Evaluation of TLR2 targeting approaches for improvement of breast cancer therapies

Breast cancer is the major cause of cancer-related death in women worldwide, despite substantial advancements in its diagnosis and treatment. Routine screening and improved therapeutic interventions have significantly enhanced early detection and survival rates. However, therapy resistance and poor outcomes persist, underscoring the urgent need for continued research to address these challenges. Among the key contributors to therapy resistance, breast cancer stem cells (CSCs) have a crucial role. CSCs possess unique phenotypic plasticity, metabolic reprogramming, and resistance mechanisms, which makes them able to drive metastasis and evade conventional treatments. The aim of my thesis is to set up new therapies able to effectively target CSCs, thus overcoming therapy resistance. Building on prior research by our group, this work explores the therapeutic targeting of Toll-like receptor 2 (TLR2), a molecule expressed in breast cancer and upregulated in breast CSCs. TLR2 plays a dual role in cancer progression, activating pro-tumorigenic pathways while promoting immune evasion. This thesis identifies a novel non-immunological role for TLR2 in breast cancer. Public dataset analyses revealed that high TLR2 expression correlates with poor prognosis and chemoresistance, establishing it as a promising therapeutic target. When activated by damageassociated molecular patterns (DAMPs) such as High Mobility Group Box 1 (HMGB1) within the tumor microenvironment, TLR2 triggers NF-κB signaling and induces pro-tumoral cytokine production. These cytokines establish cancer cell-intrinsic survival loops, drive neoangiogenesis, and recruit immunosuppressive cells such as regulatory T cells (Tregs). Furthermore, chemotherapeutic agents like doxorubicin inadvertently hyperactivate TLR2 via DAMPs release, exacerbating therapy resistance. To counteract these effects, this thesis demonstrates the efficacy of TLR2 inhibition using the small molecule CU-CPT22. In preclinical models, TLR2 inhibition reverses doxorubicin resistance, enhances therapeutic efficacy, and synergistically reduces tumor progression and CSC frequency. To refine this approach, targeted nanoparticle-based drug delivery systems were developed. Poly lacticco- glycolic acid (PLGA) nanoparticles encapsulating CU-CPT22 and liposomes delivering doxorubicin were coated with an integrin αvβ3-targeting cyclic Arg-Gly-Asp (RGD) tripeptide. This strategy enhances tumor-specific drug accumulation, while minimizing off-target effects, thus significantly reducing chemotherapy-associated toxicity. Additionally, this thesis explores a multi-targeted approach by combining TLR2 inhibition and chemotherapy with a vaccination-based immunotherapy targeting xCT, a protein implicated in CSC 4 maintenance and metastasis. This combinatorial strategy exhibited remarkable therapeutic efficacy in preclinical models, aligning with emerging trends that integrate diverse pharmacological approaches. In conclusion, the findings presented in this thesis highlight TLR2 as a critical driver of breast cancer progression and resistance, providing a robust foundation for novel therapeutic interventions. The development of multi-disciplinary, multi-targeted strategies holds promise for improving breast cancer treatment and patient outcomes. Future research will extend the applicability of TLR2 and xCT targeting to other tumor types, advancing the clinical potential of this approach.