Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling ...of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal‐associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease.
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This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue‐24
The use of β-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether β-blockers could be used ...in combination with chemotherapy for the treatment of neuroblastoma.
Seven β-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma.
Three β-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. β-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, β-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, β-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01).
β-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.
Neuroblastoma, the most common solid tumor of young children, frequently presents with aggressive metastatic disease and for these children the 5-year survival rates are dismal. Metastasis, the ...movement of cancer cells from one site to another, involves remodeling of the cytoskeleton including altered microtubule dynamics. The microtubule-destabilizing protein, stathmin, has recently been shown to mediate neuroblastoma metastasis although precise functions remain poorly defined. In this study we investigated stathmin's contribution to the metastatic process and potential mechanism(s) by which it exerts these effects. Stathmin suppression significantly reduced neuroblastoma cell invasion of 3D tumor spheroids into an extracellular matrix. Moreover, inhibiting stathmin expression significantly reduced transendothelial migration in two different neuroblastoma cell lines in vitro. Inhibition of ROCK, a key regulator of cell migration, in neuroblastoma cells highlighted that stathmin regulates transendothelial migration through ROCK signaling. Reduced stathmin expression in neuroblastoma cells significantly increased the activation of the RhoA small GTPase. Notably, re-expression of either wild type or a phospho-mimetic stathmin mutant (4E) made defective in tubulin binding returned cell migration and transendothelial migration back to control levels, indicating that stathmin may influence these processes in neuroblastoma cells independent of tubulin binding. Finally, stathmin suppression in neuroblastoma cells significantly reduced whole body, lung, kidney and liver metastases in an experimental metastases mouse model. In conclusion, stathmin suppression interferes with the metastatic process via RhoA/ROCK signaling in neuroblastoma cells. These findings highlight the importance of stathmin to the metastatic process and its potential as a therapeutic target for the treatment of neuroblastoma.
Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA ...delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes βIII-tubulin, Polo-Like Kinase 1 (PLK1) which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of βIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.
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Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in ...this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.
The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with ...glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.
Abstract Pancreatic cancer (PC) is a lethal disease which is characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. βIV-tubulin is one such component ...that has two isotypes—βIVa and βIVb. βIVa and βIVb isotypes only differ in two amino acids at their C-terminus. Studies have implicated βIVa-tubulin or βIVb-tubulin expression with chemoresistance in prostate, breast, ovarian and lung cancer. However, no studies have examined the role of βIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity. We aimed to determine the role of βIVa- or βIVb-tubulin on PC growth and chemosensitivity. PC cells (MiaPaCa-2, HPAF-II, AsPC1) were treated with siRNA (control, βIVa-tubulin or βIVb-tubulin). The ability of PC cells to form colonies in the presence or absence of chemotherapy was measured by clonogenic assays. Inhibition of βIVa-tubulin in PC cells had no effect chemosensitivity. In contrast, inhibition of βIVb-tubulin in PC cells sensitized to vinca alkaloids (Vincristine, Vinorelbine and Vinblastine), which was accompanied by increased apoptosis and enhanced cell cycle arrest. We show for the first time that βIVb-tubulin, but not βIVa-tubulin, plays a role in regulating vinca alkaloid chemosensitivity in PC cells. The results from this study suggest βIVb-tubulin may be a novel therapeutic target and predictor of vinca alkaloid sensitivity for PC and warrants further investigation.
Natural product drugs that target the tubulin/microtubule system remain an important component in the therapeutic arsenal to treat many types of malignancies. Agents such as the taxanes and vinca ...alkaloids bind to beta-tubulin and disrupt microtubule dynamics by inducing a potent mitotic block and subsequent cell death. Understanding why certain cancers do not respond to treatment or develop resistance has been the subject of numerous studies in recent years. An increasing body of evidence suggests that alterations in the drug target, such as tubulin mutations, altered microtubule dynamics, altered tubulin isotype expression, and modifications in microtubule regulatory proteins, are key mechanisms of antimicrotubule drug resistance. In addition, recent work indicates that other cytoskeletal proteins that can regulate microtubule dynamics through signaling or structural interactions may be important determinants of antimicrotubule resistance. As our understanding of drug action and resistance mechanisms has increased, we can now begin to exploit these to design strategies that overcome, or counteract resistance, hence improving the efficacy of antimicrotubule agents for the treatment of cancer. This review highlights the major areas of investigation as they relate to the tubulin/microtubule system and discusses opportunities that potentially exist for improved therapeutic benefit in the treatment of drug resistant disease.
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