012).
The role of miRNAs in the effects of the common BRAFV600E activating mutation, which drives the mitogen activated protein kinase (MAPK) pathway, is also poorly understood. This mutation is not only a critical predictive marker of sensitivity to effective systemic therapy (Long et al., 2011) but is also negatively correlated (in a BRAF-inhibitor treatment naïve setting) with patient survival in metastatic melanoma (Barbour et al., 2014; Jayawardana et al., 2014; Long et al., 2011; Mann et al., 2013). Studies leveraging clinical samples to evaluate miRNA associations with BRAF mutation status are scarce (Caramuta et al., 2010). The present study, which This article is protected by copyright. All rights reserved.
Accepted Article
relates miRNA expression to both patient clinical outcome and BRAF mutation status, was therefore intended to point to new prognostic tools, and to inform new avenues for novel therapeutic strategies by characterising the miRNA networks affected by BRAF mutation (Völler et al., 2013).
We have also examined how miRNAs exert their regulatory effects on important melanoma target genes and pathways. miRNAs have the potential to regulate multiple inter-related pathways due to their binding efficiency (primarily in the 3’UTR) to target genes and, as such, have the predicted ability to bind to thousands of genes. Recent evidence suggests that complex diseases such as melanoma can be characterized by the cooperative effects of multiple dysfunctional miRNAs rather than a single miRNA per se (Couts et al., 2013). However, while there have been several candidate-driven studies i.e., hypothesis-driven work focussed on exploring relationships between one miRNA and one or a few target genes (reviewed in (Bonazzi et al., 2012)), there has been no large-scale analysis of such networks to date. We therefore performed the first global integration of miRNA and mRNA microarray expression data derived from the same clinical specimens in American Joint Committee on Cancer (AJCC) stage III melanomas. Here, we employed both an unsupervised approach (the identification of associations between computationally predicted groups of miRNA and mRNA transcripts informed by their relative expression values) and a pathway-supervised approach (in which putative miRNAmRNA relationships were identified based on an intersection of prior knowledge in miRBase (Griffiths-Jones et al., 2008) and KEGG (Kanehisa et al., 2012)).
This article is protected by copyright. All rights reserved.
Accepted Article
Surprisingly, the deregulated network of miRNAs identified in association with reduced survival was largely anti-proliferative in the tumors from patients with worse prognosis, as judged by the effects of their target mRNA products.
RESULTS miRNA microarray expression profiling in metastatic melanoma – discovery cohort Global miRNA expression profiles were evaluated in 45 metastatic melanomas from AJCC stage III patients surviving more than 4 years after resection of lymph node metastatic disease with no sign of relapse (good prognosis (GP), nGP=22) and from patients who died of melanoma within 12 months of same (poor prognosis (PP), nPP=23) (Figure 1). Gene (mRNA) expression profiling in this cohort has been previously described (Jayawardana et al., 2014; Mann et al., 2013) and correlated with detailed clinico-pathologic, and molecular data including BRAF mutation status. Discovery analysis was restricted to clinical stage III patients to reduce the potential for confounding effects due to sample heterogeneity, which would be expected with the inclusion of tissue from AJCC stage IV disease. It is also (arguably) the more clinically urgent research setting for the prognostic stratification of patients, based on the wide range of survival outcomes indicated by the current set of prognostic markers for that group (Balch et al., 2009). Tissue levels of 12 miRNAs were associated with patient clinical outcome in AJCC stage III metastatic melanoma We examined which miRNAs (nprobes=1347) were differentially expressed (DE) between patients with GP and PP. Twelve miRNAs were significantly (adjusted
This article is protected by copyright. All rights reserved.
Accepted Article
P1.5-fold, in good compared with poor prognosis patients (adjusted P2 years) compared with short (
