KANSL2 and MBNL3 are regulators of pancreatic ductal adenocarcinoma invasion.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer. One major reason for this is that PDAC quickly metastasizes to other organs, thereby making its treatment difficult. The molecular machinery driving PDAC metastasis is still poorly understood. In this study, we applied an unbiased approach using CRISPR screening to identify genes that strongly regulate invasion (based on an in vitro assessment of their metastatic potential) in PANC-1, a PDAC cell line. Through CRISPR screening, we identified MBNL3 and KANSL2 as strong regulators of invasion in PANC-1 cells. We further validated MBNL3 and KANSL2 as regulators of PANC-1 cell invasion by using the doxycycline-inducible shRNA system. We also showed that MBNL3 and KANSL2 do not affect cell proliferation. Through our efforts, we have established a process to identify genes that regulate cell invasion and can be further investigated as potential targets for therapeutic intervention.

Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV-visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme-clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.

A high-throughput screening identifies histone deacetylase inhibitors as therapeutic agents against medulloblastoma.

BACKGROUND: Medulloblastoma is the most frequently occurring malignant brain tumor in children. Current treatment strategies for medulloblastoma include aggressive surgery, cranio-spinal irradiation and adjuvant chemotherapy. Because current treatments can cause severe long-term side effects and are not curative, successful treatment remains a challenge. METHODS: In this study, we employed a high-throughput cell viability assay to screen 12,800 compounds and to identify drug candidates with anti-proliferative properties for medulloblastoma cells. We also tested these compounds for attenuating medulloblastoma tumor development using mouse xenografts. RESULTS: We identified two histone deacetylase inhibitors (dacinostat and quisinostat) with anti-proliferative properties for medulloblastoma cells. We showed that both compounds induce cytotoxicity, trigger cell apoptosis, and block cell cycle progression at the G2/M phase. In addition, dacinostat and quisinostat attenuated xenograft medulloblastoma growth in mice. CONCLUSIONS: Our findings suggest that histone deacetylase inhibitors are potent therapeutic agents against medulloblastoma.

Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells.

BACKGROUND: Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. METHODS: We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. RESULTS: The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. CONCLUSIONS: Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.

RNA interference screen identifies NAA10 as a regulator of PXR transcription.

The pregnane X receptor (PXR) is a principal xenobiotic receptor crucial in the detection, detoxification, and clearance of toxic substances from the body. PXR plays a vital role in the metabolism and disposition of drugs, and elevated PXR levels contribute to cancer drug resistance. Therefore, to modulate PXR activity and mitigate drug resistance, it is imperative to fully understand its regulation. To this end, we screened a transcription factor siRNA library in pancreatic cancer cells that express high levels of PXR. Through a comprehensive deconvolution process, we identified N-alpha-acetyltransferase 10 (NAA10) as a factor in the transcriptional machinery regulating PXR transcription. Because no one single factor has 100% operational control of PXR transcriptional regulation, our results together with other previous findings suggest that the transcriptional regulation of PXR is complex and that multiple factors contribute to the process including NAA10.

PXR: More Than Just a Master Xenobiotic Receptor.

Pregnane X receptor (PXR) is a nuclear receptor considered to be a master xenobiotic receptor that coordinately regulates the expression of genes encoding drug-metabolizing enzymes and drug transporters to essentially detoxify and eliminate xenobiotics and endotoxins from the body. In the past several years, the function of PXR in the regulation of xenobiotic metabolism has been extensively studied, and the role of PXR as a xenobiotic sensor has been well established. It is now clear, however, that PXR plays many other roles in addition to its xenobiotic-sensing function. For instance, recent studies have discovered previously unidentified roles of PXR in inflammatory response, cell proliferation, and cell migration. PXR also contributes to the dysregulation of these processes in diseases states. These recent discoveries of the role of PXR in the physiologic and pathophysiologic conditions of other cellular processes provides the possibility of novel targets for drug discovery. This review highlights areas of PXR regulation that require further clarification and summarizes the recent progress in our understanding of the nonxenobiotic functions of PXR that can be explored for relevant therapeutic applications.

Glucose-dependent regulation of pregnane X receptor is modulated by AMP-activated protein kinase.

Pregnane X receptor (PXR) is a xenobiotic receptor that regulates the detoxification and clearance of drugs and foreign compounds from the liver. There has been mounting evidence of crosstalk between the drug metabolism pathway and the energy metabolism pathway, but little is known about this cross-regulation. To further delineate the energy metabolism and drug metabolism crosstalk in this study, we exposed HepG2 cells to varying glucose concentrations. We observed that PXR activity was induced under high-glucose conditions. This finding is consistent with previous clinical reports of increased drug clearance in patients with untreated diabetes. We demonstrated that AMP-activated protein kinase (AMPK) modulates PXR transcriptional activity and that pharmacologically manipulated AMPK activation exhibits an inverse relation to PXR activity. Activation of AMPK was shown to downregulate PXR activity and, consistent with that, potentiate the response of cells to the drug. Taken together, our results delineate a hitherto unreported axis of regulation that involves the energy status of the cell, PXR regulation, and drug sensitivity.

Regulation of PXR and CAR by protein-protein interaction and signaling crosstalk.

INTRODUCTION: Protein-protein interaction and signaling crosstalk contribute to the regulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) and broaden their cellular function. AREA COVERED: This review covers key historic discoveries and recent advances in our understanding of the broad function of PXR and CAR and their regulation by protein-protein interaction and signaling crosstalk. EXPERT OPINION: PXR and CAR were first discovered as xenobiotic receptors; however, it is clear that PXR and CAR perform a much broader range of cellular functions through protein-protein interaction and signaling crosstalk, which typically mutually affect the function of all the partners involved. Future research on PXR and CAR should, therefore, look beyond their xenobiotic function.

Synergistic Activation of ERα by Estrogen and Prolactin in Breast Cancer Cells Requires Tyrosyl Phosphorylation of PAK1.

Serine/threonine kinase PAK1 is activated by estrogen and plays an important role in breast cancer. However, the integration of PAK1 into the estrogen response is not fully understood. In this study, we investigated the mechanisms underlying the hormone-induced activation of estrogen receptor (ERα, ESR1). We show that estrogen activated PAK1 through both the ERα and GPER1 membrane receptors. Estrogen-dependent activation of PAK1 required the phosphorylation of tyrosine residues by Etk/Bmx and protein kinase A (PKA) within an assembled signaling complex comprising pTyr-PAK1, Etk/Bmx, the heterotrimer G-protein subunits Gß1, Gγ2, and/or Gγ5, PAK-associated guanine nucleotide exchange factor (ßPIX, ARHGEF7), and PKA. Moreover, the PKA RIIß subunit is a direct target of PAK1, and thus in response to estrogen, the activated pTyr-PAK1 complex reciprocally potentiated PKA activity, suggesting a positive feedback mechanism. We also demonstrate that PKA phosphorylated Ser305-ERα in response to estrogen, but pTyr-PAK1 phosphorylated Ser305-ERα in response to prolactin (PRL), implying that maximal ERα phosphorylation is achieved when cells are exposed to both PRL and estrogen. Furthermore, S305-ERα activation led to enhanced phosphorylation of Ser118-ERα and promoted cell proliferation and tumor growth. Together, these data strongly support a critical interplay between PRL and estrogen via PAK1 and suggest that ligand-independent activation of ERα through PRL/PAK1 may impart resistance to anti-estrogen therapies. Cancer Res; 76(9); 2600-11. ©2016 AACR.

PAK1 translocates into nucleus in response to prolactin but not to estrogen.

Tyrosyl phosphorylation of the p21-activated serine-threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmed these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm.

A Derivative of Differentiation-Inducing Factor-3 Inhibits PAK1 Activity and Breast Cancer Cell Proliferation.

Differentiation-inducing factors 1-3 (DIFs 1-3), chlorinated alkylphenones identified in the cellular slime mold Dictyostelium discoideum, are considered anti-tumor agents because they inhibit proliferation of a variety of mammalian tumor cells in vitro. Although the anti-proliferative effects of DIF-1 and DIF-3 are well-documented, the precise molecular mechanisms underlying the actions of DIFs have not been fully elucidated. In this study, we examined the effects of DIFs and their derivatives on PAK1, a key serine-threonine kinase, which is activated by multiple ligands and regulates cell proliferation. We examined the effect of DIF derivatives on PAK1 kinase activity in cells. We also examined the effect of DIF-3(+1) derivative on PAK1 kinase activity in vitro, cyclin D1 promoter activity and breast cancer cell proliferation. It was found that some derivatives strongly inhibited PAK1 kinase activity in human breast cancer MCF-7 cells stably over expressing PAK1. Among the derivatives, DIF-3(+1) was most potent, which directly inhibited kinase activity of recombinant purified PAK1 in an in vitro kinase assay. Furthermore, DIF-3(+1) strongly inhibited both cyclin D1 promoter activity and proliferation of MCF-7 and T47D breast cancer cells stably over expressing PAK1 in response to prolactin, estrogen, epidermal growth factor and heregulin. In the present study we propose PAK1 as DIF-3(+1) target mediating its anti-proliferative effect.

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.

PAK1 regulates breast cancer cell invasion through secretion of matrix metalloproteinases in response to prolactin and three-dimensional collagen IV.

p21-Activated serine-threonine kinase (PAK1) is implicated in breast cancer. We have shown previously that PAK1 is tyrosyl phosphorylated by prolactin (PRL)-activated Janus tyrosine kinase (JAK2). Although a role for both PRL and PAK1 in breast cancer is widely acknowledged, the mechanism remains poorly understood. In the present study, PRL-activated PAK1 stimulates the invasion of TMX2-28 human breast cancer cells through Matrigel. Three-dimensional (3D) collagen IV stimulates the secretion of the matrix proteases, metalloproteinase (MMP)-1 and -3 that is further enhanced by the PRL-dependent tyrosyl phosphorylation of PAK1. 3D collagen IV also stimulates the expression and secretion of MMP-2, but in contrast to MMP-1 and -3, PRL/PAK1 signaling down-regulates MMP-2 expression and secretion. In contrast, MMP-9 expression and secretion are stimulated by 3D collagen I, not collagen IV, and are not affected by PRL but are down-regulated by PAK1. MMP-1 and -3 are required and MMP-2 contributes to PRL-dependent invasion. ERK1/2 signaling appears to be required for the enhanced expression and secretion of MMP-1 and -3 and enhanced PRL-dependent invasion. p38 MAPK and c-Jun N-terminal kinase 1/2 pathways participate in production of MMP-1 and -3 as well as in PRL/PAK1-dependent cell invasion. Together, these data illustrate the complex interaction between the substratum and PRL/PAK1 signaling in human breast cancer cells and suggest a pivotal role for PRL-dependent PAK1 tyrosyl phosphorylation in MMP secretion.

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.

Identification of novel mitosis regulators through data mining with human centromere/kinetochore proteins as group queries.

BACKGROUND: Proteins functioning in the same biological pathway tend to be transcriptionally co-regulated or form protein-protein interactions (PPI). Multiple spatially and temporally regulated events are coordinated during mitosis to achieve faithful chromosome segregation. The molecular players participating in mitosis regulation are still being unravelled experimentally or using in silico methods. RESULTS: An extensive literature review has led to a compilation of 196 human centromere/kinetochore proteins, all with experimental evidence supporting the subcellular localization. Sixty-four were designated as "core" centromere/kinetochore components based on peak expression and/or well-characterized functions during mitosis. By interrogating and integrating online resources, we have mined for genes/proteins that display transcriptional co-expression or PPI with the core centromere/kinetochore components. Top-ranked hubs in either co-expression or PPI network are not only enriched with known mitosis regulators, but also contain candidates whose mitotic functions are not yet established. Experimental validation found that KIAA1377 is a novel centrosomal protein that also associates with microtubules and midbody; while TRIP13 is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31(comet). CONCLUSIONS: Transcriptional co-expression and PPI network analyses with known human centromere/kinetochore proteins as a query group help identify novel potential mitosis regulators.

PAK1-Nck regulates cyclin D1 promoter activity in response to prolactin.

Prolactin (PRL) is critical for alveolar proliferation and differentiation in normal mammary development and is also implicated in breast cancer. PRL influences cell proliferation and growth by altering the expression of cyclin D1. Cyclin D1 expression is directly regulated by PRL through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5-mediated transcriptional activation of the cyclin D1 promoter. A p21-activated serine-threonine kinase (PAK)1 has also been implicated in the regulation of cyclin D1 gene expression. We have previously demonstrated that JAK2 directly phosphorylates PAK1 and extend these data here to demonstrate that PAK1 activates the cyclin D1 promoter in response to PRL. We show that mutation of PAK1 Tyr 153, 201, and 285 (sites of JAK2 phosphorylation; PAK1 Y3F) decreases both PAK1 nuclear translocation in response to PRL and PRL-induced cyclin D1 promoter activity by 55%. Mutation of the PAK1 nuclear localization signals decreases PRL-induced cyclin D1 promoter activity by 46%. A PAK1 Y3F mutant lacking functional nuclear localization signals decreases PRL-induced cyclin D1 activity by 68%, suggesting that there is another PAK1-dependent mechanism to activate the cyclin D1 promoter. We have found that adapter protein Nck sequesters PAK1 in the cytoplasm and that coexpression of both PAK1 and Nck inhibits the amplifying effect of PRL-induced PAK1 on cyclin D1 promoter activity (95% inhibition). This inhibition is partially abolished by disruption of PAK1-Nck binding. We propose two PAK1-dependent mechanisms to activate cyclin D1 promoter activity in response to PRL: via nuclear translocation of tyrosyl-phosphorylated PAK1 and via formation of a Nck-PAK1 complex that sequesters PAK1 in the cytoplasm.