Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

The Microtubule Destabilizer Eribulin Synergizes with STING Agonists to Promote Antitumor Efficacy in Triple-Negative Breast Cancer Models.

Eribulin is a microtubule destabilizer used in the treatment of triple-negative breast cancer (TNBC). Eribulin and other microtubule targeted drugs, such as the taxanes, have shared antimitotic effects, but differ in their mechanism of microtubule disruption, leading to diverse effects on cellular signaling and trafficking. Herein, we demonstrate that eribulin is unique from paclitaxel in its ability to enhance expression of the immunogenic cytokine interferon beta (IFNß) in combination with STING agonists in both immune cells and TNBC models, including profound synergism with ADU-S100 and E7766, which are currently undergoing clinical trials. The mechanism by which eribulin enhances STING signaling is downstream of microtubule disruption and independent of the eribulin-dependent release of mitochondrial DNA. Eribulin did not override the requirement of ER exit for STING activation and did not inhibit subsequent STING degradation; however, eribulin significantly enhanced IRF3 phosphorylation and IFNß production downstream of the RNA sensing pathway that converges on this transcription factor. Additionally, we found that eribulin enhanced the population of activated CD4+ T-cells in vivo when combined with either a STING agonist or tumor, demonstrating the ability to function as an immune adjuvant. We further interrogated the combination of eribulin with ADU-S100 in the MMTV-PyVT spontaneous murine mammary tumor model where we observed significant antitumor efficacy with combination treatment. Together, our findings demonstrate that microtubule targeted chemotherapeutics have distinct immunological effects and that eribulin's ability to enhance innate immune sensing pathways supports its use in combination with immunotherapies, such as STING agonists, for the more effective treatment of TNBC and other malignancies.

Design, Synthesis, and Biological Evaluation of 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines as Microtubule Targeting Agents.

A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and SNAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC50 values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC50 values of 53-125 nM). Additionally, compounds 4-8, 10 and 12-13 circumvented Pgp and ßIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI50 of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma.

A screening program designed to identify natural products with selective cytotoxic effects against cell lines representing different types of pediatric solid tumors led to the identification of altertoxin II as a highly potent and selective cytotoxin against Ewing sarcoma cell lines. Altertoxin II, but not the related compounds altertoxin I and alteichin, was highly effective against every Ewing sarcoma cell line tested, with an average 25-fold selectivity for these cells as compared to cells representing other pediatric and adult cancers. Mechanism of action studies revealed that altertoxin II causes DNA double-strand breaks, a rapid DNA damage response, and cell cycle accumulation in the S phase. Our studies also demonstrate that the potent effects of altertoxin II are partially dependent on the progression through the cell cycle, because the G1 arrest initiated by a CDK4/6 inhibitor decreased antiproliferative potency more than 10 times. Importantly, the cell-type-selective DNA-damaging effects of altertoxin II in Ewing sarcoma cells occur independently of its ability to bind directly to DNA. Ultimately, we found that altertoxin II has a dose-dependent in vivo antitumor efficacy against a Ewing sarcoma xenograft, suggesting that it has potential as a therapeutic drug lead and will be useful to identify novel targets for Ewing-sarcoma-specific therapies.

Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells.

Extracellular vesicles play a central role in intercellular communication and contribute to cancer progression, including the epithelial-to-mesenchymal transition (EMT). Microtubule targeting agents (MTAs) including eribulin and paclitaxel continue to provide significant value in cancer therapy and their abilities to inhibit oncogenic signaling pathways, including eribulin's capacity to reverse EMT are being revealed. Because microtubules are involved in the intracellular trafficking required for the formation and cargo loading of small extracellular vesicles (sEVs), we investigated whether MTA-mediated disruption of microtubule-dependent transport would impact sEV release and their cargo. Eribulin and paclitaxel caused an intracellular accumulation of CD63, a tetraspanin component of sEVs, in late/multivesicular endosomes of triple-negative breast cancer cells, consistent with the disruption of endosomal sorting and exosome cargo loading in these cells. While the concentrations of sEVs released from MTA-treated cells were not significantly altered, levels of CD63 and the CD63-associated cargos, ILK and ß-integrin, were reduced in sEVs isolated from eribulin-treated HCC1937 cells as compared to vehicle or paclitaxel-treated cells. These results show that eribulin can reduce specific sEV cargos, including ILK, a major transducer of EMT in the tumor microenvironment, which may contribute to eribulin's ability to reverse EMT to promote anticancer efficacy.

Yuanhuacine Is a Potent and Selective Inhibitor of the Basal-Like 2 Subtype of Triple Negative Breast Cancer with Immunogenic Potential.

The heterogeneity of triple negative breast cancer (TNBC) has led to efforts to further subtype this disease with the hope of identifying new molecular liabilities and drug targets. Furthermore, the finding that TNBC is the most inherently immunogenic type of breast cancer provides the potential for effective treatment with immune checkpoint inhibitors and immune adjuvants. Thus, we devised a dual screen to identify compounds from natural product extracts with TNBC subtype selectivity that also promote the expression of cytokines associated with antitumor immunity. These efforts led to the identification of yuanhuacine (1) as a potent and highly selective inhibitor of the basal-like 2 (BL2) subtype of TNBC that also promoted an antitumor associated cytokine signature in immune cells. The mechanism of action of yuanhuacine for both phenotypes depends on activation of protein kinase C (PKC), defining a novel target for the treatment of this clinical TNBC subtype. Yuanhuacine showed potent antitumor efficacy in animals bearing BL2 tumors further demonstrating that PKC could function as a potential pharmacological target for the treatment of the BL2 subtype of TNBC.

Eribulin Activates the cGAS-STING Pathway via the Cytoplasmic Accumulation of Mitochondrial DNA.

Microtubule-targeting agents (MTAs), including both microtubule stabilizers and destabilizers are highly effective chemotherapeutic drugs used in the treatment of solid tumors and hematologic malignancies. In addition to the shared ability of all MTAs to block cell cycle progression, growing evidence shows that different agents of this class can also have mechanistically distinct effects on nonmitotic microtubule-dependent cellular processes, including cellular signaling and transport. Herein, we test the biologic hypothesis that MTAs used in the treatment of triple-negative breast cancer (TNBC) can differentially affect innate immune signaling pathways independent of their antimitotic effects. Our data demonstrate that the microtubule destabilizer eribulin, but not the microtubule stabilizer paclitaxel, induces cGAS-STING-dependent expression of interferon-ß in both myeloid and TNBC cells. Activation of the cGAS-STING pathway by eribulin was further found to be mediated by the accumulation of cytoplasmic mitochondrial DNA. Together, these findings provide mechanistic insight into how eribulin can induce innate immune signaling independent of its antimitotic or cytotoxic effects. SIGNIFICANCE STATEMENT: Microtubule-targeting agents (MTAs) are often used in the treatment of breast cancer and have been used in combination with immune checkpoint inhibitors to improve efficacy. Although all clinically approved MTAs share an antimitotic mechanism of action, their distinct effects on interphase microtubules can promote differential downstream signaling consequences. This work shows that the microtubule destabilizer eribulin, but not the microtubule stabilizer paclitaxel, activates the cGAS-STING innate immune signaling pathway through the accumulation of mitochondrial DNA in the cytoplasm.

Potential of substituted quinazolines to interact with multiple targets in the treatment of cancer.

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-ß. Low nanomolar inhibition of EGFR was observed for 1-3 and 9-10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

The 3-D conformational shape of N-naphthyl-cyclopenta[d]pyrimidines affects their potency as microtubule targeting agents and their antitumor activity.

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6'-methoxynaphthyl-1'-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5'-methoxynaphthyl-2'-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.

An Integrated Strategy for the Detection, Dereplication, and Identification of DNA-Binding Biomolecules from Complex Natural Product Mixtures.

A fundamental factor in natural product drug discovery programs is the necessity to identify the active component(s) from complex chemical mixtures. Whereas this has traditionally been accomplished using bioassay-guided fractionation, we questioned whether alternative techniques could supplement and, in some cases, even supplant this approach. We speculated that a combination of ligand-fishing methods and modern analytical tools (e.g., LC-MS and online natural product databases) offered a route to enhance natural product drug discovery. Herein, a candidate solution referred to as the lickety-split ligand-affinity-based molecular angling system (LLAMAS) is described. This approach utilizes an ultrafiltration-based LC-PDA-MS/MS-guided DNA-binding assay in combination with the (i) Global Natural Products Social Molecular Networking, (ii) Dictionary of Natural Products, and (iii) SciFinder platforms to identify DNA binders in complex chemical mixtures. LLAMAS was initially vetted in tests using known small-molecule DNA binders and then optimized to a 96-well plate-based format. A set of 332 plant samples used in traditional Chinese medicine was screened for DNA-binding activity with LLAMAS, resulting in the identification of seven DNA-binding molecules, including berberine (12), palmatine (13), coptisine (14), fangchinoline (15), tetrandrine (16), daurisoline (17), and dauricine (18). These results demonstrate that LLAMAS is an effective natural product discovery platform for the efficient identification and dereplication of DNA-binding molecules from complex mixtures.

CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells.

There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17ß-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.

Triple-Negative Breast Cancer Cells Exhibit Differential Sensitivity to Cardenolides from Calotropis gigantea.

Triple-negative breast cancers (TNBC) are aggressive and heterogeneous cancers that lack targeted therapies. We implemented a screening program to identify new leads for subgroups of TNBC using diverse cell lines with different molecular drivers. Through this program, we identified an extract from Calotropis gigantea that caused selective cytotoxicity in BT-549 cells as compared to four other TNBC cell lines. Bioassay-guided fractionation of the BT-549 selective extract yielded nine cardenolides responsible for the selective activity. These included eight known cardenolides and a new cardenolide glycoside. Structure-activity relationships among the cardenolides demonstrated a correlation between their relative potencies toward BT-549 cells and Na+/K+ ATPase inhibition. Calotropin, the compound with the highest degree of selectivity for BT-549 cells, increased intracellular Ca2+ in sensitive cells to a greater extent than in the resistant MDA-MB-231 cells. Further studies identified a second TNBC cell line, Hs578T, that is also highly sensitive to the cardenolides, and mechanistic studies were conducted to identify commonalities among the sensitive cell lines. Experiments showed that both cardenolide-sensitive cell lines expressed higher mRNA levels of the Na+/Ca2+ exchanger NCX1 than resistant TNBC cells. This suggests that NCX1 could be a biomarker to identify TNBC patients that might benefit from the clinical administration of a cardiac glycoside for anticancer indications.

Leucinostatins from Ophiocordyceps spp. and Purpureocillium spp. Demonstrate Selective Antiproliferative Effects in Cells Representing the Luminal Androgen Receptor Subtype of Triple Negative Breast Cancer.

The structures of four leucinostatin analogues (1-4) from Ophiocordyceps spp. and Purpureocillium spp. were determined together with six known leucinostatins [leucinostatins B (5), A (6), B2 (7), A2 (8), F (9), and D (10)]. The structures of the metabolites were established using a combination of analytical methods including HRESIMS and MS/MS experiments, 1D and 2D NMR spectroscopy, chiral HPLC, and advanced Marfey's analysis of the acid hydrolysate, as well as additional empirical and chemical methods. Compounds 1-10 were evaluated for their biological effects on triple negative breast cancer (TNBC) cells. Leucinostatins 1-10 showed selective cytostatic activities in MDA-MB-453 and SUM185PE cells representing the luminal androgen receptor subtype of TNBC. This selective activity motivated further investigation into the mechanism of action of leucinostatin B (5). The results demonstrate that this peptidic fungal metabolite rapidly inhibits mTORC1 signaling in leucinostatin-sensitive TNBC cell lines, but not in leucinostatin-resistant cells. Leucinostatins have been shown to repress mitochondrial respiration through inhibition of the ATP synthase, and we demonstrated that both the mTORC1 signaling and LAR-selective activities of 5 were recapitulated by oligomycin. Thus, inhibition of the ATP synthase with either leucinostatin B or oligomycin is sufficient to selectively impede mTORC1 signaling and inhibit the growth of LAR-subtype cells.

Using the Cancer Dependency Map to Identify the Mechanism of Action of a Cytotoxic Alkenyl Derivative from the Fruit of Choerospondias axillaris.

An extract prepared from the fruit of Choerospondias axillaris exhibited differential cytotoxic effects when tested in a panel of pediatric cancer cell lines [Ewing sarcoma (A-673), rhabdomyosarcoma (SJCRH30), medulloblastoma (D283), and hepatoblastoma (Hep293TT)]. Bioassay-guided fractionation led to the purification of five new hydroquinone-based metabolites, choerosponols A-E (1-5), bearing unsaturated hydrocarbon chains. The structures of the natural products were determined using a combination of 1D and 2D NMR, HRESIMS, ECD spectroscopy, and Mosher ester analyses. The purified compounds were evaluated for their antiproliferative and cytotoxic activities, revealing that 1, which contains a benzofuran moiety, exhibited over 50-fold selective antiproliferative activity against Ewing sarcoma and medulloblastoma cells with growth inhibitory (GI50) values of 0.19 and 0.07 µM, respectively. The effects of 1 were evaluated in a larger panel of cancer cell lines, and these data were used in turn to interrogate the Project Achilles cancer dependency database, leading to the identification of the MCT1 transporter as a functional target of 1. These data highlight the utility of publicly available cancer dependency databases such as Project Achilles to facilitate the identification of the mechanisms of action of compounds with selective activities among cancer cell lines, which can be a major challenge in natural products drug discovery.

Eribulin rapidly inhibits TGF-ß-induced Snail expression and can induce Slug expression in a Smad4-dependent manner.

BACKGROUND: Evidence shows that the anticancer effects of microtubule targeting agents are not due solely to their antimitotic activities but also their ability to impair microtubule-dependent oncogenic signalling. METHODS: The effects of microtubule targeting agents on regulators of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) were evaluated in breast cancer cell lines using high content imaging, gene and protein expression, siRNA-mediated knockdown and chromatin immunoprecipitation. RESULTS: Microtubule targeting agents rapidly and differentially alter the expression of Snail and Slug, key EMT-promoting transcription factors in breast cancer. Eribulin, vinorelbine and in some cases, ixabepalone, but not paclitaxel, inhibited TGF-ß-mediated Snail expression by impairing the microtubule-dependent nuclear localisation of Smad2/3. In contrast, eribulin and vinorelbine promoted a TGF-ß-independent increase in Slug in cells with low Smad4. Mechanistically, microtubule depolymerisation induces c-Jun, which consequently increases Slug expression in cells with low Smad4. CONCLUSION: These results identify a mechanism by which eribulin-mediated microtubule disruption could reverse EMT in preclinical models and in patients. Furthermore, high Smad4 levels could serve as a biomarker of this response. This study highlights that microtubule targeting drugs can exert distinct effects on the expression of EMT-regulating transcription factors and that identifying differences among these drugs could lead to their more rational use.

Correction: Regulation of E-cadherin localization by microtubule targeting agents: rapid promotion of cortical E-cadherin through p130CAS/Src inhibition by eribulin.

[This corrects the article DOI: 10.18632/oncotarget.23798.].

Triple-negative breast cancer cell line sensitivity to englerin A identifies a new, targetable subtype.

PURPOSE: Triple-negative breast cancers (TNBCs) represent a heterogeneous group of tumors. The lack of targeted therapies combined with the inherently aggressive nature of TNBCs results in a higher relapse rate and poorer overall survival. We evaluated the heterogeneity of TNBC cell lines for TRPC channel expression and sensitivity to cation-disrupting drugs. METHODS: The TRPC1/4/5 agonist englerin A was used to identify a group of TNBC cell lines sensitive to TRPC1/4/5 activation and intracellular cation disruption. Quantitative RT-PCR, the sulforhodamine B assay, pharmacological inhibition, and siRNA-mediated knockdown approaches were employed. Epifluorescence imaging was performed to measure intracellular Ca2+ and Na+ levels. Mitochondrial membrane potential changes were monitored by confocal imaging. RESULTS: BT-549 and Hs578T cells express high levels of TRPC4 and TRPC1/4, respectively, and are exquisitely, 2000- and 430-fold, more sensitive to englerin A than other TNBC cell lines. While englerin A caused a slow Na+ and nominal Ca2+ accumulation in Hs578T cells, it elicited rapid increases in cytosolic Ca2+ levels that triggered mitochondrial depolarization in BT-549 cells. Interestingly, BT-549 and Hs578T cells were also more sensitive to digoxin as compared to other TNBC cell lines. Collectively, these data reveal TRPC1/4 channels as potential biomarkers of TNBC cell lines with dysfunctional mechanisms of cation homeostasis and therefore sensitivity to cardiac glycosides. CONCLUSIONS: The sensitivity of BT-549 and Hs578T cells to englerin A and digoxin suggests a subset of TNBCs are highly susceptible to cation disruption and encourages investigation of TRPC1 and TRPC4 as potential new biomarkers of sensitivity to cardiac glycosides.

In Situ Ring Contraction and Transformation of the Rhizoxin Macrocycle through an Abiotic Pathway.

A Rhizopus sp. culture containing an endosymbiont partner ( Burkholderia sp.) was obtained through a citizen-science-based soil-collection program. An extract prepared from the pair of organisms exhibited strong inhibition of Ewing sarcoma cells and was selected for bioassay-guided fractionation. This led to the purification of rhizoxin (1), a potent antimitotic agent that inhibited microtubule polymerization, along with several new (2-5) and known (6) analogues of 1. The structures of 2-6 were established using a combination of NMR data analysis, while the configurations of the new stereocenters were determined using ROESY spectroscopy and comparison of GIAO-derived and experimental data for NMR chemical shift and 3 JHH coupling values. Whereas compound 1 showed modest selectivity for Ewing sarcoma cell lines carrying the EWSR1/ FLI1 fusion gene, the other compounds were determined to be inactive. Chemically, compound 2 stands out from other rhizoxin analogues because it is the first member of this class that is reported to contain a one-carbon-smaller 15-membered macrolactone system. Through a combination of experimental and computational tests, we determined that 2 is likely formed via an acid-catalyzed Meinwald rearrangement from 1 because of the mild acidic culture environment created by the Rhizopus sp. isolate and its symbiont.

Microtubule-Targeting Drugs: More than Antimitotics.

Nature has yielded numerous compounds that bind to tubulin/microtubules and disrupt microtubule function. Even with the advent of targeted therapies for cancer, natural products and their derivatives that target microtubules are some of the most effective drugs used in the treatment of solid tumors and hematological malignancies. For decades, these drugs were thought to work solely through their ability to inhibit mitosis. Accumulating evidence demonstrates that their actions are much more complex, in that they also have significant effects on microtubules in nondividing cells that inhibit a diverse range of signaling events important for carcinogenesis. The abilities of these drugs to inhibit oncogenic signaling likely underlies their efficacy, especially in solid tumors. In this review, we describe the role of microtubules in cells, the proliferation paradox of cells in culture as compared to cancers in patients, and evidence that microtubule-targeting drugs inhibit cellular signaling pathways important for tumorigenesis. The potential mechanisms behind differences in the clinical indications and efficacy of these natural-product-derived drugs are also discussed. Microtubules are an important target for structurally diverse natural products, and a fuller understanding of the mechanisms of action of these drugs will promote their optimal use.