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Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-?B inhibition.

ABSTRACT: Detyrosinated tubulin, a post-translational modification of ?-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-?B) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments.Using metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-?B inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-?B inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-?B inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study.Sesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-?B. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network.Our data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-?B inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.

SUBMITTER: Whipple RA

PROVIDER: S-EPMC3979133 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

Whipple Rebecca A RA Vitolo Michele I MI Boggs Amanda E AE Charpentier Monica S MS Thompson Keyata K Martin Stuart S SS

Breast cancer research : BCR 20130101 5

<h4>Introduction</h4>Detyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilita ...[more]

PMID: 24028602

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Project description:Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.

Dataset Information

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-?B inhibition.

Publications

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

Similar Datasets

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