Prolactin-induced tyrosyl phosphorylation of PAK1 facilitates epithelial-mesenchymal transition.

PAK1 and prolactin (PRL) regulate breast cancer. Prolactin-activated JAK2 tyrosyl phosphorylates PAK1 (pTyr-PAK1). We demonstrate here that pTyr-PAK1 regulates epithelial-mesenchymal transition (EMT) in breast cancer cells. PRL treatment of T47D PAK1 WT cells leads to downregulation of E-cadherin surface expression and "ectodomain shedding" (extracellular cleavage of E-cadherin). pTyr-PAK1 increases mRNA levels of Snail, Slug, and Twist2, transcriptional factors implicated in E-cadherin repression. pTyr-PAK1 also significantly increases PRL-dependent Slug activity leading to expression of vimentin, a hallmark of EMT. Thus, our current data on pTyr-PAK1 regulation of EMT bring insight into the role of PAK1 and PRL in human breast cancer.

The calcar femorale: A new perspective.

It is the role of the hip joint to support the superincumbent body weight while its complex range of movement, under a variety of stresses, allows the individual to undertake a wide range of physical activities. It is then perhaps not surprising that a recent anatomical study shows that the upper femur has a structure commensurate with the complexity of its movements. Mechanically, the upper femur consists of two separate bones: the combined femoral head and neck and the femoral shaft. Both bones consist of a mixture of two different types: cancellous bone and cortical bone, each with its own characteristics. Both bones are subjected to two different forces: those of weight bearing and movement and those of bone modelling. In the face of these complexities, this article tenders some theoretical considerations as to how this region functions and deals with these particular problems. From an anatomical viewpoint, the calcar femorale plays a central role in the mechanical structure of the femoral neck and is pivotal in the management of the stresses to which the region is subjected.

Prolactin-induced PAK1 tyrosyl phosphorylation promotes FAK dephosphorylation, breast cancer cell motility, invasion and metastasis.

BACKGROUND: The serine/threonine kinase PAK1 is an important regulator of cell motility. Both PAK1 and the hormone/cytokine prolactin (PRL) have been implicated in breast cancer cell motility, however, the exact mechanisms guiding PRL/PAK1 signaling in breast cancer cells have not been fully elucidated. Our lab has previously demonstrated that PRL-activated tyrosine kinase JAK2 phosphorylates PAK1 on tyrosines 153, 201, and 285, and that tyrosyl phosphorylated PAK1 (pTyr-PAK1) augments migration and invasion of breast cancer cells. RESULTS: Here we further investigate the mechanisms by which pTyr-PAK1 enhances breast cancer cell motility in response to PRL. We demonstrate a distinct reduction in PRL-induced FAK auto-phosphorylation in T47D and TMX2-28 breast cancer cells overexpressing wild-type PAK1 (PAK1 WT) when compared to cells overexpressing either GFP or phospho-tyrosine-deficient mutant PAK1 (PAK1 Y3F). Furthermore, pTyr-PAK1 phosphorylates MEK1 on Ser298 resulting in subsequent ERK1/2 activation. PRL-induced FAK auto-phosphorylation is rescued in PAK1 WT cells by inhibiting tyrosine phosphatases and tyrosine phosphatase inhibition abrogates cell motility and invasion in response to PRL. siRNA-mediated knockdown of the tyrosine phosphatase PTP-PEST rescues FAK auto-phosphorylation in PAK1 WT cells and reduces both cell motility and invasion. Finally, we provide evidence that PRL-induced pTyr-PAK1 stimulates tumor cell metastasis in vivo. CONCLUSION: These data provide insight into the mechanisms guiding PRL-mediated breast cancer cell motility and invasion and highlight a significant role for pTyr-PAK1 in breast cancer metastasis.

Src tyrosyl phosphorylates cortactin in response to prolactin.

The hormone/cytokine prolactin (PRL) is implicated in breast cancer cell invasion and metastasis. PRL-induced pathways are mediated by two non-receptor tyrosine kinases, JAK2 and Src. We previously demonstrated that prolactin stimulates invasion of breast cancer cells TMX2-28 through JAK2 and its target serine/threonine kinase PAK1. We hypothesize herein that the actin-binding protein cortactin, a protein involved in invadopodia formation and cell invasion, is activated by PRL. We demonstrate that TMX2-28 cells are more invasive than T47D breast cancer cells in response to PRL. We determine that cortactin is tyrosyl phosphorylated in response to PRL in a time and dose-dependent manner in TMX2-28 cells, but not in T47D cells. Furthermore, we show that PRL mediates cortactin tyrosyl phosphorylation via Src, but not JAK2. Finally, we demonstrate that maximal PRL-mediated TMX2-28 cell invasion requires both Src and JAK2 kinase activity, while T47D cell invasion is JAK2- but not Src-dependent. Thus PRL may induce cell invasion via two pathways: through a JAK2/PAK1 mediated pathway that we have previously demonstrated, and Src-dependent activation and tyrosyl phosphorylation of cortactin.

Tyrosyl phosphorylated serine-threonine kinase PAK1 is a novel regulator of prolactin-dependent breast cancer cell motility and invasion.

Despite efforts to discover the cellular pathways regulating breast cancer metastasis, little is known as to how prolactin (PRL) cooperates with extracellular environment and cytoskeletal proteins to regulate breast cancer cell motility and invasion. We implicated serine-threonine kinase p21-activated kinase 1 (PAK1) as a novel target for PRL-activated Janus-kinase 2 (JAK2). JAK2-dependent PAK1 tyrosyl phosphorylation plays a critical role in regulation of both PAK1 kinase activity and scaffolding properties of PAK1. Tyrosyl phosphorylated PAK1 facilitates PRL-dependent motility via at least two mechanisms: formation of paxillin/GIT1/ßPIX/pTyr-PAK1 complexes resulting in increased adhesion turnover and phosphorylation of actin-binding protein filamin A. Increased adhesion turnover is the basis for cell migration and phosphorylated filamin A stimulates the kinase activity of PAK1 and increases actin-regulating activity to facilitate cell motility. Tyrosyl phosphorylated PAK1 also stimulates invasion of breast cancer cells in response to PRL and three-dimensional (3D) collagen IV via transcription and secretion of MMP-1 and MMP-3 in a MAPK-dependent manner. These data illustrate the complex interaction between PRL and the cell microenvironment in breast cancer cells and suggest a pivotal role for PRL/PAK1 signaling in breast cancer metastasis.

JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein.

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.

Phosphorylation of tyrosine 285 of PAK1 facilitates ßPIX/GIT1 binding and adhesion turnover.

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to ß-PAK1-interacting guanine-nucleotide exchange factor (ßPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced ßPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.

Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A.

The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

The structure of the femoral neck: A physical dissection with emphasis on the internal trabecular system.

In an attempt to determine the position and course of the horizontal trabecular column within the femoral neck a series of femoral necks were dissected using a physical method. Unexpectedly, the findings of the study show that the internal structure of the femoral neck, overall, is more complex than has been previously described. Both trabecular columns blend intimately with the overlying cortex in the manner of a composite structure, which may have implications as to their strength. It was noted, as previously described in the literature, that the horizontal column has a spiral configuration. In addition it was found, in this study, that this column shows wide variation in its inclination between different specimens. With increasing osteoporosis, the remnant of the horizontal column comes to lie progressively more antero-superiorly within the femoral neck. While fairly uniform in cross-sectional area throughout the femoral neck, this column is found to arise from a large, internal buttress within the upper anterior femoral shaft. It is also found that the femoral calcar, initially, has widespread attachments to all the internal structures. It is accepted that the formation of these trabeculae, and their retention with age, is due to the forces acting through them. The findings of the study, however, suggest that the horizontal trabeculae are responsible primarily for the conduction of compression forces, rather than tension forces as described in the Trajectorial Theory. A suggestion as to how the compression forces may be conducted through both these columns in the upper femur is given in light of the findings. These concepts, if proven, may require a modification of the approach to the biomechanical analysis of this region, particularly when using the various imaging techniques, as well as with physical testing of prostheses and other such hip devices.

Canine digital tumors: a veterinary cooperative oncology group retrospective study of 64 dogs.

We compared clinical characteristics and outcomes for dogs with various digital tumors. Medical records and histology specimens of affected dogs from 9 veterinary institutions were reviewed. Risk factors examined included age, weight, sex, tumor site (hindlimb or forelimb), local tumor (T) stage, metastases, tumor type, and treatment modality. The Kaplan-Meier product limit method was used to determine the effect of postulated risk factors on local disease-free interval (LDFI), metastasis-free interval (MFI), and survival time (ST). Outcomes were thought to differ significantly between groups when P < or = .003. Sixty-four dogs were included. Squamous cell carcinoma (SCC) accounted for 33 (51.6%) of the tumors. Three dogs presented with or developed multiple digital SCC. Other diagnoses included malignant melanoma (MM) (n = 10; 15.6%), osteosarcoma (OSA) (n = 4; 6.3%), hemangiopericytoma (n = 3; 4.7%), benign soft tissue tumors (n = 5; 7.8%), and malignant soft tissue tumors (n = 9; 14%). Fourteen dogs with malignancies had black hair coats, including 5 of the 10 dogs with MM. Surgery was the most common treatment and, regardless of the procedure, had a positive impact on survival. None of the patient variables assessed, including age, sex, tumor type, site, and stage, had a significant impact on ST. Both LDFI and MFI were negatively affected by higher T stage, but not by type of malignancy. Although metastasis at diagnosis correlated with a shorter LDFI, it did not have a significant impact on ST. On the basis of these findings, early surgical intervention is advised for the treatment of dogs with digital tumors, regardless of tumor type or the presence of metastatic disease.

Localized venous plexi in the spine simulating prolapse of an intervertebral disc: a report of six cases.

STUDY DESIGN: Six cases are reported in which the clinical presentation of a prolapsed intervertebral disc was found to be caused by a localized venous plexus. OBJECTIVES: To emphasize the fact that the clinical presentation of a localized plexus of epidural veins in the lumbar spine can resemble that of an acute disc prolapse. SUMMARY OF BACKGROUND DATA: The finding of enlarged epidural veins during lumbar disc decompression is relatively common, but it is only recently that they have been implicated as the cause of the presenting symptoms. METHODS AND RESULTS: Six individuals presented with severe pain in the lower back accompanied by sciatica, which had begun acutely. Physical examination in most of these patients showed the presence of neurologic signs in the affected leg. The initial clinical assessment was that of an acute prolapse of a lumbar disc. This diagnosis appeared to be borne out by the MRI scans, which demonstrated a "prolapsed disc" at the relevant level of the spine. However, at surgery, the intervertebral disc appeared to be relatively normal, but at the spine was found a large, localized plexus of epidural veins whose configuration matched the MRI image. The symptoms were relieved by decompression of the spine and ablation of the veins. CONCLUSIONS: Any pathologic process in the lumbar spine compressing a nerve root can cause localized pain in the back accompanied by sciatica. Most of these conditions can be differentiated by means of an MRI scan. The MRI image of a localized plexus of epidural veins, however, closely resembles that of a prolapsed intervertebral disc, which may be diagnostically misleading.

Triangular structure of the proximal femur.

The normal human skeleton is remarkably regular in form. Its constancy is demonstrated by the projection of well-recognized lines onto plain radiographs to define many of its relationships. The finding of the geometric symmetry of the normal proximal femur, demonstrated by the projection of an isosceles triangle onto its anteroposterior radiographic view, is presented. The base of this triangle extends between the trochanters and its apex lies at the center of the femoral head. This triangle may reflect the balance of bone modeling that takes place in the proximal femur as a result of the forces acting in the coronal plane. If so, it may then allow for the development of a mathematical expression for bone modeling in this plane.