Katarzyna Bialkowska

The miR200 Family of MicroRNAs Regulates WAVE3-dependent Cancer Cell Invasion

MicroRNAs are small non-coding RNAs that are directly involved in the regulation of gene expression by either translational repression or degradation of target mRNAs. Because of the high level of conservation of the target motifs, known as seed sequences, within the 3′-untranslated regions, a single microRNA can regulate numerous target genes simultaneously, making this class of RNAs a powerful regulator of gene expression. The miR200 family of microRNAs has recently been shown to regulate the process of epithelial to mesenchymal transition during tumor progression and metastasis. Here, we report that the expression of WAVE3, an actin cytoskeleton remodeling and metastasis promoter protein, is regulated by miR200 microRNAs. We show a clear inverse correlation between expression levels of WAVE3 and miR200 microRNAs in invasive versus non-invasive cancer cells. miR200 directly targets the 3′-untranslated regions of the WAVE3 mRNA and inhibits its expression. The miR200-mediated down-regulation of WAVE3 results in a significant reduction in the invasive phenotype of cancer cells, which is specific to the loss of WAVE3 expression. Re-expression of a miR200-resistant WAVE3 reverses miR200-mediated inhibition of cancer cell invasion. Loss of WAVE3 expression downstream of miR200 also results in a dramatic change in cell morphology resembling that of a mesenchymal to epithelial transition. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties and morphology of cancer cells associated with their metastatic potential.

The integrin co-activator kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell

Integrin activation is crucial for numerous cellular responses, including cell adhesion, migration, and survival. Recent studies in mice have specifically emphasized the vital role of kindlin-3 in integrin activation. Kindlin-3 deficiency in humans also has now been documented and includes symptoms of bleeding, frequent infections, and osteopetrosis, which are consequences of an inability to activate β1, β2, and β3 integrins. To date, kindlin-3 was thought to be restricted to hematopoietic cells. In this article, we demonstrate that kindlin-3 is present in human endothelial cells derived from various anatomical origins. The mRNA and protein for KINDLIN-3 was detected in endothelial cells by reverse transcription-PCR and Western blots. When subjected to sequencing by mass spectrometry, the protein was identified as authentic kindlin-3 and unequivocally distinguished from KINDLIN-1 and KINDLIN-2 or any other known protein. By quantitative real time PCR, the level of kindlin-3 in endothelial cells was 20–50% of that of kindlin-2. Using knockdown approaches, we show that kindlin-3 plays a role in integrin-mediated adhesion of endothelial cells. This function depends upon the integrin and substrate and is distinct from that of kindlin-2. Formation of tube-like structures in Matrigel also was impaired by kindlin-3 knockdown. Mechanistically, the distinct functions of the kindlins can be traced to differences in their subcellular localization in integrin-containing adhesion structures. Thus, the prevailing view that individual kindlins exert their functions in a cell type-specific manner must now be modified to consider distinct functions of the different family members within the same cell type.

miR-31 is a broad regulator of β1-integrin expression and function in cancer cells

Integrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathologic conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs (miRNA) has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell-based, genetic, biochemical, flow cytometry, and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5, and αV) of β1 integrins and also β3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV, and β3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3′ untranslated region of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. Although different reports have shown that a single integrin can be regulated by several miRNAs, here we show that a single miRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis. Mol Cancer Res; 9(11); 1500–8. ©2011 AACR.

The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY759 motif in the β3 CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β3 CT interacted well with kindlin-2, whereas the Tyr759-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β3 CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.

Spatial Coordination of Kindlin-2 with Talin Head Domain in Interaction with Integrin β Cytoplasmic Tails

Background: The talin and kindlin play indispensable roles in integrin activation. Results: The C-terminal 12 amino acids of β1 and β3 integrins mediate kindlin-2 binding. Conclusion: Kindlin-2 binding to the extreme C terminus allows β subunits to accommodate both kindlin-2 and talin. Significance: Binding of talin and kindlin-2 to distinct sites in integrins regulates receptor activation, a pivotal event in cellular responses. Both talin head domain and kindlin-2 interact with integrin β cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to β cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin β tails. Kindlin-2 interacted well with β1 and β3 tails but poorly with the β2 cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the β3 tail, and the sequence in β2 was non-permissive. The region at the C termini of integrin β1 and β3 tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin β3 tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/β tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.

SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes.

In this study, we used cultured cells spreading on β3 integrin substrates to examine the possibility that spectrin is involved in signal transduction. Spectrin clustered with specialized calpain-induced β3 integrin signaling complexes that mediate the initial attachment of cells and initiate Rac activation and lamellipodia extension. It was absent from focal complexes and focal adhesions, the integrin complexes that mediate adhesion in lamellipodia and fully spread cells. Spectrin contains a Src homology (SH3) domain of unknown function. Cells overexpressing this domain adhered and calpain-induced integrin signaling complexes formed. However, Rac activation, lamellipodia extension and cell spreading were inhibited. Spreading was restored by overexpression of constitutively active Rac. These studies point to a previously unrecognized role for spectrin and its SH3 domain in initiating Rac activation in the specialized integrin clusters that initiate cell adhesion and spreading. Thus, spectrin may have a pivotal role in initiating integrin-induced physiological and pathological events such as development, proliferation, cell survival, wound healing, metastasis and atherosclerosis.

Tiam1 is recruited to β1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility

14‐3‐3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin‐induced Rac1 activation and spreading were inhibited by sequestration of 14‐3‐3ζ and restored by its overexpression. Here, we determined whether 14‐3‐3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1‐activating integrin complexes. We showed that GST‐14‐3‐3ζ recruited the Rac1‐GEF, Tiam1, from cell lysates through Tiam1 residues 1–182 (N1–182 Tiam1). The physiological relevance of this interaction was examined in serum‐starved Hela cells plated on fibronectin. Both Tiam1 and N1–182 Tiam1 were recruited to 14‐3‐3‐containing β1‐integrin complexes, as shown by co‐localization and co‐immunoprecipitation. Integrin‐induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N1–182 Tiam1, which was incorporated into 14‐3‐3/β1‐integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin‐induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14‐3‐3ζ S58D, which was unable to recruit Tiam1 from lysates, co‐immunoprecipitate with Tiam1, or mediate its incorporation into β1‐integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin‐induced Rac1 activation in which 14‐3‐3 dimers localize Tiam1 to integrin complexes, where it mediates integrin‐dependent Rac1 activation, thus initiating motility‐inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH‐CC‐EX domain, the present findings show that a mechanism involving its N‐terminal 182 residues is utilized to recruit Tiam1 to motility‐inducing integrin complexes. J. Cell. Physiol. 226: 2965–2978, 2011.

Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis

The FERM domain containing protein Kindlin‐3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin‐3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ~7‐fold elevation in Kindlin‐3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin‐3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5‐ and 3‐fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin‐3‐overexpressing cells displayed a 2.2‐fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin‐3‐overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin‐3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.—Sossey‐Alaoui, K., Pluskota, E., Davuluri, G., Bialkowska, K., Das, M., Szpak, D., Lindner, D. J., Downs‐Kelly, E., Thompson, C. L., Plow, E. F. Kindlin‐3 enhances breast cancer progression and metastasis by activating Twist‐mediated angiogenesis. FASEB J. 28, 2260–2271 (2014). www.fasebj.org

Kindlin-2 directly binds actin and regulates integrin outside-in signaling

Bledzka et al. show that kindlin-2 binds actin via its F0 domain, and mutation of this site diminishes cell spreading, revealing a new mechanism by which kindlin-2 regulates cellular responses.