Journal: Cancer research

Article Title: The role of metastasis-associated protein 1 in prostate cancer progression.

doi: 10.1158/0008-5472.can-03-2755

Figure Lengend Snippet: Fig. 1. A, heatmap of the cDNA microarray data for benign prostatic tissue (Benign), localized pros- tate cancer (PCA), and metastatic prostate cancer (METS) samples [modified from Varambally et al. (2)]. Metastasis-associated protein 1 (MTA1) was among several genes up-regulated in METS when compared with PCA or benign prostate tissue. B, error bars with 95% confidence intervals (CIs) demonstrating the expression level of MTA1 in benign prostatic tissue, PCA, and METS cases. The level of MTA1 expression in PCA was signifi- cantly increased compared with benign prostate tissue. METS showed the highest level of MTA1 expression. C, Western blot analysis demonstrating MTA1 protein expression. Fifteen g of protein extract of normal, PCA, and METS tissue was separated in a 10% polyacrylamide gel. Incubation with anti-MTA1 antibody 1:50 overnight at 4°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to demonstrate equal protein loading. The bar graph shows normalized expres- sion as MTA1/GAPDH ratio. Normal samples showed only little MTA1 expression, whereas the majority of localized prostate cancer and almost all of the metastatic prostate cancer samples have a strong increase in MTA1 expression. D, represent- ative images of tissue microarray samples staining absent, weak, moderate, and strong for MTA1 ex- pression. MTA1 expression was confined to the nuclei of cells as demonstrated by immunohisto- chemistry (200 magnification). E, error bars with 95% CIs demonstrating MTA1 protein expres- sion levels in a wide spectrum of prostate tissue samples. MTA1 staining intensity was evaluated along a scale ranging from negative expression (score 1) to strong staining (score 4). F, Kaplan-Meier analyses of MTA1 status and pros- tate-specific antigen (PSA)-defined recurrence-free survival after radical prostatectomy for clinically localized prostate cancer. Biochemical failure was defined as a PSA elevation 0.2 ng/ml after rad- ical prostatectomy. Negative MTA1 expression was associated with a 2.8 relative risk (95% CI: 1.2–6.7, P 0.02) of PSA recurrence as compared with moderate/strong MTA1 expression.

Article Snippet: The primary antibody against MTA1 [anti-MTA1 monoclonal (A11), sc-17773; Santa Cruz Biotechnology] was incubated overnight in a 1:5 dilution at 4°C.

Techniques: Microarray, Modification, Expressing, Western Blot, Incubation, Staining, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 and MTA1 levels negatively correlate in human cancers. ( A ) mRNA expression pattern from Oncomine and TCGA data sets for prostate, breast and colon cancer tissues. For prostate cancer: red solid circle represents Magee et al , 2001 , blue solid triangle represents Grasso et al , 2012 , green solid square represents Tomlins et al , 2007 and solid orange hexagon represents Wallace et al , 2008 data sets respectively. For breast cancer: red solid circle represents Curtis et al , 2012 , solid blue triangle represents TCGA study for invasive breast carcinoma, solid green square represents TCGA data set for invasive ductal breast carcinoma, solid orange hexagon represents TCGA data set for mucinous breast carcinoma and solid magenta star represents Radvanyi et al , 2005 data sets. Similarly, for colon cancer: solid red dot represents TCGA data set for rectal adenocarcinoma, solid blue triangle represents TCGA data set for colon adenocarcinoma, solid green square represents TCGA data set for cecum adenocarcinoma and solid magenta diamond represents Notterman et al , 2001 data set. ( B ) To define expression correlation between PKD1 and MTA1, western blot analysis was performed using protein extracts of prostate (LNCaP, C4-2) and colon (SW480, SW48) cancer cells. ( C ) Confocal microscopy images to show subcellular localisation of PKD1 (Green) and MTA1 (Red) in LNCaP, C4-2, SW480 and SW48 cells. DAPI (blue) is used for nuclear staining. Original magnifications × 400, scale bar=10 μ m. ( D ) C4-2 and SW480 cells were stably transfected with either PKD1-overexpressing vector (C4-2-PKD1, SW480-PKD1) or GFP-tagged vector (C4-2-GFP, SW480-GFP) (control) and protein lysates were subjected to western blot analysis. ( E ) Quantitative PCR analysis of MTA1 mRNA levels in PKD1-overexpressing (C4-2-PKD1, SW480-PKD1) and vector control (C4-2-GFP, SW480-GFP) cells. ( F ) Western blot analysis of PKD1 and MTA1 in PKD1-expressing (PKD1-control siRNA) and PKD1 knockdown (PKD1-siRNA) LNCaP cells. LNCaP cells were transiently transfected with control and PKD1 siRNAs for 48 h. ( G ) Similarly, cells were transiently transfected with control and MTA1 siRNA and protein lysates were examined for MTA1 and PKD1 expression by western blot analysis. MTA1=metastasis-associated protein 1; PKD1=protein kinase D1. * P <0.01.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Expressing, Western Blot, Confocal Microscopy, Staining, Stable Transfection, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Knockdown

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 induces nucleo-cytoplasmic translocation and degradation of MTA1. ( A and B ) Immunofluorescence analysis of PKD1 and MTA1 expression in vector control (C4-2-GFP and SW480-GFP) and PKD1-overexpressing C4-2 (C4-2-PKD1 and SW480-PKD1) cells. Control and PKD1 vectors were detected using GFP tag (Green), whereas MTA1 was detected using CY3-labelled secondary antibody (Red) and DAPI was used for nuclear staining. Original magnifications × 400. ( C and D ) Western blot analysis of MTA1 and pPKD1 expression in vector control and PKD1-overexpressing C4-2 cells that were treated with indicated concentration of cyclohexamide (CHX for 2, 2.5 and 3 h) or Bryostatin-1 (20 and 30 n M ) for 24 h. ( E–G ) Confocal microscopy images Bryostatin-1 treated C4-2 cells at 3, 12 and 24 h. At 3 h localisation of MTA1 (Red) into Golgi as detected by GM130 (Green), a Golgi marker, at 12 h in trans -Golgi network (TGN) using P230 (Green) as TGN marker and at 24 h MTA1 (Green) in lysosomes using Red lysotracker (Red) as a marker for lysosome. Original magnifications × 400, scale bar=10 μ m. MTA1=metastasis-associated protein 1; PKD1=protein kinase D1.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Translocation Assay, Immunofluorescence, Expressing, Plasmid Preparation, Control, Staining, Western Blot, Concentration Assay, Confocal Microscopy, Marker

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 degrades MTA1 protein via ubiquitin mechanism. ( A ) MTA1 (Red) co-localisation with Ubiquitin (UBX) marker (Green) in response to Bryostatin-1, a PKD1 activator treatment in confocal microscopy analysis. Bryostatin-1 treatment (24 h) translocates MTA1 to lysosome via ubiquitin-dependent mechanism. ( B ) MTA1 (Red) co-localisation with Ubiquitin (UBX) marker (Green) in cells that were treated with Bryostatin-1 and MG132, a proteasome inhibitor (10 μ M ) for 24 h. MG132, abrogated the co-localisation of MTA1 and UBX. Original magnifications × 400. ( C ). Western blot analysis for MTA1 analysis in lysates of C4-2 cells that were treated with Bryostatin-1, MG132 (10 μ M ) and combination of Bryostatin-1 and MG132 for 24 h. β -Actin was used as a loading control. ( D ) Protein lysates of transiently transfected C4-2-PKD1 and C4-2-GFP cells were subjected to immunoprecipitation with K48-specific polyubiquitin antibody followed by western blotting to detect interaction of MTA1 with K48 protein. IgG was used as an internal control. Original magnifications × 400, scale bar=10 μ m. IP=immunoprecipitation; MTA1=metastasis-associated protein 1; PKD1=protein kinase D1; WB=western blotting.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Ubiquitin Proteomics, Marker, Confocal Microscopy, Western Blot, Control, Transfection, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 interacts and phosphorylates MTA1. ( A ) Protein lysates of C4-2-PKD1, C4-2-GFP and C4-2-PKD1-KD cells were subjected to immunoprecipitation (IP) with PKD1 antibody followed by western blot analysis with MTA1- and PKD1-specific antibodies, to detect the interaction between PKD1 and MTA1. ( B ) Proximity ligation assay (PLA) for interaction of PKD1 with MTA1 in C4-2-PKD1 cells, which is showing PKD1-MTA1 interaction in peri-nuclear space as indicated by red dots (i). Proximity ligation assay (PLA) image of PKD1 only and HER2/MUC13 in HPAF-II cells serves as negative (ii) and positive controls (iii), respectively. (iv) represent the mean fluorescence intensity of nuclear and cytoplasmic MTA1 in C4-2-PKD-overexpressing cells. * P <0.01. (iv). Original magnifications × 400, scale bar=10 μm. ( C ) To determine which domain of PKD1 is responsible for interaction MTA1, C4-2 cells were transiently transfected with different deletion mutants of PKD1 (ΔAC (acidic region deleted), ΔC1a (Cysteine-rich region a deleted), ΔC1b (Cysteine-rich region b deleted), ΔCRD (Cysteine-rich region a and b deleted) ΔPH (pleckstrin homology domain deletion), ΔN (N-terminal domain deletion), PKD1-KD (Kinase dead domain)) and full-length PKD1. Protein lysates were subjected to IP with PKD1 antibody followed by western blot analysis for MTA1 and PKD1 proteins. ( D ) To determine phosphorylation of MTA1 by PKD1, kinase assay was performed using protein lysates from C4-2-PKD1 and C4-2-GFP cells. Protein lysates were subjected to IP with phospho-PKD substrate antibody followed by western blot analysis with MTA1 and PKD1 antibodies. IgG was used as negative control. IP=immunoprecipitation; MTA1=metastasis-associated protein 1; PKD1=protein kinase D1; WB=western blotting.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Fluorescence, Transfection, Phospho-proteomics, Kinase Assay, Negative Control

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 inhibits metastatic and growth phenotypes cancer cells. ( A and B ) Cellular invasion and migration of C4-2-PKD1 (red line) and C4-2-GFP (green line) were determined using real-time xCeLLigence system. Red bar indicates significantly less invasion and migration of C4-2-PKD1 cells compared with C4-2-GFP. ( C ) C4-2-PKD1 and C4-2-GFP cells (2 × 10 6 ) were ectopically transplanted into the dorsal flank of nude mice ( n =4). Representative images of tumour bearing mice (i) and tumour volume (ii) of C4-2-GFP ( n =2) and C4-2-PKD1 ( n =0) implanted cells are shown at day 29 post injection. Asterisk represents statistical significance. ( D ) C4-2-GFP and C4-2-PKD1 cells (1 × 10 5 ) were intra-tibially transplanted in nude mice. Representative images of tumour growth and bone metastasis are shown in mice and in X-Ray images. C4-2-GFP implanted mice showed tumour growth and bone metastasis, whereas C4-2-PKD1 implanted mice failed to show growth and bone metastasis (i). Histopathological analysis of bone of C4-2-GFP and C4-2-PKD1-implanted mice for the expression of PKD1, MTA1 and RANK proteins as analysed by immunohistochemistry (ii). MTA1=metastasis-associated protein 1; PKD1=protein kinase D1. Scale bar=10 μ m.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Migration, Injection, Expressing, Immunohistochemistry

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: PKD1 negatively correlates with MTA1 in prostate tumour tissues of TRAMP and PTEN-KO mice. ( A – C ) PKD1 and MTA1 expression in the prostate tissues of WT and TRAMP mice at different weeks by immunohistochemical ( A ), immunoblotting ( B ) and qPCR ( C ) analyses. Result indicate decreased expression of PKD1 and subsequent increased of MTA1 in the prostate tumour of TRAMP mice compared to the WT control mice. ( D – F ). Similarly, the expression of PKD1 and MTA1 in the prostate tissues of wild-type (WT) and PTEN-KO mice was analysed at different weeks by immunohistochemical ( D ), immunoblotting ( E ) and qPCR ( F ) analyses. Values in bar graphs are shown as mean±s.d. obtained from three experimental replicates. MTA1=metastasis-associated protein 1; PKD1=protein kinase D1; PTEN-KO=PTEN-knockout; WT=wild type. * P <0.01; Student’s t -test, scale bar=10 μ m.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Control, Knock-Out

Journal: British Journal of Cancer

Article Title: Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

doi: 10.1038/bjc.2017.431

Figure Lengend Snippet: Clinical significance of PKD1 and MTA1 negative correlation in human cancer. ( A ) Expression of PKD1 and MTA1 in prostate tissue microarray (TMA: total 96 spots; adjacent normal; n =32, grade 7; n =30, grade 8; n =10, grade 9; n =24), which suggest loss of PKD1 and gain of MTA1 with the progression of cancer (Gleason grade 7 to 9). Similar pattern of PKD1 and MTA1 was observed in colon cancer TMA (total of 65 spots; adjacent normal; n =30, stage 2A; n =8, stage 2B; n =15, stage 3B; n =12) ( B ) and breast cancer TMA (total 81 spots; adjacent normal; n=45, stage 2A; n =23, stage 2B; n =9, stage 3B; n =4). ( C ) Original magnifications × 400. ( D ) Schematic diagram depicting molecular mechanism of PKD1-mediated regulation of MTA1 expression in cancer cells. MTA1=metastasis-associated protein 1; PKD1=protein kinase D1.

Article Snippet: Rabbit polyclonal MTA1 (A300-280A) was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA).

Techniques: Expressing, Microarray