AKT drives sustained motility following MEK inhibition via promoting SNAIL and AXL in MDA-MB-231 LM2.

The adaptive activation of alternative signaling pathways contributes to acquired resistance against targeted cancer therapies. Our previous research has shown that blocking Ras/ERK signaling promotes PI3K/AKT signaling in the lung metastatic derivative of MDA-MB-231 (LM2). Because AKT activation was required to drive sustained cell motility following MEK suppression, we extend our research to elucidate how activation of the PI3K/AKT signaling drives sustained motility following MEK inhibition. Reverse phase protein array (RPPA) revealed that SNAIL (SNAI1) was upregulated in U0126 (MEK inhibitor)-treated LM2 cells. Importantly, LM2 cells simultaneously treated with U0126 and PI3K inhibitor LY294002 exhibited reduced expression of SNAIL. Furthermore, depletion of SNAIL led to reduced cell motility in U0126-treated LM2 cells. In addition, we identified AXL as another downstream effector of AKT. These results suggest that SNAIL and AXL are key factors mediating sustained motility of LM2 cells following MEK suppression. Because AKT mediates motile behavior under MEK suppression, our results suggest that AKT and AXL may be targeted to overcome resistance against drugs targeting the Ras/ERK pathway.

Up-regulated fibronectin in 3D culture facilitates spreading of triple negative breast cancer cells on 2D through integrin ß-5 and Src.

Using MDA-MB-231 cells as a model of triple negative breast cancer (TNBC) and its metastatic sub-cell lines that preferentially metastasize to lung, bone or brain, we found that the mRNA and protein levels of fibronectin (FN) are increased in MDA-MB-231 cells and its lung metastatic derivative, when cultivated in three-dimensional (3D) suspension cultures. The increase of FN expression in 3D was dependent on p38 mitogen-activated protein kinase (MAPK) because it was prevented by treatment of cells with SB203580, an inhibitor of p38MAPK. The up-regulated FN was converted into fibrils, and it enhanced cell spreading when cells cultured in 3D were transferred to two-dimensional (2D) culture. The arginine-glycine-aspartate (RGD) peptides and siRNAs targeting of integrin ß-5 inhibited spreading of cells regardless of the presence of FN on 2D culture dishes. In addition, the levels of phosphorylated Src were found to be increased in 3D and the treatment of cells with SU6656, an inhibitor of Src, decreased the rate of cell spreading on FN. Collectively, these studies demonstrate that increased cellular FN in 3D suspension culture facilitates cancer cell attachment and spreading via integrin ß-5 and Src, suggesting that the increased FN promotes initial attachment of cancer cells to secondary organs after circulation during metastasis.

Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells.

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and ß-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and ß-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Loss of Tpm4.1 leads to disruption of cell-cell adhesions and invasive behavior in breast epithelial cells via increased Rac1 signaling.

Here we report the identification and characterization of a novel high molecular weight isoform of tropomyosin, Tpm4.1, expressed from the human TPM4 gene. Tpm4.1 expression is down-regulated in a subset of breast cancer cells compared with untransformed MCF10A breast epithelial cells and in highly metastatic breast cancer cell lines derived from poorly metastatic MDA-MD-231 cells. In addition, patients with invasive ductal breast carcinoma show decreased TPM4 expression compared with patients with ductal breast carcinoma in situ, and low TPM4 expression is associated with poor prognosis. Loss of Tpm4.1 using siRNA in MCF10A cells increases cell migration in wound-healing and Boyden chamber assays and invasion out of spheroids as well as disruption of cell-cell adhesions. Down-regulation of Tpm4.1 in MDA-MB-231 cells leads to disruption of actin organization and increased cell invasion and dissemination from spheroids into collagen gels. The down-regulation of Tpm4.1 induces Rac1-mediated alteration of myosin IIB localization, and pharmacologic inhibition of Rac1 or down-regulation of myosin IIB using siRNA inhibits the invasive phenotypes in MCF10A cells. Thus Tpm4.1 plays an important role in blocking invasive behaviors through Rac1-myosin IIB signaling and our findings suggest that decreased expression of Tpm4.1 might play a crucial role during tumor progression.

Integrin ß1, myosin light chain kinase and myosin IIA are required for activation of PI3K-AKT signaling following MEK inhibition in metastatic triple negative breast cancer.

The effectiveness of targeted therapies against the Ras-ERK signaling pathway are limited due to adaptive resistance of tumor cells. Inhibition of the Ras-ERK pathway can result in activation of the PI3K-AKT pathway, thereby diminishing the therapeutic effects of targeting ERK signaling. Here we investigated the crosstalk between the Ras-ERK and PI3K-AKT pathways in MDA-MB-231 breast cancer cell lines that have a preference to metastasize to lung (LM2), brain (BrM2) or bone (BoM2). Inhibition of the Ras-ERK pathway reduced motility in both parental and BoM2 cells. In contrast, inhibition of the Ras-ERK pathway in BrM2 and LM2 cells resulted in activation of PI3K-AKT signaling that was responsible for continued cell motility. Analysis of the cross talk between Ras-ERK and PI3K-AKT signaling pathways revealed integrin ß1, myosin light chain kinase (MLCK) and myosin IIA are required for the activation of PI3K-AKT following inhibition of the Ras-ERK pathway. Furthermore, feedback activation of the PI3K-AKT pathway following MEK suppression was independent of the epidermal growth factor receptor. Thus, integrin ß1, MLCK, and myosin IIA are factors in the development of resistance to MEK inhibitors. These proteins could provide an opportunity to develop markers and therapeutic targets in a subgroup of triple negative breast cancer (TNBC) that exhibit resistance against MEK inhibition.

Modeling of Menkes disease via human induced pluripotent stem cells.

Menkes disease (MD) is a copper-deficient neurodegenerative disorder that manifests severe neurologic symptoms such as seizures, lethargic states, and hypotonia. Menkes disease is due to a dysfunction of ATP7A, but the pathophysiology of neurologic manifestation is poorly understood during embryonic development. To understand the pathophysiology of neurologic symptoms, molecular and cellular phenotypes were investigated in Menkes disease-derived induced pluripotent stem cells (MD-iPSCs). MD-iPSCs were generated from fibroblasts of a Menkes disease patient. Abnormal reticular distribution of ATP7A was observed in MD-fibroblasts and MD-iPSCs, respectively. MD-iPSCs showed abnormal morphology in appearance during embryoid body (EB) formation as compared with wild type (WT)-iPSCs. Intriguingly, aberrant switch of E-cadherin (E-cad) to N-cadherin (N-cad) and impaired neural rosette formation were shown in MD-iPSCs during early differentiation. When extracellular copper was chelated in WT-iPSCs by treatment with bathocuprione sulfate, aberrant switch of E-cad to N-cad and impaired neuronal differentiation were observed, like in MD-iPSCs. Our results suggest that neurological defects in Menkes disease patients may be responsible for aberrant cadherin transition and impaired neuronal differentiation during early developmental stage.

From skeletal muscle to cancer: insights learned elucidating the function of tropomyosin.

The tropomyosins (Tms) are a family of actin filament binding proteins that possess a simple dimeric α-helical coiled-coil structure along their entire length. Our knowledge of Tm structure and function has greatly expanded since they were first discovered in skeletal muscle almost 65 years ago. In multicellular organisms they exhibit extensive cell type specific isoform diversity. In this essay we discuss the genetic mechanisms by which this diversity is generated and its significance to actin-based cellular functions.

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin.

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.

HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway.

HGAL is a germinal center (GC)-specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism - direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.

Non-muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC).

Non-muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF-alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property.

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.

HGAL, a lymphoma prognostic biomarker, interacts with the cytoskeleton and mediates the effects of IL-6 on cell migration.

HGAL is a newly identified germinal center (GC)-specific gene whose expression by the tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. The function of HGAL is unknown. Previous studies demonstrated that HGAL is dispensable for GC formation, immunoglobulin gene class-switch recombination, and somatic hypermutation. Herein, we identify a role for HGAL in the regulation of cell motility. We demonstrate that IL-6 induces the phosphorylation of the C-terminal tyrosine residue of the HGAL protein via the Lyn kinase, and promotes its relocalization from the cytoplasm to podosome-like structures. Further, IL-6-induced HGAL phosphorylation increases its interaction with myosin II and is associated with inhibition of cell migration. Knockdown of endogenous HGAL ameliorates IL-6-induced inhibition of cell migration, whereas overexpression of HGAL imparts inhibitory effects of IL-6 on cell migration. Taken together, our results suggest that HGAL is involved in negative regulation of lymphocyte migration, thus constraining lymphocytes to the GC. Inhibition of lymphocyte migration might contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas.

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.

Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras.

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility.

A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells.

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.

Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation.

Cell-substratum interactions trigger key signaling pathways that modulate growth control and tissue-specific gene expression. We have previously shown that abolishing adhesive interactions by suspension culture results in G(0) arrest of myoblasts. We report that blocking intracellular transmission of adhesion-dependent signals in adherent cells mimics the absence of adhesive contacts. We investigated the effects of pharmacological inhibitors of acto-myosin contractility on growth and differentiation of C2C12 myogenic cells. ML7 (5-iodonaphthalene-1-sulfonyl homopiperazine) and BDM (2,3, butanedione monoxime) are specific inhibitors of myosin light chain kinase, and myosin heavy chain ATPase, respectively. ML7 and BDM affected cell shape by reducing focal adhesions and stress fibers. Both inhibitors rapidly blocked DNA synthesis in a dose-dependent, reversible fashion. Furthermore, both ML7 and BDM suppressed expression of MyoD and myogenin, induced p27(kip1) but not p21(cip1), and inhibited differentiation. Thus, as with suspension-arrest, inhibition of acto-myosin contractility in adherent cells led to arrest uncoupled from differentiation. Over-expression of inhibitors of the small GTPase RhoA (dominant negative RhoA and C3 transferase) mimicked the effects of myosin inhibitors. By contrast, wild-type RhoA induced arrest, maintained MyoD and activated myogenin and p21 expression. The Rho effector kinase ROCK did not appear to mediate Rho's effects on MyoD. Thus, ROCK and MLCK play different roles in the myogenic program. Signals regulated by MLCK are critical, since inhibition of MLCK suppressed MyoD expression but inhibition of ROCK did not. Inhibition of contractility suppressed MyoD but did not reduce actin polymer levels. However, actin depolymerization with latrunculin B inhibited MyoD expression. Taken together, our observations indicate that actin polymer status and contractility regulate MyoD expression. We suggest that in myoblasts, the Rho pathway and regulation of acto-myosin contractility may define a control point for conditional uncoupling of differentiation and the cell cycle.

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.

Cytoplasmic p21Cip1 is involved in Ras-induced inhibition of the ROCK/LIMK/cofilin pathway.

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.

Characterization of the metastasis-associated protein, S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin.

Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimers and interacts with several proteins in a calcium-dependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancer-derived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calcium-independent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.