Taxol-induced Cell Death Research Articles

E3 Ubiquitin Ligase TRIP12 Controls Exit from Mitosis via Positive Regulation of MCL-1 in Response to Taxol.

Chemotherapy resistance is a major hurdle in cancer treatment. Taxol-based chemotherapy is widely used in the treatment of cancers including breast, ovarian, and pancreatic cancer. Loss of function of the tumor suppressor F-box WD-40 domain containing 7 (FBW7) mutations leads to the accumulation of its substrate MCL-1 which is associated with Taxol resistance in human cancers. We recently showed that E3 ubiquitin ligase TRIP12 is a negative regulator of FBW7 protein. In this study, we find that Taxol-induced mitotic block in cancer cells is partly controlled by TRIP12 via its positive regulation of MCL-1 protein. Genetic inhibition of TRIP12 accelerates MCL-1 protein degradation in mitosis. Notably, introducing double-point mutations in lysines 404/412 of FBW7 to arginine which makes it resistant to proteasomal degradation, leads to the sharp reduction of MCL-1 protein levels and sensitizes cancer cells to Taxol-induced cell death. Finally, TRIP12 deletion leads to enhanced mitotic arrest and cell death in an FBW7 and MCL-1 dependent manner in multiple cell lines including colorectal and ovarian cancer but not in breast cancer. Thus, the TRIP12/FBW7/MCL-1 axis may provide a therapeutic target to overcome Taxol-associated chemotherapy resistance in cancer.

The role of Caspase-1/GSDMD-mediated pyroptosis in Taxol-induced cell death and a Taxol-resistant phenotype in nasopharyngeal carcinoma regulated by autophagy.

Taxol has been widely used as a first-line chemotherapeutic agent for the treatment of advanced nasopharyngeal carcinoma (NPC). However, acquired drug resistance has caused great difficulties in clinical treatment. Pyroptosis is a newly discovered programmed cell death pathway, and Caspase-1 and gasdermin D (GSDMD) play key roles in driving canonical pyroptosis. Increasing evidence suggests that pyroptosis is associated with the development of cancer; however, the function and mechanism of pyroptosis in NPC remain obscure. In this study, we observed that Taxol treatment caused pyroptotic cell death, along with activation of Caspase-1 and maturation of IL-1β, as well as cleavage of GSDMD, which is the canonical pyroptosis executor. Furthermore, Taxol-induced pyroptotic cell death could be suppressed by Caspase-1 inhibitor (Z-YVAD-FMK) and GSDMD knockout. Moreover, NPC parental cells demonstrated higher levels of pyroptosis than Taxol-resistant cells, and pyroptosis mediated by Caspase-1/GSDMD suppression induced by a Caspase-1 inhibitor and GSDMD knockout could induce a Taxol-resistant phenotype in vitro and in vivo. By transfecting an siRNA targeting Beclin-1 into NPC Taxol-resistant cells, we discovered that autophagy could negatively regulate pyroptosis by inhibiting Caspase-1/GSDMD activation. Taken together, our results indicated that Caspase-1/GSDMD mediated Taxol-induced pyroptosis and a Taxol-resistant phenotype in NPC cell lines, which may be regulated by autophagy. Thus, we provide novel insight into the mechanisms of Taxol-induced cell death and a promising approach to improve the therapeutic outcomes of patients with advanced NPC.

PO-062 BAX and BAK interaction with the mitochondrial permeability transition pore (MPTP) is required for taxol-mediated apoptosis

IntroductionMicrotubule Interfering Agents (MIA’s), such as Taxol (Paclitaxel) are used in the treatment of cancers such as breast, ovarian and non–small cell lung cancer. MIA’s arrest cells in mitosis by activating the mitotic checkpoint. Prolonged mitotic arrest results in apoptotic cell death although the precise mechanism of Taxol-induced cell death is unclear. Previous studies have shown that Taxol activates the pro-apoptotic effector proteins, Bak and Bax, which accumulate at the mitochondrial outer membrane. At the mitochondrion Bax and Bak are thought to oligomerize and/or interact with the Mitochondrial Permeability Transition Pore (MPTP) to increase the permeability of the mitochondrial outer membrane which then leads to cytochrome c release and apoptosis. In this study we have examined the requirement for Bax and Bak in Taxol-induced apoptosis and their possible interaction with components of the MPTP.Material and methodsHuman cervical carcinoma (HeLa) cells were treated with either Bax, Bak or both Bax and Bak siRNA, synchronised and treated with Taxol (60 nM) for varying times (0–24 hour). Apoptosis was assessed using either cytokeratin 18 cleavage (M30 antibody), poly (ADP-ribose) polymerase (PARP) cleavage or activation of pro-caspases (3 and 9). Bak and Bax were immunoprecipitated using anti-active Bak (N-20) and anti-active Bax (6A7) antibodies in CHAPS lysis buffer. To identify interacting proteins, Bak and Bax IPs were subjected to peptide mass fingerprint analysis by mass spectrometry. Confocal microscopy was used to examine the intracellular localization of Bax and Bak following Taxol treatment.Results and discussionsThe results of our siRNA studies indicate that although Bax and Bak can form homo-oligomers in the absence of each other, both proteins are required for Taxol-induced apoptosis. Our immunofluorescence study shows that Bak and Bax co-localise at mitochondria in the Taxol-arrested cells. Our proteomic and co-IP analyses indicate that Bak and Bax form a complex specifically in the Taxol-arrested cells that also includes components of the MPTP such as the voltage dependent anion channel (VDAC), the adenine nucleotide translocator (ANT2) and Bcl-2.ConclusionWe conclude that the oligomerisation of Bax and Bak is insufficient for Taxol-mediated apoptosis. However, the interaction of activated Bax and Bak with the MPTP appears to be necessary for Taxol-induced apoptosis.

Highlights of This Issue

Aberrant glycosylation is one of the hallmarks of cancer, and cancer cell glycans serve as tumor biomarkers. Glycosylation modulates the functions of glycoproteins; however, the functional significance of protein glycosylation in cancer progression remains poorly defined. Here, β1,6GlcNAc-branched N-glycan–suppressing and N-glycan–defective β4-integrin expressing cell model systems were used to demonstrate that N-glycosylation of β4-integrin contributes to tumor progression. It was determined that β4-integrin clustering by galectin-3–mediated cross-linking of β1,6GlcNAc-branched N-glycans on β4-integrin activates PI3K/Akt signaling, thereby promoting cancer cell behavior. Thus, N-glycosylation of β4-integrin represents a potential therapeutic target for cancer.Adaptive cellular responses induced by cancer therapeutics are emerging as an important mechanism for acquired resistance to chemotherapies. In this study, Fang and colleagues identified olaparib-induced FOXM1 expression as an adaptive response that induces the expression of homologous recombination (HR) repair genes. Knockdown of olaparib-induced FOXM1 expression by RNAi or pharmacologic inhibition by thiostrepton downregulates the expression of HR repair genes, increases DNA damage and PARP1 trapping, and enhances sensitivity to olaparib. This study highlights the important role of FOXM1 in the adaptive response to olaparib, and that targeting the adaptive response mediated by FOXM1 extends the efficacy of PARP inhibitors.A major problem that limits the effectiveness of cancer chemotherapeutics is the development of drug resistance. Recently, dysregulation of the Hippo pathway core component LATS has been implicated in this process, but its underlying mechanisms remain unknown. Now, Yeung and colleagues functionally characterize a novel signaling pathway in which anti-tubulin drugs activate Cdk1 and phosphorylate LATS, which then binds prolyl isomerase Pin1 and inhibits its anti-apoptotic function. Importantly, loss of LATS phosphorylation reduces taxol-induced cell death. Together, this work provides new insight into the anti-tubulin drug response and demonstrates the Cdk1-LATS-Pin1 signaling axis as a promising target for treatment.ATP-binding cassette (ABC) transporters are a known mechanism of drug resistance, however multiple clinical trials targeting ABCB1 have failed. Thus, it is hypothesized here, that ABC transporters aggravate cancer biology by exporting endogenous lipid mediator sphingosine-1-phosphate (S1P) regardless of drugs. S1P generated by SphK1 and exported via ABCC1 aggravated cancer in a malicious feed-forward manner by amplifying the S1P axis in vitro, and worsened cancer progression in vivo. Patients with high levels of both SphK1 and ABCC1 have worse survival. While, ABCB1, which does not export S1P, did not demonstrate these findings. This suggests a critical role for the S1P axis in breast cancer progression.

Charactor of JNK and P53 in Taxol-Induced Apoptotic Signaling in SKOV3 Human Ovarian Cancer Cell Proliferation

C-Jun N-terminal kinase (JNK) represents a group of mitogen-activated protein kinases (MAPKs) involved in many cellular responses including apoptosis. We have previously reported that taxol, a microtubule-interfering therapeutic agent widely used against various cancers, induces caspase-independent but apoptotic inducing factor (AIF)-dependent apoptosis in human ovarian cancer cell line SKOV3 cells. In the present study, we add to this report a detailed analysis of the taxol-induced apoptotic mechanisms in SKOV3 cells, particularly focusing on JNK and p53. In line with the previous report, we found that taxol induced caspase-independent apoptosis with concurrent activation of JNK, phosphorylation of Bcl-2, Bax translocation to the mitochondria, and AIF release from the mitochondria. Restoration of p53 functionality into SKOV3 cells, which are p53-null cells, by transfection of wild-type p53, however, induced caspase-dependent apoptosis in response to taxol treatment as evidenced by increasing PARP cleavage and the emergence of processed, active caspase-3 and -7. More to the point, treatment with a JNK inhibitor SP600125 blocked taxol-induced apoptotic cell death in both parental SKOV3 cells (p53-deficient) and p53-transfectant cells. Collectively, the aforementioned findings lend support to the view that taxol-induced apoptotic cell death in SKOV3 cells is executed by different mechanisms depending on the presence of p53 but commonly mediated by ASK1-JNK and/or -p38 axes.

Abstract 2896: 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity

Abstract Taxane based chemotherapy is commonly used to treat a wide variety of solid tumors including breast, prostate, lung and ovarian. Taxanes bind to and stabilize microtubules inhibiting their function in interphase and mitosis, and leading to cancer cell apoptosis. Although taxanes are widely used clinically and their target, tubulin, is ubiquitously expressed, patient response is heterogeneous and the molecular mechanism of clinical drug resistance remains elusive. In addition to their antimitotic effects, taxanes affect several signaling pathways whose perturbation by microtubule stabilization may determine cell sensitivity or resistance. Our recent work has identified one such pathway by showing that Taxol treatment inhibits the translation of HIF-1α mRNA by inducing its sequestration to P-bodies where it is silenced. However, the signaling cascade that is initiated in response to microtubule damage leading to translation suppression of HIF-1α is not understood. Using polysome analysis we showed that Taxol treatment inhibited translation at the initiation step of protein synthesis, evidenced by the increase in the 80S peak. Post-translational modifications of various translation initiation factors are implicated in the regulation of translation initiation. Hence, we analyzed phosphorylation states of several initiation factors, including eIF4E, eIF2α, 4E-BP1, 4E-BP2 and rpS6. We didn't observe any change in the phosphorylation state of these initiation factors except for 4E-BP1. We observed that Taxol treatment led to 4E-BP1 hyper-phosphorylation, at a unique previously unrecognized, site. Using isogenic cell lines with acquired mutations at the taxane binding site we conclusively showed that microtubule disruption was required for the hyper-phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 is known to activate cap-dependent translation. Instead, our data using polysome profiling revealed that Taxol-induced 4E-BP1 hyper-phosphorylation was associated with translation suppression. We confirmed these results by performing the cap-binding assay, which demonstrated that Taxol treatment had no effect on cap-dependent translation. Taken together these results suggest a novel biological function for 4E-BP1 hyper-phosporylation. Further, treatment with the mTOR inhibitor, Torin-1, had no effect on Taxol-induced 4E-BP1 hyper-phosphorylation, suggesting mTOR pathway independence. Interestingly, treatment with the CDK inhibitor Roscovitine, prevented both Taxol-induced 4E-BP1 hyper-phosphorylation and Taxol-induced cell death. Although we do not yet know which of the three CDKs exerts the inhibitory effect on taxanes, our data suggest that there is a functional link between 4E-BP1 hyper-phosphorylation and taxane activity. We are currently investigating the role of CDK1, CDK2 and CDK5 in this pathway to decipher the novel biological function of 4E-BP1 hyper-phosphorylation. Citation Format: Prashant Khade, Paraskevi Giannakakou. 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2896.

Mitochondrial dysfunction-mediated apoptosis resistance associates with defective heat shock protein response in African-American men with prostate cancer.

Background:African–American (AA) patients with prostate cancer (PCa) respond poorly to current therapy compared with Caucasian American (CA) PCa patients. Although underlying mechanisms are not defined, mitochondrial dysfunction is a key reason for this disparity.Methods:Cell death, cell cycle, and mitochondrial function/stress were analysed by flow cytometry or by Seahorse XF24 analyzer. Expression of cellular proteins was determined using immunoblotting and real-time PCR analyses. Cell survival/motility was evaluated by clonogenic, cell migration, and gelatin zymography assays.Results:Glycolytic pathway inhibitor dichloroacetate (DCA) inhibited cell proliferation in both AA PCa cells (AA cells) and CA PCa cells (CA cells). AA cells possess reduced endogenous reactive oxygen species, mitochondrial membrane potential (mtMP), and mitochondrial mass compared with CA cells. DCA upregulated mtMP in both cell types, whereas mitochondrial mass was significantly increased in CA cells. DCA enhanced taxol-induced cell death in CA cells while sensitising AA cells to doxorubicin. Reduced expression of heat shock proteins (HSPs) was observed in AA cells, whereas DCA induced expression of CHOP, C/EBP, HSP60, and HSP90 in CA cells. AA cells are more aggressive and metastatic than CA cells.Conclusions:Restoration of mitochondrial function may provide new option for reducing PCa health disparity among American men.

Protective effects of fucoidan on the cell death of spleen cells induced by taxol

Taxol is an anti-cancer agent that stabilizes the microtubules of cancer cells, resulting in inhibition of mitosis and thus preventing the proliferation of cancer cells. However, many anti-cancer agents including taxol work on normal cells as well as cancer cells, resulting in side effects such as immunosuppression. A marine algae-derived sulfated polysaccharide, fucoidan, an anti-cancer agent, also showed immunostimulating effects. This study investigated the effects of fucoidan on taxol-treated spleen cells. Spleen cells were treated with taxol in a concentration-dependent manner and in combination with fucoidan. MTT assay and flow cytometry analysis were performed to measure the viability and activity of treated cells. Two assays demonstrated that taxol induced the death of spleen cells. Fucoidan clearly inhibited the cell death induced by taxol. In addition, fucoidan enhanced the production of nitric oxide in spleen cells, which was decreased by taxol. Taken together, taxol can induce the cell death of spleen cells, a major type of immune cells and fucoidan protects spleen cells from taxol-induced cell death. This finding suggests that taxol and fucoidan can be used in combination for lowering the immunosuppressive effects of taxol.

Abstract 2936: Mitochondria dysfunction-mediated apoptosis resistance associates with defective heat shock protein response in African American prostate cancer

Abstract African American (AA) patients with prostate cancer (PCa) respond poorly to current therapy compared to Caucasian American (CA) patients. Although underlying mechanisms are not clearly defined, mitochondria dysfunction is a key reason for PCa disparity. We observed that glycolytic pathway inhibitor dichloroacetate (DCA) inhibited cell proliferation in both AA PCa cells (AA cells) and CA PCa cells (CA cells). CA cells were arrested at G1 and G2/M phases whereas S-phase arrest was observed in AA cells. AA cells showed resistance to cell death upon DCA exposure in spite of higher caspase activation than in CA cells. AA cells possess reduced level of reactive oxygen species (ROS) and mitochondrial membrane potential (mtMP) compared to CA cells. DCA induced cellular and mitochondrial ROS in AA cells but were unchanged in CA cells. DCA upregulated the levels of mtMP in both cell types. DCA enhanced taxol-induced cell death only in CA cells, but DCA sensitized doxorubicin-induced AA cell death. Expression of heat shock proteins (HSPs) were inhibited only in AA cells upon doxorubicin alone or combined treatment of DCA with doxorubicin, whereas Taxol alone or in combination with DCA did not show any changes in the levels of HSPs. DCA suppressed the metastatic features and reduced the population of cancer stem cell-like cells in CA cells but had a little effect on AA cells. Therefore, anticancer agents that can effectively restore mitochondria function may provide new therapeutic potential in reducing PCa health disparity and poor prognosis in AA men with PCa. Citation Format: Ajay K. Chaudhary, Tariq A. Bhatt, Sandeep Kumar, Willie Underwood, Shahriar Koochekpour, Mojgan Shourideh, Neelu Yadav, Dhyan Chandra. Mitochondria dysfunction-mediated apoptosis resistance associates with defective heat shock protein response in African American prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2015-2936

Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA–MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.

TAXOL INDUCES CELL DEATH WITH CYTOPLASM VACUOLIZATION IN PARAPTOSIS-LIKE BUT NOT ONCOSIS FASHION IN ASTC-a-1 CELLS

Recently, we found that high concentration of taxol (70 μM) induced cell death with cytoplasm vacuolization, the typical characteristic of both paraptosis and oncosis, in human lung carcinoma (ASTC-a-1) cells. This report was designed to further determine the form of taxol-induced cell death with cytoplasm vacuolization. It is generally considered that the cytoplasm vacuolization in oncosis due to the swelling of endoplasmic reticulum (ER), mitochondria, lysosomes and nuclei occurs after the loss of mitochondrial membrane potential (ΔΨm). However, flow cytometry (FCM) analysis showed that taxol-induced cytoplasm vacuolization preceded the loss of ΔΨm. Moreover, taxol treatment did not induce the collapse of microtubule, the typical characteristic of oncosis. These data demonstrated that taxol-induced cell death with cytoplasm vacuolization is not oncosis. FCM analysis by Annexin V-FITC/PI apoptosis detection kit further demonstrated that taxol-induced cell death with cytoplasm vacuolization is not apoptosis. In conclusion, in combination with our recent in vitro and in vivo data, this report further demonstrates that high concentration of taxol induces cell death with cytoplasm vacuolization in paraptosis-like but not oncosis fashion.

Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation

Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

Cyclin B1 interacts with the BH3-only protein Bim and mediates its phosphorylation by Cdk1 during mitosis

Protracted mitotic arrest leads to cell death; however, the molecular signals that link these distinct processes remain poorly understood. Here we report that the pro-apoptotic BH3-only family member Bim undergoes phosphorylation in K562 cells following treatment with the microtubule targeting agents Taxol and Nocodazole. The phosphorylation of two Bim isoforms, BimEL and BimL, at the mitochondria correlates with mitotic arrest and precedes cell death induced by Taxol. It was also found that Bim undergoes transient phosphorylation during normal mitosis in K562 cells. In addition, siRNA silencing of Bim reduces sensitivity to Taxol-induced cell death. The transition of K562 cells from mitosis to G1 results in the loss of BimEL and BimL phosphorylation and correlates with the degradation of cyclin B1. The Cdk1 inhibitors, RO-3306 and Purvalanol A, block Bim phosphorylation in mitotically arrested cells. Importantly, it was found that cyclin B1 co-immunoprecipitates with endogenous Bim in mitotic extracts. Furthermore, active recombinant Cdk1/cyclin B1 phosphorylates BimEL and BimL in vitro and Serine 44 on BimL has been identified as a Cdk1 phosphorylation site. Collectively, these results suggest that Cdk1/cyclin B1-dependent hyper-phosphorylation of Bim during prolonged mitotic arrest is an important cell death signal.

Lysophosphatidate Induces Chemo-Resistance by Releasing Breast Cancer Cells from Taxol-Induced Mitotic Arrest

BackgroundTaxol is a microtubule stabilizing agent that arrests cells in mitosis leading to cell death. Taxol is widely used to treat breast cancer, but resistance occurs in 25–69% of patients and it is vital to understand how Taxol resistance develops to improve chemotherapy. The effects of chemotherapeutic agents are overcome by survival signals that cancer cells receive. We focused our studies on autotaxin, which is a secreted protein that increases tumor growth, aggressiveness, angiogenesis and metastasis. We discovered that autotaxin strongly antagonizes the Taxol-induced killing of breast cancer and melanoma cells by converting the abundant extra-cellular lipid, lysophosphatidylcholine, into lysophosphatidate. This lipid stimulates specific G-protein coupled receptors that activate survival signals.Methodology/Principal FindingsIn this study we determined the basis of these antagonistic actions of lysophosphatidate towards Taxol-induced G2/M arrest and cell death using cultured breast cancer cells. Lysophosphatidate does not antagonize Taxol action in MCF-7 cells by increasing Taxol metabolism or its expulsion through multi-drug resistance transporters. Lysophosphatidate does not lower the percentage of cells accumulating in G2/M by decreasing exit from S-phase or selective stimulation of cell death in G2/M. Instead, LPA had an unexpected and remarkable action in enabling MCF-7 and MDA-MB-468 cells, which had been arrested in G2/M by Taxol, to normalize spindle structure and divide, thus avoiding cell death. This action involves displacement of Taxol from the tubulin polymer fraction, which based on inhibitor studies, depends on activation of LPA receptors and phosphatidylinositol 3-kinase.Conclusions/SignificanceThis work demonstrates a previously unknown consequence of lysophosphatidate action that explains why autotaxin and lysophosphatidate protect against Taxol-induced cell death and promote resistance to the action of this important therapeutic agent.

The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein

The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Abstract 696: BRCA1-mediated ubiquitination of tubulin may serve as a signal for taxol-induced apoptosis

Abstract The taxanes are among the most effective chemotherapeutic agents used for the treatment of solid tumors, yet eventually most tumors become resistant to taxane chemotherapy. Mechanisms responsible for clinical resistance to taxanes have not been fully elucidated. Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene, whose expression has been clearly correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the relationship between BRCA1 expression and taxane activity remains unclear. BRCA1 is a nuclear shuffling protein with many more described nuclear actions than cytoplasmic. BRCA1 has been shown to regulate the cell's ability to undergo apoptosis and that increased BRCA1 cytoplasmic localization correlates with increased apoptotic functions. In association with BARD1, the BRCA1/BARD1 complex functions as an E3 ubiquitin ligase. Recent literature suggests γ-tubulin is a target of this complex resulting in mono-ubiquitination of γ-tubulin which regulates its centrosomal localization and function. Here, we test the hypothesis that either α- or β-tubulin also serve as a target for the BRCA1/BARD1 E3 ubiquitin ligase activity which regulates the cell's sensitivity to Taxol. To approach this, we generated a stable-BRCA1 knockdown (B1-KD) of the NSCLC cell line A549 in which an 80% knockdown of BRCA1 results in resistance to Taxol when compared to the parental line, phenocopying the clinical data correlating low BRCA1 expression with taxane resistance. The B1-KD line exhibited aberrant centrosomal amplification and activity likely due to the γ-tubulin mislocalization. Then, we looked at the ability of BRCA1 to ubiquinate α- or β- tubulin subunits that form the microtubule cytoskeleton. Tubulin immunoprecipitation experiments revealed that the B1-KD results in a loss of tubulin ubiquitination when compared to the parental line. Taxol treatment in both cell lines seems to increase tubulin ubiquitination suggesting that the residual BRCA1 in the knockdown line is enough to ubiquitinate tubulin. Currently, we are looking at mechanisms linking tubulin ubiquitination to cell death, mainly capthesin-B-, calpain- or caspase-mediated apoptosis. Initial experiments suggest that capthesin-B inhibition does not inhibit Taxol-induced cell death in both the parental and B1-KD A549 cell line. We propose a model in which Taxol-induced tubulin ubiquitination, via the BRCA1/BARD1 complex, serves as the signal to initiate downstream cell death signaling pathways in the presence of Taxol treatment. This model would assign a strong cytoplasmic role for BRCA1 and show BRCA1 regulation of apoptosis may exist outside of the nucleus. It might also provide insight into how lung cancer cells become resistant to taxanes with the potential to disrupt these resistance mechanisms and enhance therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2011-696

MKK7γ1 reverses nerve growth factor signals: Proliferation and cell death instead of neuritogenesis and protection

c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54 kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1, which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances taxol-induced cell death in breast cancer

The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) enhances taxol-induced antitumor effects against some human cancer cells. The aim of this study is to investigate whether SAHA can enhance taxol-induced cell death against human breast cancer cells and to illustrate the mechanism in detail. A panel of eight human breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the inhibitory effects of SAHA, taxol, or their combination by MTT assay. The effects of SAHA with or without taxol on cell cycle distributions, apoptosis, and protein expressions were also examined. The inhibitory effects on tumor growth were characterized in vivo in BALB/c nude mice bearing a breast cancer xenograft model. Taxol-resistant and multi-resistant breast cancer cells were as sensitive to SAHA as taxol-sensitive breast cancer cells. A dose-dependent synergistic growth inhibition was found in all the tested breast cancer cell lines treated with the SAHA/taxol combinations. The synergetic effect was also observed in the in vivo xenograft tumor model. The cell cycle analysis and apoptosis assay showed that the synergistic effects resulted from enhanced G2/M arrest and apoptosis. SAHA increased the anti-tumor effects of taxol in breast cancer in vitro and in vivo. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.

TIP47 confers resistance to taxol-induced cell death by preventing the nuclear translocation of AIF and Endonuclease G

Tail-interacting protein (TIP47, also named PP17) has been implicated in lipid droplet metabolism and in the development of late endosomes, to date however, no data about its possible role in regulating cell death processes has been available. Here, we provide evidence for the role of TIP47 in the regulation of mitochondrial membrane stability and cell death. Overexpression of TIP47 protected NIH3T3 cells from taxol-induced cell death, while suppression of TIP47 by siRNA facilitated cell death. TIP47, but not its truncated form, t-TIP47, decreased taxol-induced cell death as determined by propidium iodide and fluorescent Annexin V staining. Recombinant TIP47, but not t-TIP47, partially prevented taxol-induced depolarization of mitochondria in vitro. Overexpression of TIP47, but not its truncated form, prevented the taxol-induced nuclear and cytoplasmic translocation of AIF and Endonuclease G, as well as the taxol-induced depolarization of mitochondria in NIH3T3 cells. Furthermore, overexpression of TIP47 facilitated Bcl-2 expression and suppressed Bax expression in taxol-treated cells. These data show that besides its previously known functions, TIP47 is involved in the regulation of mitochondria-related cell death by directly stabilizing the mitochondrial membrane system and by favorably affecting the expression of Bcl-2 homologues. Since TIP47 is overexpressed in certain tumors, it is possible that TIP47 contributes to the development of cytostatic resistance.

Estrogen Suppresses MLK3-Mediated Apoptosis Sensitivity in ER+ Breast Cancer Cells

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.