Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is among the most aggressive breast cancer subtypes. Despite being initially responsive to chemotherapy, patients develop drug-resistant and metastatic tumors. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a secreted protein with a tumor suppressor function due to its anti-proteolytic activity. Nevertheless, evidence indicates that TIMP-1 binds to the CD63 receptor and activates noncanonical oncogenic signaling in several cancers, but its role in mediating TNBC chemoresistance is still largely unexplored. Here, we show that mesenchymal-like TNBC cells express TIMP-1, whose levels are further increased in cells generated to be resistant to cisplatin (Cis-Pt-R) and doxorubicin (Dox-R). Moreover, public dataset analyses indicate that high TIMP-1 levels are associated with a worse prognosis in TNBC subjected to chemotherapy. Knock-down of TIMP-1 in both Cis-Pt-R and Dox-R cells reverses their resistance by inhibiting AKT activation. Consistently, TNBC cells exposed to recombinant TIMP-1 or TIMP-1-enriched media from chemoresistant cells, acquire resistance to both cisplatin and doxorubicin. Importantly, released TIMP-1 reassociates with plasma membrane by binding to CD63 and, in the absence of CD63 expression, TIMP-1-mediated chemoresistance is blocked. Thus, our results identify TIMP-1 as a new biomarker of TNBC chemoresistance and lay the groundwork for evaluating whether blockade of TIMP-1 signal is a viable treatment strategy.

Aptamer-Based Strategies to Boost Immunotherapy in TNBC.

The immune system (IS) may play a crucial role in preventing tumor development and progression, leading, over the last years, to the development of effective cancer immunotherapies. Nevertheless, immune evasion, the capability of tumors to circumvent destructive host immunity, remains one of the main obstacles to overcome for maximizing treatment success. In this context, promising strategies aimed at reshaping the tumor immune microenvironment and promoting antitumor immunity are rapidly emerging. Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with poor outcomes, is highly immunogenic, suggesting immunotherapy is a viable strategy. As evidence of this, already, two immunotherapies have recently become the standard of care for patients with PD-L1 expressing tumors, which, however, represent a low percentage of patients, making more active immunotherapeutic approaches necessary. Aptamers are short, highly structured, single-stranded oligonucleotides that bind to their protein targets at high affinity and specificity. They are used for therapeutic purposes in the same way as monoclonal antibodies; thus, various aptamer-based strategies are being actively explored to stimulate the IS's response against cancer cells. The aim of this review is to discuss the potential of the recently reported aptamer-based approaches to boost the IS to fight TNBC.

Aptamer-Functionalized Nanoparticles Mediate PD-L1 siRNA Delivery for Effective Gene Silencing in Triple-Negative Breast Cancer Cells.

Small interfering RNA (siRNA) therapies require effective delivery vehicles capable of carrying the siRNA cargo into target cells. To achieve tumor-targeting, a drug delivery system would have to incorporate ligands that specifically bind to receptors expressed on cancer cells to function as portals via receptor-mediated endocytosis. Cell-targeting and internalizing aptamers are the most suitable ligands for functionalization of drug-loaded nanocarriers. Here, we designed a novel aptamer-based platform for the active delivery of siRNA targeting programmed cell death-ligand 1 (PD-L1) to triple-negative breast cancer (TNBC) cells. The generated nanovectors consist of PLGA-based polymeric nanoparticles, which were loaded with PD-L1 siRNA and conjugated on their surface with a new RNA aptamer, specific for TNBC and resistant to nucleases. In vitro results demonstrated that these aptamer-conjugated nanoparticles promote siRNA uptake specifically into TNBC MDA-MB-231 and BT-549 target cells, along with its endosomal release, without recognizing non-TNBC BT-474 breast cancer cells. Their efficiency resulted in an almost complete suppression of PD-L1 expression as early as 90 min of cell treatment. This research provides a rational strategy for optimizing siRNA delivery systems for TNBC treatments.

Optimization of Short RNA Aptamers for TNBC Cell Targeting.

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2'Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.

Optimizing cisplatin delivery to triple-negative breast cancer through novel EGFR aptamer-conjugated polymeric nanovectors.

BACKGROUND: Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC. METHODS: Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses. RESULTS: We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs. CONCLUSIONS: Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.