Ratna K. Vadlamudi

Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor.

Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-β1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-α. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

Heregulin Regulates Cytoskeletal Reorganization and Cell Migration through the p21-activated Kinase-1 via Phosphatidylinositol-3 Kinase

The mechanisms through which heregulin (HRG) regulates the activities of breast cancer cells are currently unknown. We demonstrate that HRG stimulation of noninvasive breast cancer cells enhanced the conversion of globular to filamentous actin and the formation of membrane ruffles, stress fibers, filopodia, and lamellipodia and accompanied by increased cell migration. In addition, HRG triggered a rapid stimulation of p21-activated kinase1 (PAK1) activity and its redistribution into the leading edges of motile cells. The HRG-induced stimulation of PAK1 kinase activity followed phosphatidylinositol-3 kinase (PI-3 kinase) activation. Inhibition of PI-3 kinase activity blocked the activation of PAK1 kinase and also blocked cell migration in response to HRG. Furthermore, direct inhibition of PAK1 functions by the dominant-negative mutant suppressed the capacity of HRG to reorganize actin cytoskeleon structures. We also demonstrated that HRG stimulation promoted physical interactions between PAK1, actin, and human epidermal growth factor receptor 2 (HER2) receptors, and these interactions were dependent on the activation of PI-3 kinase. The blockade of HER2 receptor by an anti-HER2 monoclonal antibody resulted in the inhibition of HRG-mediated stimulation of PI-3 kinase/PAK pathway and also the formation of motile actin cytoskeleton structures but not extracellular signal-regulated kinases. These findings suggest a role of PI-3 kinase/PAK1-dependent reorganization of the cortical actin cytoskeleton in HRG-mediated increased cell migration, and these changes may have significant consequences leading to enhanced invasion by breast cancer cells.

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1.

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.

p62, a Phosphotyrosine-independent Ligand of the SH2 Domain of p56lck, Belongs to a New Class of Ubiquitin-binding Proteins

p62 is a novel cellular protein which was initially identified as a phosphotyrosine-independent ligand of the SH2 domain of p56lck. In the yeast two-hybrid system, p62 specifically interacted with ubiquitin in vivo. Furthermore, p62 bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. The interaction was independent of ATP hydrolysis, and its dissociation did not require a reducing agent. Thus, p62 binds to ubiquitin noncovalently. Further analysis showed that the C-terminal 80 amino acids of p62 were indispensable for its interaction with ubiquitin. However, p62 has homology neither with ubiquitin C-terminal hydrolases nor with the S5a subunit of the 26 S proteasome complex, the only proteins known to bind to ubiquitin noncovalently. These results suggest that p62 belongs to a new class of ubiquitin-binding proteins and that p62 affects signal transduction at least partly through ubiquitination-mediated protein degradation.

Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells

Stimulation of growth factor signaling has been implicated in the development of invasive phenotypes and the activation of p21-activated kinase (Pak1) in human breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238–28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000)J. Biol. Chem. 275, 12041–12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft agar in a preferential mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of Pak1 kinase activity and base-line invasiveness of human breast cancer cells and breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.

Regulation of cyclooxygenase-2 pathway by HER2 receptor.

Emerging lines of evidence suggest that in addition to growth factors, the process of colorectal tumorigenesis may also be driven by the upregulation of the inducible form of cyclooxygenase-2 (COX-2), an enzyme responsible for the conversion of arachidonic acid to PGEs. The present study was undertaken to investigate the expression and activation of the HER family members, and to explore the regulation of COX-2 expression by the HER2 pathway in human colorectal cancer cells. Here, we report that human colorectal cancer cell lines express abundant levels of HER2 and HER3 receptors, and are growth-stimulated by recombinant neu-differentiation factor-beta 1 (NDF). NDF-treatment of colorectal cancer cells was accompanied by increased tyrosine phosphorylation and heterodimerization of HER3 with HER2. In addition, we demonstrated that HER2 and HER3 receptors in colorectal cancer cells are constitutively phosphorylated on tyrosine residues and form heterodimeric complexes in the absence of exogenous NDF. Inhibition of HER2/HER3 signaling by an anti-HER3 mAb against the ligand binding site resulted in a decrease in the levels of constitutively activated HER2/HER3 heterodimers, and the unexpected reduction of COX-2 expression. Activation of the HER2/HER3 pathway by NDF induced the activation of COX-2 promoter, expression of COX-2 mRNA, COX-2 protein and accumulation of prostaglandin E2 in the culture medium. Finally, we demonstrated that NDF promotes the ability of colorectal cancer cells to survive in an extracellular matrix milieu, such as Matrigel, and also to invade through a 8 μm porous membrane. These biological activities of NDF and its stimulation of cell proliferation are blocked by a specific inhibitor of COX-2. Taken together, our findings provide the first biochemical evidence of a possible role of the COX-2 pathway in the mitogenic action of NDF in colorectal cancer cells where it may be constitutively upregulated due to the autocrine/paracrine activation of HER2/HER3 heterodimers.

A naturally occurring MTA1 variant sequesters oestrogen receptor-α in the cytoplasm

Oestrogen receptor (ER) is a good prognostic marker for the treatment of breast cancers. Upregulation of metastatic tumour antigen 1 (MTA1) is associated with the invasiveness and metastatic potential of several human cancers and acts as a co-repressor of nuclear ER-α. Here we identify a naturally occurring short form of MTA1 (MTA1s) that contains a previously unknown sequence of 33 amino acids with an ER-binding motif, Leu-Arg-Ile-Leu-Leu (LRILL). MTA1s localizes in the cytoplasm, sequesters ER in the cytoplasm, and enhances non-genomic responses of ER. Deleting the LRILL motif in MTA1s abolishes its co-repressor function and its interaction with ER, and restores nuclear localization of ER. Dysregulation of human epidermal growth factor receptor-2 in breast cancer cells enhances the expression of MTA1s and the cytoplasmic sequestration of ER. Expression of MTA1s in breast cancer cells prevents ligand-induced nuclear translocation of ER and stimulates malignant phenotypes. MTA1s expression is increased in human breast tumours with no or low nuclear ER. The regulation of the cellular localization of ER by MTA1s represents a mechanism for redirecting nuclear receptor signalling by nuclear exclusion.

Pak1 Phosphorylation of Snail, a Master Regulator of Epithelial-to-Mesenchyme Transition, Modulates Snail's Subcellular Localization and Functions

The process of epithelial-mesenchymal transition plays a pivotal role in the conversion of early stage tumors into invasive malignancies, and has been shown to be regulated by the zinc finger phosphoprotein, Snail; however, no upstream signaling kinases have been shown to modulate Snail functions. Since the invasiveness of breast cancer cells is also influenced by p21-activated kinase 1 (Pak1) signaling, we investigated Pak1s potential mechanistic role in the regulation of Snail functions. We found for the first time that Pak1 promotes transcription repression activity of Snail from E-cadherin, occludin, and aromatase promoters. Pak1 regulates the repressor activity of Snail by phosphorylating on Ser(246). Pak1 phosphorylation of Snail supports Snails accumulation in the nucleus as well as its repressor functions. A Ser(246)Ala substitution in Snail or Pak1 knockdown by short interference RNA blocked Pak1-mediated Snail phosphorylation, leading to increased cytoplasmic accumulation of Snail and attenuation of Snail repressor activity in breast cancer cells. The regulation of phosphorylation and function of Snail by Pak1 represents a novel mechanism by which a signaling kinase might contribute to the process of epithelial-mesenchymal transition.

P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-α and promotes hyperplasia in mammary epithelium

Stimulation of p21‐activated kinase‐1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant‐negative fragment or by short interference RNA markedly reduced the estrogen receptor‐α (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the β‐lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen‐activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function‐2 domain of the ER at the N‐terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1‐mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1–ER pathway in promoting hyperplasia in mammary epithelium.

Rapid estrogen signaling in the brain.

Estrogen has multiple actions in the brain to modulate homeostasis, synaptic plasticity/cognition and neuroprotection. While many of these actions undoubtedly involve mediation via the classical genomic mechanism of regulation of transcription of genes via estrogen nuclear receptors, there has been growing interest in the rapid nongenomic effects of estrogen and the role they may play in the neural actions of estrogen. In this review, we will focus on these rapid nongenomic actions of estrogen in the brain and discuss the potential physiological significance of these actions. The evidence for rapid estrogen regulation of cell signaling pathways, including calcium, ion channel and kinase signaling pathways in the brain will be reviewed, as will evidence derived from plasma-membrane impermeable estrogen-peptide conjugates in the regulation of these cell signaling pathways. Evidence supporting classical and nonclassical estrogen receptor localization to the plasma membrane of neurons will also be reviewed, including the putative new membrane estrogen G-protein-coupled receptor, GPR30. Precisely how membrane estrogen receptors couple to kinase signaling pathways is unclear, but we will discuss the latest findings on estrogen receptor-interacting scaffold proteins, such as MNAR/PELP1, striatin and p130Cas, which are capable of linking estrogen receptors and kinases such as Src and PI3K, to potentially mediate estrogen-induced kinase signaling. Finally, we will review the growing evidence that rapid membrane-mediated effects of estrogen play an important physiological role in the neural actions of estrogen in the brain, including estrogen feedback control and modulation of homeostasis, regulation of synaptic plasticity/cognition, and estrogen-mediated neuroprotection.