Roberto E. Favoni

Different Response of Human Glioma Tumor-initiating Cells to Epidermal Growth Factor Receptor Kinase Inhibitors

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

17β-Estradiol Promotes Breast Cancer Cell Proliferation-Inducing Stromal Cell-Derived Factor-1-Mediated Epidermal Growth Factor Receptor Transactivation: Reversal by Gefitinib Pretreatment

The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17β-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1′-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

Imatinib Mesylate Enhances Therapeutic Effects of Gemcitabine in Human Malignant Mesothelioma Xenografts

Purpose: Platelet-derived growth factor receptor β (PDGFRβ), frequently activated in malignant mesothelioma, is a promising cancer therapeutic target. Imatinib mesylate (STI571; Glivec) is a selective inhibitor of tyrosine kinases as bcr-abl, c-kit, c-fms, and PDGFRβ and enhances tumor drug uptake by reducing the interstitial fluid pressure. We previously showed that imatinib mesylate synergizes with gemcitabine and pemetrexed in PDGFRβ-positive mesothelioma cells. Here, we aimed at investigating these combined treatments in a novel mesothelioma model. Experimental Design: REN mesothelioma cells, infected with a lentiviral vector carrying the luciferase gene, were injected in the peritoneum of severe combined immunodeficient mice. This model allowed imaging of live animals treated with pemetrexed or gemcitabine chemotherapeutics, or with imatinib mesylate alone, as well as with a combination of gemcitabine and imatinib mesylate. Results: We show here that, consistent with our previous in vitro studies, gemcitabine inhibited tumor growth, whereas pemetrexed was ineffective, even at the highest dosage tested. Compared with monotreatment, the combination of gemcitabine with imatinib mesylate led to a further tumor growth inhibition and improved mice survival, by a decrease rate of tumor cell proliferation and an increase in number of apoptotic tumor cells. Conclusions: Imatinib mesylate enhances the therapeutic response to gemcitabine, in accordance with our previous in vitro data. These in vivo results validate imatinib mesylate and gemcitabine as a combination treatment of malignant mesothelioma, also in view of its known positive effects on tumor drug uptake. These evidences provide the rationale for the currently ongoing clinical trials.

Steroidal and nonsteroidal oestrogen antagonists in breast cancer: basic and clinical appraisal

Endogenous sex hormones, such as oestrogens, control the development and growth of breast carcinoma. Blocking the binding between the steroid hormone and its receptor using anti-oestrogenic compounds is one of the possible ways of impeding the action of oestrogens at the level of their target tissues. The nonsteroidal, triphenylethylenic, anti-oestrogen tamoxifen has so far been the standard compound for endocrine treatment of breast cancer. Nevertheless, it forms a complex with the oestrogen receptor which gives rise to a range of biopharmacological events, ranging from full oestrogen antagonism to partial agonism. Moreover, its use is limited by the possible onset of drug resistance in many patients. In spite of these restrictions, tamoxifen has proved to be very helpful as a starting point for the development of new, more efficacious compounds. In this article, Roberto Favoni and Alessandra de Cupis review and discuss tamoxifen, several of its derivatives and the steroidal-like anti-oestrogens in terms of laboratory development, pharmacology and clinical evaluation.

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.

Preliminary data suggestive of a novel translational approach to mesothelioma treatment: imatinib mesylate with gemcitabine or pemetrexed

Background: Malignant mesothelioma is a cancer which is refractory to current treatments. Imatinib mesylate is a selective inhibitor of tyrosine kinases such as bcr-abl, c-Kit, c-Fms and platelet derived growth factor receptor β (PDGFRβ). PDGFRβ is often overexpressed in mesothelioma cells and is a therapeutic target for imatinib in some solid tumours. A study was undertaken to assess whether imatinib alone or combined with chemotherapeutic agents may be effective for treating mesothelioma. Methods: Cultures from mesothelioma MMP, REN and ISTMES2 cell lines were treated with imatinib alone or in combination with a chemotherapeutic agent. Results: Imatinib induced cytotoxicity and apoptosis selectively on PDGFRβ positive mesothelioma cells via blockade of receptor phosphorylation and interference with the Akt pathway. Of the chemotherapeutic agents tested in combination with imatinib, a synergistic effect was obtained with gemcitabine and pemetrexed. Conclusions: This study provides a rationale for a novel translational approach to the treatment of mesothelioma which relies on enhancement of tumour chemosensitivity by inhibition of Akt.

Comparison between novel steroid‐like and conventional nonsteroidal antioestrogens in inhibiting oestradiol‐ and IGF‐I‐induced proliferation of human breast cancer‐derived cells

1 This study has two specific aims: (a) to compare the antioestrogenic activity of two steroidal analogues of 17β‐oestradiol, the 7α‐alkylamide, ICI 164, 384 and the 7α‐alkylsulphinylamide, ICI 182, 780, with that of the triphenylethylene‐derived compound 4OH‐tamoxifen on a pool of human breast cancer cell lines (HBCCL) with a range of hormonal responsiveness and acquired antioestrogen resistance and (b) to investigate the ability of such antioestrogens to modulate the potent breast carcinoma growth‐stimulatory activity of the ‘IGF‐I system’. 2 For the chemosensitivity investigations we used a long‐term colorimetric and the short‐term thymidine incorporation assay; we analysed IGF‐I in conditioned media by a radioimmunoassay, IGF‐I mRNA in the cells by RT‐PCR and molecular species of IGF‐I‐binding proteins, secreted in conditioned media, by Western ligand blot. IGF‐I receptors were assayed on cell monolayers by binding studies and by Scatchard analysis, we calculated KD, Bmax and sites/cell. 3 Our results indicate that ICI 182, 780 and ICI 164, 384 are 1.5‐5.5 fold more potent than 40H‐tamoxifen in inhibiting the basal proliferation of oestrogen‐receptor positive (ER +) breast cancer cell lines. Moreover we demonstrate the capacity of ICI 182, 780 and ICI 164, 384 to reduce, in a time‐dependent fashion, oestrogen‐ and/or IGF‐I‐stimulated growth of ER + cell lines, possibly by negatively interfering with an IGF‐I‐like material secretion and IGF‐I‐receptor number. 4 Our data provide the first evidence that, on ER+ human breast carcinoma cell lines, steroidal antioestrogens inhibit cell growth and modulate the IGF‐I mitogenic system. The mechanism of this latter effect has yet to be identified.

Oestrogen/growth factor cross-talk in breast carcinoma: a specific target for novel antioestrogens

Breast cancer cells express receptors for and are sensitive to a variety of steroids, polypeptide hormones and growth factors; the blocking of and/or the interference with their biochemical pathways could represent a new approach to breast tumour therapy. Antioestrogens achieve such a goal by competing with oestradiol for binding to the oestrogen receptors through which intracellular effects of the hormone are mediated. Tamoxifen has undergone the most extensive clinical evaluations and represents the treatment of choice for the endocrine management of breast cancer. Nevertheless, it is well known that tamoxifen retains agonist activity both in vitro and in vivo. To circumvent this disadvantage, new molecules with steroid-like structure, represented by ICI 164,384 and ICI 182,780, have been synthesized. In this review, Alessandra de Cupis and Roberto Favoni review data about the cross-talk between the two major families of breast cancer growth regulator: oestrogens and growth factors, focusing on the use of nonsteroidal antioestrogens and the new generation of steroidal antioestrogens as possible specifically targeted inhibitors of breast tumour proliferation.

Interaction between novel anticancer agents and radiation in non-small cell lung cancer cell lines.

Integration of chemotherapy and radiation is the standard practice in the management of locally advanced inoperable NSCLC. To assess the biological interaction between third generation chemotherapeutic agents and radiation in non-small cell lung cancer (NSCLC) in vitro, we tested a number of different drugs (paclitaxel, docetaxel, gemcitabine, topotecan, SN-38 and cisplatin) combined with radiation, in lung cancer cell lines. Cellular chemosensitivity was determined, using the semi-automated colorimetric MTT assay, after 48, 72 and 96 h of exposure to increasing drug concentrations, (0.001-100 microM) and radiation doses (100-400 cGy). Cell lines used were the adenocarcinoma (ADK), A-549, and the squamous-cell carcinoma (SCC), LX-1. Cells were pre-treated with anticancer agents at 24, 12 and 0 h before irradiation. Cytofluorimetric cell cycle analysis was performed. A significant S-phase block or a G(2)/M block was seen with gemcitabine and topotecan or paclitaxel pre-treatment, respectively. Apoptosis was seen only after paclitaxel exposure in the A-549 cell line. Despite a similar pattern of cell-kinetic changes induced by chemotherapy pre-treatment in all cell lines, the adenocarcinoma A-549 cell line was not radiosensitized by any of the anticancer agents tested, whereas synergism was observed in the LX-1 squamous carcinoma cell line, when exposed to gemcitabine, SN-38, topotecan and cisplatin. Paclitaxel, despite a favourable cell cycle effect, was not found to be synergistic with radiotherapy in our experimental model. In conclusion, the observed synergism appears to be dose- and timing-independent and seems to be related to the histological subtype being present in SCC only. Favourable perturbation of the cell cycle is evident with all the new agents tested in both cell types, but was not sufficient to produce synergism with radiation.

Preclinical studies identify novel targeted pharmacological strategies for treatment of human malignant pleural mesothelioma

The incidence of human malignant pleural mesothelioma (hMPM) is still increasing worldwide. hMPM prognosis is poor even if the median survival time has been slightly improved after the introduction of the up‐to‐date chemotherapy. Nevertheless, large phase II/III trials support the combination of platinum derivatives and pemetrexed or raltitrexed, as preferred first‐line schedule. Better understanding of the molecular machinery of hMPM will lead to the design and synthesis of novel compounds targeted against pathways identified as crucial for hMPM cell proliferation and spreading. Among them, several receptors tyrosine kinase show altered activity in subsets of hMPM. This observation suggests that these kinases might represent novel therapeutic targets in this chemotherapy‐resistant disease. Over these foundations, several promising studies are ongoing at preclinical level and novel molecules are currently under evaluation as well. Yet, established tumour cell lines, used for decades to investigate the efficacy of anticancer agents, although still the main source of drug efficacy studies, after long‐term cultures tend to biologically diverge from the original tumour, limiting the predictive potential of in vivo efficacy. Cancer stem cells (CSCs), a subpopulation of malignant cells capable of self‐renewal and multilineage differentiation, are believed to play an essential role in cancer initiation, growth, metastasization and relapse, being responsible of chemo‐ and radiotherapy refractoriness. According to the current carcinogenesis theory, CSCs represent the tumour‐initiating cell (TIC) fraction, the only clonogenic subpopulation able to originate a tumour mass. Consequently, the recently described isolation of TICs from hMPM, the proposed main pharmacological target for novel antitumoural drugs, may contribute to better dissect the biology and multidrug resistance pathways controlling hMPM growth.