Breast cancer stem cells (CSCs) are a small population of tumour cells with self-renewal and differentiation capabilities that are associated with tumour progression, metastasis, and therapy resistance.

These cells rely heavily on mitochondrial biogenesis and oxidative metabolism, making mitochondrial function a potential metabolic vulnerability.

A new research paper on the topic was published in the journal Ageing.

The study was led by first author Chiara Chinigò from the Italian National Research Centre on Ageing (IRCCS INRCA), Cosenza, Italy.

Corresponding authors Federica Sotgia and Michael P. Lisanti are affiliated with the University of Salford, United Kingdom, and Lunella Biotech in Ottawa, Canada, with Lisanti additionally affiliated with the Institute of Mental Health Research at the University of Ottawa and The Royal Ottawa Hospital, and Université Laval in Québec.

The researchers focused on mitochondrial DNA polymerase gamma (POLG), the enzyme responsible for mitochondrial DNA (mtDNA) replication and repair.

POLG contains a catalytic subunit encoded by POLG1 and an accessory subunit encoded by POLG2.

Using oestrogen receptor-positive MCF-7 breast cancer cells, the researchers genetically silenced POLG1 and POLG2 to determine whether disrupting mtDNA maintenance would affect cancer stemness.

POLG1 knockdown reduced mtDNA content by approximately 80%, while POLG2 knockdown produced an approximately 70% reduction.

Both interventions impaired mitochondrial function, lowering the mtDNA-encoded respiratory-chain protein MTCO2, mitochondrial membrane potential, respiration, and ATP production.

Importantly, these metabolic changes were accompanied by a reduction in stemness-related properties without substantially affecting short-term two-dimensional cell growth.

POLG1 silencing reduced OCT4 expression and decreased mammosphere formation by approximately 70%, while also markedly suppressing colony formation.

POLG2 knockdown similarly reduced OCT4 expression, mammosphere formation, and clonogenic growth.

These findings suggest that POLG-dependent mitochondrial metabolism is particularly important for the long-term self-renewal and anchorage-independent growth associated with breast CSCs.

The investigators next tested whether POLG could also be targeted pharmacologically.

Alovudine, a nucleoside reverse transcriptase inhibitor originally developed for HIV treatment that can inhibit POLG as an off-target effect, reduced mammosphere and colony formation in MCF-7 cells while lowering MTCO2 expression, mitochondrial respiration, and ATP generation.

However, the authors emphasise that Alovudine previously showed haematological toxicities during antiviral development, so the compound serves primarily as a proof-of-concept tool rather than an established breast cancer therapy.

To strengthen the findings, the researchers used a second POLG inhibitor, Zalcitabine (ddC), and expanded testing to additional breast cancer models.

ddC impaired mitochondrial respiration, reduced MTCO2 expression, and suppressed mammosphere and colony formation in MCF-7 cells.

Across T47D, MDA-MB-231, MDA-MB-436, and MDA-MB-453 breast cancer cells, ddC consistently reduced mammosphere formation, although its effects on conventional monolayer growth varied between cell types.

Non-tumoural MCF10A mammary epithelial cells showed limited effects on viability under the tested conditions.

The researchers also examined stemness-related transcriptional programmes.

In MCF-7 cells, ddC markedly reduced expression of SOX2 and NANOG, while MDA-MB-453 cells showed reduced SOX2 with a more modest effect on NANOG.

These differences suggest that the transcriptional response to POLG inhibition may vary according to the molecular and metabolic characteristics of different breast cancer subtypes.

“Collectively, these additional findings obtained using an independent POLG inhibitor and multiple breast cancer models substantially reinforce the broader biological relevance of POLG inhibition as a potential mitochondrial-targeting strategy in breast cancer.”

To explore potential clinical relevance, the researchers performed preliminary Kaplan–Meier analyses in 458 patients with high-risk, oestrogen receptor-positive, lymph node-positive luminal A breast cancer.

Higher POLG1 expression was associated with poorer overall survival, with a hazard ratio of 1.34, as well as shorter relapse-free, distant metastasis-free, and post-progression survival.

The authors caution that these findings require validation in independent cohorts using multivariable analyses that account for established clinicopathological factors.

The study remains preliminary.

Much of the genetic work was conducted in MCF-7 cells; potential shRNA-related off-target effects cannot be completely excluded, and the pharmacological inhibitors may have additional molecular targets.

Most importantly, the findings have not yet been validated in animal models or patients.

Future studies using additional genetic approaches, xenograft or orthotopic models, and larger clinical cohorts will be needed to determine whether POLG inhibition can reduce tumour initiation, CSC frequency, treatment resistance, and tumour growth while maintaining acceptable systemic safety.

Overall, this pilot study identifies POLG-dependent mitochondrial maintenance as an important contributor to breast cancer stem-cell properties.

Genetic disruption of POLG1 or POLG2 and pharmacological interference with POLG-dependent mitochondrial function consistently impaired mitochondrial metabolism and reduced CSC-associated phenotypes.

Together with the preliminary association between elevated POLG1 expression and poorer patient outcomes, the findings support further investigation of POLG as a potential mitochondrial therapeutic target and prognostic biomarker in breast cancer.

Source: Ageing