Sandra Cascio

Increased expression of leptin and the leptin receptor as a marker of breast cancer progression : Possible role of obesity-related stimuli

Purpose: Recent in vitro studies suggested that the autocrine leptin loop might contribute to breast cancer development by enhancing cell growth and survival. To evaluate whether the leptin system could become a target in breast cancer therapy, we examined the expression of leptin and its receptor (ObR) in primary and metastatic breast cancer and noncancer mammary epithelium. We also studied whether the expression of leptin/ObR in breast cancer can be induced by obesity-related stimuli, such as elevated levels of insulin, insulin-like growth factor-I (IGF-I), estradiol, or hypoxic conditions. Experimental Design: The expression of leptin and ObR was examined by immunohistochemistry in 148 primary breast cancers and 66 breast cancer metastases as well as in 90 benign mammary lesions. The effects of insulin, IGF-I, estradiol, and hypoxia on leptin and ObR mRNA expression were assessed by reverse transcription-PCR in MCF-7 and MDA-MB-231 breast cancer cell lines. Results: Leptin and ObR were significantly overexpressed in primary and metastatic breast cancer relative to noncancer tissues. In primary tumors, leptin positively correlated with ObR, and both biomarkers were most abundant in G3 tumors. The expression of leptin mRNA was enhanced by insulin and hypoxia in MCF-7 and MDA-MB-231 cells, whereas IGF-I and estradiol stimulated leptin mRNA only in MCF-7 cells. ObR mRNA was induced by insulin, IGF-I, and estradiol in MCF-7 cells and by insulin and hypoxia in MDA-MB-231 cells. Conclusions: Leptin and ObR are overexpressed in breast cancer, possibly due to hypoxia and/or overexposure of cells to insulin, IGF-I, and/or estradiol.

miR-20b modulates VEGF expression by targeting HIF-1α and STAT3 in MCF-7 breast cancer cells†

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR‐20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF‐7 breast cancer cells under normoxic and hypoxia‐mimicking conditions (CoCl2 exposure). Using immunoblotting, ELISA, and quantitative real‐time PCR, we demonstrated that miR‐20b decreased VEGF protein levels at 4 and 24 h following CoCl2 treatment, and VEGF mRNA at 4 h of treatment. In addition, miR‐20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre‐miR‐20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF‐1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR‐20b. Downregulation of VEGF mRNA by miR‐20b under a 4 h of CoCl2 treatment was associated with reduced levels of nuclear HIF‐1α subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF‐1α, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl2 treatment. This effect was inhibited by transfection of cells with pre‐miR‐20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl2‐mediated HIF‐1α nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF‐1 and STAT3 in a miR‐20b‐dependent manner. J. Cell. Physiol. 224:242–249, 2010

Nuclear insulin receptor substrate 1 interacts with estrogen receptor α at ERE promoters

Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptor α (ERα)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-β-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERα; (4) the IRS-1:ERα complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERα-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERα. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERα activity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERα and that this interaction might influence ERα transcriptional activity.

Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1alpha.

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1α (HIF-1α) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1α on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1α in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.

Insulin-Dependent Leptin Expression in Breast Cancer Cells

Pathologic conditions associated with hyperinsulinemia, such as obesity, metabolic syndrome, and diabetes, seem to increase the risk of breast cancer. Here, we studied molecular mechanisms by which insulin activates the expression of leptin, an obesity hormone that has been shown to promote breast cancer progression in an autocrine or paracrine way. Using MDA-MB-231 breast cancer cells, we found that (a) insulin stimulated leptin mRNA and protein expression, which was associated with increased activation of the leptin gene promoter; (b) insulin increased nuclear accumulation of transcription factors hypoxia inducible factor (HIF)-1alpha and Sp1 and their loading on the leptin promoter; (c) small interfering RNA (siRNA)-mediated knockdown of either HIF-1alpha or Sp1 significantly down-regulated insulin-induced leptin mRNA and protein expression; further inhibition of leptin expression was observed under the combined HIF-1alpha and Sp1 siRNA treatment; (d) inhibition of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol-3-OH kinase (PI-3K) pathways significantly, albeit partially, decreased insulin-dependent leptin mRNA and protein expression, which coincided with reduced association of HIF-1alpha and/or Sp1 with specific leptin promoter regions; and (e) inhibition of ERK1/2 reduced recruitment of both HIF-1alpha and Sp1 to the leptin promoter, whereas down-regulation of PI-3K influenced only HIF-1alpha binding. In summary, our data suggest that hyperinsulinemia could induce breast cancer progression through leptin-dependent mechanisms. In MDA-MB-231 cells, this process requires Sp1- and HIF-1alpha-mediated leptin gene transcription and is partially regulated by the PI-3K and ERK1/2 pathways.

Insulin-like growth factor 1 differentially regulates estrogen receptor-dependent transcription at estrogen response element and AP-1 sites in breast cancer cells.

Cross-talk between insulin-like growth factor 1 (IGF-1) and estrogen receptor α (ER) regulates gene expression in breast cancer cells, but the underlying mechanisms remain unclear. Here, we studied how 17-β-estradiol (E2) and IGF-1 affect ER transcriptional machinery in MCF-7 cells. E2 treatment stimulated ER loading on the estrogen response element (ERE) in the pS2 promoter and on the AP-1 motif in the cyclin D1 promoter. On ERE, similar amounts of liganded ER were found at 1–24-h time points, whereas on AP-1, ER binding fluctuated over time. At 1 h, liganded ER was recruited to ERE together with histone acetyltransferases SRC-1 and p300, ubiquitin ligase E6-AP, histone methyltransferase Carm1 (Carm), and polymerase (pol) II. This coincided with increased histone H3 acetylation and up-regulation of pS2 mRNA levels. At the same time, E2 moderately increased cyclin D1 expression, which was associated with the recruitment of liganded ER, SRC-1, p300, ubiquitin ligase E6-AP (E6L), Mdm2, and pol II, but not other regulatory proteins, to AP-1. In contrast, at 1 h, IGF-1 increased the recruitment of the ER·SRC-1·p300·E6L·Mdm2·Carm·pol II complex on AP-1, but not on ERE, and induced cyclin D1, but not pS2, mRNA expression. Notably, ER knockdown reduced the association of ER, E6L, Mdm2, Carm, and pol II with AP-1 and resulted in down-regulation of cyclin D1 expression. IGF-1 potentiated the effects of E2 on ERE but not to AP-1 and increased E2-dependent pS2, but not cyclin D1, mRNA expression. In conclusion, E2 and IGF-1 differentially regulate ER transcription at ERE and AP-1 sites.

MUC1 Protein Expression in Tumor Cells Regulates Transcription of Proinflammatory Cytokines by Forming a Complex with Nuclear Factor-κB p65 and Binding to Cytokine Promoters IMPORTANCE OF EXTRACELLULAR DOMAIN

Background: Human MUC1 is overexpressed and abnormally glycosylated in adenocarcinomas, correlating with inflammation and tumor growth. Results: MUC1 activates NF-κB in tumors and, in complex with NF-κB p65, migrates to the nucleus, where it promotes transcription of proinflammatory cytokines. Conclusion: MUC1 is an important mediator of tumor-induced inflammation. Significance: Targeting the MUC1-p65 complex could reduce inflammation and inhibit tumor growth. MUC1 is a transmembrane glycoprotein abnormally expressed in all stages of development of human adenocarcinomas. Overexpression and hypoglycosylation of MUC1 in cancer cells compared with normal epithelial cells are likely to alter its function and affect the behavior of cancer cells. The extracellular domain, specifically the highly O-glycosylated VNTR (variable number of tandem repeats) region, plays an important role in cell-cell communication; however, we show here that it also participates intracellularly in activation of the NF-κB pathway. Transfection of MUC1− tumor cells with cDNA encoding MUC1 with 22 tandem repeats (MUC1/22TR) or two tandem repeats (MUC1/2TR) or two isoforms that lack the VNTR region (MUC1/Z and MUC1/Y) showed that the highest expression levels of NF-κB family members correlated with the presence of VNTR and the highest number of tandem repeats. Because expression of MUC1 with VNTR on tumors was previously associated with chemotactic activity for cells of the innate immune system, we investigated the influence of MUC1 expression on the NF-κB-dependent transcriptional regulation of proinflammatory cytokines. ChIP and real-time PCR experiments revealed that MUC1/22TR up-regulated IL-6 and TNF-α expression by binding to their promoter regions in a NF-κB p65-dependent manner in both MUC1-transfected and human breast cancer cells that express endogenous MUC1. This newly detected complex of MUC1 and p65 is a novel mechanism that tumors can use to promote inflammation and cancer development.

Expression of Angiogenic Regulators, VEGF and Leptin, Is Regulated by the EGF/PI3K/STAT3 Pathway in Colorectal Cancer Cells

Both leptin and vascular endothelial growth factor (VEGF) are growth and angiogenic cytokines that are upregulated in different types of cancer and have been implicated in neoplastic progression. Here we investigated the molecular mechanism by which leptin and VEGF expression are regulated in colon cancer by epidermal growth factor (EGF). In colon cancer cell line HT‐29, EGF induced the binding of signal transducer and activator transcription 3 (STAT3) to STAT3 consensus motifs within the VEGF and leptin promoters and stimulated leptin and VEGF mRNA and protein synthesis. All these EGF effects were significantly blocked when HT‐29 cells were treated with an inhibitor of the phosphoinositide 3‐kinase (PI3K) pathway, LY294002, or with small interfering RNA (siRNA) targeting STAT3. Thus, our study identified the EGF/PI3K/STAT3 signaling as an essential pathway regulating VEGF and leptin expression in EGF‐responsive colon cancer cells. This suggests that STAT3 pathways might constitute attractive pharmaceutical targets in colon cancer patients where anti‐EGF receptor drugs are ineffective. J. Cell. Physiol. 221: 189–194, 2009.

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.

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas.

The putative role of TP53 and p16INK4A tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex‐PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific‐PCR (MS‐PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H‐Ras and K‐Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K‐Ras. p16INK4A promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16INK4A promoter methylation, but not alterations in the H‐Ras and K‐Ras genes, might be involved in the malignant progression of PA into carcinoma.