David M. Helfman

Cytoskeletal changes in cell transformation and tumorigenesis

Research during the past couple of years has provided important new information as to how the actin cytoskeleton contributes to growth control in both normal and transformed cells. The cytoskeleton can no longer be viewed as simply a structural framework playing a role in cell shape and motile events such as cell movement, intracellular transport, contractile-ring formation and chromosome movement. More recent experiments show that the cytoskeleton plays a critical role in the regulation of various cellular processes linked to transformation including proliferation, contact inhibition, anchorage-independent cell growth, and apoptosis.

FUNCTIONAL PROPERTIES OF NON-MUSCLE TROPOMYOSIN ISOFORMS

Tropomyosins are a family of actin filament binding proteins. They have been identified in many organisms, including yeast, nematodes, Drosophila, birds and mammals. In metazoans, different forms of tropomyosin are characteristic of specific cell types. Most non-muscle cells, such as fibroblasts, express five to eight isoforms of tropomyosins. The various isoforms exhibit distinct biochemical properties that appear to be required for specific cellular functions.

Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon.

ABSTRACT Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

RSK1 drives p27Kip1 phosphorylation at T198 to promote RhoA inhibition and increase cell motility.

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G1. As for many kinase–substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G1. Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.

Tropomyosin Localization Reveals Distinct Populations of Microfilaments in Neurites and Growth Cones

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

Post-transcriptional down-regulation of ROCKI/Rho-kinase through an MEK-dependent pathway leads to cytoskeleton disruption in Ras-transformed fibroblasts.

Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.

Alterations in tropomyosin isoform expression in human transitional cell carcinoma of the urinary bladder

Previous studies of transformed rodent fibroblasts have suggested that specific isoforms of the actin‐binding protein tropomyosin (TM) could function as suppressors of transformation, but an analysis of TM expression in patient tumor tissue is limited. The purpose of our study was to characterize expression of the different TM isoforms in human transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. We found that TM1 and TM2 protein levels were markedly reduced and showed >60% reduction in 61% and 55% of tumor samples, respectively. TM5, which was expressed at very low levels in normal bladder mucosa, exhibited aberrant expression in 91% of tumor specimens. The Western blot findings were confirmed by immunohistochemical analysis in a number of tumors. We then investigated the mechanism underlying TM expression deregulation, in the T24 human bladder cancer cell line. We showed that levels of TM1, TM2 and TM3 are reduced in T24 cells, but significantly upregulated by inhibition of the mitogen‐activated protein kinase‐signaling pathway. In addition, inhibition of this pathway was accompanied by restoration of stress fibers. Overall, changes in TM expression levels seem to be an early event during bladder carcinogenesis. We conclude that alterations in TM isoform expression may provide further insight into malignant transformation in transitional cell carcinomas of the bladder and may be a useful target for early detection strategies.

Distinct localizations of tropomyosin isoforms in LLC‐PK1 epithelial cells suggests specialized function at cell–cell adhesions

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.

Tropomyosin as a regulator of cancer cell transformation.

Tropomyosins (Tms) are among the most studied structural proteins of the actin cytoskeleton that are implicated in neoplastic-specific alterations in actin filament organization. Decreased expression of specific nonmuscle Tm isoforms is commonly associated with the transformed phenotype. These changes in Tm expression appear to contribute to the rearrangement of microfilament bundles and morphological alterations, increased cell motility and oncogenic signaling properties of transformed cells. Below we review aspects of Tm biology as it specifically relates to transformation and cancer including its expression in culture models of transformed cells and human tumors, mechanisms that regulate Tm expression and the role of Tm in oncogenic signaling.

Myosin light chain kinase plays a role in the regulation of epithelial cell survival

Myosin II activation is essential for stress fiber and focal adhesion formation, and is implicated in integrin-mediated signaling events. In this study we investigated the role of acto-myosin contractility, and its main regulators, i.e. myosin light chain kinase (MLCK) and Rho-kinase (ROCK) in cell survival in normal and Ras-transformed MCF-10A epithelial cells. Treatment of cells with pharmacological inhibitors of MLCK (ML-7 and ML-9), or expression of dominant-negative MLCK, led to apoptosis in normal and transformed MCF-10A cells. By contrast, treatment of cells with a ROCK inhibitor (Y-27632) did not induce apoptosis in these cells. Apoptosis following inhibition of myosin II activation by MLCK is probably meditated through the death receptor pathway because expression of dominant-negative FADD blocked apoptosis. The apoptosis observed after MLCK inhibition is rescued by pre-treatment of cells with integrin-activating antibodies. In addition, this rescue of apoptosis is dependent on FAK activity, suggesting the participation of an integrin-dependent signaling pathway. These studies demonstrate a newly discovered role for MLCK in the generation of pro-survival signals in both untransformed and transformed epithelial cells and supports previous work suggesting distinct cellular roles for Rho-kinase- and MLCK-dependent regulation of myosin II.