Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

A New Model of Esophageal Cancers by Using a Detergent-Free Decellularized Matrix in a Perfusion Bioreactor.

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Characterization of huntingtin interactomes and their dynamic responses in living cells by proximity proteomics.

Huntingtin (Htt) is a large protein without clearly defined molecular functions. Mutation in this protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Identification of Htt-interacting proteins by the traditional approaches including yeast two-hybrid systems and affinity purifications has greatly facilitated the understanding of Htt function. However, these methods eliminated the intracellular spatial information of the Htt interactome during sample preparations. Moreover, the temporal changes of the Htt interactome in response to acute cellular stresses cannot be easily resolved with these approaches. Ascorbate peroxidase (APEX2)-based proximity labeling has been used to spatiotemporally investigate protein-protein interactions in living cells. In this study, we generated stable human SH-SY5Y cell lines expressing full-length Htt23Q and Htt145Q with N-terminus tagged Flag-APEX2 to quantitatively map the spatiotemporal changes of Htt interactome to a mild acute proteotoxic stress. Our data revealed that normal and mutant Htt (muHtt) are associated with distinct intracellular microenvironments. Specifically, mutant Htt is preferentially associated with intermediate filaments and myosin complexes. Furthermore, the dynamic changes of Htt interactomes in response to stress are different between normal and mutant Htt. Vimentin is identified as one of the most significant proteins that preferentially interacts with muHtt in situ. Further functional studies demonstrated that mutant Htt affects the vimentin's function of regulating proteostasis in healthy and HD human neural stem cells. Taken together, our data offer important insights into the molecular functions of normal and mutant Htt by providing a list of Htt-interacting proteins in their natural cellular context for further studies in different HD models.

Impaired Restoration of Global Protein Synthesis Contributes to Increased Vulnerability to Acute ER Stress Recovery in Huntington's Disease.

Neurons are susceptible to different cellular stresses and this vulnerability has been implicated in the pathogenesis of Huntington's disease (HD). Accumulating evidence suggest that acute or chronic stress, depending on its duration and severity, can cause irreversible cellular damages to HD neurons, which contributes to neurodegeneration. In contrast, how normal and HD neurons respond during the resolution of a cellular stress remain less explored. In this study, we challenged normal and HD cells with a low-level acute ER stress and examined the molecular and cellular responses after stress removal. Using both striatal cell lines and primary neurons, we first showed the temporal activation of p-eIF2α-ATF4-GADD34 pathway in response to the acute ER stress and during recovery between normal and HD cells. HD cells were more vulnerable to cell death during stress recovery and were associated with increased number of apoptotic/necrotic cells and decreased cell proliferation. This is also supported by the Gene Ontology analysis from the RNA-seq data which indicated that "apoptosis-related Biological Processes" were more enriched in HD cells during stress recovery. We further showed that HD cells were defective in restoring global protein synthesis during stress recovery and promoting protein synthesis by an integrated stress response inhibitor, ISRIB, could attenuate cell death in HD cells. Together, these data suggest that normal and HD cells undergo distinct mechanisms of transcriptional reprogramming, leading to different cell fate decisions during the stress recovery.

Multiple channels with interconnected pores in a bioceramic scaffold promote bone tissue formation.

Insufficient nutrition exchange and limited transportation of blood supply in a porous only scaffold often hinder bone formation, even though the porous scaffold is loaded with cells or growth factors. To overcome these issues, we developed a cell- and growth factor-free approach to induce bone formation in a critical-size bone defect by using an interconnected porous beta-tricalcium phosphate (ß-TCP) scaffold with multiple channels. In vitro cell experimental results showed that multiple channels significantly promoted cell attachment and proliferation of human bone marrow mesenchymal stem cells, stimulated their alkaline phosphatase activity, and up-regulated the osteogenic gene expression. Multiple channels also considerably stimulated the expression of various mechanosensing markers of the cells, such as focal adhesion kinase, filamentous actin, and Yes-associated protein-1 at both static and dynamic culturing conditions. The in vivo bone defect implantation results demonstrated more bone formation inside multiple-channeled scaffolds compared to non-channeled scaffolds. Multiple channels prominently accelerated collagen type I, bone sialoprotein and osteocalcin protein expression. Fluorochrome images and angiogenic marker CD31 staining exhibited more mineral deposition and longer vasculature structures in multiple-channeled scaffolds, compared to non-channeled scaffolds. All the findings suggested that the creation of interconnected multiple channels in the porous ß-TCP scaffold is a very promising approach to promote bone tissue regeneration.

RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells.

BACKGROUND: Huntingtin (Htt) protein is the product of the gene mutated in Huntington's disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt. METHODS: To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion. RESULTS: In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR. CONCLUSIONS: The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.

Development of a Decellularized Porcine Esophageal Matrix for Potential Applications in Cancer Modeling.

Many decellularized extracellular matrix-derived whole organs have been widely used in studies of tissue engineering and cancer models. However, decellularizing porcine esophagus to obtain decellularized esophageal matrix (DEM) for potential biomedical applications has not been widely investigated. In this study a modified decellularization protocol was employed to prepare a porcine esophageal DEM for the study of cancer cell growth. The cellular removal and retention of matrix components in the porcine DEM were fully characterized. The microstructure of the DEM was observed using scanning electronic microscopy. Human esophageal squamous cell carcinoma (ESCC) and human primary esophageal fibroblast cells (FBCs) were seeded in the DEM to observe their growth. Results show that the decellularization process did not cause significant loss of mechanical properties and that blood ducts and lymphatic vessels in the submucosa layer were also preserved. ESCC and FBCs grew on the DEM well and the matrix did not show any toxicity to cells. When FBS and ESCC were cocultured on the matrix, they secreted more periostin, a protein that supports cell adhesion on matrix. This study shows that the modified decellularization protocol can effectively remove the cell materials and maintain the microstructure of the porcine esophageal matrix, which has the potential application of studying cell growth and migration for esophageal cancer models.

Aberrant subcellular localization of SQSTM1/p62 contributes to increased vulnerability to proteotoxic stress recovery in Huntington's disease.

Proteotoxic stress plays an important role in the pathogenesis of Huntington's disease (HD). Autophagy is proposed as a compensatory mechanism to remove protein aggregates under proteotoxic stress by up-regulating p62 expression. In the present study, we investigated the molecular action of p62 to proteotoxic stress in HD cells. Using two different HD cellular models, STHdhQ7 and STHdhQ111 cells derived from wild type and HD knock-in mice and human fibroblasts from healthy and HD patients, we found that HD cells are more vulnerable to cell death under proteotoxic stress and during stress recovery. We further showed that P62 was up-regulated in both STHdhQ7 and STHdhQ111 cells in response to the stress with distinct subcellular localization patterns. While dispersed p62 puncti were found in STHdhQ7 cells, p62 bodies were initially present in the lysosomes and accumulated to the juxtanuclear regions of STHdhQ111 cells as MG132 incubation continued. Unlike in STHdhQ7 cells, p62 puncti were not associated with K48-linked polyubiquitinated protein aggregates or proteasomal components in STHdhQ111. Interestingly, addition of cysteine during MG132 incubation rescued cell death in STHdhQ111 cells caused by stress recovery and altered the subcellular distribution of p62. Our data suggest that aberrant positioning of p62 affects the proteasomal clearance of protein aggregates and may contribute to the increased vulnerability to proteotoxic stress-induced cell death in HD cells.

Altered lysosomal positioning affects lysosomal functions in a cellular model of Huntington's disease.

Huntington's disease (HD) is a hereditary and devastating neurodegenerative disorder caused by a mutation in the huntingtin protein. Understanding the functions of normal and mutant huntingtin protein is the key to revealing the pathogenesis of HD and developing therapeutic targets. Huntingtin plays an important role in vesicular and organelle trafficking. Lysosomes are dynamic organelles that integrate several degradative pathways and regulate the activity of mammalian target of rapamycin complex 1 (mTORC1). In the present study, we found that the perinuclear accumulation of lysosomes was increased in a cellular model of HD derived from HD knock-in mice and primary fibroblasts from an HD patient. This perinuclear lysosomal accumulation could be reversed when normal huntingtin was overexpressed in HD cells. When we further investigated the functional significance of the increased perinuclear lysosomal accumulation in HD cells, we demonstrated that basal mTORC1 activity was increased in HD cells. In addition, autophagic influx was also increased in HD cells in response to serum deprivation, which leads to premature fusion of lysosomes with autophagosomes. Taken together, our data suggest that the increased perinuclear accumulation of lysosomes may play an important role in HD pathogenesis by altering lysosomal-dependent functions.

SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication.

UNLABELLED: Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by >200-fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA-binding domains but not the arginine serine-rich activator domain can downregulate viral replication by >2,000-fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA-binding domains. IMPORTANCE: Most drugs utilized to treat the HIV-1 infection are based on compounds that directly target proteins encoded by the virus. However, given the high viral mutation rate, the appearance of novel drug-resistant viral strains is common. Thus, there is a need for novel therapeutics with diverse mechanisms of action. In this study, we show that the cellular protein SRSF1 is a strong inhibitor of viral replication. Furthermore, expression of the SRSF1 RNA-binding domains alone can inhibit viral replication by >2,000-fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein, the RNA-binding domains, and the minimal effects observed on cell metabolism, further studies are warranted to assess the therapeutic potential of peptides derived from these sequences.

A truncated hnRNP A1 isoform, lacking the RGG-box RNA binding domain, can efficiently regulate HIV-1 splicing and replication.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. hnRNP A1 is localized prevalently in the nucleus but it can relocate to the cytoplasm in response to specific stimuli shuttling between nuclear and cytoplasmic compartments. The cellular localization of this protein is regulated by a short C-terminus motif (M9) and other less defined sequences. The RNA binding specificity of this protein is dependent on multiple RNA binding domains (RBDs), which regulate its role in RNA processing and expression. hnRNP A1 plays multiple roles in gene expression by regulating the biogenesis and translation of messengers RNAs, the processing of miRNAs, affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease, cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that the three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein, lacking one of the RBDs, the RGG-box, can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild-type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform, which had not been previously experimentally characterized.

Direct interaction between AR and PAK6 in androgen-stimulated PAK6 activation.

A p21-activated kinase 6 (PAK6) was previously identified to be an androgen receptor (AR) interacting protein through a yeast two-hybrid screening. We used hormone responsive prostate cancer LAPC4 and LNCap cell lines as models to study the signaling events associated with androgen stimulation and PAK6. An androgen-stimulated PAK6 kinase activation was observed in LAPC4 cells expressing endogenous PAK6 and in LNCap cells ectopically expressing a wild type PAK6. This activation was likely mediated through a direct interaction between AR and PAK6 since siRNA knock-down of AR in LAPC4 cells downregulated androgen-stimulated PAK6 activation. In addition, LNCap cells expressing a non-AR-interacting PAK6 mutant exhibited dampened androgen-stimulated kinase activation. As a consequence of androgen-stimulated activation, PAK6 was phosphorylated at multiple serine/threonine residues including the AR-interacting domain of PAK6. Furthermore, androgen-stimulation promoted prostate cancer cell motility and invasion were demonstrated in LNCap cells ectopically expressing PAK6-WT. In contrast, LNCap expressing non-AR-interacting mutant PAK6 did not respond to androgen stimulation with increased cell motility and invasion. Our results demonstrate that androgen-stimulated PAK6 activation is mediated through a direct interaction between AR and PAK6 and PAK6 activation promotes prostate cancer cells motility and invasion.

Increased PAK6 expression in prostate cancer and identification of PAK6 associated proteins.

BACKGROUND: PAK6 is a member of the p21-activated kinase (PAK) family of serine/threonine kinases that was originally cloned from prostate cancer (PCa) cells as an androgen receptor interacting protein, but its cellular distribution and functions have not been established. METHODS: An affinity purified rabbit anti-PAK6 antiserum was generated to assess PAK6 protein expression. PAK6 associated proteins were identified by immunopurification of 3xFlag-tagged PAK6 followed by LC/MS/MS. RESULTS: We confirmed that PAK6 protein is expressed in prostate and breast cancer cell lines. PAK6 expression in LNCaP PCa cells was not directly androgen regulated, but was markedly increased when the cells were cultured for 6-8 weeks in steroid hormone depleted medium. By immunohistochemistry, PAK6 was weakly expressed in normal prostate epithelium. Its expression was increased in primary and metastatic PCa, and was further increased in tumors that relapsed after androgen deprivation therapy. LC/MS/MS identified IQ motif containing GTPase activating protein 1 (IQGAP1) and protein phosphatase 1B (PP1B) as candidate PAK6 interacting proteins, and these findings were confirmed by coimmunoprecipitation. CONCLUSIONS: These results indicate that PAK6 contributes to PCa development and progression after androgen deprivation therapy, and that it may play roles in the regulation of motility and in stress responses.

SOX9 is expressed in human fetal prostate epithelium and enhances prostate cancer invasion.

SOX9 is a transcription factor that plays a critical role in the development of multiple tissues. We previously reported that SOX9 in normal human adult prostate was restricted to basal epithelium. SOX9 was also expressed in a subset of prostate cancer (PCa) cells and was increased in relapsed hormone-refractory PCa. Moreover, SOX9 expression in PCa cell lines enhanced tumor cell proliferation and was beta-catenin regulated. Here we report additional in vivo results showing that SOX9 is highly expressed during fetal prostate development by epithelial cells expanding into the mesenchyme, suggesting it may contribute to invasive growth in PCa. Indeed, SOX9 overexpression in LNCaP PCa xenografts enhanced growth, angiogenesis, and invasion. Conversely, short hairpin RNA-mediated SOX9 suppression inhibited the growth of CWR22Rv1 PCa xenografts. These results support important functions of SOX9 in both the development and maintenance of normal prostate, and indicate that these functions contribute to PCa tumor growth and invasion.

SOX9 is expressed in normal prostate basal cells and regulates androgen receptor expression in prostate cancer cells.

SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of HMG DNA-binding domain transcription factors and is required for the development and differentiation of multiple cell lineages. This report shows that basal epithelial cells express SOX9 in normal prostate, with no detectable expression in luminal epithelial cells. In contrast, SOX9 is expressed in primary prostate cancers in vivo, at a higher frequency in recurrent prostate cancer and in prostate cancer cell lines (LNCaP, CWR22, PC3, and DU145). SOX9 message and protein levels in prostate cancer cells were increased by treatment with glycogen synthase kinase 3beta inhibitor (SB415286), and SOX9 was reduced when beta-catenin was down-regulated by small interfering RNA (siRNA), indicating that SOX9 expression in prostate cancer is regulated by Wnt/beta-catenin signaling. SOX9 bound specifically to androgen receptor (AR) DNA-binding domain glutathione S-transferase fusion proteins, and this interaction was dependent on a short peptide immediately COOH-terminal to the DNA-binding domain (the C-terminal extension), which is required for interactions between steroid hormone receptors and the architectural HMG proteins. Exogenous SOX9 expressed at high nonphysiologic levels decreased AR expression and activity; however, at lower levels, SOX9 increased AR protein expression. Significantly, down-regulation of SOX9 by siRNA in prostate cancer cells reduced endogenous AR protein levels, and cell growth indicating that SOX9 contributes to AR regulation and decreased cellular proliferation. These results indicate that SOX9 in prostate basal cells supports the development and maintenance of the luminal epithelium and that a subset of prostate cancer cells may escape basal cell requirements through SOX9 expression.

Androgen receptor remains critical for cell-cycle progression in androgen-independent CWR22 prostate cancer cells.

The majority of prostate cancers (PCa) that relapse after androgen deprivation therapy (androgen-independent PCa) continue to express androgen receptor (AR). To study the functional importance of AR in these tumors, we derived androgen-independent CWR22 PCa xenografts in castrated mice and generated a cell line from one of these xenografts (CWR22R3). Similarly to androgen-independent PCa in patients, the relapsed xenografts and cell line expressed AR and were resistant to treatment with bicalutamide. However, expression of the AR-regulated PSA gene in the CWR22R3 cell line was markedly decreased compared to the relapsed xenografts in vivo. Transfections with androgen-regulated reporter genes further indicated that the cells lacked androgen-independent AR transcriptional activity and were not hypersensitive to low androgen concentrations despite constitutive activation of the Erk/MAP kinases. Nonetheless, AR remained essential for androgen-independent growth because retroviral shRNA-mediated AR down-regulation resulted in marked long-term growth suppression. This was associated with increased levels of p27(kip1) and hypophosphorylation of retinoblastoma protein but not with decreases in D-type cyclin levels or MAP kinase activation. These results reveal a potentially critical function of AR in androgen-independent PCa that is distinct from its previously described transcriptional or nontranscriptional functions.

Rho/ROCK-dependent pseudopodial protrusion and cellular blebbing are regulated by p38 MAPK in tumour cells exhibiting autocrine c-Met activation.

BACKGROUND INFORMATION: The c-Met-dependent, beta-actin-rich, blebbed pseudopodia of MSV-MDCK-INV (invasive Moloney-sarcoma-virus-transformed Madin-Darby canine kidney) cells are induced by Rho/ROCK (Rho kinase) activation, and are morphologically distinct from flat extended lamellipodia. RESULTS: Microtubules were shown to extend to these actin-rich pseudopodial domains, and microtubule depolymerization by nocodazole treatment resulted in progressive cellular blebbing, initiating in the pseudopodial domains and resulting in transient cellular rounding and blebbing after 30 min. The blebbing response was dependent on autocrine HGF (hepatocyte growth factor) activation of c-Met and prevented by inhibition of RhoA, ROCK and p38 MAPK (p38 mitogen-activated protein kinase), but not ERK (extracellular-signal-regulated kinase) or PI3K (phosphoinositide 3-kinase). Phospho-p38 MAPK was present in pseudopodia, localizing activation of this signalling pathway to this protrusive membrane structure. In serum-starved cells, LPA (lysophosphatidic acid) activation of RhoA induced p38 MAPK-dependent pseudopodial protrusions, and inhibition of p38 MAPK prevented pseudopodial protrusion and displacement of MSV-MDCK-INV cells. MSV-MDCK-INV cells exhibited intermittent blebbing and rounding, which may represent an integral part of their motile behaviour. CONCLUSIONS: The localized activation of an autocrine HGF/c-Met loop regulates Rho/ROCK activation of p38 MAPK signalling to stimulate both membrane blebbing and pseudopod formation.

Membrane rafts as potential sites of nongenomic hormonal signaling in prostate cancer.

Recent evidence indicates that nuclear receptors for steroid hormones can signal by nongenomic mechanisms that operate independently of their transcription function. These signal-transduction processes occur within seconds to minutes after initiation with agonist and involve interactions between nuclear receptors and other signaling proteins in the cytoplasm and at membrane surfaces. This review provides an overview of published information on possible nongenomic activities of the androgen receptor (AR) and other nuclear receptors, focusing on the potential involvement of these processes in prostate cancer. We discuss the hypothesis that the cholesterol-rich lipid-raft compartment(s) of cancer-cell membranes might provide privileged sites for nongenomic signals mediated by the AR.

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase.

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.

Integrin-dependent protein tyrosine phosphorylation is a key regulatory event in collagen-IV-mediated adhesion and proliferation of human lung tumor cell line, Calu-1.

BACKGROUND: The clinical phenomenon of lung cancer metastasis to specific target organs is believed to be a direct interaction between tumor cells and extracellular matrix components. During invasion, tumor cells attach to the basement membrane protein, collagen type IV, degrade it, migrate through blood vessels, and adhere to extracellular matrix proteins. METHODS: Four nonsmall-cell lung cancer cells were tested for adhesion, proliferation, migration and morphologic alterations on collagen type IV matrix by immunoprecipitation, Western blotting, phase contrast and time lapse video microscopy. RESULTS: Collagen type IV promoted Calu-1 cell adhesion (< 15 minutes) and motility (< 6 hours) without any significant effect on proliferation. beta(1)-integrin is essential for collagen type IV adhesion and 8 to 10 fold higher expression of beta1-integrin on the surface of Calu-1 cells was identified. A protein tyrosine phosphatase inhibitor, peroxyvanadate, caused 50% inhibition in the adhesion process within 5 minutes but exposure to 60 micromol/L genistein for 72 hours, a protein tyrosine kinase inhibitor, drastically inhibits Calu-1 cell proliferation (> 70%). We examined the influence of beta1-integrin, peroxyvanadate and genistein on the spreading morphogenesis of Calu-1 cells on Collagen type IV. Use of either 1 microg of anti beta1-integrin inhibitory antibody (P5D2), 100 micromol/L peroxyvanadate or 60 micromol/L genistein had dramatic influence on the spreading of Calu-1 cells. Finally, Focal adhesion kinase was identified as a phosphoprotein target. CONCLUSIONS: We have characterized an in vitro model of matrix-specific binding of a lung cancer cell line, Calu-1 to Coll IV. Calu-1 cells use primarily a beta1-integrin mediated intracellular tyrosine phosphorylation phenomenon as the key regulatory mechanism for its binding advantage to Coll IV matrix.