Nanoparticles with active-targeting and stimuli-responsive behavior are a promising class of engineered materials able to recognize the site of cancer disease, targeting the drug release and limiting side effects in the healthy organs. In this work, new dual pH/redox-responsive nanoparticles with affinity for folate receptors were prepared by the combination of two amphiphilic dextran (DEX) derivatives. DEXFA conjugate was obtained by covalent coupling of the polysaccharide with folic acid (FA), whereas DEXssPEGCOOH derived from a reductive amination step of DEX was followed by condensation with polyethylene glycol 600. After self-assembling, nanoparticles with a mean size of 50 nm, able to be destabilized in acidic pH and reducing media, were obtained. Doxorubicin was loaded during the self-assembling process, and the release experiments showed the ability of the proposed system to modulate the drug release in response to different pH and redox conditions. Finally, the viability and uptake experiments on healthy (MCF-10A) and metastatic cancer (MDA-MB-231) cells proved the potential applicability of the proposed system as a new drug vector in cancer therapy.

Adiponectin is the major adipocytes‐secreted protein involved in obesity‐related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα‐positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα‐coactivator. Here, we showed that adiponectin‐mediated ERα transactivation enhances E‐cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E‐cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E‐cadherin expression in ERα‐positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E‐cadherin gene promoter by ERα/LKB1‐complex. The impact of E‐cadherin on ERα‐positive breast cancer cell proliferation comes from the evidence that in the presence of E‐cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E‐cadherin connects cell polarity and growth, we investigated if the adiponectin‐enhanced E‐cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin‐treated MCF‐7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF‐7 cells revealed an enhanced E‐cadherin‐mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF‐7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin‐treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E‐cadherin expression, alters cell polarity and stimulates ERα‐positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.

Introduction: Breast cancer is the most diagnosed neoplasia and the second leading cause of malignancy death in women. Drug resistance is still the major challenge in the clinical management of breast cancer patients. Indeed, despite the improvements in the early diagnosis and in the therapeutic approaches, many breast cancer patients experience disease relapse due to de novo or acquired drug resistance. Growing evidence recognized a small population of breast cancer cells, named Breast Cancer Stem Cells (BCSCs), as the leading cause of tumor progression, metastasis formation and resistance against conventional therapy. BCSCs have some specific properties such as self-renewal, differentiation into different cell types, migration, tumorsphere formation, antioxidative activity, that make tumors more aggressive. Tumor microenvironment plays a crucial role in the regulation of stem cell proliferation and resistance to apoptosis through the secretion of cytokines and growth factors. Adipocytes represent the most abundant cellular component of mammary microenvironment. The excessive fat accumulation in obesity leads to the development of a dysfunctional adipose tissue, producing an unbalance in adipokines secretion. Among the secreted factors, adiponectin plays a crucial role in breast cancer development and progression. The aim of the present study was to investigate the effects of low adiponectin level (5 μl/ml), hallmark of obese status, on BCSCs activity in hormone-resistant cells. Methods: We tested the ability of MCF-7 wild type (WT) and tamoxifen-resistant (TR) to grow as mammospheres (Mammospheres Forming Efficiency, MFE), measuring the ability to maintain cell viability (self-renewal) upon serial non-adherent passages. mRNA levels of stemness, EMT and cell cycle markers were evaluated by qRT-PCR. CD44+/24- cell ratio, ALDH expression, ROS production, cell cycle, were analyzed by flow cytometry. Results: Adiponectin treatment significantly enhanced MFE and self-renewal capacity in TR-MCF-7 cells compared to MCF-7 WT cells. To identify the presence of BCSCs into mammospheres it has been evaluated the CD44+/CD24– biomarker signature. Flow cytometry revealed an enrichment of CD44, a trans-membrane glycoprotein which regulates growth signals in stem cells, in TR-MCF-7 mammospheres, whereas expression levels of the differentiation marker CD24 were decreased. The gene expression of these biomarkers was also analyzed by qRT-PCR. The increased BCSCs subpopulation in adiponectin-treated TR-MCF-7 mammospheres was also confirmed by the enhanced ALDH-expressing cells. qRT-PCR revealed that adiponectin increased the mRNA levels of stemness and EMT markers in TR-MCF-7 cells mammospheres. Interestingly, cell cycle analysis showed in adiponectin-treated TR-MCF-7 mammospheres a reduction of apoptosis. Moreover, flow cytometry analysis displayed a reduction of ROS levels, which generally are assumed as cues determining DNA damage-induced cell death, in adiponectin-treated TR-MCF-7 mammospheres. Conclusions: Our results demonstrated that low adiponectin level, as it occurs in obese breast cancer microenvironment, driving EMT, enhances stem-like features in TR-MCF-7 cells to sustain tumor progression. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-18.

Introduction: Breast cancer is the second leading cause of death among women after lung cancer. Despite the increase and improvement in preventive screening, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the rapid development of resistance to drug treatments, which recalls the complicity of multiple factors. Breast cancer prognosis can be correlated with specific histological subtypes and their receptor status. Depending on stage and invasiveness, ER + and PR + breast cancers are treated with surgery and adjuvant chemotherapy, which includes endocrine-based agents, such as SERMS (Tamoxifen), SERDs (Fulvestrant) and aromatase inhibitors (Letrazole and Anastrazole). Resistance to endocrine therapy can be explained by various mechanisms, such as mutations of the estrogen receptor, epigenetic modifications of the same receptor, alterations in hormone synthesis and metabolism, aberrant activation of signaling transduction pathways (up-regulation of PI3K, MAPK, CDKs). To reduce pharmacological resistance, it is possible to use histone deacetylase inhibitors, such as valproic acid, as the epigenetic alterations play an important role in the oncogenesis of breast cancer. Epigenetic modifications of genes are a mechanism used by cancer cells to silence the expression of tumor suppressor genes, driven by epigenetic mutations of regulator factors. The valproic acid, used as an antiepileptic drug and for the treatment of bipolar disorders, is also a selective inhibitor of class I and class II HDAC isoforms, involved in the progression of different types of tumors. In this study, we tested the effects of valproic acid on the signaling involved in apoptosis and in the generation of Reactive Oxygen Species (ROS) in MCF-7 breast cancer cells. Methods: MTT assay was performed to evaluate cell proliferation. Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, while protein levels were detected by Western Blotting (WB). Results: Cell treatment with valproic acid reduced cell proliferation and induced G0/G1 cell cycle arrest. In addition, the drug enhanced the generation of ROS by the mitochondria and addressed breast cell death by apoptosis. Indeed, the valproic acid was found to downregulate the anti-apoptotic marker Bcl-2 and to upregulate the expression of pro-apoptotic markers, such as Bax and Bad, leading to release of cytochrome C into the cytosol and PARP cleavage. The drastic reduction in mitochondrial membrane potential and in mitochondrial mass was observed following treatment with valproic acid, indeed the ratio between the two parameters, index of functionality per mitochondrion, resulted to be lowered, thus emphasizing the metabolic perturbation in the cells. Conclusion: In summary, our results have demonstrated that in estrogen receptor positive breast cancer cells the valproic acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. This study encourages that valproic acid, as an epigenetic drug, is a promising class of antineoplastic agent to be further explored also in combination with other classical chemotherapeutics in breast tumors. Citation Format: Alessandro Paolì, Martina Forestiero, Giuseppina Daniela Naimo, Loredana Mauro, Maria Luisa Panno, Francesca Giordano. Valproic acid induces apoptosis and mitochondrial perturbations in breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-02.

Obesity has a noteworthy role in breast tumor initiation and progression. Among the mechanisms proposed, the most validated is the development of chronic low-grade inflammation, supported by immune cell infiltration along with dysfunction in adipose tissue biology, characterized by an imbalance in adipocytokines secretion and alteration of their receptors within the tumor microenvironment. Many of these receptors belong to the seven-transmembrane receptor family, which are involved in physiological features, such as immune responses and metabolism, as well as in the development and progression of several malignancies, including breast cancer. These receptors are classified as canonical (G protein-coupled receptors, GPCRs) and atypical receptors, which fail to interact and activate G proteins. Among the atypical receptors, adiponectin receptors (AdipoRs) mediate the effect of adiponectin, the most abundant adipocytes-derived hormone, on breast cancer cell proliferation, whose serum levels are reduced in obesity. The adiponectin/AdipoRs axis is becoming increasingly important regarding its role in breast tumorigenesis and as a therapeutic target for breast cancer treatment. The objectives of this review are as follows: to point out the structural and functional differences between GPCRs and AdipoRs, and to focus on the effect of AdipoRs activation in the development and progression of obesity-dependent breast cancer.