Growth factor receptor signaling induces mitophagy through epitranscriptomic regulation

ABSTRACT

Aberrant growth factor receptor signaling is among the most common oncogenic drivers in cancer biology. Receptor signaling classically induces cancer growth through signaling cascades that mediate effects largely through transcriptional control. Recently, post-transcriptional RNA modifications, collectively designated as epitranscriptomics, have emerged as a critical layer of dysregulation in cancer biology. We recently reported that PDGFR (platelet-derived growth factor receptor) activity in cancer stem cells (CSCs) derived from glioblastoma patients displays increased post-transcriptional mRNA methylation (N6-methyladenosine [m6A]), which promotes CSC maintenance through regulation of mitophagy. Specifically, PDGF-PDGFRB signaling upregulates the expression of the m6A methyltransferase METTL3, which then decorates the mitophagy regulator OPTN (optineurin) mRNA with m6A, thereby promoting OPTN mRNA degradation. Glioblastomas express lower levels of OPTN than normal brain, and forced expression of OPTN reduces tumor growth, supporting a tumor suppressive role for OPTN. Pharmacological targeting of METTL3 with PDGFR or activation of mitophagy demonstrates a combinatorial benefit. Collectively, our results suggest that upstream regulation of mitophagy in lethal cancers is mediated through growth factor receptor control of post-transcriptional RNA regulation, offering novel therapeutic paradigms.

N6-methyladenosine (m6A), a methylated form of adenosine, is the most frequent modification in mRNA, which affects nuclear processes such as splicing and mRNA degradation in the cytoplasm linked to certain biological processes, including macroautophagy/autophagy. N6-methyladenosine modification has been associated with various cellular responses in many cancers, including glioblastoma. However, the upstream regulation of aberrant m6A levels is not clear. We recently identified oncogenic processes positively correlated with the expression of molecular m6A regulators in glioblastoma, revealing a tight correlation with growth factor receptor pathways [1]. PDGF and other growth factor receptor pathways are dysregulated nearly universally in glioblastomas, yet growth factor receptor inhibitors have failed to show activity in clinical trials, suggesting that additional targets will be required for precision oncology to succeed against glioblastoma.

Supporting the functional connection between PDGF signaling and m6A regulation, activation of PDGF induces the expression METTL3 through the EGR1 transcription factor, which is an essential component of the methyltransferase complex that decorates mRNAs with m6A. Reciprocally, PDGFR inhibitors reduce m6A levels and METTL3 expression. Targeting METTL3 blocks the proliferative effects of PDGFR, and METTL3 expression rescues the loss of PDGFR, suggesting that much of the impact of PDGF signaling is mediated through the effects on RNA regulation. While many mRNAs are modified by METTL3, we found that mitophagy-related gene expression is negatively correlated with METTL3 expression. Targeting METTL3 promotes MAP1LC3/LC3 (microtubule associated protein 1 light chain 3)-II levels as well as colocalization of mitochondria to lysosomes, suggesting that m6A inhibits mitophagy activation. Functional analysis of METTL3 supports its methyltransferase activity as being essential to its mitophagy regulation. Further, targeting METTL3 induces changes in the mitochondrial membrane potential and respiration capacity of CSCs. Thus, METTL3 regulates the clearance of defective mitochondria and subsequent programmed cell death. As METTL3 inhibitors are being developed, the connection between epitranscriptomics and mitophagy suggests that METTL3 and other m6A regulators may function, in part, through regulation of autophagy.

To discover the molecular mediators of m6A regulation on mitophagy, we investigated transcripts that were inversely correlated with METTL3 and methylated, revealing OPTN as the top hit. OPTN binds the autophagosome protein LC3, recruiting the phagophore to damaged mitochondria. We explored the role of OPTN-regulated mitophagy in CSC, with the genetic introduction of OPTN rescuing the effects of genetic modulation of PDGFR or METTL3 on mitophagy and cell viability.

As molecularly targeted therapies have not shown sustained efficacy against glioblastoma in clinical trials, we sought potential therapeutic combinations based on the molecular interactions that we defined. Drugs modulating PDGFR, METTL3, or mitophagy each inhibit CSC proliferation and self-renewal as monotherapy, but display synergy in combination. These results support a pro-tumorigenic role for m6A regulation whereby PDGF-PDGFRB signaling induces METTL3 expression, which promotes m6A-mediated OPTN control with the PDGFRB-METTL3-OPTN axis representing a potential therapeutic target with clinical impact for glioblastoma patients.

In conclusion, growth factor signaling regulates mitophagy in CSCs through the induction of global m6A levels and the methylation of OPTN mRNA specifically. PDGF promotes OPTN m6A modification via METTL3 to increase OPTN mRNA decay and reduce OPTN protein levels (Figure 1). The connection between signal transduction, epitranscriptomics, and metabolism reveals complex regulation in tumor biology amenable to novel combinatorial therapeutic interventions.

Figure 1.

Growth factor receptor activity regulates mitophagy through m6a regulation. PDGFR upregulates the methyltransferase METTL3 expression via EGR1 to induce m6a modifications, including on OPTN to inhibit mitophagy in GSCs.

Funding Statement

Support was provided by NIH grants [CA197718, CA238662, and NS103434 to J.N.R.], the Computational Genomic Epidemiology of Cancer (CoGEC) program at Case Comprehensive Cancer Center [T32CA094186 to K.Y], the Young Investigator Award in Glioblastoma from ASCO Conquer Cancer Foundation [to K.Y.], and RSNA Research Resident Grant [to K.Y.].

Disclosure statement

No potential conflict of interest was reported by the author(s).