Adriana Eramo

Identification and expansion of the tumorigenic lung cancer stem cell population.

Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133+ cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 104 lung cancer CD133+ cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133+ cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133+ cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.

Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1.

The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.

Cells with Characteristics of Cancer Stem/Progenitor Cells Express the CD133 Antigen in Human Endometrial Tumors

Purpose: Cancer stem cells represent an attractive therapeutic target for tumor eradication. The present study aimed to determine whether CD133 expression may identify cells with characteristics of cancer stem/progenitor cells in human endometrial tumors. Experimental Design: We analyzed 113 tumor samples for CD133/1 expression by flow cytometry, immunohistochemistry, and semiquantitative reverse transcription–PCR. CD133+ cells were isolated and used to assess phenotypic characteristics, self-renewal capacity, ability to maintain CD133 expression and form sphere-like structures in long-term cultures, sensitivity to chemotherapeutic agents, gene expression profile, and ability to initiate tumors in NOD/SCID mice. Results: Primary tumor samples exhibited a variable degree of immunoreactivity for CD133/1, ranging from 1.3% to 62.6%, but stained negatively for other endothelial and stem cell–associated markers. Isolated CD133+ cells expanded up to 4.6-fold in serum-replenished cultures and coexpressed the GalNAcα1-O-Ser/Thr MUC-1 glycoform, a well-characterized tumor-associated antigen. Dissociated bulk tumors formed sphere-like structures; cells grown as tumor spheres maintained CD133 expression and could be propagated for up to 12 weeks. CD133+ cells purified from endometrioid adenocarcinomas were resistant to cisplatin-induced and paclitaxel-induced cytotoxicity and expressed a peculiar gene signature consisting of high levels of matrix metalloproteases, interleukin-8, CD44, and CXCR4. When serially transplanted into NOD/SCID mice, CD133+ cells were capable of initiating tumor formation and recapitulating the phenotype of the original tumor. Conclusions: CD133 is expressed by human endometrial cancers and might represent a valuable tool to identify cells with cancer stem cell characteristics.

Caspase activation without death.

Since molecular cloning of the C. elegans ced-3 gene revealed its homology with mammalian IL-1β-converting enzyme,* 14 members of the caspase family have been identified, which have often been involved as mediators of one or more phases of the apoptotic process., However, an over-simplified role of these proteases may be insufficient to explain the usually constitutive expression of such a large and complex family of enzymes, many of which display overlapping specificity. In addition to the well-established role of caspase-1 in the production of active IL-1β and IL-18 in inflammation,* an increasing number of reports has recently suggested that caspases may have a function outside of apoptosis. In this review, the situations in which cells survive despite the presence of activated caspases in their cytoplasm will be examined and discussed, with the intent to gather all recent advances in this new field that promises to be a focus for caspase research in the near future.

CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase‐8. We found that CD95 was preferentially located inlipid rafts of type I cells, while it was present both in raft and non‐raft plasma membrane sub‐domains of type II cells. After stimulation, CD95 located in phospholipid‐rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross‐linking resulted in caspase‐independent translocation of FADD/MORT1 and caspase‐8 to the lipid rafts, which was prevented by a death domain‐defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas‐associated death domain (FADD)and caspase‐8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase‐8 underwent massive processing. Taken together, our data demonstrate that CD95 death‐inducing signaling complex formation and internalization in type I and type II cells occur in lipidrafts, which are a major site of caspase‐8 activation.

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Lung cancer stem cells: tools and targets to fight lung cancer

Cancer stem cell (CSC) theory states that tumors are organized in a similar hierarchical manner as normal tissues, with a sub-population of tumorigenic stem-like cells that generate the more differentiated nontumorigenic tumor cells. CSCs are chemoresistant and seem to be responsible for tumor recurrence and formation of metastases. Therefore, the study of these cells may lead to crucial advances in the understanding of tumor biology as well as to innovative and more effective therapies. Lung cancer represents the leading cause of cancer-related mortality worldwide. Despite improvements in medical and surgical management, patient survival rates remain stable at ∼15%, calling for innovative strategies that may contribute to improve patient outcome. The discovery of lung CSCs and the possibility to characterize their biological properties may provide powerful translational tools to improve the clinical outcome of patients with lung cancer. In this report, we review what is known about lung CSCs and discuss the diagnostic, prognostic and therapeutic prospective of these findings.

Inhibition of DNA Methylation Sensitizes Glioblastoma for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Destruction

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments.

Pro-inflammatory gene expression in solid glioblastoma microenvironment and in hypoxic stem cells from human glioblastoma

BackgroundAdaptation to hypoxia and consequent pro-inflammatory gene expression of prostate and breast carcinomas have been implicated in the progression toward cancer malignant phenotype. Only partial data are available for the human tumor glioblastoma multiforme (GBM). The aim of our study was to analyze the hypoxic and pro-inflammatory microenvironment in GBMs and to demonstrate that in a stem/progenitor cell line derived from human glioblastoma (GBM-SCs), hypoxia activates a coordinated inflammatory response, evidencing an invasive and migratory phenotype.MethodsFrom each of 10 human solid glioblastomas, clinically and histopathologically characterized, we obtained three surgical samples taken from the center and the periphery of the tumor, and from adjacent host normal tissue. Molecular and morphological analyses were carried out using quantitative real-time PCR and western blot (WB). GBM stem and differentiated cells were incubated under hypoxic conditions and analyzed for pro-inflammatory gene expression and for invasive/migratory behavior.ResultsA panel of selected representative pro-inflammatory genes (RAGE and P2X7R, COX2, NOS2 and, PTX3) were analyzed, comparing tumor, peritumor and host normal tissues. Tumors containing leukocyte infiltrates (as assessed using CD45 immunohistochemistry) were excluded. Selected genes were overexpressed in the central regions of the tumors (i.e. in the more hypoxic areas), less expressed in peripheral regions, and poorly expressed or absent in adjacent normal host tissues. Western blot analysis confirmed that the corresponding pro-inflammatory proteins were also differently expressed. Hypoxic stem cell lines showed a clear time-dependent activation of the entire panel of pro-inflammatory genes as compared to differentiated tumor cells. Biological assays showed that invasive and migratory behavior was strengthened by hypoxia only in GBM stem cells.ConclusionsIn human solid glioblastoma we have observed a coordinated overexpression of a panel of pro-inflammatory genes as compared to host normal tissue. We have also evidenced a similar pattern of overexpressed genes in GBM-SCs after hypoxic treatment, showing also a gain of invasive and migratory function that was lost when these stem cells differentiated. We suggest that, as has been previously described for prostatic and mammary carcinoma, in human glioblastoma acquisition of a proinflammatory phenotype may be relevant for malignant progression.

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-XL is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-XL inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-XL. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-XL for their survival and indicate Bcl-XL inhibition as a potential therapeutic avenue in NSCLC.