Catia Morelli

PEG-templated mesoporous silica nanoparticles exclusively target cancer cells

Mesoporous silica nanoparticles (MSNs) have been proposed as DNA and drug delivery carriers, as well as efficient tools for fluorescent cell tracking. The major limitation is that MSNs enter cells regardless of a target-specific functionalization. Here we show that non functionalized MSNs, synthesized using a PEG surfactant-based interfacial synthesis procedure, do not enter cells, while a highly specific, receptor mediated, cellular internalization of folic acid (FOL) grafted MSNs (MSN-FOL), occurs exclusively in folate receptor (FR) expressing cells. Neither the classical clathrin pathway nor macropinocytosis is involved in the MSN endocytic process, while fluorescent MSNs (MSN-FITC) enter cells through aspecific, caveolae-mediated, endocytosis. Moreover, internalized particles seem to be mostly exocytosed from cells within 96 h. Finally, cisplatin (Cp) loaded MSN-FOL were tested on cancerous FR-positive (HeLa) or normal FR-negative (HEK293) cells. A strong growth arrest was observed only in HeLa cells treated with MSN-FOL-Cp. The results presented here show that our mesoporous nanoparticles do not enter cells unless opportunely functionalized, suggesting that they could represent a promising vehicle for drug targeting applications.

Inhibition of cyclin D1 expression by androgen receptor in breast cancer cells—identification of a novel androgen response element

Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at −570 to −556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.

Estrogen receptor-α regulates the degradation of insulin receptor substrates 1 and 2 in breast cancer cells

In breast cancer cells, 17-β-estradiol (E2) upregulates the expression of insulin receptor substrate 1 (IRS-1), a molecule transmitting insulin-like growth factor-I (IGF-I) signals through the PI-3K/Akt survival pathways. The stimulation of IRS-1 by E2 has been documented on the transcriptional level. Here we studied whether the expression of estrogen receptor (ER)-α affects IRS molecules post-transcriptionally. We used ER-α-negative MDA-MB-231 breast cancer cells and MDA-MB-231 cells with re-expressed ER-α. In MDA-MB-231 cells cultured under serum-free conditions, IRS-1 and IRS-2 were degraded through the 26S proteasome and calpain pathways. Re-expression of ER-α in MDA-MB-231 cells correlated with enhanced stability of IRS molecules. This effect coincided with significantly reduced ubiquitination of IRS-1 and IRS-2, but did not involve increased IRS-1 and IRS-2 transcription. The interference of ER-α with IRS-1 and IRS-2 turnover could rely on the competition for common degradation pathways, as in MDA-MB-231/ER cells, ER-α processing was blocked by proteasome and calpain inhibitors. Notably, a fraction of the cytosolic ER-α colocalized and coprecipitated with IRS-1 and IRS-2, indicating a possible common destination for these proteins. The stabilization of IRS-1 in MDA-MB-231/ER cells was paralleled by the upregulation of the IRS-1/Akt/GSK-3 pathway and improved survival in the presence of IGF-I, whereas IRS-2 was not involved in IGF-I signaling.

Interaction between estrogen receptor alpha and insulin/IGF signaling in breast cancer.

Estrogens and insulin/Insulin like growth factor 1 (IGF-I) have potent positive effects on the proliferation of mammary epithelial cells and estrogen-dependent breast cancer cells. A cooperative crosstalk between estrogens and insulin/IGF-I signaling pathways exists and it plays a critical role in breast carcinogenesis, tumor cell proliferation, differentiation and survival through the modulation of multiple biological events. The biological effects of estrogens are mainly mediated by the activation of estrogen receptor (ERalpha) whose activity is deeply influenced by the insulin/IGF-I signaling pathway. On the other hand, estrogens enhance insulin signaling by increasing the expression and/or the functional activity of some proteins involved in the insulin/IGF-I pathway. This review will focus on the critical node of the IGF-I network involved in the crosstalk with ERalpha and implicated in breast cancer development and progression.

Retinoic acid mediates degradation of IRS-1 by the ubiquitin–proteasome pathway, via a PKC-dependant mechanism

Insulin receptor substrate-1 (IRS-1) mediates signaling from the insulin-like growth factor type-I receptor. We found that all-trans retinoic acid (RA) decreases IRS-1 protein levels in MCF-7, T47-D, and ZR75.1 breast cancer cells, which are growth arrested by RA, but not in the RA-resistant MDA-MB-231 and MDA-MB-468 cells. Based on prior reports of ubiquitin-mediated degradation of IRS-1, we investigated the ubiquitination of IRS-1 in RA-treated breast cancer cells. Two proteasome inhibitors, MG-132 and lactacystin, blocked the RA-mediated degradation of IRS-1, and RA increased ubiquitination of IRS-1 in the RA-sensitive breast cancer cells. In addition, we found that RA increases serine phosphorylation of IRS-1. To elucidate the signaling pathway responsible for this phosphorylation event, pharmacologic inhibitors were used. Two PKC inhibitors, but not a MAPK inhibitor, blocked the RA-induced degradation and serine phosphorylation of IRS-1. We demonstrate that RA activates PKC-δ in the sensitive, but not in the resistant cells, with a time course that is consistent with the RA-induced decrease of IRS-1. We also show that: (1) RA-activated PKC-δ phosphorylates IRS-1 in vitro, (2) PKC-δ and IRS-1 interact in RA-treated cells, and (3) mutation of three PKC-δ serine sites in IRS-1 to alanines results in no RA-induced in vitro phosphorylation of IRS-1. Together, these results indicate that RA regulates IRS-1 levels by the ubiquitin–proteasome pathway, involving a PKC-sensitive mechanism.

Magnetic molecularly imprinted polymers (MMIPs) for carbazole derivative release in targeted cancer therapy

The synthesis of an innovative delivery system for targeted cancer therapy which combines the drug controlled release ability of Molecularly Imprinted Polymers (MIPs) with magnetic properties of magnetite is described herein. In the present study, an easy and smart synthetic strategy, involving new engineered precipitation photo-polymerization, was developed with the aim to obtain Magnetic Molecularly Imprinted Polymers (MMIPs) for 9H-carbazole derivative sustained delivery in cancer treatment. Both in vitro drug release and cytotoxicity studies on different cancer cell lines, such as HeLa and MCF-7, were performed in order to evaluate the controlled release ability and the potential application as a drug carrier in targeted cancer therapy. The synthesized polymeric materials have shown not only good selective recognition and controlled release properties, but also high magnetic responding capacity. The performed cytotoxicity studies highlighted the high inhibitory activity against the tested cell lines due to a dramatic growth arrest, compared to controls, by triggering apoptosis. These results clearly indicate the potential application of synthesized MMIPs as a magnetic targeted drug delivery nanodevice.

The estrogen receptor α is the key regulator of the bifunctional role of FoxO3a transcription factor in breast cancer motility and invasiveness

The role of the Forkhead box class O (FoxO)3a transcription factor in breast cancer migration and invasion is controversial. Here we show that FoxO3a overexpression decreases motility, invasiveness, and anchorage-independent growth in estrogen receptor α-positive (ERα+) cancer cells while eliciting opposite effects in ERα-silenced cells and in ERα-negative (ERα−) cell lines, demonstrating that the nuclear receptor represents a crucial switch in FoxO3a control of breast cancer cell aggressiveness. In ERα+ cells, FoxO3a-mediated events were paralleled by a significant induction of Caveolin-1 (Cav1), an essential constituent of caveolae negatively associated to tumor invasion and metastasis. Cav1 induction occurs at the transcriptional level through FoxO3a binding to a Forkhead responsive core sequence located at position −305/−299 of the Cav1 promoter. 17β-estradiol (E2) strongly emphasized FoxO3a effects on cell migration and invasion, while ERα and Cav1 silencing were able to reverse them, demonstrating that both proteins are pivotal mediators of these FoxO3a controlled processes. In vivo, an immunohistochemical analysis on tissue sections from patients with ERα+ or ERα− invasive breast cancers or in situ ductal carcinoma showed that nuclear FoxO3a inversely (ERα+) or directly (ERα−) correlated with the invasive phenotype of breast tumors. In conclusion, FoxO3a role in breast cancer motility and invasion depends on ERα status, disclosing a novel aspect of the well-established FoxO3a/ERα interplay. Therefore FoxO3a might become a pursuable target to be suitably exploited in combination therapies either in ERα+ or ERα− breast tumors.

Akt2 Inhibition Enables the Forkhead Transcription Factor FoxO3a To Have a Repressive Role in Estrogen Receptor α Transcriptional Activity in Breast Cancer Cells

ABSTRACT Estrogen receptor alpha (ER) and the insulin-like growth factor I receptor (IGF-IR) pathways are engaged in a functional cross talk in breast cancer, promoting tumor progression and increased resistance to anticancer treatments and radiotherapy. Here, we introduce new mechanisms through which proteins of the IGF-I/IGF-IR signaling pathway may regulate ER function in the absence of ligand. Our results indicate that in ER-positive breast cancer cells, Akt2 modulates ER transcriptional activity at multiple levels, including (i) the regulation of ER expression and its nuclear retention and (ii) the activation of one of its downstream targets, the Forkhead transcription factor FoxO3a. FoxO3a colocalizes and coprecipitates with ER in the nucleus, where it binds to Forkhead-responsive sequences on the ER target pS2/TFF-1 promoter; in addition, FoxO3a silencing leads to an increase of ER transcriptional activity, suggesting a repressive role of the Forkhead transcription factor in ER function. Moreover, 17β-estradiol upregulates FoxO3a levels, which could represent the basis for an ER-mediated homeostatic mechanism. These findings provide further evidence of the importance of mediators of the growth factor signaling in ER regulation, introducing the Akt2/FoxO3a axis as a pursuable target in therapy for ER-positive breast cancer.

Expression of nuclear insulin receptor substrate 1 in breast cancer

Background: Insulin receptor substrate 1 (IRS-1), a cytoplasmic protein transmitting signals from the insulin and insulin-like growth factor 1 receptors, has been implicated in breast cancer. Previously, it was reported that IRS-1 can be translocated to the nucleus and modulate oestrogen receptor α (ERα) activity in vitro. However, the expression of nuclear IRS-1 in breast cancer biopsy specimens has never been examined. Aims: To assess whether nuclear IRS-1 is present in breast cancer and non-cancer mammary epithelium, and whether it correlates with other markers, especially ERα. Parallel studies were carried out for the expression of cytoplasmatic IRS-1. Methods: IRS-1 and ERα expression was assessed by immunohistochemical analysis. Data were evaluated using Pearson’s correlation, linear regression and receiver operating characteristic analysis. Results: Median nuclear IRS-1 expression was found to be low in normal mammary epithelial cells (1.6%) and high in benign tumours (20.5%), ductal grade 2 carcinoma (11.0%) and lobular carcinoma (∼30%). Median ERα expression in normal epithelium, benign tumours, ductal cancer grade 2 and 3, and lobular cancer grade 2 and 3 were 10.5, 20.5, 65.0, 0.0, 80 and 15%, respectively. Nuclear IRS-1 and ERα positively correlated in ductal cancer (p<0.001) and benign tumours (p<0.01), but were not associated in lobular cancer and normal mammary epithelium. In ductal carcinoma, both nuclear IRS-1 and ERα negatively correlated with tumour grade, size, mitotic index and lymph node involvement. Cytoplasmic IRS-1 was expressed in all specimens and positively correlated with ERα in ductal cancer. Conclusions: A positive association between nuclear IRS-1 and ERα is a characteristic for ductal breast cancer and marks a more differentiated, non-metastatic phenotype.

Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation

Endometrial cancer exhibits a strong incidence in western developed countries mainly due to fat-rich diet and obesity. Processing of dietary lipids is triggered by bile acids, amphipathic detergents that are synthesized in the liver and stored in the gallbladder. In addition to their well-recognized role in dietary lipid absorption and cholesterol homeostasis, bile acids can also act as signaling molecules with systemic endocrine functions. In the present study we investigated the biological effects of the primary bile chenodeoxycholic acid (CDCA) on a human endometrial cancer cell line, Ishikawa. Low concentrations of CDCA are able to stimulate Ishikawa cell growth by inducing a significant increase in Cyclin D1 protein and mRNA expression through the activation of the membrane G protein-coupled receptor (TGR5)-dependent pathway. Dissecting the molecular mechanism underlying this effect by mutagenesis, EMSA and ChIP analysis revealed that CDCA-induced Cyclin D1 expression requires the enhanced recruitment of the transcription factor CREB on the cyclic AMP-responsive element motif within the Cyclin D1 gene proximal promoter. Our results suggest a novel molecular mechanism explaining the potential contribution of high-fat diet and obesity to endometrial cancer growth and progression opening the rationale for strategies to prevent the risk of this obesity-related cancer in women.