Antonella De Luca

Cripto-1 Activates Nodal- and ALK4-Dependent and -Independent Signaling Pathways in Mammary Epithelial Cells

ABSTRACT Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor β family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs): Simple drugs with a complex mechanism of action?

A range of target‐based agents for the treatment of solid tumors are in development. The epidermal growth factor receptor (EGFR) has been identified as a relevant target as it is involved in regulating several cellular functions important in the proliferation and survival of cancer cells, is commonly expressed at high levels in a range of tumors, and high expression is often related to poor prognosis. EGFR is a member of the ErbB family of receptors which also includes ErbB‐2, ErbB‐3, and ErbB‐4. These receptors form dimers of the same type (homodimers) or with other family members (heterodimers), each combination resulting in different downstream effects. Some of the most advanced targeted agents in development are the EGFR tyrosine kinase inhibitors (EGFR‐TKIs), of which ZD1839 (‘Iressa’) is an example. In Phase II monotherapy trials, oral ZD1839 was well tolerated and demonstrated clinically meaningful antitumor activity and symptom relief in pretreated patients with advanced NSCLC. Preclinical studies have suggested that the antitumor activity of ZD1839 does not depend on the level of EGFR expression. Furthermore, in addition to an effect on EGFR signaling, treatment with ZD1839 as well as with other quinazoline EGFR‐TKIs, may also affect signaling of other ErbB family members. EGFR‐TKIs have been shown in preclinical studies to increase the efficacy of cytotoxic drugs and Phase III trials of such combinations are ongoing. On the basis that different signal transduction pathways contribute to the control of tumor growth, future therapeutic approaches are likely to involve combination of different targeted agents.

Simultaneous blockade of different EGF-like growth factors results in efficient growth inhibition of human colon carcinoma xenografts

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor α (TGFα), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFα, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFα, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFα, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.

EGF‐related peptides are involved in the proliferation and survival of MDA‐MB‐468 human breast carcinoma cells

A majority of human breast carcinomas co‐express the epidermal growth factor (EGF)‐like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor α (TGF‐α). MDA‐MB‐468 breast carcinoma cells express CR, AR and TGFα, while SK‐BR‐3 cells express CR and TGF‐α. Anti‐sense phosphorothioate oligodeoxynucleotides (AS S‐oligos) directed against either CR or TGF‐α inhibit the proliferation of both cell lines. A 40–50% growth inhibition was observed at a 2‐μM concentration of each AS S‐oligo. Treatment with the AR AS S‐oligo also resulted in a significant inhibition of MDA‐MB‐468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA‐MB‐468 or SK‐BR‐3 cells were treated with a mis‐sense S‐oligo. The AS S‐oligos inhibited the expression of AR, CR or TGF‐α proteins and mRNAs, as assessed by immuno‐cytochemistry and semi‐quantitative RT‐PCR. An additive growth‐inhibitory effect was observed when MDA‐MB‐468 cells were treated with a combination of EGF‐related AS S‐oligos. Indeed, treatment of MDA‐MB‐468 cells with a combination of AR, CR and TGF‐α AS S‐oligos resulted in about 70% growth inhibition at a concentration of 0.7 μM each. Finally, treatment of MDA‐MB‐468 cells with a combination either of the 3 AS S‐oligos or of an EGF receptor‐blocking antibody (MAb 225) and either CR, AR or TGFα AS S‐oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF‐related peptides are involved in the proliferation and survival of breast carcinoma cells. Int. J. Cancer 80:589–594, 1999.

Anti-sense oligonucleotides directed against EGF-related growth factors enhance anti-proliferative effect of conventional anti-tumor drugs in human colon-cancer cells

We have demonstrated that anti‐sense phosphorothioate oligodeoxynucleotides (AS S‐oligos) directed against the EGF‐like growth factors CRIPTO (CR), amphiregulin (AR) or transforming‐growth‐factor‐α(TGFα) mRNA, are equipotent in their ability to inhibit the growth of human colon‐carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S‐oligos and conventional anti‐tumor drugs, such as 5‐fluorouracil (5‐FU), adriamycin (ADR), mitomycin C (MIT) and cis‐platinum (CDDP), on GEO cell growth. Dose‐dependent growth inhibition was observed by treatment either with AS S‐oligos or with anti‐tumor drugs, using a clonogenic assay. Furthermore, an additive growth‐inhibitory effect occurred when GEO cells were exposed to the AS S‐oligos after treatment with different concentrations of either 5‐FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5‐FU and any of the 3 AS S‐oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S‐oligos before exposure to 5‐FU or CDDP resulted in reduced efficacy of both drugs. Flow‐cytometric analysis of DNA content demonstrated that treatment with the AS S‐oligos caused a slight reduction of the percentage of cells in the S‐phase of the cell cycle. These data suggest that combinations of AS S‐oligos directed against EGF‐related growth factors and of conventional anti‐tumor drugs may result in efficient inhibition of colon‐carcinoma cell growth. Int. J. Cancer 73:277–282, 1997.

EGFR mutations in lung cancer: from tissue testing to liquid biopsy

ABSTRACT  The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.

Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

A live biopsy in a small-cell lung cancer patient by detection of circulating tumor cells

A 71-year-old patient with a pulmonary lesion was diagnosed with a low-grade neuroendocrine tumor following examination of a fine needle aspiration biopsy. Analysis of a peripheral blood sample with the CellSearch system revealed the presence of putative circulating tumor cells (CTC) that were positive for EpCAM and cytokeratin (CK) expression. Since EpCAM is not usually expressed in neuroendocrine tumors, we performed a biopsy of liver metastases. Morphological and immunophenotypical characterization revealed that the patient had an EpCAM and CK positive small-cell lung cancer (SCLC). By using the CellSearch apparatus, EpCAM/CK positive CTC were detected in peripheral blood samples from 3 out of 4 additional SCLC patients. This study is the first to demonstrate that CTC can be identified in SCLC patients by using the CellSearch system.

Detection and localization of Cripto-1 binding in mouse mammary epithelial cells and in the mouse mammary gland using an immunoglobulin–cripto-1 fusion protein†

Human Cripto‐1 (CR‐1), a member of the epidermal growth factor‐CFC (EGF‐CFC) family of peptides, is expressed in the developing mouse mammary gland and can modulate mammary epithelial cell migration, branching morphogenesis and milk protein expression in vitro. In order to screen for a CR‐1 receptor and to identify potential CR‐1 target tissues, we constructed a fusion protein comprising the EGF‐like domain of CR‐1 and the Fc domain of a human IgG1. The recombinant CR‐1 fusion protein (CR‐1‐Fc) was biologically active as it was able to activate the ras/raf/mitogen activated protein kinase (MAPK) pathway and to inhibit transcription of the milk protein β‐casein in NMuMG and HC‐11 mouse mammary epithelial cells. By using immunocytochemistry and by an in situ enzyme‐linked immunosorbent assay (ELISA), CR‐1‐Fc was found to specifically bind to NMuMG and HC‐11 cells. Finally, immunohistochemical analysis using CR‐1‐Fc showed a specific localization of CR‐1 binding to tissue sections from mouse mammary gland. In particular, more than 60% of the epithelial cells were intensely stained with the CR‐1‐Fc fusion protein in the lactating mouse mammary gland, whereas approximately 25% of the mammary epithelial cells were stained in the gland from pregnant mouse. Since expression of mouse cripto‐1 (Cr‐1) in the pregnant and lactating mouse mammary gland as well as its presence in milk has been previously demonstrated, these data strongly suggest that an autocrine pathway involving Cr‐1 and its putative receptor is operating in the mouse mammary gland during pregnancy and lactation. J. Cell. Physiol. 190: 74–82, 2002. Published 2002 Wiley‐Liss, Inc.