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α-Tubulin detyrosination, Δ2, and acetylation can be uncoupled in cancer cells. (A) Correlation between TTL protein (our screen) and mRNA (z scores; CellMiner database) levels in NCI-60 cancer cells. (B−D) Correlation between levels of α-tubulin detyrosination and Δ2-tubulin, TTL, and α-tubulin acetylation in NCI-60 cancer cells. (B9−D9) Proportion of cancer cell lines of the panel with high and low Δ2-tubulin, TTL, and α-tubulin acetylation in the two groups with high and low α-tubulin detyrosination. From the tubulin PTM level values, the fourth and fifth quintiles (>60th percentile) were defined as the high group and the first and second quintiles (<40th percentile) as the low group. Spearman correlation coefficient (r) and P values are indicated in the graphs.
Source publication
α/β-Tubulin posttranslational modifications (PTMs) generate microtubule diversity, but whether they account for cancer cell resistance to microtubule-targeting drugs remains unknown. Here, we performed a pilot dissection of the “cancer tubulin code” using the NCI-60 cancer cell panel. We found that acetylated, detyrosinated, and ∆2-α-tubulin that t...
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... S1) and compared its expression with the respective TTL mRNA levels obtained from the CellMiner database, a public web-based suite compiling transcriptomic data from the NCI-60 cancer cell panel (Reinhold et al., 2012). A strong correlation (r = 0.8272, P <0.0001) between TTL protein and mRNA levels among the different NCI-60 cell lines was found (Fig. 2 A), thereby validating our approach. To compare different cancer cell lines between immunoblots, the leukemia cell line HL-60, which showed intermediate levels of α-tubulin acetylation and detyrosination, was included in each immunoblot as an internal reference (Fig. S1). The nontransformed human telomerase reverse transcriptase ...
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... lines (including within the same tissue) that in some cases was higher than 10-fold (Fig. 1, A−D). Importantly, these differences could not be explained by cell cycle regulation of tubulin PTMs and TTL or cell confluency, since their respective levels did not change more than 1.5-fold throughout the cell cycle or at distinct cell density stages (Fig. S2, ...
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... and PC-3 (prostate), showing no obvious relationship between these PTMs (Fig. 1, B and C, Fig. 2 B, and Fig. S1). In general, α-tubulin detyrosination showed a poor correlation with TTL protein expression (negative correlation) and α-tubulin acetylation levels (positive correlation) among the different cancer cell lines of the NCI-60 panel (Fig. 2, C and D). Interestingly, a more detailed analysis focusing on the cell lines with higher α-tubulin detyrosination levels (>60th percentile) revealed that 68.4% were found to express low TTL protein levels, 76.5% showed high Δ2-tubulin, and 61.1% had high α-tubulin acetylation levels. In agreement, for the cell lines with lower (<40th ...
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... 68.4% were found to express low TTL protein levels, 76.5% showed high Δ2-tubulin, and 61.1% had high α-tubulin acetylation levels. In agreement, for the cell lines with lower (<40th percentile) α-tubulin detyrosination levels, 78.6% expressed high TTL protein levels, 76.5% had low Δ2-α-tubulin, and 64.3% showed low α-tubulin acetylation levels (Fig. 2, B−D). Next, we investigated whether α-tubulin detyrosination and TTL expression is a function of the expression of the α-tubulin carboxypeptidases VASH1, VASH2, and MATCAP using mRNA levels obtained from the CellMiner database (Fig. S3, A−M). We only found a weak correlation between α-tubulin detyrosination and VASH2 expression ( Fig. S3 ...
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... Timelapse microscope (Leica Microsystems), equipped with a Hamamatsu FLASH4.0 camera (Hamamatsu), using the objective HCX PL FLUOTAR L 20×/0.40 CORR Ph1 (Leica Microsystems). Images were analyzed in ImageJ software. Online supplemental material Fig. S1 shows representative immunoblots of the screen of tubulin PTMs in the NCI-60 cancer cell panel. Fig. S2 shows that tubulin PTMs only vary slightly throughout the cell cycle or distinct cell confluency. Fig. S3 shows that VASH2 expression weakly correlates with α-tubulin detyrosination, while VASH1 and MATCAP weakly correlates with TTL levels. Fig. S4 shows that α-tubulin detyrosination, Δ2, TTL, and MCAK levels do not correlate with ...
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... S1) and compared its expression with the respective TTL mRNA levels obtained from the CellMiner™ database, a public web-based suite compiling transcriptomic data from the NCI-60 cancer cell panel (Reinhold et al., 2012). A strong correlation (r=0.8272, p<0.0001) between TTL protein and mRNA levels among the different NCI-60 cell lines was found ( Fig. 2A), thereby validating our approach. To compare different cancer cell lines between immunoblots, the leukemia cell line HL-60, which showed intermediate levels of α-tubulin acetylation and detyrosination, was included in each immunoblot as an internal reference (Fig. S1). The human nontransformed hTERT-immortalized RPE1 cell line was also ...
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... lines (including within the same tissue) that in some cases was higher than 10-fold (Fig. 1A-D). Importantly, these differences could not be explained by cell cycle regulation of tubulin PTMs and TTL or cell confluency, since their respective levels did not change more than 1.5-fold throughout the cell cycle or at distinct cell density stages ( Fig. ...
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... KM12 (colon) and PC-3 (prostate), showing no obvious relationship between these PTMs (Fig. 1B, C, Fig. 2B and Fig. S1). In general, α-tubulin detyrosination showed a poor correlation with TTL protein expression (negative correlation) and α-tubulin acetylation levels (positive correlation) among the different cancer cell lines of the NCI-60 panel (Fig. 2C, D). Interestingly, a more detailed analysis focusing on the cell lines with higher α-tubulin detyrosination levels (>60 th percentile), revealed that 68.4% were found to express low TTL protein levels, 76.5% showed high Δ2-tubulin and 61.1% had high α-tubulin acetylation levels. In agreement, for the cell lines with lower (<40 th ...
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... 68.4% were found to express low TTL protein levels, 76.5% showed high Δ2-tubulin and 61.1% had high α-tubulin acetylation levels. In agreement, for the cell lines with lower (<40 th percentile) α-tubulin detyrosination levels, 78.6% expressed high TTL protein levels, 76.5% had low Δ2-α-tubulin and 64.3% showed low α-tubulin acetylation levels ( Fig. 2B'-D'). Next, we investigated whether α-tubulin detyrosination and TTL expression is a function of the expression of the α-tubulin carboxypeptidases VASH1, VASH2 and MATCAP using mRNA levels obtained from the CellMiner™ database ( Fig. S3A-M). We only found a weak correlation between α-tubulin detyrosination and VASH2 expression (Fig. S3C), ...
Citations
... Paclitaxel can attach to microtubules and stabilize them by encouraging the assembly of αand β-tubulin subunits. It interferes with microtubule dynamics by reducing the shortening of Tubulin polymer, resulting in the destruction of the cell's ability to divide those arrests at G2/M phase and cell apoptosis [21,22]. Unfortunately, most BC patients have inherited or acquired resistance to paclitaxel treatments and relapsed [23]. ...
Paclitaxel, a natural anticancer drug, is widely recognized and extensively utilized in the treatment of breast cancer (BC). However, it may lead to certain side effects or drug resistance. Fortunately, combination therapy with another anti-tumor agent has been explored as an option to improve the efficacy of paclitaxel in the treatment of BC. Herein, we first evaluated the synergistic effects of paclitaxel and flubendazole through combination index (CI) calculations. Secondly, flubendazole was demonstrated to synergize paclitaxel-mediated BC cell killing in vitro and in vivo. Moreover, we discovered that flubendazole could reverse the drug resistance of paclitaxel-resistant BC cells. Mechanistically, flubendazole was demonstrated to enhance the inhibitory effect of paclitaxel via HIF1α/PI3K/AKT signaling pathways. Collectively, our findings demonstrate the effectiveness of flubendazole in combination with paclitaxel for treating BC, providing an insight into exploiting more novel combination therapies for BC in the future.
... Abnormal levels of α-tubulin detyrosination have been correlated with various pathological states, such as increased tumor aggressiveness [10] , the onset of neuronal disorders [11] , heart failure [12] , and cardiomyopathy [13] . This post-translational modi cation, mediated by microtubule carboxypeptidase, is also intricately connected to cancer drug resistance and progression [14,15,16] . ...
... reported that have elucidated that AGBL2 contributes to the growth of cancer cells by regulating IRGM-controlled autophagy and augmenting Aurora A activity. Zhang et al. [14] have marked AGBL2 as an independent prognostic determinant for breast cancer, while similar conclusions were reached by He et al [37] . for ovarian carcinoma. ...
... Surprisingly, we detected high AGBL2 expression in RCC cells' nucleus. Based on the ndings of Lopes et al [14] , we theorize that AGBL2-mediated α-tubulin detyrosination could affect kinetochore-telomeres (KTs) and MTs complexes, leading to impaired mitotic error correction and an increased frequency of mitotic errors. These mechanisms might play critical roles in the initiation and progression of renal cancer tumorigenesis. ...
Background
AGBL2's role in tumorigenesis and cancer progression has been reported in several cancer studies, and it is closely associated with α-tubulin detyrosination. The roles of AGBL2 and α-tubulin detyrosination in renal cell carcinoma (RCC) pathogenesis remain unclear and require further investigation.
Methods
In this study, we conducted an analysis of AGBL2 expression differences between renal clear cell carcinoma tissues and normal tissues using data from The Cancer Genome Atlas (TCGA). We performed a comprehensive prognostic analysis of AGBL2 in Kidney Renal Clear Cell Carcinoma(KIRC) using univariate and multivariate Cox regression. Based on the results of the Cox analysis, we constructed a prognostic model to assess its predictive capabilities. Receiver Operating Characteristic (ROC) analysis confirmed the diagnostic value of AGBL2 in renal cancer. We conducted further validation by analyzing cancer tissue samples and renal cancer cell lines, which confirmed the role of AGBL2 in promoting RCC cell proliferation and migration through in vitro experiments. Additionally, we verified the impact of AGBL2's detyrosination on α-tubulin using the tubulin carboxypeptidase (TCP) inhibitor parthenolide. Finally, we performed sequencing analysis on AGBL2 knockdown 786-O cells to investigate the correlation between AGBL2, immune infiltration, and AKT phosphorylation. Moreover, we experimentally demonstrated the enhancing effect of AGBL2 on AKT phosphorylation.
Results
TCGA analysis revealed a significant increase in AGBL2 expression in RCC patients, which was correlated with poorer overall survival (OS), disease-specific survival (DSS), and progression-free intervals (PFI). According to the analysis results, we constructed column-line plots to predict the 1-, 3-, and 5-year survival outcomes in RCC patients. Additionally, the calibration plots assessing the model's performance exhibited favorable agreement with the predicted outcomes. And the ROC curves showed that AGBL2 showed good diagnostic performance in KIRC (AUC = 0.836)). Cell phenotyping assays revealed that AGBL2 knockdown in RCC cells significantly inhibited cell proliferation and migration. Conversely, overexpression of AGBL2 resulted in increased cell proliferation and migration in RCC cells. We observed that AGBL2 is predominantly located in the nucleus and can elevate the detyrosination level of α-tubulin in RCC cells. Moreover, the enhancement of RCC cell proliferation and migration by AGBL2 was partially inhibited after treatment with the TCP inhibitor parthenolide. Analysis of the sequencing data revealed that AGBL2 is associated with a diverse array of biological processes, encompassing signal transduction and immune infiltration. Interestingly, AGBL2 expression exhibited a negative correlation with the majority of immune cell infiltrations. Additionally, AGBL2 was found to enhance the phosphorylation of AKT in RCC cells.
Conclusion
Our study suggests that AGBL2 fosters RCC cell proliferation and migration by enhancing α-tubulin detyrosination. Moreover, elevated AGBL2 expression increases phosphorylation of AKT in RCC cells
... Combining MCAK inhibitors with taxanes could significantly reduce these side effects, improving patient outcomes and quality of life. MCAK function has been further linked with taxane cytotoxicity through tubulin post-translational modifications that inhibit the enzyme [76]. As a mediator of DNA repair, MCAK inhibition may synergize with taxanes to increase DNA damage in the cell, as taxanes have been shown to synergize with certain DNA damaging agents [77]. ...
Standard of care for triple-negative breast cancer (TNBC) involves the use of microtubule poisons such as paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug-resistant tumors. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple-negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50 k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target.
... Although both of these modifications are associated with "stable" microtubules [2,13,52] and often occur on the same subset of microtubules [52,53,86,87], we have discovered that specific cellular conditions select for one PTM over the other, indicating that these modifications can be separated and utilized for specific cellular purposes. Our results are consistent with prior studies that show acetylation and detyrosination modifications are not always tightly linked, such as in cancer cells, in migrating cells, and upon microtubule regrowth after depolymerization [52,88,89]. Therefore, although these modifications are not mutually exclusive, we show here that MAP7 association has the ability to promote acetylation and limit detyrosination, thus decoupling them. ...
Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of acute perturbations in cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs), unraveling the molecular underpinnings of cellular adaptation via the microtubule cytoskeleton. We find that cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association, without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification, and find that MAP7 promotes acetylation by biasing the conformation of the microtubule lattice, and directly inhibits detyrosination. Acetylation and detyrosination can therefore be decoupled and utilized for distinct cellular purposes. Our data reveal that the MAP code dictates the tubulin code, resulting in remodeling of the microtubule cytoskeleton and alteration of intracellular transport as an integrated mechanism of cellular adaptation.
... Combining MCAK inhibitors with taxanes could significantly reduce these side effects, improving patient outcomes and quality of life. MCAK function has been further linked with taxane cytotoxicity through tubulin post-translational modifications that inhibit the enzyme [76]. As a mediator of DNA repair, MCAK inhibition may synergize with taxanes to increase DNA damage in the cell, as taxanes have been shown to synergize with certain DNA damaging agents [77]. ...
Standard of care for triple negative breast cancer (TNBC) involves the use of microtubule poisons like paclitaxel, which are proposed to work by inducing lethal levels of aneuploidy in tumor cells. While these drugs are initially effective in treating cancer, dose-limiting peripheral neuropathies are common. Unfortunately, patients often relapse with drug resistant tumors. Identifying agents against targets that limit aneuploidy may be a valuable approach for therapeutic development. One potential target is the microtubule depolymerizing kinesin, MCAK, which limits aneuploidy by regulating microtubule dynamics during mitosis. Using publicly available datasets, we found that MCAK is upregulated in triple negative breast cancer and is associated with poorer prognoses. Knockdown of MCAK in tumor-derived cell lines caused a two- to five-fold reduction in the IC 50 for paclitaxel, without affecting normal cells. Using FRET and image-based assays, we screened compounds from the ChemBridge 50k library and discovered three putative MCAK inhibitors. These compounds reproduced the aneuploidy-inducing phenotype of MCAK loss, reduced clonogenic survival of TNBC cells regardless of taxane-resistance, and the most potent of the three, C4, sensitized TNBC cells to paclitaxel. Collectively, our work shows promise that MCAK may serve as both a biomarker of prognosis and as a therapeutic target.
Simple Summary
Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype with few treatment options available. Standard of care for TNBC involves the use of taxanes, which are initially effective, but dose limiting toxicities are common, and patients often relapse with resistant tumors. Specific drugs that produce taxane-like effects may be able to improve patient quality of life and prognosis. In this study we identify three novel inhibitors of the Kinesin-13 MCAK. MCAK inhibition induces aneuploidy; similar to cells treated with taxanes. We demonstrate that MCAK is upregulated in TNBC and is associated with poorer prognoses. These MCAK inhibitors reduce the clonogenic survival of TNBC cells, and the most potent of the three inhibitors, C4, sensitizes TNBC cells to taxanes, similar to the effects of MCAK knockdown. This work will expand the field of precision medicine to include aneuploidy-inducing drugs that have the potential to improve patient outcomes.
... Noteworthy, the significant increase in cell length was independent of the width of the line micropattern (SI Appendix, Fig. S1C). Importantly, changes in detyrosination levels achieved by siRNA-mediated depletion of VASHs and TTL in RPE-1 cells did not affect acetylated tubulin levels (SI Appendix, Fig. S1A), in line with recent observations of mechanistic uncoupling of αtubulin acetylation and detyrosination (56). ...
To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.
... Therefore, although it can be taken from some studies that αTAT1/HDAC6-dependent control of tubulin acetylation levels may have a key role in the regulation of cancer cell migration, invasion, and cancer metastasis, recent contradictory results have shown that further research is needed to clarify the role of α-tubulin acetylation in cancer. Additionally, a recent study using the NCI-60 cancer cell panel showed that, although high levels of acetylated α-tubulin correlate with, they were not required for, taxol cytotoxicity [390]. ...
... For example, Rho GTPases, critical molecules in actin cytoskeleton reorganization, mediate the downregulation of HDAC6 [405,413], and vimentin, an intermediate filaments protein, decreases tubulin acetylation levels via αTAT1 degradation [411]. Illustrating this are the heterogeneous tubulin acetylation patterns in different cell types and also in distinct cancer cells from different origins [390]. Additionally, the consequences of tubulin acetylation will be modulated by the content (identity and abundance) of MAPs and motor proteins in each cell type. ...
Microtubules (MTs), dynamic polymers of α/β-tubulin heterodimers found in all eukaryotes, are involved in cytoplasm spatial organization, intracellular transport, cell polarity, migration and division, and in cilia biology. MTs functional diversity depends on the differential expression of distinct tubulin isotypes and is amplified by a vast number of different post-translational modifications (PTMs). The addition/removal of PTMs to α- or β-tubulins is catalyzed by specific enzymes and allows combinatory patterns largely enriching the distinct biochemical and biophysical properties of MTs, creating a code read by distinct proteins, including microtubule-associated proteins (MAPs), which allow cellular responses. This review is focused on tubulin-acetylation, whose cellular roles continue to generate debate. We travel through the experimental data pointing to α-tubulin Lys40 acetylation role as being a MT stabilizer and a typical PTM of long lived MTs, to the most recent data, suggesting that Lys40 acetylation enhances MT flexibility and alters the mechanical properties of MTs, preventing MTs from mechanical aging characterized by structural damage. Additionally, we discuss the regulation of tubulin acetyltransferases/desacetylases and their impacts on cell physiology. Finally, we analyze how changes in MT acetylation levels have been found to be a general response to stress and how they are associated with several human pathologies.
Cancer treatment faces many hurdles and resistance is one among them. Anti-cancer treatment strategies are evolving due to innate and acquired resistance capacity, governed by genetic, epigenetic, proteomic, metabolic, or microenvironmental cues that ultimately enable selected cancer cells to survive and progress under unfavorable conditions. Although the mechanism of drug resistance is being widely studied to generate new target-based drugs with better potency than existing ones. However, due to the broader flexibility in acquired drug resistance, advanced therapeutic options with better efficacy need to be explored. Combination therapy is an alternative with a better success rate though the risk of amplified side effects is commonplace. Moreover, recent groundbreaking precision immune therapy is one of the ways to overcome drug resistance and has revolutionized anticancer therapy to a greater extent with the only limitation of being individual-specific and needs further attention. This review will focus on the challenges and strategies opted by cancer cells to withstand the current therapies at the molecular level and also highlights the emerging therapeutic options -like immunological, and stem cell-based options that may prove to have better potential to challenge the existing problem of therapy resistance.
