The omega-3 long chain polyunsaturated fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), elicit anti-proliferative effects in cancer cell lines and in animal models. Dietary DHA and EPA can be converted to their ethanolamide derivatives, docosahexaenoyl ethanolamine (DHEA), and eicosapentaenoyl ethanolamine (EPEA), respectively; however, few studies are reported on their anti-cancer activities. Here, we demonstrated that DHEA and EPEA were able to reduce cell viability in MCF-7 breast cancer cells whereas they did not elicit any effects in MCF-10A non-tumorigenic breast epithelial cells. Since DHA and EPA are ligands of peroxisome proliferator-activated receptor gamma (PPARγ), we sought to determine whether PPARγ may also mediate DHEA and EPEA actions. In MCF-7 cells, both compounds enhanced PPARγ expression, stimulated a PPAR response element-dependent transcription as confirmed by the increased expression of its target gene PTEN, resulting in the inhibition of AKT-mTOR pathways. Besides, DHEA and EPEA treatment induced phosphorylation of Bcl-2 promoting its dissociation from beclin-1 which resulted in autophagy induction. We also observed an increase of beclin-1 and microtubule-associated protein 1 light chain 3 expression along with an enhanced autophagosomes formation as revealed by mono-dansyl-cadaverine staining. Finally, we demonstrated the involvement of PPARγ in DHEA- and EPEA-induced autophagy by using siRNA technology and a selective inhibitor. In summary, our data show that the two omega-3 ethanolamides exert anti-proliferative effects by inducing autophagy in breast cancer cells highlighting their potential use as breast cancer preventive and/or therapeutic agents.

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Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis. In this study, we show, in different human breast cancer cell lines, that leptin enhanced the expression of a chaperone protein Hsp90 resulting in increased HER2 protein levels. Silencing of Hsp90 gene expression by RNA interference abrogated leptin-mediated HER2 up-regulation. Leptin effects were dependent on JAK2/STAT3 activation, since inhibition of this signaling cascade by AG490 or ectopic expression of a STAT3 dominant negative abrogated leptin-induced HER2 and Hsp90 expressions. Functional experiments showed that leptin treatment significantly up-regulated human Hsp90 promoter activity. This occurred through an enhanced STAT3 transcription factor binding to its specific responsive element located in the Hsp90 promoter region as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Analysis of HER2, Akt and MAPK phosphorylation levels revealed that leptin treatment amplified the responsiveness of breast cancer cells to growth factor stimulation. Furthermore, we found that long-term leptin exposure reduced sensitivity of breast cancer cells to the antiestrogen tamoxifen. In the same experimental conditions, the combined treatment of tamoxifen with the Hsp90 inhibitor 17-AAG completely abrogated leptin-induced anchorage-independent breast cancer cell growth. In conclusion, our results highlight, for the first time, the ability of the adipocyte-secreted factor leptin to modulate Hsp90/HER2 expressions in breast cancer cells providing novel insights into the molecular mechanism linking obesity to breast cancer growth and progression.

Ligand activation of peroxisome proliferator-activated receptor (PPAR)␥ and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPAR␥ ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21 WAF1/Cip1 . Functional experiments indicate that the nuclear factor-B site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. Breast cancer is the leading cause of death among women in the world. The principal effective endocrine therapy for advanced treatment on this type of cancer is anti-estrogens, but therapeutic choices are limited for estrogen receptor (ER)␣-negative tumors, which are often aggressive. The development of cancer cells that are resistant to chemotherapeutic agents is a major clinical obstacle to the successful treatment of breast cancer, providing a strong stimulus for exploring new approaches in vitro. Using ligands of nuclear hormone receptors to inhibit tumor growth and progression is a novel strategy for cancer therapy. An example of this is the treatment of acute promyelocytic leukemia using all-trans retinoic acid, the specific ligand for retinoic acid receptors.1-3 A further paradigm for the use of retinoids in cancer therapy is for early lesions of head and neck cancer 4 and squamous cell carcinoma of the cervix. 5The retinoic acid receptor, retinoid X receptor (RXR), and peroxisome proliferator receptor (PPAR)␥, ligandactivated transcription factors belonging to the nuclear hormone receptor superfamily, are able to modulate gene networks involved in controlling growth and cellular differentiation.6 Particularly, heterodimerization of PPAR␥ with RXR by their own ligands greatly enhances DNA binding to the direct-repeated consensus sequence AGGTCA, which leads to transcriptional activation.7 Previous data show that PPAR␥, poorly expressed in normal breast epithelial cells, 8 is present at higher levels in Supported by AIRC, MURST, and Ex 60%.Portions of this work were presented as an Abstract at Società Italiana di Patologia XXIX

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor α (ERα) positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study we show that activation of Farnesoid X Receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an “in vitro” model of acquired Tam-resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage–independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from Western blot analysis and real-time RT-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 MAPK phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment down-regulated basal HER2 promoter activity. This occurred through an inhibition of NF-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome antiestrogen resistance in human breast cancer cells, and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Our study demonstrates for the first time that renal tubular cells express and secrete ADPN, and their concentration increases upon inflammatory stimulus. These results suggest that in renal inflammatory diseases, tubular cells may contribute to the increase in circulating ADPN levels, triggering a feedback response in order to self-mitigate the inflammatory process.

Collectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.

Obesity is a major risk factor for the development and progression of breast cancer. Leptin, a cytokine mainly produced by adipocytes, plays a crucial role in mammary carcinogenesis and is elevated in hyperinsulinemia and insulin resistance. The antidiabetic thiazolidinediones inhibit leptin gene expression through ligand activation of the peroxisome proliferator-activated receptor-γ (PPARγ) and exert antiproliferative and apoptotic effects on breast carcinoma. In this study, we investigated the ability of PPARγ ligands to counteract leptin stimulatory effects on breast cancer growth in either in vivo or in vitro models. The results show that activation of PPARγ prevented the development of leptin-induced MCF-7 tumor xenografts and inhibited the increased cell-cell aggregation and proliferation observed on leptin exposure. PPARγ ligands abrogated the leptin-induced up-regulation of leptin gene expression and its receptors in breast cancer. PPARγ-mediated repression of leptin gene involved the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors corepressors on the glucocorticoid responsive element site in the leptin gene expression regulatory region in the presence of glucocorticoid receptor and PPARγ. In addition, PPARγ ligands inhibited leptin signaling mediated by MAPK/STAT3/Akt phosphorylation and counteracted leptin stimulatory effect on estrogen signaling. These findings suggest that PPARγ ligands may have potential therapeutic benefits in the treatment of breast cancer.