Cancer Letters

Cancer Letters

Volume 373, Issue 1, 1 April 2016, Pages 77-87
Cancer Letters

Original Articles
MED27 promotes melanoma growth by targeting AKT/MAPK and NF-κB/iNOS signaling pathways

https://doi.org/10.1016/j.canlet.2016.01.005Get rights and content

Highlights

  • Med27 was highly expressed in melanoma cells and tumor tissues.

  • The silencing of Med27 led to melanoma cell proliferation inhibition, cell cycle arrest and apoptosis.

  • The silencing of Med27 resulted in the inactivation of PI3K/AKT and MAPK/ERK signaling and the activation of Bax/Cyto C/Caspase apoptotic pathway in melanoma cells.

  • The silencing of Med27 led to the decrease of iNOS expression through inhibiting the activation of a serial of upstream key proteins of NF-κB signaling pathway and the final translocation of p50/p65 from cytoplasm to nucleus.

  • NF-κB and p300 interacted with Med27 and p300 mediated the acetylation of Med27 in melanoma cells.

  • MED27 knockdown inhibited melanoma progression in vivo in a melanoma mouse model.

Abstract

The inhibitors of BRAF and MEK targeting MAPK signaling pathway provide a comparatively effective therapeutic strategy for melanoma caused by BRAF mutation. However, melanoma, especially metastatic melanoma, has become one of the most threatening malignancies. Thus, the identification of exact molecular mechanisms and the key components involved in such mechanisms is urgently needed in order to provide new therapeutic options for patients with melanoma. Here, we identified MED27 as a potential melanoma target and explored its role and the associated molecular mechanism involved in melanoma progression. MED27 was found to be highly expressed in melanoma cells and tumor tissues. Its silencing led to melanoma cell proliferation inhibition, cell cycle arrest and apoptosis induction accompanied by the inactivation of PI3K/AKT and MAPK/ERK signaling and the activation of Bax/Cyto-C/Caspase-dependent apoptotic pathway. In addition, silencing of MED27 led to the decrease of iNOS expression through inhibiting the activation of a serial of upstream key proteins of NF-κB signaling pathway and the translocation of p50/p65 from cytoplasm to nucleus. MED27 was also found to be able to interact with NF-κB and p300 and to be acetylated by p300. Furthermore, the results in a xenograft tumor model indicated that melanoma progression was effectively suppressed by MED27 knockdown accompanied by the down-regulation of p-AKT, p-ERK, p-MEK1/2, MMP-9, Bcl-2 and iNOS expressions in the tumor tissues. Taken together, our study not only demonstrated the new function of MED27 as an oncogenic protein and the associated molecular mechanisms involved in melanoma progression, but also provided a possibility for the development of MED27 as a new anticancer target in melanoma therapy.

Introduction

Melanoma is an aggressive type of cutaneous malignancy. It is more serious than other skin cancers because of its aggressiveness and poses a great threat to the health of people in the world, especially in the Western world. Although most melanomas can be cured with surgery, the prerequisite is early detection. In case of metastasis, it usually causes serious illnesses and even death [1], [2], [3], [4]. About 76,000 new cases of melanoma are diagnosed in the United States every year and more than 9000 deaths happened in 2014 [5]. Furthermore, the risk of melanoma seems to be increased towards a younger trend in people under 40 and the averaged survival rate for patients with metastasis is currently only 6–12 months. Although the inhibitors targeting BRAF and/or MEK pathways provide a therapeutic option for non-resectable melanoma driven by BRAF mutation [6], [7], [8], a fair proportion of melanomas are BRAF wild type, or NRAS-mutant, or TERT-mutant and are insensible to the above mutation-targeting therapy [9], [10], [11], [12]. Collectively, based on the high morbidity, high mortality and high complexity of molecular mechanisms associated with metastatic melanoma, more attention should be paid to clarify the underlying molecular mechanisms involved in the disease progression and identify newly potential therapeutic targets to improve the treatment.

The human mediator complex (MED), composed of 28 elements, represents a fundamental component of the transcription machinery and interacts with the RNA polymerase II enzyme to regulate its ability to control gene expression [13]. As a component of these 28 elements, MED27 is an enzyme ubiquitously expressed in humans and universally required in transcriptional initiation [14]. It is reported as a subunit of CRSP complex (cofactor required for SP1 activation), which is recruited by Sp1 through direct interactions to promoters and serves as a scaffold for the assembly of other regulatory proteins to promote the transcription of SP1-driven down-stream target genes [15]. MED27 is also found to interact with thyroid hormone receptor (TR) to facilitate TR function by conjunction with other transcriptional factors and co-factors [16]. In addition, the new findings demonstrated its involvement in HIV-1 transcription. Knockdown of MED27 protein, combined with the knockdown of other MED proteins, significantly impaired viral replication without affecting cell viability [17]. However, to date, unlike other members of MED family, the functional role of MED27 in human diseases is still rarely reported, and its function in respect to tumorigenesis and development is especially poorly characterized, or even nearly unknown.

Our research based on siRNA library screening has identified a serial of new proteins implicated in melanoma progression [18]. Here, we chose one of these proteins, MED27, as the main study object and provided an evaluation of its activity on melanoma cell growth and apoptosis. Moreover, we explored its potential molecular mechanisms associated with melanoma survival and its clinical significance in melanoma. Our results have shown that knockdown of MED27 leads to reduced cell proliferation, increased cell cycle repression and apoptosis in melanoma, and such proliferation-inhibiting effects of MED27 knockdown is mediated by inactivating PI3K/AKT, MEK/Erk pathway, elevating Bcl-2/Cyt C/Caspase 3 signaling pathway and inhibiting NF-κB/iNOS signaling pathway. Our researches not only demonstrated the role of MED27 in melanoma progress at first time, but also provided a potential candidate therapeutic target for melanoma treatment.

Section snippets

Cell lines and cell culture

Human melanoma cell lines MeWo, SK-Mel-28, A375, C8161 and immortalized human epidermal cell line HaCaT,normal skin cell SK were all obtained from the American Type Culture Collection (ATCC). These cells were all cultured in Dulbecco,s Modified Eagle Medium supplemented with 10% FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The cultures were maintained in an incubator with 5% CO2 at 37 °C under standard culture condition.

Transient transfection

To overexpress MED27 in A375 or SK-Mel-28 cells, cells were

MED27 was highly expressed in melanoma cells and tumor tissues

To analyze the expression level of MED27 in human melanoma, we first detected the protein level of MED27 in human normal skin cell line SK, Knot (K) and fibroblasts (F), immortalized human epidermal cell line (HaCaT) and melanoma cell lines (MeWo, A375, SK-Mel-28, C8161) by Western blotting. MED27 was expressed in all the tested cell lines and a comparatively low expression was shown in normal skin cells (SK, Knot and fibroblasts) compared to the melanoma cells (Fig. 1A). We next tested the

Discussion

Given the demonstration of the mutations or overexpression of some mediator complex subunits (MED) in various human cancer [26], [27], [28], [29], [30], MED proteins have been recognized to play an increasingly important role in tumorigenesis and development. However, MED subunits most recently reported to be involved in tumor growth have been limited to MED1, 12, 14, 15, 19 ,24, and 28 [31], [32], [33], [34], [35], and little is known about the functional role of other subunits of Med complex

Conflict of interest

We declare there are not any competing financial interests in relation to the work described.

Acknowledgements

This work was supported by the funds from the National Natural Science Foundation of China (81301721, 81472178, 81071687, 81272195), the State “863 Program” of China (SS2012AA020403), the State “973 Program” of China (2014CB542005), the Education Department of Liaoning Province, China (the “Program for Pan-Deng Scholars”), the Natural Science Foundation of Liaoning Province (2014023009).

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