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 CD133/1expression 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 from1.3% 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 GalNAca1-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 paclitaxelinduced 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.

We reported previously that Bcl-2 is paradoxically downregulated in paclitaxel-resistant cancer cells. We reveal here that paclitaxel directly targets Bcl-2 in the loop domain, thereby facilitating the initiation of apoptosis. Molecular modeling revealed an extraordinary similarity between the paclitaxel binding sites in Bcl-2 and B-tubulin, leading us to speculate that paclitaxel could be mimetic of an endogenous peptide ligand, which binds both proteins. We tested the hypothesis that paclitaxel mimics Nur77, which, like paclitaxel, changes the function of Bcl-2. This premise was confirmed by Nur77 interacting with both paclitaxel targets (Bcl-2 and B-tubulin) and a peptide sequence mimicking the Nur77 structural region, thus reproducing the paclitaxel-like effects of tubulin polymerization and opening the permeability transition pore channel in mitochondria. This discovery could help in the development of novel anticancer agents with nontaxane skeleton as well as in identifying the clinical subsets responsive to paclitaxel-based therapy.

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Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of ⌬ m after stimulation of the respiratory state. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.Taxanes are natural products derived from trees of the genus Taxoidaceae. The first taxane introduced in cancer therapy was paclitaxel, firstly isolated from Taxus brevifolia (Schiff et al., 1979). The clinical success of taxanes is dependent on the excellent response rate in second-line treatment of relapsing/resistant cancers and on the efficacy of taxanes in the multichemotherapeutic approach of ovarian and breast cancer (Verweij et al., 1994).In earlier studies, the microtubule network appeared as the main target of paclitaxel (Schiff et al., 1979;Manfredi et al., 1982). In fact, taxanes bind to ␤-tubulin subunits, thereby disrupting normal turnover of the microtubules. The final consequence is the arrest of the cell cycle in M phase with formation of aberrant mitosis and the activation of cell death pathways (Jordan et al., 1993). Along with arrest in M phase of the cell cycle, taxanes have also been reported to induce post-translational serine phosphorylation of the Bcl-2 protein (Haldar et al., 1995). The BCL2 gene is the homologous of the nematode CED-9 gene product (Hengartner and Horvitz, 1994) and is capable of prolonging cell survival by inhibiting apoptotic cell death. Overexpression of Bcl-2 has been observed in follicular lymphoma, where this protein is deregulated by chromosomal translocation, and in a large number of human tumors, including breast, lung, and prostate cancer.Disagreement exists on the levels of Bcl-2 and resistance to taxanes. A strong suggestion for a direct role of Bcl-2 in mediating paclitax...

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This study was aimed at determining whether high-grade endometrioid carcinomas (grade 3 International Federation of Gynecology and Obstetrics) might overlap, at least partially, non-endometrioid carcinomas (type II). To this end, a panel of clinical-pathological and immunohistochemical parameters was evaluated in three different populations: low-grade endometrioid carcinomas (LGECs; n = 57), high-grade endometrioid carcinomas (HGECs; n = 26), and non-endometrioid carcinomas (NECs; n = 30). Besides morphological appearance, HGECs appeared similar to LGECs in p53 immunostaining profile; features different from LGECs included a higher local aggressiveness, a higher invasion of lymph-vascular spaces, a lower expression of ERalpha and PR, and a higher proliferative index. HGECs were similar to NECs for local aggressiveness, invasion rate of lymph-vascular spaces, lymph node metastasis incidence, and proliferative index. HGECs, however, showed a lower rate of extra-nodal metastases, a lower incidence of p53 overexpression, and a higher positivity for ERalpha and PR. In conclusion, results from this study show that HGECs exhibit overlapping morphological and immunohistochemical features of both type I and type II endometrial carcinomas. Further research is needed to clarify the clinical value of this observation.

A prominent mechanism of drug resistance to taxanes is the overexpression of class III B-tubulin. The seco-taxane IDN5390 was chosen for its selective activity in paclitaxel-resistant cells with an overexpression of class III B-tubulin. Moreover, the combined treatment paclitaxel/IDN5390 yielded a strong synergism, which was also evident in cell-free tubulin polymerization assays. In the presence of an anti-class III Btubulin as a blocking antibody, tubulin polymerization induced by paclitaxel and IDN5390 was enhanced and not affected, respectively, whereas synergism was abolished, thereby indicating that IDN5390 activity is not modulated by class III B-tubulin levels. Such properties can be explained by taking into consideration the composition of class III Btubulin paclitaxel binding site; in fact, Ser 277 interacting with paclitaxel C group in class I is replaced by an Arginine in class III. IDN5390 that has an open and flexible C ring and an acidic A-unsaturated enol-keton moiety better fits with class III Btubulin than paclitaxel at the binding site. Taken altogether, these findings indicate that the concomitant treatment IDN5390/paclitaxel is able to successfully target class I and III B-tubulin and the combined use of two taxanes with diverse spectrum activity against tubulin isotypes could represent a novel approach to overcome paclitaxel resistance. (Cancer Res 2005; 65(6): 2397-405)

Patupilone is an epothilone in advanced clinical development that has shown promising efficacy in heavily pretreated patients. This study aimed at characterizing the mechanisms of patupilone activity in resistant patients. To this end, we generated patupilone-resistant cells using two cellular models, the first characterized by high chemosensitivity and low class III B-tubulin (TUBB3) expression (A2780), and the second by low chemosensitivity and high TUBB3 expression (OVCAR-3). The obtained cell lines were named EPO3 and OVCAR-EPO, respectively. The same selection procedure was done in A2780 cells to generate a paclitaxel-resistant cell line (TAX50). Factors of resistance are expected to increase in the drugresistant cell lines, whereas factors of drug sensitivity will be down-regulated. Using this approach, we found up-regulation of TUBB3 in TAX50, but not EPO3, cells, showing that TUBB3 mediates the resistance to paclitaxel but not to patupilone. Moreover, TUBB3 was a factor of patupilone sensitivity because OVCAR-EPO cells exhibited a dramatic reduction of TUBB3 and a concomitant sensitization to hypoxia and cisplatin-based chemotherapy. To identify the mechanisms underlying patupilone resistance, tubulin genes were sequenced, thereby revealing that a prominent mechanism of drug resistance is represented by point mutations in class I B-tubulin. Overall, these results suggest that paclitaxel and patupilone have nonoverlapping mechanisms of resistance, thus allowing the use of patupilone for those patients relapsing after paclitaxel-based chemotherapy. Furthermore, patupilone represents a promising first-line option for the treatment of high-risk ovarian cancer patients, who exhibit high TUBB3 levels and poor response to standard paclitaxelplatin chemotherapy.