Identification of Metastasis-Suppressive microRNAs in Primary Melanoma

Doug Hanniford

Miguel Segura

Judy Zhong

Elliot Philips

Anna Pavlick+3 authors

Background:

Surgical management of primary melanoma is curative for most patients with clinically localized disease at diagnosis; however, a substantial number of patients recur and progress to advanced disease. Understanding molecular alterations that influence differential tumor progression of histopathologically similar lesions may lead to improved prognosis and therapies to slow or prevent metastasis.

Methods:

We examined microRNA dysregulation by expression profiling of primary melanoma tumors from 92 patients. We screened candidate microRNAs selected by differential expression between recurrent and nonrecurrent tumors or associated with primary tumor thickness (Student’s t test, Benjamini-Hochberg False Discovery Rate [FDR] < 0.05), in in vitro invasion assays. We performed in vivo metastasis assays, matrix remodeling experiments, and molecular studies to identify metastasis-regulating microRNAs and their cellular and molecular mechanisms. All statistical tests were two-sided.

Results:

We identified two microRNAs (hsa-miR-382, hsa-miR-516b) whose expression was lower in aggressive vs nonaggressive primary tumors, which suppressed invasion in vitro and metastasis in vivo (mean metastatic foci: control: 37.9, 95% confidence interval [CI] = 25.6 to 50.2; miR-382: 19.5, 95% CI = 12.2 to 26.9, P = .009; miR-516b: 12.5, 95% CI = 7.7 to 17.4, P < .001, Student’s t test). Mechanistically, miR-382 overexpression inhibits extracellular matrix degradation by melanoma cells. Moreover, we identified actin regulators CTTN, RAC1, and ARPC2 as direct targets of miR-382. Depletion of CTTN partially recapitulates miR-382 effects on matrix remodeling, invasion, and metastasis. Inhibition of miR-382 in a weakly tumorigenic melanoma cell line increased tumor progression and metastasis in vivo.

Conclusions:

Aberrant expression of specific microRNAs that can functionally impact progression of primary melanoma occurs as an early event of melanomagenesis.

Funding

This study was funded by the National Institutes of Health (NIH)/National Cancer Institute (NCI) (1R01CA155234;; PI: DH). DH has been supported by NIH/NCI 5 T32 CA009161-37 (Training Program in Molecular Oncology and Immunology). JZ has been supported by NIH/National Institute of General Medical Sciences (1R21 GM110450-01; PI: JZ).

Supplementary Material

Supplementary Data:

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Affiliations of authors:Department of Pathology (DH, MFS, EP, XJS, FD, EH), Interdisciplinary Melanoma Cooperative Group (DH, MFS, JZ, XJS, FD, RSB, RLS, AP, IO, EH), Department of Environmental Medicine (JZ), Department of Dermatology (AP, IO), Department of Medicine (AP, IO), Department of Urology (IO); Department of Surgery (RSB, RLS), NYU Langone Medical Center, New York, NY; Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai, New York, NY (BB).

^{Corresponding author.}

Correspondence to: Eva Hernando, PhD, 522 First Ave, Department of Pathology, Floor 3 Rm 305, Smilow Research Building, New York, NY 10016 (e-mail: gro.cmuyn@egnom-odnanreh.ave).

Received 2014 Apr 27; Revised 2014 Aug 12; Accepted 2014 Dec 16.

Abstract

Background:

Methods:

Results:

Conclusions:

Aberrant expression of specific microRNAs that can functionally impact progression of primary melanoma occurs as an early event of melanomagenesis.

Abstract

Metastasis, which is the cause of approximately 90% of tumor deaths (1), is a multistep cascade requiring diverse biological processes, including local invasion/matrix remodeling, intravasation/extravasation, survival in circulation, and colonization, survival, and growth in secondary sites. Regulation of these processes in different cancer contexts has begun to be elucidated (2); however, further understanding of the molecular mechanisms exploited by metastasizing cells will aid the development of more informed therapeutic strategies.

Melanoma is a prototypical example of a solid tumor with a propensity to spread throughout the body, even at early stages of tumorigenesis (3). Metastasis is the essential event dictating poor outcome for patients diagnosed with a localized primary melanoma. Staging of primary melanomas incorporates histopathological features (thickness, mitotic index, ulceration, and lymph node status) and is generally prognostic of clinical outcome (4–8). However, frequently melanoma cell dissemination occurs from primary tumors that are histologically equivalent to nonmetastasizing lesions at diagnosis. Approximately 7% and 30% of patients with localized melanoma at diagnosis (stage I and II, respectively) will suffer a recurrence (9), and most will then progress to metastatic disease and eventual death (10). These outcomes suggest that histopathologically similar melanomas may have divergent underlying molecular features influencing their potential to metastasize. Consistent with this concept, messenger RNA (mRNA) or microRNA (miRNA) expression in a variety of cancers is associated with or predicts disease recurrence, progression to metastasis, and other outcome measures (11–20). Collectively, these studies suggest that measurable populations of metastasis-initiating cells are present in a subset of primary tumors. Identifying molecular alterations at cancer diagnosis that are functionally involved in metastatic progression will further elucidate mechanisms used during initiation of the metastatic cascade, which could yield novel therapeutic strategies to disrupt tumor cell dissemination and/or new prognostic biomarkers.

miRNAs are short RNAs that control complex cellular processes through post-transcriptional regulation of target mRNA (21). Recent studies have shown that altered expression of specific miRNAs is associated with patient outcomes in melanoma and that perturbation of individual miRNAs functionally impacts melanoma cell metastasis (22–26). These and other studies have analyzed global miRNA expression comparing nevi to primary melanoma, primary to metastatic melanoma, or metastatic melanomas with differing outcomes. However, miRNA expression profiling of primary melanoma tissues of different outcomes has not been examined. Moreover, the mechanisms underlying metastatic propensity of a subset of primary melanomas are unclear.

Here, we identified miRNAs associated with aggressive, cutaneous primary melanomas (associated with tumor thickness or recurrence status) by expression profiling of a cohort of clinically well-annotated primary melanoma tissues (n = 92). To discover metastasis-relevant miRNAs, we screened candidates differentially expressed in aggressive vs nonaggressive primary melanomas in a high-throughput in vitro invasion assay and subsequently an in vivo metastasis model. Mechanistically, we examined the effects of invasion- and metastasis-regulating miRNAs on matrix remodeling, a key process during initiation of tumor cell dissemination. Finally, we identified a network of target genes that are potential direct downstream mediators of these phenotypes through regulation of the actin cytoskeleton.

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