Leptin Signaling Contributes to Aromatase Inhibitor Resistant Breast Cancer Cell Growth and Activation of Macrophages.

Obesity represents a risk factor for breast cancer development and therapy resistance, but the molecular players underling these links are unclear. Here, we identify a role for the obesity-cytokine leptin in sustaining aromatase inhibitor (AI) resistant growth and progression in breast cancer. Using as experimental models MCF-7 breast cancer cells surviving long-term treatment with the AI anastrozole (AnaR) and Ana-sensitive counterparts, we found that AnaR cells expressed higher levels of leptin and its receptors (ObR) along with a constitutive activation of downstream effectors. Accordingly, leptin signaling inhibition reduced only AnaR cell growth and motility, highlighting the existence of an autocrine loop in mechanisms governing drug-resistant phenotypes. In agreement with ObR overexpression, increasing doses of leptin were able to stimulate to a greater extent growth and migration in AnaR than sensitive cells. Moreover, leptin contributed to enhanced crosstalk between AnaR cells and macrophages within the tumor microenvironment. Indeed, AnaR, through leptin secretion, modulated macrophage profiles and increased macrophage motility through CXCR4 signaling, as evidenced by RNA-sequencing, real-time PCR, and immunoblotting. Reciprocally, activated macrophages increased AnaR cell growth and motility in coculture systems. In conclusion, acquired AI resistance is accompanied by the development of a leptin-driven phenotype, highlighting the potential clinical benefit of targeting this cytokine network in hormone-resistant breast cancers, especially in obese women.

N-heterocyclic carbene complexes of silver and gold as novel tools against breast cancer progression.

AIM: Metal carbenic complexes have received considerable attention in both the catalysis and biological fields for their potential applications in cancer and antimicrobial therapies. RESULTS: A small series of new silver and gold N-heterocyclic carbene complexes has been designed and synthesized. Among the tested complexes, one compound was particularly active in inhibiting anchorage-dependent and -independent breast cancer proliferation, and inducing cell apoptosis via a mitochondria-related process. The antitumor activity was associated to the transcriptional activation of the tumor suppressor gene p53 in an Sp1-dependent manner, as evidenced by biological and docking studies. CONCLUSION: Our results highlight the importance and the versatility of N-heterocyclic carbene complexes of gold and silver as useful tools against breast cancer progression.

Expression and Function of Phosphodiesterase Type 5 in Human Breast Cancer Cell Lines and Tissues: Implications for Targeted Therapy.

PURPOSE: By catalyzing cGMP hydrolysis, phosphodiesterase (PDE) 5 is a critical regulator of its concentration and effects in different (patho)physiologic processes, including cancers. As PDE5 is a known druggable target, we investigated the clinical significance of its expression in breast cancer and the underlying mechanisms by which it may contribute to tumor progression. EXPERIMENTAL DESIGN: PDE5 expression was evaluated in seven breast cancer cell lines by RT-PCR and immunoblotting. To examine the impact of PDE5 on cancer phenotype, MCF-7 cells expressing lower levels of the enzyme were engineered to stably overexpress PDE5. Proliferation was evaluated by MTT assays, motility and invasion by wound-healing/transmigration/invasion assays, transcriptome-profiling by RNA-sequencing, and Rho GTPase signaling activation by GST-pulldown assays and immunoblotting. Clinical relevance was investigated by IHC on tissues and retrospective studies from METABRIC cohort. RESULTS: PDE5 is differentially expressed in each molecular subtype of both breast cancer cell lines and tissues, with higher levels representing a startling feature of HER2-positive and triple-negative breast cancers. A positive correlation was established between elevated PDE5 levels and cancers of high histologic grade. Higher PDE5 expression correlated with shorter patient survival in retrospective analyses. On molecular level, stable PDE5 overexpression in Luminal-A-like MCF-7 cells resulted in enhanced motility and invasion through Rho GTPase signaling activation. Treatment of PDE5-stable clones with selective ROCK or PDE5 inhibitors completely restored the less motile and weak invasive behavior of control vector cells. CONCLUSIONS: PDE5 expression enhances breast cancer cell invasive potential, highlighting this enzyme as a novel prognostic candidate and an attractive target for future therapy in breast cancers. Clin Cancer Res; 22(9); 2271-82. ©2015 AACR.

Leptin as a mediator of tumor-stromal interactions promotes breast cancer stem cell activity.

Breast cancer stem cells (BCSCs) play crucial roles in tumor initiation, metastasis and therapeutic resistance. A strict dependency between BCSCs and stromal cell components of tumor microenvironment exists. Thus, novel therapeutic strategies aimed to target the crosstalk between activated microenvironment and BCSCs have the potential to improve clinical outcome. Here, we investigated how leptin, as a mediator of tumor-stromal interactions, may affect BCSC activity using patient-derived samples (n = 16) and breast cancer cell lines, and determined the potential benefit of targeting leptin signaling in these model systems. Conditioned media (CM) from cancer-associated fibroblasts and breast adipocytes significantly increased mammosphere formation in breast cancer cells and depletion of leptin from CM completely abrogated this effect. Mammosphere cultures exhibited increased leptin receptor (OBR) expression and leptin exposure enhanced mammosphere formation. Microarray analyses revealed a similar expression profile of genes involved in stem cell biology among mammospheres treated with CM and leptin. Interestingly, leptin increased mammosphere formation in metastatic breast cancers and expression of OBR as well as HSP90, a target of leptin signaling, were directly correlated with mammosphere formation in metastatic samples (r = 0.68/p = 0.05; r = 0.71/p = 0.036, respectively). Kaplan-Meier survival curves indicated that OBR and HSP90 expression were associated with reduced overall survival in breast cancer patients (HR = 1.9/p = 0.022; HR = 2.2/p = 0.00017, respectively). Furthermore, blocking leptin signaling by using a full leptin receptor antagonist significantly reduced mammosphere formation in breast cancer cell lines and patient-derived samples. Our results suggest that leptin/leptin receptor signaling may represent a potential therapeutic target that can block the stromal-tumor interactions driving BCSC-mediated disease progression.

A novel leptin antagonist peptide inhibits breast cancer growth in vitro and in vivo.

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I-III. Site I is crucial for the formation of an active leptin-leptin receptor complex and in its subsequent activation. Amino acids 39-42 (Leu-Asp-Phe-Ile- LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild-type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin-induced anchorage-dependent and -independent growth as well as the migration of ERα-positive (MCF-7) and -negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin-mediated up-regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women.

Androgens inhibit aromatase expression through DAX-1: insights into the molecular link between hormone balance and Leydig cancer development.

Leydig cell tumors (LCTs) of the testis are steroid-secreting tumors associated with various steroid biosynthetic abnormalities and endocrine dysfunctions. Despite their overall rarity, LCTs are still of substantial interest owing to the paucity of information regarding their exact nature and malignant potential. In the present study, we disclose the ability of androgens to inhibit Leydig tumor cell proliferation by opposing to self-sufficient in situ estrogen production. In rat Leydig tumor cells, R2C, androgen treatment significantly decreases the expression and the enzymatic activity of cytocrome P450 aromatase, responsible for the local conversion of androgens into estrogens. This inhibitory effect relies on androgen receptor (AR) activation and involves negative regulation of the CYP19 gene transcriptional activity through the nuclear orphan receptor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1). Ligand-activated AR up-regulates the expression of DAX-1 and promotes its increased recruitment within the steroidogenic factor-1 site-containing region of the aromatase proximal promoter II in association with the nuclear receptor corepressor. The biological relevance in LCTs of the newly highlighted functional interplay between AR, DAX-1, and aromatase is underlined by our in vivo observations, revealing a marked down-regulation of AR and DAX-1 expression and a strong increase in aromatase levels in testes tissues from old Fischer rats with spontaneously developed Leydig cell neoplasia, compared with normal testes tissues from younger animals. In elucidating a mechanism by which androgens modulate the growth of Leydig tumor cells, our finding support the hypothesis that maintaining the adequate balance between androgen and estrogens may represent the key for blocking estrogen-secreting Leydigioma development, opening new prospects for therapeutic intervention.