Anti-microtubule Drugs Research Articles

A nanoprodrug derived from branched poly (ethylene glycol) recognizes prostate-specific membrane antigen to precisely suppress prostate cancer progression

Prostate-specific membrane antigen (PSMA) is overexpressed in 80–90 % of prostate cancers (PCa) and is widely used as a diagnostic and therapeutic biomarker. Docetaxel (DTX), an FDA-approved anti-microtubule drug, is commonly employed to manage metastatic castration-resistant PCa; however, DTX therapy is often associated with severe side effects. One promising strategy to mitigate these side effects is the development of nanomedicine by loading small molecules into biocompatible vectors. Poly (ethylene glycol) (PEG) has been extensively used in clinical settings for this purpose, with PEGylated drugs demonstrating significant success. Compared to linear PEG, branched PEG (multi-arm PEG) provides enhanced stability for nanomedicines. In this study, we developed a novel nanoprodrug 4armPEG-Docetaxel DCL (4armPEG-DD) by conjugating a 4-arm PEG with DTX via a reduction-sensitive disulfide bond and further modifying it with 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (DCL), a PSMA-targeting ligand. Both in vitro and in vivo results demonstrated that the designed nanoprodrug specifically recognized PSMA-positive PCa cells and effectively released DTX in response to the intracellular reducing environment, leading to potent cytotoxic effects on PSMA-positive prostate tumors. Importantly, 4armPEG-DD exhibited improved in vivo safety compared to small-molecule DTX. Thus, we propose that 4armPEG-DD represents a promising candidate for the clinical treatment of PSMA-positive PCa.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7144.

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

STNM1 in human cancers: role, function and potential therapy sensitizer

STMN1 belongs to the stathmin gene family, it encodes a cytoplasmic phosphorylated protein, stathmin1, which is commonly observed in vertebrate cells. STMN1 is a structural microtubule-associated protein (MAP) that binds to microtubule protein dimers rather than microtubules, with each STMN1 binding two microtubule protein dimers and preventing their aggregation, leading to microtubule instability. STMN1 expression is elevated in a number of malignancies, and inhibition of its expression can interfere with tumor cell division. Its expression can change the division of tumor cells, thereby arresting cell growth in the G2/M phase. Moreover, STMN1 expression affects tumor cell sensitivity to anti-microtubule drug analogs, including vincristine and paclitaxel. The research on MAPs is limited, and new insights on the mechanism of STMN1 in different cancers are emerging. The effective application of STMN1 in cancer prognosis and treatment requires further understanding of this protein. Here, we summarize the general characteristics of STMN1 and outline how STMN1 plays a role in cancer development, targeting multiple signaling networks and acting as a downstream target for multiple microRNAs, circRNAs, and lincRNAs. We also summarize recent findings on the function role of STMN1 in tumor resistance and as a therapeutic target for cancer.

Co-administration of JQ1, a bromodomain-containing protein 4 inhibitor, enhances the antitumor effect of combretastatin A4, a microtubule inhibitor, while attenuating its cardiotoxicity

Combretastatin A4 (CA4) inhibits microtubule polymerization, and clinical trials of the prodrug, CA4 disodium phosphate (CA4DP), as an anti-cancer agent have been conducted. However, CA4DP has not been marketed to date because the margin between the effective dose and the cardiotoxic dose is insufficient. Meanwhile, bromodomain-containing protein 4 (BRD4) has been reported to be required for recovery from mitotic arrests induced by anti-microtubule drugs. BRD4 has also been reported to be involved in the progression of heart failure. Therefore, we hypothesized that the combined use of CA4DP with BRD4 inhibitors can enhance the antitumor effect and attenuate CA4DP-induced cardiotoxicity. In this study, the antitumor effect and cardiotoxicity caused by the co-administration of CA4DP with JQ1, a BRD4 inhibitor, were evaluated.CA4 or JQ1 alone reduced the viability of cultured canine mammary tumor cells (CHMp-13a). Viability was further reduced by co-administration, through the suppression of c-Myc. BRD4 positivity in CHMp-13a cytoplasm showed a significant increase when treated with CA4 alone, while the increase was not significant following co-administration. In CHMp-13a xenograft-transplanted mice, co-administration of CA4DP and JQ1 suppressed tumor growth significantly.In CA4DP-induced cardiac injury model rats, echocardiography showed a CA4DP-induced decrease in cardiac function and histopathology showed cardiomyocyte necrosis. Meanwhile, these cardiac changes tended to be milder following the co-administration of CA4DP and JQ1.These results suggest that CA4DP-JQ1 co-administration enhances the antitumor effect of CA4DP while attenuating its cardiotoxicity and therefore potentially open the doors to the development of a novel cancer chemotherapy with reduced cardiotoxicity risks.

Eribulin normalizes pancreatic cancer-associated fibroblasts by simulating selected features of TGFβ inhibition

BackgroundLess than 11% of pancreatic cancer patients survive 5-years post-diagnosis. The unique biology of pancreatic cancer includes a significant expansion of its desmoplastic tumor microenvironment, wherein cancer-associated fibroblasts (CAFs) and their self-produced extracellular matrix are key components. CAF functions are both tumor-supportive and tumor-suppressive, while normal fibroblastic cells are solely tumor-suppressive. Knowing that CAF-eliminating drugs are ineffective and can accelerate cancer progression, therapies that “normalize” CAF function are highly pursued. Eribulin is a well-tolerated anti-microtubule drug used to treat a plethora of neoplasias, including advanced/metastatic cancers. Importantly, eribulin can inhibit epithelial to mesenchymal transition via a mechanism akin to blocking pathways induced by transforming growth factor-beta (TGFβ). Notably, canonical TGFβ signaling also plays a pivotal role in CAF activation, which is necessary for the development and maintenance of desmoplasia. Hence, we hypothesized that eribulin could modulate, and perhaps “normalize” CAF function.MethodsTo test this premise, we used a well-established in vivo-mimetic fibroblastic cell-derived extracellular matrix (CDM) system and gauged the effects of eribulin on human pancreatic CAFs and cancer cells. This pathophysiologic fibroblast/matrix functional unit was also used to query eribulin effects on CDM-regulated pancreatic cancer cell survival and invasive spread.ResultsDemonstrated that intact CAF CDMs modestly restricted eribulin from obstructing pancreatic cancer cell growth. Nonetheless, eribulin-treated CAFs generated CDMs that limited nutrient-deprived pancreatic cancer cell survival, similar to reported tumor-suppressive CDMs generated by TGFβ-deficient CAFs.ConclusionsData from this study support the central proposed premise suggesting that eribulin could be used as a CAF/matrix-normalizing drug.

The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

During prolonged mitotic arrest induced by anti-microtubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death, the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SCF (SKP1-CUL1-F-Box) E3 ubiquitin ligase complex promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5, a component of the mixed lineage leukemia (MLL) complex of Histone 3 Lysine 4 (H3K4) methyltransferases, is a substrate of FBXW7. We determined by co-immunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SCF-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss-of-function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation which might contribute to prevent chemotherapy resistance in patients after anti-microtubule drug treatment.

Paclitaxel-Induced Pneumonitis in Trinidad: A Case Report

Paclitaxel-induced pneumonitis (PIP) is an immune-mediated disease resulting from a delayed hypersensitivity reaction (type IV) to paclitaxel, an anti-microtubule chemotherapeutic drug commonly used to treat breast cancer in both neoadjuvant and adjuvant settings. PIP is diagnosed by exclusion utilizing laboratory work-up, imaging, biopsy studies, and results of antibiotic therapy because there is no single diagnostic test. Ground-glass opacifications on CT, coupled with minimal restrictive disturbance with decreased diffusion on pulmonary function tests (PFTs), negative bronchoalveolar lavage (BAL), and bronchoscopy cultures, may assist physicians in diagnosing paclitaxel-induced pneumonitis. In this report, we describe a case of PIP present in Trinidad, West Indies, which has not been described previously in this region.

How COVID-19 Hijacks the Cytoskeleton: Therapeutic Implications.

The SARS-CoV-2 virus invades and replicates within host cells by “hijacking” biomolecular machinery, gaining control of the microtubule cytoskeleton. After attaching to membrane receptors and entering cells, the SARS-CoV-2 virus co-opts the dynamic intra-cellular cytoskeletal network of microtubules, actin, and the microtubule-organizing center, enabling three factors that lead to clinical pathology: (1) viral load due to intra-cellular trafficking, (2) cell-to-cell spread by filopodia, and (3) immune dysfunction, ranging from hyper-inflammatory cytokine storm to ineffective or absent response. These factors all depend directly on microtubules and the microtubule-organizing center, as do cell functions such as mitosis and immune cell movement. Here we consider how the SARS-CoV-2 virus may “hijack” cytoskeletal functions by docking inside the microtubule-organizing center’s centriole “barrels”, enabling certain interactions between the virus’s positively charged spike (“S”) proteins and negatively charged C-termini of the microtubules that the centriole comprises, somewhat like fingers on a keyboard. This points to the potential benefit of therapies aimed not directly at the virus but at the microtubules and microtubule-organizing center of the host cell on which the virus depends. These therapies could range from anti-microtubule drugs to low-intensity ultrasound (megahertz mechanical vibrations) externally applied to the vagus nerve at the neck and/or to the spleen (since both are involved in mediating inflammatory response). Given that ultrasound imaging machines suitable for vagal/splenic ultrasound are available for clinical trials in every hospital, we recommend an alternative therapeutic approach for COVID-19 based on addressing and normalizing the host cell microtubules and microtubule-organizing centers co-opted by the SARS-CoV-2 virus.

KANSER TEDAVİSİNDE KULLANILAN ANTİ-MİKROTÜBÜL İLAÇLARDAN VİNCA ALKALOİDLER VE PAKLİTAKSEL’İN DNA BAĞLANMA AKTİVİTELERİ

OBJECTIVE: Microtubules are an essential part of the intracellular cytoskeletal structure and possess unique polymerization dynamics that are critical for many cellular functions, including cell division. Anti-microtubule drugs that interfere with microtubule formation are important chemotherapeutic agents for the treatment of various cancer. These drugs that block mitosis seem to work by a common mechanism, which suppresses the dynamics of microtubules, slows cells, induces apoptosis and subsequently kills tumor cells. Vinca alkaloids (vinblastine, vincristine and vinorelbine) and Taxanes (paclitaxel) are two different classes of anti-microtubule drugs that cause microtubule dysfunction and inhibit cancer cell proliferation. The main activity of vinca alkaloids and taxanes result from their binding interactions with tubulin proteins. However, studies on DNA interactions of these anti-microtubule drugs are not sufficient. In this study, it was aimed to investigate the DNA binding activities of the vinca alkaloids (vinblastine, vincristine, vinorelbine) and paclitaxel.MATERIAL AND METHODS: The interactions of the drugs with DNA were analyzed by agarose gel electrophoresis assay. Three types of DNA were used in each experiment, including 100bp marker DNA, pUC19 plasmid DNA (2686 bp), and pBR322 plasmid DNA (4361 bp). After the DNAs were incubated with different concentrations of the drugs under certain conditions, agarose gel electrophoresis was performed. DNA band distributions were analyzed with a gel analysis system so that the drugs-DNA interactions could be interpreted.RESULTS: According to our results, it was found that among the vinca alkaloids, especially vinorelbine binds to DNA with higher activity than vincristine and vinblastine. The Vinca alkaloids have structural properties required for DNA binding activity and there is a similarity in their DNA binding models. However, the results showed that paclitaxel, which is from the taxane group, did not have DNA binding activity. This may be because the chemical structure of paclitaxel is not suitable for binding to DNA.CONCLUSIONS: The interaction of drugs with DNA play an important role in determining the pathways of drugs action and their ability to cause DNA damage. Consequently, the findings of our study will contribute to elucidating the effect mechanisms and the genotoxic potentials of these drugs, which are microtubule inhibitors.

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets.

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

Abstract 1045: Combined targeting of actin/tropomyosin and microtubules underlies a potential treatment strategy of epithelial ovarian cancer with cell-cycle dependent synergy

Abstract Anti-microtubule agents are widely used to treat ovarian cancers in the first line and recurrent setting either in combination with platinum or as single agents. However, the majority of patients will experience a recurrence, and most will die with drug resistant disease. We investigated co-targeting the actin cytoskeleton in combination with anti-microtubule agents to increase efficacy of treatment in epithelial ovarian cancers and potentially overcome resistance mechanisms. We examined the presence of actin/tropomyosin 3.1 (Tpm3.1) filaments in a large cohort of clinical specimens from patients with epithelial ovarian cancer of all histotypes using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound (ATM) with both vinorelbine and paclitaxel were evaluated in three ovarian cancer cell lines using cell viability and apoptosis assays. The mechanisms of synergy of both combinations were established using live-cell imaging, fluorescent microscopy, and pathway analysis. We found that Tpm3.1 is abundant and overexpressed in 97% of ovarian cancers examined (558 of 577) representing all histotypes of epithelial ovarian cancer. High levels of Tpm3.1 were also present in all sites sampled and similar at primary diagnosis and at recurrence. ATM displayed both single agent activity as well as synergy with both anti-microtubule drugs to reduce cell viability in all ovarian cancer cell lines tested, including one with platinum resistance. Only vinorelbine, however, synergised with ATM in the induction of apoptosis. Vinorelbine-induced mitotic arrest was significantly prolonged by ATM with elevated activity of the spindle assembly checkpoint, leading to almost one third of total cells dying in mitosis. In contrast, ATM showed minor impact on paclitaxel-induced mitotic defects. Both combinations resulted in a substantial increase in cells arrested in the subsequent G1 phase with a large decrease of both cyclin D1 and E1 as compared to single agents. Upregulation of p21Cip and p27Kip were associated with both combinations. In summary, targeting Tpm3.1-associated actin filaments in combination with anti-microtubule drugs is a promising treatment strategy that should be tested in clinical trials and is potentially applicable to all histotypes of ovarian cancer. Citation Format: Xing Xu, Yao Wang, Nicole Bryce, Katrina Tang, Nicola S. Meagher, Eun Young Kang, Linda E. Kelemen, Martin Köbel, Susan J. Ramus, Michael Friedlander, Caroline E. Ford, Edna C. Hardeman, Peter W. Gunning. Combined targeting of actin/tropomyosin and microtubules underlies a potential treatment strategy of epithelial ovarian cancer with cell-cycle dependent synergy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1045.

Inhibition of cell expansion enhances cortical microtubule stability in the root apex of Arabidopsis thaliana

BackgroundCortical microtubules regulate cell expansion by determining cellulose microfibril orientation in the root apex of Arabidopsis thaliana. While the regulation of cell wall properties by cortical microtubules is well studied, the data on the influence of cell wall to cortical microtubule organization and stability remain scarce. Studies on cellulose biosynthesis mutants revealed that cortical microtubules depend on Cellulose Synthase A (CESA) function and/or cell expansion. Furthermore, it has been reported that cortical microtubules in cellulose-deficient mutants are hypersensitive to oryzalin. In this work, the persistence of cortical microtubules against anti-microtubule treatment was thoroughly studied in the roots of several cesa mutants, namely thanatos, mre1, any1, prc1-1 and rsw1, and the Cellulose Synthase Interacting 1 protein (csi1) mutant pom2-4. In addition, various treatments with drugs affecting cell expansion were performed on wild-type roots. Whole mount tubulin immunolabeling was applied in the above roots and observations were performed by confocal microscopy.ResultsCortical microtubules in all mutants showed statistically significant increased persistence against anti-microtubule drugs, compared to those of the wild-type. Furthermore, to examine if the enhanced stability of cortical microtubules was due to reduced cellulose biosynthesis or to suppression of cell expansion, treatments of wild-type roots with 2,6-dichlorobenzonitrile (DCB) and Congo red were performed. After these treatments, cortical microtubules appeared more resistant to oryzalin, than in the control.ConclusionsAccording to these findings, it may be concluded that inhibition of cell expansion, irrespective of the cause, results in increased microtubule stability in A. thaliana root. In addition, cell expansion does not only rely on cortical microtubule orientation but also plays a regulatory role in microtubule dynamics, as well. Various hypotheses may explain the increased cortical microtubule stability under decreased cell expansion such as the role of cell wall sensors and the presence of less dynamic cortical microtubules.

Identification of a novel Bax-Cdk1 signalling complex that links activation of the mitotic checkpoint to apoptosis.

In eukaryotes, entry into and exit from mitosis is regulated, respectively, by the transient activation and inactivation of Cdk1. Taxol, an anti-microtubule anti-cancer drug, prevents microtubule-kinetochore attachments to induce spindle assembly checkpoint (SAC; also known as the mitotic checkpoint)-activated mitotic arrest. SAC activation causes mitotic arrest by chronically activating Cdk1. One consequence of prolonged Cdk1 activation is cell death. However, the cytoplasmic signal(s) that link SAC activation to the initiation of cell death remain unknown. We show here that activated Cdk1 forms a complex with the pro-apoptotic proteins Bax and Bak (also known as BAK1) during SAC-induced apoptosis. Bax- and Bak-mediated delivery of activated Cdk1 to the mitochondrion is essential for the phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-xL (encoded by BCL2L1) and the induction of cell death. The interactions between a key cell cycle control protein and key pro-apoptotic proteins identify the Cdk1-Bax and Cdk1-Bak complexes as the long-sought-after cytoplasmic signal that couples SAC activation to the induction of apoptotic cell death.

The Frequent Sampling of Wound Scratch Assay Reveals the "Opportunity" Window for Quantitative Evaluation of Cell Motility-Impeding Drugs.

Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs’, nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10–15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10–100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

Abstract PS16-10: Tao kinase 3 augment the resistance to microtubule-disrupting agents in breast cancer cells

Abstract Breast cancer is the leading type of cancer in women worldwide. It is a heterogeneous disease that contains several subtypes with substantial differences in pathology and clinical outcome. With the advancements in imaging and targeted therapy, many patients with early stages of this disease have a great opportunity to be cured. However, recurrence or metastasis of tumor still occurs in patients even after successful primary treatment. Chemotherapy remains one of the most important treatment for advanced breast cancer. Anti-microtubule agents including taxanes, eribulin and vinca-alkaloids constitutes the major anti-breast cancer chemotherapeutic category, while chemoresistance remains a thorny issue for advanced disease. We aimed to discover novel candidate regulators for chemoresistance in breast cancer. A lentiviral-based, high-throughput shRNA platform was developed for screening the variation of global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The underlying molecular mechanisms was further explored by global phosphoprotein array and expression microarray. The serine/threonine kinase, TAO Kinase 3 (TAOK3), was identified from 724 kinase genes. Knockdown of TAOK3 exhibited the most profound reduction of IC50 values in response to paclitaxel treatment in breast cancer cells. High expression of TAOK3 was related to poor prognosis in breast cancer patients after adjuvant chemotherapy. Furthermore, the expression of TAOK3 also decreased the sensitivity to other anti-microtubule drugs, including eribulin and vinorelbine. Expression microarray analysis revealed that NF-κB signaling plays the major regulation roles in TAOK3-associated resistance. Taken together, our results showed that TAOK3 increases resistance to anti-microtubule drug through upregulating the NF-κB signaling in breast cancer. Inhibitors that disrupt TAOK3 and NF-κB signaling pathway may overcome the drug resistance for patients treated with anti-microtubule agents. Citation Format: Chih-Yeu Fang, Tsung-Ching Lai, Michael Hsiao. Tao kinase 3 augment the resistance to microtubule-disrupting agents in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-10.

ABT-751 Induces Multiple Anticancer Effects in Urinary Bladder Urothelial Carcinoma-Derived Cells: Highlighting the Induction of Cytostasis through the Inhibition of SKP2 at Both Transcriptional and Post-Translational Levels.

The objective was to investigate the anti-cancer effects and underlying molecular mechanisms of cytostasis which were activated by an anti-microtubule drug, ABT-751, in two urinary bladder urothelial carcinoma (UBUC)-derived cell lines, BFTC905 and J82, with distinct genetic backgrounds. A series of in vitro assays demonstrated that ABT-751 induced G2/M cell cycle arrest, decreased cell number in the S phase of the cell cycle and suppressed colony formation/independent cell growth, accompanied with alterations of the protein levels of several cell cycle regulators. In addition, ABT-751 treatment significantly hurdled cell migration and invasion along with the regulation of epithelial–mesenchymal transition-related proteins. ABT-751 triggered autophagy and apoptosis, downregulated the mechanistic target of rapamycin kinase (MTOR) and upregulated several pro-apoptotic proteins that are involved in extrinsic and intrinsic apoptotic pathways. Inhibition of autophagosome and autolysosome enhanced apoptosis was also observed. Through the inhibition of the NFκB signaling pathway, ABT-751 suppressed S-phase kinase associated protein 2 (SKP2) transcription and subsequent translation by downregulation of active/phospho-AKT serine/threonine kinase 1 (AKT1), component of inhibitor of nuclear factor kappa B kinase complex (CHUK), NFKB inhibitor alpha (NFKBIA), nuclear RELA proto-oncogene, NFκB subunit (RELA) and maintained a strong interaction between NFKBIA and RELA to prevent RELA nuclear translocation for SKP2 transcription. ABT-751 downregulated stable/phospho-SKP2 including pSKP2(S64) and pSKP2(S72), which targeted cyclin-dependent kinase inhibitors for degradation through the inactivation of AKT. Our results suggested that ABT-751 may act as an anti-cancer drug by inhibiting cell migration, invasion yet inducing cell cycle arrest, autophagy and apoptosis in distinct UBUC-derived cells. Particularly, the upstream molecular mechanism of its anticancer effects was identified as ABT-751-induced cytostasis through the inhibition of SKP2 at both transcriptional and post-translational levels to stabilize cyclin dependent kinase inhibitor 1A (CDKN1A) and CDKN1B proteins.

Cross-resistance of cisplatin selected cells to anti-microtubule agents: Role of general survival mechanisms

Although the first line of therapy for epithelial ovarian cancer typically consists of taxane-platinum combination therapy, many patients develop a platinum-resistant tumor within a year. Several previous studies have looked at this cross-resistance between cisplatin and anti-microtubule drugs, but their findings have been somewhat conflicting. Here, we developed cisplatin-resistant cell lines that are resistant to low and high levels of cisplatin and explored the effects of three anti-microtubule drugs (paclitaxel, vincristine, and colchicine) on the parental and cisplatin-resistant cells. We found that cells resistant to lower levels of cisplatin were no more resistant to anti-microtubule drugs than parental cells, while cells that were resistant to higher levels of cisplatin had a subpopulation of cells that were cross-resistant to anti-microtubule drugs, clarifying discrepancies within the field. We then isolated this subpopulation by applying selective pressure with anti-microtubule drugs and performed RNA sequencing and gene set enrichment analysis to identify resistance mechanisms. This subpopulation was found to express increased levels of pro-survival TNF/NFκB signaling, among other enriched pathways, suggesting that cross-resistance was due to more general survival mechanisms found in the cisplatin-selected cells.

Kinase shRNA screening reveals that TAOK3 enhances microtubule-targeted drug resistance of breast cancer cells via the NF-\u03baB signaling pathway

BackgroundChemotherapy is currently one of the most effective treatments for advanced breast cancer. Anti-microtubule agents, including taxanes, eribulin and vinca-alkaloids are one of the primary major anti-breast cancer chemotherapies; however, chemoresistance remains a problem that is difficult to solve. We aimed to discover novel candidate protein targets to combat chemoresistance in breast cancer.MethodsA lentiviral shRNA-based high-throughput screening platform was designed and developed to screen the global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The phenotypes were confirmed with alternative expression in vitro and in vivo. Molecular mechanisms were investigated using global phosphoprotein arrays and expression microarrays. Global microarray analysis was performed to determine TAOK3 and genes that induced paclitaxel resistance.ResultsA serine/threonine kinase gene, TAOK3, was identified from 724 screened kinase genes. TAOK3 shRNA exhibited the most significant reduction in IC50 values in response to paclitaxel treatment. Ectopic downregulation of TAOK3 resulted in paclitaxel-resistant breast cancer cells sensitize to paclitaxel treatment in vitro and in vivo. The expression of TAOK3 also was correlated to sensitivity to two other anti-microtubule drugs, eribulin and vinorelbine. Our TAOK3-modulated microarray analysis indicated that NF-κB signaling played a major upstream regulation role. TAOK3 inhibitor, CP43, and shRNA of NF-κB both reduced the paclitaxel resistance in TAOK3 overexpressed cells. In clinical microarray databases, high TAOK3 expressed breast cancer patients had poorer prognoses after adjuvant chemotherapy.ConclusionsHere we identified TAOK3 overexpression increased anti-microtubule drug resistance through upregulation of NF-κB signaling, which reduced cell death in breast cancer. Therefore, inhibition of the interaction between TAOK3 and NF-κB signaling may have therapeutic implications for breast cancer patients treated with anti-microtubule drugs.7Jqasva6SWS6RazCnN3hPhVideo abstract