miR-1273g silences MAGEA3/6 to inhibit human colorectal cancer cell growth via activation of AMPK signaling

doi:10.1016/j.canlet.2018.07.031

Cancer Letters

Volume 435, 28 October 2018, Pages 1-9

https://doi.org/10.1016/j.canlet.2018.07.031 Get rights and content

Highlights

•
miR-1273g induces MAGEA3/6 downregulation and AMPK activation in colorectal cancer cells.
•
miR-1273g-3p downregulation in colon cancer tissues correlates with AMPK in-activation.
•
miR-1273g-induced cytotoxicity in HT-29 cells is nullified by AMPKα1 knockout.
•
miR-1273g-expressing HT-29 tumors grew significantly slower than control tumors.

Abstract

AMP-activated protein kinase (AMPK) is a metabolic regulator that acts to limit the growth of cancer cells. AMPK is downregulated by melanoma antigens A3/6 (MAGEA3/6), which are cancer-specific proteins that enhance the activity of specific E3 ubiquitin ligases to ubiquitinate and degrade AMP-activated protein kinase α1 (AMPKα1). Here, using a bioinformatic approach, we identified a microRNA, miR-1273g-3p, that is predicted to target the 3′ untranslated region (UTR) of MAGEA3/6. Analyzing miR-1273g-3p expression in human colon cancer tissues, we found a reduction in miR-1273g-3p expression correlating with increased MAGEA3/6 expression and AMPKα1 downregulation. Expression of miR-1273g in HT-29 cells and primary human colon cancer cells down-regulated MAGEA3/6, leading to AMPKα1 upregulation, inhibition of proliferation and cell apoptosis. The anti-CRC activity of miR-1273g was blocked by AMPKα1 knockout. MAGEA3/6 shRNA silencing mimicked and abolished miR-1273g-induced actions in HT-29 cells. In vivo, miR-1273g- or MAGEA3/6 shRNA-expressing HT-29 tumors grew significantly slower than control tumors. We propose a novel miRNA-based mechanism, whereby miR-1273g represses MAGEA3/6 expression in human CRC cells and tissues, which may provide a novel cancer-specific therapeutic.

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related human mortalities [1]. Despite developments in CRC treatment there is no effective therapy for patients with advanced metastatic and/or recurrent CRC [2]. Due to heterogeneity, which impedes treatment with specific molecularly-targeted agents, it is important to further explore the pathological mechanisms of CRC [2].

AMP-activated protein kinase (AMPK) is a metabolic regulator activated in response to cellular stress [3]. Although the role of AMPK in controlling tumorigenesis remains controversial [4], AMPK acts as a negative regulator of aerobic glycolysis and cellular biosynthesis to limit the growth of cancer cells [5]. AMPK restrains growth through downregulating the mTOR complex 1 (mTORC1) [6,7], cell cycle arrest [8,9], and promoting autophagy [10,11]. Conversely, a number of studies suggest that reduced AMPK activity or expression favor tumor growth. Thus, the phosphorylation status of AMPKα1 at Thr-172, essential for AMPK activation, has been reported to be downregulated in breast cancer [12], and expression levels of AMPKα protein are significantly decreased in breast and bladder cancer [13,14]. Additionally, the loss of AMPKα1 protein accelerates the development of c-Myc-driven lymphomas [5]. Further supporting a tumor suppressor role for AMPK, a number of anti-cancer agents stimulate AMPK activation to mediate CRC cell death [[15], [16], [17], [18]].

Melanoma antigen (MAGE) genes encode MAGE proteins [19], including MAGEA3 and MAGEA6. MAGEA3 and MAGEA6 proteins, ordinarily only expressed in testis, have been shown to be abnormally expressed in human cancers where they potentiate the activity of certain E3 ubiquitin ligases [20]. Recent studies in human cancers demonstrate the MAGEA3/6-TRIM28 ubiquitin ligase complex serves to ubiquitinate and degrade AMPKα1 [21,22]. MAGEA3/6 expression, and AMPKα1 downregulation, correlates with mTORC1 over-activation and cancer cell progression [23,24].

Identifying the mechanisms that regulate MAGEA3/6 expression in human cancer may provide insights into inhibiting its expression, which is anticipated to increase AMPKα1 expression and slow cancer cell progression. MicroRNAs are a family of endogenous small non-coding regulatory RNAs. They downregulate targeted genes via binding to the 3′ untranslated region (UTR) [25]. In the present study, we show that microRNA-1273g-3p (“miR-1273g-3p”) targets both MAGEA3 and MAGEA6. Significantly, expression of miR-1273g silences MAGEA3/6 to activate AMPK signaling, thereby inhibiting CRC cell growth in vitro and in vivo.

Section snippets

Chemicals and reagents

Puromycin and MTT were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture reagents were purchased from Hyclone Co. (Logan, UT). The anti-MAGEA6 antibody (ab38495) and anti-MAGEA3 antibody (ab140678) were purchased from Abcam (Shanghai, China). All other antibodies were purchased from Cell Signaling Tech (Shanghai, China) and Santa Cruz Biotech (Santa Cruz, CA).

Cell culture

HT-29 cells were provided by Dr. Lu [17]. Two different primary human colon cancer cells (“pri-Can-1/-2”) and two different

Identification of a miRNA, miR-1273g-3p, targeting the 3′UTR of MAGEA3 and MAGEA6

MicroRNAs (miRNAs) are known to control the degradation or translational repression of the target mRNAs linked to tumorigenesis [31]. Therefore, the enhanced expression of MAGEA3/6 in cancer tissues could be due to dysregulation of certain miRNAs. To explore this possibility, the human miRNA database, TargetScan (Bartel lab, http://www.targetscan.org, V7.2) was first utilized to identify possible MAGEA3/6-targeting miRNAs. These potential miRNAs were further verified by other miRNA databases,

Discussion

Increasing evidence shows that MAGEA3/6 proteins, which are expressed in many tumor types, drive tumorigenesis. Tarnowski et al., have shown that MAGEA3 and other cancer-testis antigens were upregulated in colon cancer tissues [32], which were positively correlated with cancer progression [32]. Shantha Kumara et al., found weak to moderate MAGEA3 expression in 28% of the tested CRC tumors [33]. Kim et al., show that MAGEA3 mRNA is expressed in 66% of all 32 CRC cell lines, and 77% of all 87

Conclusion

AMPK activation has proven to be a beneficial strategy to inhibit human CRC cells. The results of this study indicate that miR-1273g could have important therapeutic value for CRC treatment.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgement

This project was supported by the Natural Science Foundation of Zhejiang Province (LY15H030015), and by National Natural Science Foundation of China (81302181). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Cancer testis antigen Melanoma associated antigen A3 (MAGE-A3) has been subject of research for many years. Being expressed in various tumor types and influencing proliferation, metastasis, and tumor pathogenicity, MAGE-A3 is an attractive target for cancer therapy, particularly because in healthy tissues, MAGE-A3 is only expressed in testes and placenta. MAGE-A3 acts as a cellular master regulator by stimulating E3 ubiquitin ligase tripartite motif-containing protein 28 (TRIM28), resulting in regulation of various cellular targets. These include tumor suppressor protein p53 and cellular energy sensor AMP-activated protein kinase (AMPK). The restricted expression of MAGE-A3 in tumor cells makes MAGE-A3 an attractive target for vaccine-based immune therapy. However, although phase I and phase II clinical trials involving MAGE-A3-specific immunotherapeutic interventions were promising, large phase III studies failed. This article gives an overview about the role of MAGE-A3 as a cellular master switch and discusses approaches to improve MAGE-A3-based immunotherapies.
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Identification of ectopic gene activation in cancer cells serves as a basis for both gene signature-guided tumor targeting and unearthing of oncogenic mechanisms to expand the understanding of tumor biology/oncogenic process. Proteins expressed only in germ cells of testis and/or placenta (immunoprivileged organs) and in malignancies are called cancer testis antigens; they are antigenic because of the lack of antigen presentation by those specific cell types (germ cells), which limits the exposure of the proteins to the immune cells. Since the Cancer Testis Antigens (CTAs) are immunogenic and expressed in a wide variety of cancer types, CT antigens have become interesting target for immunotherapy against cancer. Among CT antigens MAGEA family is reported to have 12 members (MAGEA1 to MAGEA12). The current review highlights the studies on MAGEA3 which is a CT antigen and reported in almost all types of cancer. MAGEA3 is well tried for cancer immunotherapy. Recent advances on its functional and immunological aspect warranted much deliberation on effective therapeutic approach, thus making it a more interesting target for cancer therapy.
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Through binding to AMPKα1 and TRIM28, these MAGE proteins enable the ubiquitination and subsequent proteasomal degradation of AMPKα1 that leads to a reduction in overall AMPK protein levels (151). In line with MAGE-A3/6 regulating AMPK stability, MAGE-A3/6 mRNA expression inversely correlates with AMPK activity and protein levels in diverse cancer patient samples, including breast, lung, and colon cancer (134, 151). In colon cancer, MAGE-A3/6 mRNA levels inversely correlate with the expression of miR-1273g-3p, which silences MAGE-A3/6 expression and inhibits human colorectal cancer cell growth via AMPK activation (134).
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Original ArticlesmiR-1273g silences MAGEA3/6 to inhibit human colorectal cancer cell growth via activation of AMPK signaling

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Cell culture

Identification of a miRNA, miR-1273g-3p, targeting the 3′UTR of MAGEA3 and MAGEA6

Discussion

Conclusion

Conflicts of interest

Acknowledgement

Clin. Colorectal Canc.

Cell Metabol.

Mol. Cell

Mol. Cell

Cell

Transl. Oncol.

Cell. Signal.

Mol. Cell

Cell

Canc. Lett.

J. Biol. Chem.

Canc. Lett.

Cell

FEBS Lett.

Cancer statistics, 2018

CA A Cancer J. Clin.

The AMPK signalling pathway coordinates cell growth, autophagy and metabolism

Nat. Cell Biol.

AMPK: a contextual oncogene or tumor suppressor?

Canc. Res.

LKB1 and AMPK and the cancer-metabolism link - ten years after

BMC Biol.

AMPK promotes p53 acetylation via phosphorylation and inactivation of SIRT1 in liver cancer cells

Canc. Res.

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

Nat. Cell Biol.

Histological evaluation of AMPK signalling in primary breast cancer

BMC Canc.

AMP-activated protein kinase alpha 2 isoform suppression in primary breast cancer alters AMPK growth control and apoptotic signaling

Genes Canc.

NPC-26 kills human colorectal cancer cells via activating AMPK signaling

Oncotarget

Original Articles
miR-1273g silences MAGEA3/6 to inhibit human colorectal cancer cell growth via activation of AMPK signaling