A new duet in cancer biology: AMPK the typical and UBE2O the atypical

ABSTRACT

Ubiquitin-conjugating enzyme E2O (UBE2O) is upregulated in human cancers. We have demonstrated that genetic deletion or pharmacological blockade of UBE2O reduces tumorigenesis through inhibiting the mammalian target of rapamycin complex 1–hypoxia-inducible factor 1-α pathway. Critically, UBE2O targets adenosine monophosphate (AMP)-activated protein kinase-α 2 (AMPKα2) for ubiquitination and degradation. We thus suggest the UBE2O-AMPKα2 axis as a potential therapeutic target for cancer.

Aberrations or defects in ubiquitin (Ub) signaling can result in debilitating diseases, including cancer and metabolic disorders.¹ Ubiquitination requires the coordinated action of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. There are ∼35 E2 enzymes in the human genome, and most are thought to be directly involved in Ub transfer,² but very little functional information is available for the majority of them.

Ubiquitin-conjugating enzyme E2O (UBE2O) is an atypical ubiquitin enzyme with both E2 and E3 activities.^3,4 Recently, in several in vitro studies, UBE2O has been implicated in several pathways.^5,6,7 However, in vivo evaluation of UBE2O has been limited due to a lack of animal models. Recent public data set has revealed that UBE2O is frequently amplified in human cancers (∼20% in breast, bladder, liver and lung carcinoma) but its role in tumorigenesis remained unknown.

To better understand the role of UBE2O in tumorigenesis in vivo, we generated a Ube2o knockout mouse line that has been cross-bred in two transgenic mouse models of spontaneous cancer [mouse mammary tumor virus (MMTV)-polyoma virus middle T antigen (PyVT) for breast cancer and transgenic adenocarcinoma mouse prostate (TRAMP) for prostate cancer].⁸ Remarkably, Ube2o deletion in those cancer mouse models resulted in a profound delay in tumor initiation and growth, as determined by the tumor-free survival in MMTV-PyVT mice or the development of prostatic intraepithelial neoplasia in TRAMP mice. We then analyzed tumor metastasis; the rates of distant metastasis of Ube2o-deficient mice were much lower than those of Ube2o-proficient mice of breast and prostate cancers.

Using the affinity-purification mass spectrometry approach, we identified a catalytic α2 subunit of AMP-activated protein kinase (AMPK) (encoded by PRKAA2, best known as AMPKα2) as a novel UBE2O-interacting protein. We then demonstrated that UBE2O directly ubiquitinates AMPKα2 and promotes its proteasomal degradation (Fig. 1). Surprisingly, UBE2O was not involved in the modulation of AMPKα1 (encoded by PRKAA1, best known as AMPKα1) protein levels. We also discovered that UBE2O is able to ligate Ub to the AMPKα2 protein on its lysine-470 (K470) residue, but here is replaced by arginine-475 (R475) in an AMPKα1 isoform. Furthermore, the loss of UBE2O increased AMPKα2 (but not AMPKα1) protein levels leading to a strong activation of its downstream targets in vitro and in vivo.

Figure 1.

Role for the UBE2O-AMPKα2 axis in cancer and its implications for cancer therapy. Amplification or overexpression of ubiquitin-conjugating enzyme E2O (UBE2O) in cancer results in the degradation of AMP-activated protein kinase-α 2 (AMPKα2), the consequent upregulation of the mammalian target of rapamycin complex 1 (mTORC1)–hypoxia-inducible factor 1-α (HIF1α) pathway, and rewires metabolism to undergo glycolysis which is cancer promoting. Thus, its genetic deletion or pharmacological blockade impairs tumorigenesis through the restoration of AMPKα2. Ub, ubiquitin; K470, lysine-470 (K470); β, AMP-activated protein kinase-beta; γ, AMP-activated protein kinase-gamma.

Importantly, downregulation of UBE2O promoted AMPKα2-mediated suppression of the mammalian target of rapamycin complex 1 (mTORC1)–hypoxia-inducible factor 1-α (HIF1α) pathway that is essential for metabolic “reprogramming” of cancer cells. We also confirmed that the tumorigenesis induced by UBE2O was AMPKα2 dependent using allograft and xenograft models. We corroborated the propensity of AMPKα2 in UBE2O-dependent tumorigenesis in vivo using a set of mouse models of lymphomas [Eμ-Myc and phosphatase and tensin homolog (Pten)^+/−]. Indeed, lymphocytes express only AMPKα1 (and AMPKα2 is undetectable), and in these contexts, ablation of Ube2o did not impair the progression of lymphoma.

Next, we explored the clinical relevance of the UBE2O-AMPKα2-mTORC1-HIF1α axis in human cancers, and we found that UBE2O expression is inversely correlated with AMPKα2 expression and positively correlated with P-S6 and HIF1α levels in human breast tumors. As UBE2O is known to be susceptible to inhibition by arsenite, which can crosslink adjacent cysteines within its catalytic domain,⁸ we tested a UBE2O inhibitor (arsenic trioxide: ATO) in mouse models of breast and prostate cancers. As a result, UBE2O inhibition reduced tumor incidence and progression and extended survival. Future efforts should be also taken to develop the exciting drugs directed against UBE2O (e.g., small molecule inhibitors) to prevent and treat human malignant disease.

A number of AMPK agonists are currently being assessed for their potential in cancer treatment. In particular, the anti-diabetic agent metformin is widely viewed as a promising anticancer agent, and is currently being tested in several large-scale clinical trials.⁹ Thus our findings suggest that a combined treatment of UBE2O inhibitors and AMPK agonists could show even greater promise as a new therapeutic strategy.

The role of AMPK in cancer remains controversial at the moment with tumor suppressor or oncogene.¹⁰ Substantial evidence demonstrates that the loss of AMPK activity can promote tumor progression with the cooperation of oncogenic hits, such as H-RasV12, B-RAF^V600E, Myc, and PTEN-loss. Conversely, engaging AMPK signaling may also aid tumor cell survival and provide a selective advantage to tumor cells through multiple mechanisms, including induction of autophagy, fatty acid oxidation, and conversion of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH (the reduced form of NADP). Thus AMPK may exert either a positive or negative effect on cancer cell survival, depending on the context of cellular stress.

Our results reinforce the idea that AMPKα2 may be tumor suppressive. The selective modulation of UBE2O on the AMPKα2 subunit (but not AMPKα1 isoform) creates a new paradigm of AMPKα1 versus AMPKα2 function in cancer biology. Also, our findings settle an interesting question regarding the roles of the UBE2O-AMPKα2 axis in other patho-physiologic contexts, such as diabetes, obesity, infectious diseases and many others, where AMPK is well known as a key regulator.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank all Su Jung Song's and Min Sup Song's laboratory members for critical discussions.

Funding

This work was supported by a grant of the Soonchunhyang University Research Fund and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI15C2679) to S.J.S., and a Cancer Prevention Research Institute of Texas grant (RP150084), a Department of Defense grant (W81XWH-15–1–0662), and a NIH grant (CA196740) to M.S.S.