Stéphane Honoré

Safety and Efficacy of Temozolomide in Patients With Recurrent Anaplastic Oligodendrogliomas After Standard Radiotherapy and Chemotherapy

PURPOSE Most primary oligodendrogliomas and mixed gliomas (oligoastrocytoma) respond to treatment with procarbazine, lomustine, and vincristine (PCV), with response rates of approximately 80%. However, limited data on second-line treatments are available in patients with recurrent tumors. A novel second-generation alkylating agent, temozolomide, has recently demonstrated efficacy and safety in patients with recurrent glioblastoma multiforme and anaplastic astrocytoma. This study describes the effects of temozolomide in patients with recurrent anaplastic oligodendroglioma (AO) and anaplastic mixed oligoastrocytoma (AOA). PATIENTS AND METHODS Forty-eight patients with histologically confirmed AO or AOA who had received previous PCV chemotherapy were treated with temozolomide (150 to 200 mg/m2/d for 5 days per 28-day cycle). The primary end point was objective response. Secondary end points included progression-free survival (PFS), time to progression, overall survival (OS), safety, and tolerability. RESULTS Eight patients (16.7%) experienced a complete response, 13 patients (27.1%) experienced a partial response (objective response rate, 43.8%), and 19 patients (39.6%) experienced stable disease. For the entire treatment group, median PFS was 6.7 months and median OS was 10 months. For objective responders, median PFS was 13.1 months and median OS was 16 months. For complete responders, PFS was more than 11. 8 months and OS was more than 26 months. Response correlated with improved survival. Temozolomide was safe and well tolerated. Twelve patients developed grade 1/2 thrombocytopenia and three patients developed grade 3/4 thrombocytopenia. CONCLUSION Temozolomide is safe and effective in the treatment of recurrent AO and AOA.

Antiangiogenic Concentrations of Paclitaxel Induce an Increase in Microtubule Dynamics in Endothelial Cells but Not in Cancer Cells

Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis.

Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC50s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

Memo–RhoA–mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.

Antiangiogenic Concentrations of Vinflunine Increase the Interphase Microtubule Dynamics and Decrease the Motility of Endothelial Cells

Angiogenesis is a key event in tumor progression and metastasis. This complex process, which constitutes a potent target for cancer therapy, is inhibited by very low concentrations of microtubule-targeting drugs (MTD). However, the intimate mechanisms of the antiangiogenic activity of MTDs remain unclear. Recently, we have shown that low antiangiogenic and noncytotoxic concentrations of paclitaxel induced an unexpected increase in microtubule dynamics in endothelial cells. In this study, we showed that vinflunine, the newest Vinca alkaloid, increased microtubule dynamic instability in human endothelial cells after 4-hour incubation at low concentrations (29% and 54% at 0.1 and 2 nmol/L). The growth and shortening rates were increased, and the percentage of time spent in pause and the mean duration of pauses were decreased, as previously observed with paclitaxel. As opposed to paclitaxel, the transition frequencies were not significantly disturbed by vinflunine. Moreover, low concentrations of vinflunine did not affect mitotic index and anaphase/metaphase ratio. Interestingly, these low vinflunine concentrations that increased microtubule dynamics exhibited an antiangiogenic effect through the inhibition of both morphogenesis and random motility. Capillary tube formation on Matrigel was decreased up to 44%. The cell speed and the random motility coefficient were decreased (13% and 19% and 13% and 33% at 0.1 and 2 nmol/L, respectively) and the persistent time was statistically increased. Altogether, our results confirm that the increase in microtubule dynamics is involved in MTD antiangiogenic activity and highlight the crucial role of interphase microtubule dynamics in angiogenesis.

Pharmacological inhibition of LIM Kinase stabilizes microtubules and inhibits neoplastic growth

The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment.

Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites.

The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01–1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 ± 20 seconds in control cells to 38 ± 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility. [Mol Cancer Ther 2008;7(7):2080–9]

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line.

Abstract. We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. α3β1, α5β1, α6β1/β4, α8β1 and αvβ1/β5/β6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, α2β1, αvβ6 and α6β1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas αvβ5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.

Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends

Invasion of normal brain tissue by tumor cells is a major contributing factor to the recurrence of glioblastoma and its resistance to therapy. Here, we have assessed the efficacy of the microtubule (MT) targeting agent Epothilone B (patupilone) on glioblastoma cell migration, a prerequisite for invasive tumor cell behavior. At non-cytotoxic concentrations, patupilone inhibited glioblastoma cell movement, as shown by transwell cell migration, random motility and spheroid assays. This anti-migratory effect was associated with a reduced accumulation of EB1 and other MT plus end tracking proteins at MT ends and with the induction of MT catastrophes, while the MT growth rate and other MT dynamic instability parameters remained unaltered. An increase in MT catastrophes led to the reduction of the number of MTs reaching the leading edge. Analysis of the effect of patupilone on MT dynamics in a reconstituted in vitro system demonstrated that the induction of MT catastrophes and an alteration of EB1 accumulation at MT plus end are intrinsic properties of patupilone activity. We have thus demonstrated that patupilone antagonizes glioblastoma cell migration by a novel mechanism, which is distinct from suppression of MT dynamic instability. Taken together, our results suggest that EB proteins may represent a new potential target for anti-cancer therapy in highly invasive tumors.

ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics

Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study, we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression.