Misha Blagosklonny

In this new study, researchers investigated the intricate link between mitophagy and cancer stem cells. 

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Cellular quality control mechanisms like mitophagy, a specialized form of autophagy that eliminates dysfunctional mitochondria, play a pivotal role in various physiological processes. Defects in mitophagy have been linked to neurodegeneration, heart failure, cancer, and aging.

A recent study, by researchers Marta Mauro-Lizcano, Federica Sotgia, and Michael P. Lisanti from the University of Salford, has shed light on the intricate link between mitophagy and cancer stem cells (CSCs). In this study, the researchers developed an innovative fluorescence-based approach to enrich subpopulations of cancer cells exhibiting high basal levels of mitophagy. Their findings reveal that elevated mitophagy activity enhances CSC properties, including self-renewal, ATP production, proliferation, and cell migration, underscoring the potential of targeting mitophagy as a therapeutic strategy for cancer treatment. 

On June 4, 2024, their research paper was published on the cover of Aging’s Volume 16, Issue 11, entitled, “Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.”

“CSCs are responsible for cancer relapse, therapy-resistance, and metastatic dissemination. Therefore, CSC elimination is necessary to prevent cancer recurrence and improve long-term patient outcomes. The search of new targets against CSCs is essential for the success of cancer treatment.” — Mauro-Lizcano et al.

Background

Mitophagy plays a crucial role in maintaining cellular homeostasis by selectively degrading damaged or superfluous mitochondria. This process is governed by specific mitochondrial outer membrane receptors, such as BNIP3 and BNIP3L (also known as NIX), which interact with autophagy-related proteins like LC3/GABARAP to initiate mitophagy. The current study focused on the BNIP3/BNIP3L-dependent pathway, which is rapidly induced under cellular stress conditions like hypoxia and nutrient deprivation.

Cancer stem cells (CSCs) are a subpopulation of cells within a tumor that exhibit stem cell-like properties, such as self-renewal, tumor initiation capability, and drug resistance. These cells are implicated in cancer recurrence, treatment failure, and metastatic dissemination, making their elimination a critical target for effective cancer therapy. Accumulating evidence suggests that mitophagy plays a pivotal role in sustaining CSC properties, including self-renewal, cell propagation, and tumorigenic ability. Consequently, targeting mitophagy has emerged as a promising approach for CSC eradication.

The Study

To investigate the role of mitophagy in CSCs, the researchers developed a novel model system to enrich subpopulations of cancer cells with high basal levels of mitophagy. They employed a BNIP3(L)-promoter-eGFP-reporter system, where the transcriptional activity of BNIP3 and BNIP3L was linked to the expression of enhanced green fluorescent protein (eGFP). This allowed the isolation of cancer cells with high BNIP3/BNIP3L transcriptional activity, indicative of elevated mitophagy levels, using flow cytometry.

The validity of the model was confirmed through various functional assays. Immunoblotting revealed higher protein levels of BNIP3 and BNIP3L in the eGFP-high subpopulations. Additionally, these cells exhibited increased lysosomal mass and mitophagy activity, as measured by flow cytometry using specific probes. Furthermore, the researchers employed the mitochondrially-targeted red fluorescent protein (mt-Keima) to directly visualize and quantify mitophagy, providing further evidence of the model’s robustness.

Stemness & Self-Renewal: Mammosphere Formation Assays

“Mammospheres, or mammary epithelial stem cell aggregates, derived from primary breast tumors or cell lines are thought to develop from rare cancer stem cell (CSC) subpopulations within the tumor.” — Millipore Sigma

To investigate the role of mitophagy in CSC propagation, the researchers compared the mammosphere-forming ability, a functional assay for anchorage-independent growth and self-renewal, between eGFP-high and eGFP-low subpopulations. The eGFP-high cells demonstrated a statistically significant increase in mammosphere formation, indicating enhanced CSC properties. Moreover, these cells exhibited higher levels of CD44, a well-known cell surface marker of CSCs.

To further validate the mammosphere phenotype’s dependence on mitophagy, the researchers treated the eGFP-high and eGFP-low cells with chloroquine, an autophagy inhibitor, and cyclosporin A, a specific mitophagy inhibitor. Interestingly, the eGFP-low subpopulations were more sensitive to both inhibitors, suggesting that the high levels of endogenous mitophagy in the eGFP-high cells conferred resistance to these agents, further reinforcing the functional implication of mitophagy in mammosphere formation.

ATP Production & Mitochondrial Activity

To better understand the effects of mitophagy on CSCs, the researchers analyzed their metabolic profiles. The eGFP-high cells exhibited significantly higher ATP levels compared to eGFP-low cells, despite similar mitochondrial mass. Notably, the eGFP-high cells also demonstrated an increased GSH/GSSG ratio, indicating higher antioxidant capacity and better mitochondrial function.

Proliferation & Cell Cycle Progression

Cell cycle analysis revealed that the eGFP-high cells exhibited a decreased G0/G1 phase and corresponding increases in the S and G2/M phases, suggesting a more proliferative phenotype. This finding aligns with the observed increase in ATP production and mitochondrial activity, supporting the notion that mitophagy contributes to the energetic and proliferative advantages of CSCs.

Drug Resistance: Tamoxifen & Palbociclib

To assess the potential drug resistance phenotype of the eGFP-high and eGFP-low subpopulations, the researchers evaluated their sensitivity to 4-OH-Tamoxifen, an FDA-approved drug for treating estrogen receptor-positive (ER+) breast cancer, and Palbociclib, a CDK4/6 inhibitor. Remarkably, the eGFP-high cells exhibited multi-drug resistance, with significantly higher mammosphere formation compared to the eGFP-low cells upon treatment with these agents, further underscoring the aggressive nature of mitophagy-high CSCs.

Cell Migration and Metastatic Potential

Using the highly metastatic MDA-MB-231 breast cancer cell line, the researchers investigated the migratory capacity of the eGFP-high and eGFP-low subpopulations. Consistent with the observed stemness and metabolic advantages, the eGFP-high MDA-MB-231 cells exhibited higher levels of cell migration, suggesting that elevated mitophagy contributes to the metastatic potential of CSCs.

Therapeutic Implications & Future Directions

“In summary, our current work has provided a novel strategy to enrich for a sub-population of cancer cells, with high basal levels of mitophagy.” — Mauro-Lizcano et al.

The findings of this study highlight the critical role of mitophagy in driving various hallmarks of CSCs, including self-renewal, ATP production, proliferation, and cell migration. By targeting mitophagy, particularly the BNIP3/BNIP3L-dependent pathway, researchers may be able to develop novel therapeutic strategies for eliminating CSCs and improving patient outcomes in cancer treatment.

Future research should focus on exploring the molecular mechanisms underlying the observed effects of mitophagy on CSC properties and identifying specific mitophagy inhibitors or modulators with potential therapeutic applications. Additionally, further investigation into the interplay between mitophagy and other cellular processes, such as metabolic reprogramming and signaling pathways, could provide valuable insights into the complex biology of CSCs and pave the way for more effective targeted therapies.

Conclusion

The study by Mauro-Lizcano et al. represents a significant advancement in our understanding of the role of mitophagy in cancer stem cell biology. By developing an innovative model system and employing a multifaceted approach, the researchers have unveiled the energetic drivers and functional implications of mitophagy in stemness features, ATP production, proliferation, and cell migration. These findings not only deepen our knowledge of the intricate mechanisms governing CSC behavior but also highlight the potential of targeting mitophagy as a promising therapeutic strategy for combating cancer recurrence, treatment resistance, and metastatic dissemination.

Click here to read the full research paper published in Aging.

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Aging is an open-access, traditional, peer-reviewed journal that publishes high-impact papers in all fields of aging research. All papers are available to readers (at no cost and free of subscription barriers) in bi-monthly issues at Aging-US.com.

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