The RQC pathway plays an important role in managing aberrant proteins produced during translation. This study focused on understanding the effects of RQC-mediated modifications, particularly the addition of msiCAT tails, on mitochondrial proteins such as ATP5α in GBM cells. This finding revealed that GBM cells harboring msiCAT-modified ATP5α exhibit a unique metabolic profile. Despite reduced ATP synthesis, these cells maintain mitochondrial membrane potential (ΔΨm), a critical component of cell function and survival. Furthermore, they exhibit enhanced cell viability and motility, properties associated with increased tumor invasiveness and metastasis. In particular, the presence of msiCAT-modified ATP5α confers resistance to STS-induced apoptosis by modulating MPTP, a key regulator of cell death pathways. Figure 6. These identified traits contribute to increased tumor aggressiveness, suggesting that the RQC pathway plays an important role in cancer cell survival and proliferation. Encouragingly, recent studies have also demonstrated that the RQC pathway is involved in issues such as: Drosophila Notch overexpression-induced brain tumor model (Kackett et al., 2024). This finding suggests that modulation of the RQC pathway may serve as a promising strategy to complement existing chemotherapy regimens. By targeting this specific pathway, therapeutic interventions can effectively disrupt the mechanisms that allow cancer cells to avoid apoptosis and maintain energy production under stress, potentially leading to improved outcomes for patients with GBM and other cancers characterized by similar protein modifications.

Studying the behavior of ATP synthase in cancer is particularly important. During carcinogenesis, ATP synthase is frequently relocated to the cell membrane and is termed ectopic ATP synthase (eATP synthase). These eATP synthases exhibit catalytic activity and promote ATP generation in the extracellular space, promoting a favorable tumor microenvironment (Komeri et al., 2016). Studies have shown that eATP synthase is first assembled in mitochondria before being transported to the cell surface via microtubules (Chan et al., 2023). However, the specific type of ATP synthase delivered to the cell membrane remains unclear. Future studies on the localization of CAT-tailed eATP synthase may provide valuable insights into this process.

Multiple mitochondrial proteins within cancer cells can undergo CAT tailing in a similar manner. These msiCAT tail peptides may have different effects on mitochondria and cells due to differences in their base proteins. For example, CAT-tailed COX4 proteins can substantially and directly reduce mitochondrial respiratory efficiency. It is important to examine the individual roles of these proteins, as the combined effects of their defects may be important for understanding the mitochondrial changes observed in cancer. A small caveat here is that the observed effect of the presence of the CAT tail is primarily derived from the artificial CAT tail sequence with high threonine content, rather than from the endogenous CAT tail protein. Other sequence components may cause different effects (Chan et al., 2024). A recent study found that ANKZF1 knockdown inhibits GBM progression by causing abnormal protein accumulation in mitochondria (Lee et al., 2024). This combined with our data suggests that balanced ANKZF1 expression and activity is essential for cancer growth. Both excess and deficiency can alter cellular fitness. Minor deficiencies in this study were the use of a mitochondrial-localized non-paused GFP protein to induce proteostasis stress and the lack of direct biochemical evidence for CAT tail proteins. Our study focuses on endogenous proteins and dissects their effects on mitochondria. The rationale is that highly expressed nonphysiological ectopic proteins may cause general proteostasis failure and mask specific functions of endogenous proteins. Additionally, different cell lines were used in the study. GSCs, a patient-derived GBM cell line with better stemness, may have unique mitochondrial status and RQC pathway activity compared to U87 or U251 cell lines. Therefore, the conclusions of the two studies are complementary rather than contradictory, and both demonstrate the importance of RQC in tumorigenesis. Our study delves into the mechanistic role of the RQC pathway in GBM and identifies new potential targets for future treatments.

Detailed studies on the quantification of nuclear genome-encoded mitochondrial proteins modified via the msiCAT tailing mechanism using advanced mass spectrometry methods are an attractive area for future research. A recent study by Lv et al. cell reportrevealed that the cytosolic E3 ligase Pirh2 and the mitochondrial protease ClpXP cooperate with the established NEMF-ANKZF1 system to degrade mitochondrial protein aggregates generated by ribosome arrest (LV et al., 2024). The increased presence of ClpXP in various cancers may be associated with increased msiCAT tailing products in mitochondria, but further studies are required to elucidate the role of ClpXP in mitochondrial RQC (Colmio et al., 2021). Additionally, ClpXP affects the levels of multiple mitochondrial proteins. Our own experiments showed that ATP5α proteins lacking the msiCAT tail are the most difficult to express ectopically. Proteins with short tails (AT3) were more easily expressed, and proteins with long tails (AT20) had the highest expression levels, but also tended to form SDS-insoluble aggregates. This regulatory effect may be mediated by ClpXP-dependent degradation or by transcriptional regulation. PGC-1α, a peroxisome proliferator-activated receptor gamma coactivator, is an important regulator of mitochondrial biogenesis in mammals (Ventura-Clapier et al., 2008). PGC-1α binds to and activates nuclear transcription factors, leading to transcription of mitochondrial proteins encoded in the nuclear genome and the mitochondrial transcription factor Tfam. Tfam then activates transcription and replication of the mitochondrial genome (Wu et al., 1999). Future studies, including closer examination of msiCAT tail target mRNA levels and analysis of PGC-1α and Tfam binding to transcriptional elements, will be required to distinguish between these regulatory possibilities.

MPTP is a complex supramolecular channel that crosses the inner mitochondrial membrane and is characterized by its nonselective ion permeability, calcium dependence, and pleiotropic functions. Despite extensive research on the functional properties and regulatory mechanisms of MPTP, the exact molecular structure of MPTP remains elusive (Endricher et al., 2023). Several theoretical models have been proposed to elucidate the structural composition of MPTP. First, the VDAC/ANT/CypD model (Beutner et al., 1998) proposed an assembly of voltage-gated anion channels (VDACs), adenine nucleotide translocators (ANTs), and CypD as the structural basis. However, subsequent genetic analyzes have generated major controversy regarding the essential role of these proteins in the MPTP complex (Baynes et al., 2007; Gutierrez Aguilar et al., 2014; Kokoska et al., 2004; Karch et al., 2019). Second, the ATP synthase model postulates that MPTP formation involves a dimer or rearranged C-ring of ATP synthase (Arabian et al., 2014; Giorgio et al., 2013). Although this hypothesis presents an interesting perspective, empirical confirmation of the role of ATP synthase as a critical structural element of the pore remains inconclusive, and a series of contradictory studies have been conducted surrounding this proposition. Third, a currently prevailing hypothesis suggests that MPTP is composed of a large complex called the ATP synthasome, consisting of ANT and ATP synthase, and that CypD plays a regulatory role in the dynamic behavior of the complex (Beutner et al., 2017).

MPTP activity is regulated by mitochondrial membrane potential (ΔΨm) and mutually influences mitochondrial ion homeostasis and energy metabolism (Petronilli et al., 1994; Boyman et al., 2019). Our study elucidated the dual functions of msiCAT-tailed ATP5α protein in cancer cells: alleviating MPTP induction by stabilizing high membrane potential and directly inhibiting MPTP function by participating in its assembly. Although the important role of MPTP in cell death has been established, the premise that MPTP inhibition allows cancer cells to escape drug-induced programmed cell death lacks substantial evidence. This study provides empirical support for this hypothesis, demonstrating that GBM cells, particularly GSCs, exhibit a marked reduction in MPTP activity compared to control cells. This reduction in activity directly correlates with CAT tailing modification of the ATP synthase subunit. These observations are consistent with previous studies and indicate that genetic mutations or post-translational modifications in specific ATP synthase subunits can modulate MPTP activity. This finding highlights a novel mechanism by which cancer cells may acquire resistance to therapeutic intervention by manipulating mitochondrial function (Antoniel et al., 2018; Carraro et al., 2020).