Impact of metformin-based chemo-radiotherapeutic strategies on breast cancer stemness and autophagy-associated gene expression: evidence from ALDH1A1 and LC3 expression analysis.

BACKGROUND

Breast cancer remains a leading cause of cancer death worldwide, with treatment failure driven partly by cancer stem cells (CSCs) and dysregulated autophagy. Aldehyde dehydrogenase 1A1 (ALDH1A1) marks CSCs and indicates poor prognosis, whereas microtubule-associated protein 1 A/1B-light chain 3 (LC3) regulates autophagic flux. Metformin has anti-neoplastic activity, but its molecular effects during standard breast-cancer therapy remain incompletely defined. We evaluated the impact of metformin on ALDH1A1 and LC3 gene expression in patients receiving different chemo-radiotherapeutic regimens.

METHODS

In this comparative, cross-sectional study, 86 early-stage breast cancer patients treated in the peri-operative (neoadjuvant or adjuvant) setting were grouped as chemotherapy only (n = 29), chemotherapy plus radiotherapy (n = 29), or chemotherapy plus radiotherapy plus metformin (n = 28); 30 healthy individuals served as controls. All major molecular subtypes (luminal A, luminal B, luminal B/HER2-positive, HER2-enriched and triple-negative) were represented across cohorts. ALDH1A1 and LC3 expression was quantified in peripheral blood mononuclear cells (PBMCs) by SYBR Green quantitative real-time PCR with GAPDH as the endogenous reference gene, using the 2 - ΔΔCt method. Non-parametric tests were applied.

RESULTS

Fold-change values were highly skewed and are reported as medians. ALDH1A1 expression decreased progressively across the chemotherapy (2.26), chemotherapy plus radiotherapy (0.75) and metformin (0.37) groups (H = 8.577, P = 0.014), being lowest in the metformin group (adjusted P = 0.039). LC3 expression rose progressively (4.18, 13.76 and 48.84; H = 24.177, P < 0.001), being highest with metformin (adjusted P < 0.001 versus chemotherapy alone; adjusted P = 0.021 versus chemotherapy plus radiotherapy). In receiver-operating-characteristic analysis, LC3 discriminated metformin-treated patients well (AUC = 0.80), and a combined ALDH1A1-LC3 model further improved discrimination (AUC = 0.91).

CONCLUSION

The addition of metformin to chemo-radiotherapy was associated with lower ALDH1A1 and higher LC3 expression in peripheral blood mononuclear cells, consistent with engagement of cancer-stem-cell and autophagy-related pathways and supporting further evaluation of metformin as an adjuvant therapy. These associations do not by themselves establish a direct anti-tumour effect.