Does salmon calcitonin cause cancer? A review and meta-analysis.

Recently an association between the use of calcitonin and cancer has been postulated. We reviewed the biological rationale and performed an additional analysis of historical data with respect to the possibility. An association cannot be excluded, but the relationship is weak and causality is unlikely. The purpose of the present study is to review the strength of association and likelihood of a causal relationship between use of calcitonin and cancer. We reviewed the evidence for this association, including the molecular signaling mechanisms of calcitonin, preclinical data, an "experiment of nature," and the results of a previous meta-analysis which showed a weak association. We performed an additional meta-analysis to incorporate the data from a novel investigational oral formulation of salmon calcitonin. Review of the literature did not identify a cellular signaling mechanism of action which might account for a causal relationship or toxicologic or postmarketing data to support the thesis. Additional clinical results incorporated into previous meta-analyses weakened but did not completely negate the possibility of association. A causal association between calcitonin use and malignancy is unlikely, as there is little biological plausibility. The preponderance of nonclinical and clinical evidence also does not favor a causal relationship.

The role of p21-activated kinase in the initiation of atherosclerosis.

BACKGROUND: p21-activated kinase (PAK) has been implicated in the inflammatory activation of endothelial cells by disturbed fluid shear stress, which is the initiating stimulus in atherosclerosis. The study addresses whether PAK1 contributes to inflammatory marker expression in endothelial cells at atherosclerosis-susceptible regions of arteries in vivo. METHOD: Aortas from WT and PAK1-/- C57BL/6J mice on a normal chow diet were fixed, dissected and processed for immunohistochemistry using a panel of inflammatory markers. We visualized and quantified staining in the endothelium at the greater and lesser curvatures of the arch of aorta, as atherosclerosis-resistant and susceptible regions, respectively. RESULTS: Fibronectin, VCAM-1 and the activated RelA NF-κB subunit were localized to the lesser curvature and decreased in PAK1-/- mice. The activated RelB NF-κB subunit was also localized to the lesser curvature but was increased in PAK1-/- mice. Low levels of staining for ICAM-1 and the monocyte/macrophage marker Mac2 indicated that overall inflammation in this tissue was minimal. CONCLUSION: These data show that PAK1 has a significant pro-inflammatory function at atherosclerosis-prone sites in vivo. These effects are seen in young mice with very low levels of inflammation, suggesting that inflammatory activation of the endothelium is primarily biomechanical. Activation involves NF-κB, expression of leukocyte recruitment receptors and fibronectin deposition. These results support and extend in vitro studies demonstrating that PAK contributes to activation of inflammatory pathways in endothelial cells by fluid shear stress.

Emerging from the Pak: the p21-activated protein kinase family.

The p21-activated protein kinases (PAKs) are members of a growing family of regulatory enzymes that may play roles in diverse phenomena such as cellular morphogenesis, the stress response and the pathogenesis of AIDS. PAKs were initially discovered as binding partners for small (21 kDa) GTPases that regulate actin polymerization, and recent evidence has shown that some members of the PAK family may be effectors for related GTPases that are involved in intracellular vesicle trafficking. Because the downstream signalling pathways for all such GTPases are poorly understood, intense studies are under way to discern the role of PAK and its cousins. In this review, the authors highlight some of the established properties of the extended PAK family and discuss current controversies regarding their possible roles as GTPase effectors.

Plant GTPases: the Rhos in bloom.

In animal cells and in fungi, small GTP-binding proteins of the Rho family have well-established roles in morphogenesis, cell-cycle progression, gene transcription and the generation of superoxide anions. The presence of these proteins in plant cells, however, has been established only recently, and the role of Rho GTPases in plants is now coming into view. Already, it is apparent that there are both striking similarities and fascinating differences in how Rho GTPases are regulated and used in plant versus animal and fungal cells. These new findings define certain core properties that might be common to members of this protein family in all eukaryotes.

Temporal and spatial distribution of activated Pak1 in fibroblasts.

p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.

p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts.

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form of Pak1 projected multiple lamellipodia emerging from different parts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.

Regulation of human leukocyte p21-activated kinases through G protein--coupled receptors.

The Rac guanosine 5'-triphosphate (GTP)-binding proteins regulate oxidant production by phagocytic leukocytes. Two Ste20-related p21-activated kinases (PAKs) were identified as targets of Rac in human neutrophils. Activity of the approximately 65- and approximately 68-kilodalton PAKs was rapidly stimulated by chemoattractants acting through pertussis toxin-sensitive heterotrimeric GTP-binding proteins (G proteins). Native and recombinant PAKs phosphorylated the p47phox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component in a Rac-GTP-dependent manner. The action of PAKs during phagocyte activation by G protein-coupled pathways may contribute to regulation of NADPH oxidase activity.

The kinase-inhibitory domain of p21-activated kinase 1 (PAK1) inhibits cell cycle progression independent of PAK1 kinase activity.

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.

Group I Paks are essential for epithelial- mesenchymal transition in an Apc-driven model of colorectal cancer.

p21-activated kinases (Paks) play an important role in oncogenic signaling pathways and have been considered as potential therapeutic targets in various cancers. Most studies of Pak function employ gene knock-out or knock-down methods, but these approaches result in loss of both enzymatic and scaffolding properties of these proteins, and thus may not reflect the effects of small molecule inhibitors. Here we use a transgenic mouse model in which a specific peptide inhibitor of Group I Paks is conditionally expressed in response to Cre recombinase. Using this model, we show that inhibition of endogenous Paks impedes the transition of adenoma to carcinoma in an Apc-driven mouse model of colorectal cancer. These effects are mediated by inhibition of Wnt signaling through reduced ß-catenin activity as well as suppression of an epithelial-mesenchymal transition program mediated by miR-200 and Snai1. These results highlight the potential therapeutic role of Pak1 inhibitors in colorectal cancer.

Suppression of RAC1-driven malignant melanoma by group A PAK inhibitors.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small-molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1. In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1-mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

Protein tyrosine phosphatase 1B negatively regulates integrin signaling.

Protein tyrosine phosphatase (PTP) 1B has long been known to regulate cell proliferation negatively, but the mechanism by which this inhibition occurs is poorly defined. We have shown previously that PTP1B binds to, and dephosphorylates, p130(Cas) (Crk-associated substrate) [1], a protein that is thought to play a role in integrin signaling [2,3]. In this report, we present evidence that PTP1B interferes specifically with cell-adhesion-stimulated, but not growth-factor-stimulated, signaling pathways. In rat fibroblasts that overexpress PTP1B, the activation of mitogen-activated protein (MAP) kinase by growth factors was not affected, but activation by cell adhesion was markedly impaired. The inhibition of adhesion-dependent MAP kinase activation by PTP1B required an intact proline-rich region in the carboxyl terminus of PTP1B, a region we have shown to mediate binding to the Src-homology 3 (SH3) domain of p130Cas [1]. Overexpression of wild-type PTP1B, but not of a proline-to-alanine mutant form (PA-PTP1B) that is unable to bind or dephosphorylate p130Cas, interfered with cell spreading, cytoskeletal architecture, and the formation of focal adhesion complexes. Cells overexpressing wild-type PTP1B also displayed markedly reduced migration in response to a fibronectin gradient, whereas cells expressing the PA-PTP1B mutant migrated normally. These data indicate that PTP1B exerts its inhibitory effects via proline-dependent interactions with one or more critical components of the adhesion-dependent signaling apparatus, and suggest that one of these components may be p130Cas.

Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.

BACKGROUND: The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. RESULTS: Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. CONCLUSIONS: These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility.

Transformation suppression by protein tyrosine phosphatase 1B requires a functional SH3 ligand.

We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot bind p130Cas (PA-PTP1B), causes substantial tyrosine dephosphorylation of p130Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290-31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B, inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130Cas and two proteins that are associated with p130Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation suppression by PTP1B requires a functional SH3 ligand and suggest that p130Cas may represent an important physiological target of PTP1B in cells.

The fission yeast genes pyp1+ and pyp2+ encode protein tyrosine phosphatases that negatively regulate mitosis.

We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway.

Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.

Regulation of macropinocytosis by p21-activated kinase-1.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

Targeting group I p21-activated kinases to control malignant peripheral nerve sheath tumor growth and metastasis.

Malignant peripheral nerve sheath tumors (MPNSTs) are devastating sarcomas for which no effective medical therapies are available. Over 50% of MPSNTs are associated with mutations in NF1 tumor suppressor gene, resulting in activation of Ras and its effectors, including the Raf/Mek/Erk and PI3K/Akt/mTORC1 signaling cascades, and also the WNT/ß-catenin pathway. As Group I p21-activated kinases (Group I Paks, PAK1/2/3) have been shown to modulate Ras-driven oncogenesis, we asked if these enzymes might regulate signaling in MPNSTs. In this study we found a strong positive correlation between the activity of PAK1/2/3 and the stage of human MPNSTs. We determined that reducing Group I Pak activity diminished MPNST cell proliferation and motility, and that these effects were not accompanied by significant blockade of the Raf/Mek/Erk pathway, but rather by reductions in Akt and ß-catenin activity. Using the small molecule PAK1/2/3 inhibitor Frax1036 and the MEK1/2 inhibitor PD0325901, we showed that the combination of these two agents synergistically inhibited MPNST cell growth in vitro and dramatically decreased local and metastatic MPNST growth in animal models. Taken together, these data provide new insights into MPNST signaling deregulation and suggest that co-targeting of PAK1/2/3 and MEK1/2 may be effective in the treatment of patients with MPNSTs.

Effects of p21-activated kinase 1 inhibition on 11q13-amplified ovarian cancer cells.

p21-activated kinases (Paks) are Cdc42/Rac-activated serine-threonine protein kinases that regulate several key cancer-relevant signaling pathways, such as the Mek/Erk, PI3K/Akt and Wnt/b-catenin signaling pathways. Pak1 is frequently overexpressed and/or hyperactivated in different human cancers, including human breast, ovary, prostate and brain cancer, due to amplification of the PAK1 gene in an 11q13 amplicon. Genetic or pharmacological inactivation of Pak1 has been shown to reduce proliferation of different cancer cells in vitro and reduce tumor progression in vivo. In this work, we examined the roles of Pak1 in cellular and animal models of PAK1-amplified ovarian cancer. We found that inhibition of Pak1 leads to decreased proliferation and migration in PAK1-amplified/overexpressed ovarian cancer cells, and has no effect in cell that lack such amplification/overexpression. Further, we observed that loss of Pak1 function causes 11q13-amplified ovarian cancer cells to arrest in the G2/M phase of the cell cycle. This arrest correlates with activation of p53 and p21(Cip) and decreased expression of cyclin B1. These findings suggest that small-molecule inhibitors of Pak1 may have a therapeutic role in the ~25% of ovarian cancers characterized by PAK1 gene amplification.

Evidence for a role of mixed lineage kinases in neuronal apoptosis.

Superior cervical ganglion (SCG) sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It has been shown previously that the induction of apoptosis in these neurons at NGF withdrawal requires both the activity of the small GTP-binding protein Cdc42 and the activation of the c-Jun N-terminal kinase (JNK) pathway. The mixed lineage kinase 3 (MLK3) belongs to a family of mitogen-activated protein (MAP) kinase kinase kinases. MLK3 contains a Cdc42/Rac interactive-binding (CRIB) domain and activates both the JNK and the p38 MAP kinase pathways. In this study the role of MLK3 in the induction of apoptosis in sympathetic neurons has been investigated. Overexpression of an active MLK3 induces activation of the JNK pathway and apoptosis in SCG neurons. In addition, overexpression of kinase dead mutants of MLK3 blocks apoptosis as well as c-Jun phosphorylation induced by NGF deprivation. More importantly, MLK3 activity seems to increase by 5 hr after NGF withdrawal in both differentiated PC12 cells and SCG neurons. We also show that MLK3 lies downstream of Cdc42 in the neuronal death pathway. Regulation of MLK3 in neurons seems to be dependent on MLK3 activity and possibly on an additional cellular component, but not on its binding to Cdc42. These results suggest that MLK3, or a closely related kinase, is a physiological element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal death. MLK3 therefore could be an interesting therapeutic target in a number of neurodegenerative diseases involving neuronal apoptosis.