Laura Gatti

Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133− counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.

p53 Gene Status and Response to Platinum/Paclitaxel-Based Chemotherapy in Advanced Ovarian Carcinoma

PURPOSE The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P: =.008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.

Overview of Tumor Cell Chemoresistance Mechanisms

Drug resistance of tumor cells is recognized as the primary cause of failure of chemotherapeutic treatment of most human tumors. Although pharmacological factors including inadequate drug concentration at the tumor site can contribute to clinical resistance, cellular factors play a major role in chemoresistance of several tumors. Although manifestations of resistance are conventionally referred to as acquired or intrinsic on the basis of the initial response to the first therapy, a common feature is the development of a phenotype resistant to a variety of structurally and functionally distinct agents. In both manifestations, drug resistance is a multifactorial phenomenon involving multiple interrelated or independent mechanisms. A heterogeneous expression of involved mechanisms may characterize tumors of the same type or cells of the same tumor and may at least in part reflect tumor progression. The relevant mechanisms that can contribute to cellular resistance include: increased expression of defense factors involved in reducing intracellular drug concentration; alterations in drug-target interaction; and changes in cellular response, in particular increased cell ability to repair DNA damage or tolerate stress conditions, and defects in apoptotic pathways. This chapter presents an overview of the drug resistance mechanisms.

Role of apoptosis and apoptosis-related genes in cellular response and antitumor efficacy of anthracyclines.

Cellular resistance to anthracyclines is a major limitation of their clinical use in the treatment of human tumors. Resistance to doxorubicin is described as a multifactorial phenomenon involving the overexpression of defense factors and alterations in drug-target interactions. Such changes do not account for all manifestations of drug resistance, in particular intrinsic resistance of solid tumors. Since anthracyclines can induce apoptotic cell death, an alternative promising approach to drug resistance has focused on the study of cellular response to drug-induced DNA damage, with particular reference to the relationship between cytotoxicity/antitumor efficacy and apoptotic response. The evidence that a novel disaccharide analog (MEN 10755), endowed with an improved preclinical activity over doxorubicin, was also more effective as an inducer of apoptosis provided additional insights to better understand the cellular processes that confer sensitivity to anthracyclines. Although the presence or alteration of a single apoptosis-related factor (e.g., p53, bcl-2) is not predictive of the sensitivity/resistance status, the complex interplay among DNA damage-activated pathways is likely an important determinant of tumor cell sensitivity to anthracyclines

The cellular basis of the efficacy of the trinuclear platinum complex BBR 3464 against cisplatin-resistant cells

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional mononuclear platinum-based drugs. In this study we performed a comparative study of cisplatin and of the triplatinum complex BBR 3464 in a human osteosarcoma cell system (U2-OS) including an in vitro selected cisplatin-resistant subline (U2-OS/Pt). BBR 3464 was extremely potent in comparison with cisplatin in U2-OS cells and completely overcame resistance of U2-OS/Pt cells. In both cell lines, BBR 3464 accumulation and DNA-bound platinum were higher than those observed for cisplatin. On the contrary, a low frequency of interstrand cross-links after exposure to BBR 3464 was found. Differently from the increase of DNA lesions induced by cisplatin, kinetics studies indicated a low persistence of interstrand cross-link formation for BBR 3464. Western blot analysis of DNA mismatch repair proteins revealed a marked decrease of expression of PMS2 in U2-OS/Pt cells, which also exhibited microsatellite instability. Studies on DNA mismatch repair deficient and proficient colon carcinoma cells were consistent with a lack of influence of the DNA mismatch repair status on BBR 3464 cytotoxicity. In conclusion, the cytotoxic potency and the ability of the triplatinum complex to overcome cisplatin resistance appear to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared to conventional mononuclear complexes.

Increased levels and defective glycosylation of MRPs in ovarian carcinoma cells resistant to oxaliplatin

Pt compounds still represent the mainstay of the treatment of ovarian carcinoma. The aim of the present study was to investigate the molecular bases of resistance to Pt drugs using an oxaliplatin-resistant ovarian carcinoma cell model IGROV-1/OHP. These cells exhibited high levels of resistance to oxaliplatin, cross-resistance to cisplatin and topotecan and displayed a marked accumulation defect of Pt drugs. This feature was associated with increased expression and altered N-linked glycosylation of ATP binding cassette transporters MRP1 and MRP4. Pre-treatment with tunicamycin, which inhibits the biosynthesis of N-linked oligosaccharides, decreased the accumulation of Pt in sensitive cells exposed to oxaliplatin or cisplatin and increased the electrophoretic mobility of MRP1 and MRP4, reproducing the association between decreased glycosylation of MRP1 and MRP4 and decreased Pt accumulation observed in the resistant IGROV-1/OHP cells. The observed N-glycosylation defect of oxaliplatin-resistant cells was linked to reduced levels of N-acetylglucosamine-1-phosphotransferase (GNPTG) and mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase (MGAT5). This feature, observed in IGROV-1/OHP cells, was associated with decreased retention of Pt drugs. In addition, the overexpression of fully glycosylated MRP1 or MRP4 in tumor cell line of ovarian origin was associated with resistance to oxaliplatin and cisplatin. Our findings, showing that development of resistance to oxaliplatin results in up-regulation of MRPs, support that patients with oxaliplatin-refractory ovarian carcinomas may benefit from non-Pt-based regimens which do not contain MRP1 and MRP4 substrates.

Targeting the Akt Kinase to Modulate Survival, Invasiveness and Drug Resistance of Cancer Cells

The deregulation of oncogenic signaling pathways which provide survival advantages to tumor cells is mediated by multiple cellular networks. Among them, the PI3K-Akt-mTOR axis, in particular the serine/threonine kinase Akt, is recognized as a key player. The kinase is hyperactivated due to a variety of mechanisms including loss of PTEN, mutations in the PI3K catalytic subunit, receptor tyrosine kinase and Ras activation. Indeed, inappropriate activation of the Akt kinase is a common event in human tumors and Akt appears to be a critical player in cell survival that may also account for the therapeutic resistance and the invasive phenotype of tumors. Inhibition of Akt signalling results in apoptosis and growth inhibition of tumour cells with elevated Akt activity. A functional role in drug resistance is supported by evidence that tumor cells with acquired resistance to antitumor agents may display increased Akt activation and that treatment with molecularly targeted agents can activate feed-back loops involving Akt. This serine/threonine kinase may therefore represent an amenable target for modulation of sensitivity to compounds with different molecular features due to its pleiotropic role in cell survival. Different types of Akt inhibitors [i.e., ATP mimetics and pleckstrin-homology (PH) domain binders] have been generated and some of them have reached the clinical setting. The present review focuses on the i) mechanisms implicating Akt in increased survival and invasive potential of tumor cells of different tumor types and ii) on the development of Akt inhibitors as modulators of drug resistance.

Strategies to Improve the Efficacy of Platinum Compounds

Platinum drugs are widely used in antitumour therapy and are a cornerstone of the treatment of different solid tumours. The pharmacological interest of cisplatin has led to the design of many analogues to broaden the spectrum of activity, reduce side effects, and overcome resistance. Although the cis configuration was initially identified as the only active one, trans-platinum complexes have shown significant antitumour activity in preclinical models. In addition to mononuclear platinum compounds, multinuclear platinum complexes have been generated that are characterised by a different mode of interaction with DNA. Since a major limitation to the clinical efficacy of platinum compounds is drug resistance, the most important feature of nonconventional platinum drugs should be the capability of overcoming cellular resistance. However, due to the multifactorial nature of clinical resistance, which also involves pharmacological factors, the optimisation of current platinum-based therapy also includes the development of drug delivery approaches. The present review focuses on recent studies on the molecular alterations of tumour cells that are associated with resistance to platinum drugs, the development of novel platinum drugs, and approaches that may contribute to improve the efficacy of platinum-based therapy.

Development of resistance to a trinuclear platinum complex in ovarian carcinoma cells

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin‐resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross‐resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21WAF1 levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21WAF1 only in parental cells. An inverse relationship with p21WAF1 modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21WAF1 at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464.

ABC Transporters as Potential Targets for Modulation of Drug Resistance

ATP binding cassette transporters are implicated in multidrug resistant phenotypes of tumor cells and may be cancer stem cell markers. Inhibitors of drug efflux pumps represent an emerging group of potentially useful agents for the improvement of antitumor therapy. Here we provide an overview of drug transporter functions and modulation.