Journal: International Journal of Molecular Sciences

Article Title: PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression

doi: 10.3390/ijms22031275

Figure Lengend Snippet: Effect of PLEKHA7 re-expression on SUM149 cell adherens junctions. ( A ) SUM149 cells expressing LZRS ms neo (control) or LZRS PLEKHA7 were grown to confluence and immunofluorescence was performed for E-cadherin, p120-catenin, PLEKHA7, α-catenin, or β-catenin. Images were obtained by confocal microscopy. Images are maximum projection intensity. Scale bar is 10μM. Representative images are shown. ( B ) Intensity of p120-catenin or β-catenin staining is expressed as a ratio of the signal at apical AJs to cytoplasmic staining. N > 75 cells per condition per group. ** indicates p < 0.001, Student’s t -test ( p < 0.0001 for both p120-catenin and β-catenin). ( C ) Protein levels of PLEKHA7, E-cadherin, p120-catenin, β-catenin, and α-catenin in SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 cells were obtained by Western blot. A representative image is shown. No consistent change was observed for any junctional protein in repeated experiments. ( D ) A representative graph displaying Wnt/β-catenin signaling in SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 cell lines using dual luciferase reporter assay. * indicates p < 0.05, Student’s t -test ( p = 0.022).

Article Snippet: SUM149 cells tested negative for Ectromelia, LCMV, LDEV, MHV, MPV, MVM, Mycoplasma pulmonis, Mycoplasma sp, Polyoma, and TMEV (IDEXX BioResearch).

Techniques: Expressing, Control, Immunofluorescence, Confocal Microscopy, Staining, Western Blot, Luciferase, Reporter Assay

Journal: International Journal of Molecular Sciences

Article Title: PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression

doi: 10.3390/ijms22031275

Figure Lengend Snippet: Effects of PLEKHA7 re-expression on SUM149 cell growth and survival in 3D culture. ( A ) SUM149 LZRS ms neo (control) and SUM149 LZRS PLEKHA7 cells were grown in Matrigel for approximately two weeks. Representative images at 4×, 10×, and 20× magnification are shown. Scale bar in each image = 100µm. ( B ) Quantification of SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 colonies from 3A are shown as the fold change from SUM149 LZRS ms neo. * indicates p < 0.05, Student’s t -test. ( C ) Representative images from SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 suspension cultures undergoing sphere compaction over 18 h. Images taken at 4×. Scale bar in each image = 100µm. ( D ) A representative graph of SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 suspension cultures compacting into spheres over 18 h under ultralow attachment conditions. *** indicates p < 0.001, Student’s t -test ( p < 0.0001). ( E ) A representative graph of normalized ATP content produced by SUM149 LZRS ms neo and SUM149 PLEKHA7 spheres after treatment with various concentrations of DOXIL/doxorubicin for 72 h. ** indicates p < 0.01, Student’s t -test ( p = 0.002 for 1 µM doxorubicin and 10 µM doxorubicin). Each group was normalized to no treatment.

Article Snippet: SUM149 cells tested negative for Ectromelia, LCMV, LDEV, MHV, MPV, MVM, Mycoplasma pulmonis, Mycoplasma sp, Polyoma, and TMEV (IDEXX BioResearch).

Techniques: Expressing, Control, Suspension, Produced

Journal: International Journal of Molecular Sciences

Article Title: PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression

doi: 10.3390/ijms22031275

Figure Lengend Snippet: PLEKHA7 effects on SUM149 tumor growth in orthotopic xenografts. ( A ) Tumor volume of xenografts from SUM149 LZRS ms neo (control) or SUM149 LZRS PLEKHA7 cells implanted into the fourth mammary gland of NOD/SCID immunocompromised mice measured at eight weeks post-implantation. * indicates p = 0.034, Student’s t -test. n = 6 for control group, n = 8 for PLEKHA7 group. ( B ) Fraction of Ki-67 positive cells in tumors from SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 xenografts at the eight week endpoint. p = 0.082, Student’s t -test. n = 6 for control group, n = 8 for PLEKHA7 group. ( C ) Representative IHC images from SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 xenografts for H & E, PLEKHA7, and Ki67. Scale bar represents 100 µm.

Article Snippet: SUM149 cells tested negative for Ectromelia, LCMV, LDEV, MHV, MPV, MVM, Mycoplasma pulmonis, Mycoplasma sp, Polyoma, and TMEV (IDEXX BioResearch).

Techniques: Control

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 (A) and DU145 (B) cells were seeded at a density of 5 × 10 4 cells per well. Cells were exposed to various concentrations of PVSRIPO (MOI of 0, 0.1, 1 and 10 MOIs) for 0, 24, 48 and 72 hours. Cell lysis was assessed using crystal violet staining. (C) PVSRIPO propagation following infection (MOI of 10) of DU145 and SUM149 cells was assessed by plaque assay at the designated time points. Data are representative of at least 3 independent experiments; note that titers in (C) are from an assay distinct from (A) and (B).

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Lysis, Staining, Infection, Plaque Assay

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumors were collected at 7 days post injection and size and weight were assessed. (A) SUM149 tumor data are representative of five different experiments and a total of 25 mice per group. Graph represents tumor weights (grams) from one representative experiment. (B) DU145 tumor data are representative of five different experiments and a total of 25 mice per group. Graph represents tumor weights (grams) from one representative experiment. ***p<0.001.

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 (A) and DU145 (B) cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumors were collected at 24 hours post injection and innate immune-related transcript abundance was assessed. Data are representative of two different experiments and a total of 6 mice per group. Only changes of ≥8-fold are reported. (C) Cytokines/chemokines represented in (A, B) and their function in inflammation; cytokines shown in bold were induced in both tumors. Information about cytokine significance was obtained from Uniprot and NCBI databases.

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: (A) SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumors were collected at 48 hours post injection and innate immune cell infiltration was assessed by flow cytometry (CD45.2, CD11b, F480, Ly6C, Ly6G, B220, CD335). The numbers reflect percentage of cells in the gated population (boxed area) in the preceding flow quadrant as indicated. Data are representative of three different experiments and a total of 15 mice per group. (B) Tumor infiltrating immune cell percentages from one representative experiment (n=5) in the SUM149 breast tumor model are shown. ***p<0.001. (C) Tumor infiltrating immune cell percentages from one representative experiment (n=5) in the DU145 prostate tumor model are shown. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection, Flow Cytometry

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumors were collected at 7 days post injection and immune cell recruitment was assessed by histology (H&E) and immunohistochemistry (CD11b). Data are representative of two different experiments and a total of 10 mice per group. SUM149 (A) and DU145 (B) tumors demonstrate significantly increased infiltration of CD11b+ immune cells. (C, D) SUM149 tumor homogenates from mock (PBS) or PVSRIPO-treated mice were tested (C, top) for markers of neutrophil and innate immune cell inflammation by immunoblot; (C, bottom) for the presence of H 2 O 2 ; (D) for the presence of TNF-α and IFN-β by ELISA.

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumor growth was monitored daily and mice were sacrificed when tumors reached 2000 mm 3 . Data are representative of 3 different experiments (n=30). SUM149 (A, B) and DU145 (C, D) tumor growth 7 days post PVRIPO injection and overall survival up to 100 days.

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection

Journal: Oncotarget

Article Title: Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

doi: 10.18632/oncotarget.12975

Figure Lengend Snippet: SUM149 (A) and DU145 (B) cells were implanted in athymic nu/nu mice. Tumors were injected with 10 8 pfu of PVSRIPO when they reached 150-200 mm 3 . Tumors were isolated at 1, 2, 3, 4 and 7 days post virus inoculation. Pfu per mg of tumor were determined by plaque assay and plotted; data are representative of two independent experiments (n=30).

Article Snippet: SUM149 cells were grown in 10% FBS in Ham's DMEM-F12 medium (Lonza, Basel, Switzerland).

Techniques: Injection, Isolation, Virus, Plaque Assay

Journal: Nature Communications

Article Title: Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2–JNK signalling in breast cancer

doi: 10.1038/ncomms10318

Figure Lengend Snippet: ( a ) Basal cell lines chosen for their high basal score/correlation were SUM149 r =0.36 ( a ) and HCC1806 ( b ) r =0.28 and threshold was 0.15. Expression of two short hairpin RNAs abrogated VANGL2 expression in SUM149 cells by western blot analysis (upper panels). SUM149 cells (5 × 10 6 ) were subcutaneously inoculated into the right flank of 4–6-week-old female NSG mice. Tumoral volume was measured at different times (lower panels). The mean and s.e.m. values ( n =6, for shLuc and shVANGL2-transfected cells). The statistical significance between the data sets was determined using a two-way ANOVA test. * P ≤0.05, ** P ≤0.005. ( b ) Same as a using HCC1806 cells, except that 1 × 10 6 cells were inoculated into NSG mice. ( c , d ) Downregulation of VANGL2 with two different shRNAs led to a decreased proliferation of SUM149 ( c ) and HCC1806 ( d ) cells. Error bars represent mean±s.d. ( e ) COMMA-D cells were transduced with lentiviral supernatants allowing expression of GFP or GFP–VANGL2. Cell extracts were probed by western blot analysis with anti-GFP, -VANGL2 and -tubulin antibodies. An asterisk pinpoints endogenous VANGL2. ( f ) Kaplan–Meier curve of tumour-free status of mice transplanted with COMMA-D cells overexpressing GFP or GFP–VANGL2 ( n =30). The statistical significance between the data sets was determined using a log-rank test.

Article Snippet: Second, we tested anchorage-dependent and -independent proliferation and observed that loss of VANGL2 decreased the proliferation rates of SUM149 ( and ) and HCC1806 cells ( ).

Techniques: Expressing, Western Blot, Transfection, Transduction

Journal: Nature Communications

Article Title: Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2–JNK signalling in breast cancer

doi: 10.1038/ncomms10318

Figure Lengend Snippet: LC-MS/MS using LTQ-Velos-Orbitrap mass spectrometry analysis of proteins co-immunoprecipitated with VANGL2 in SUM149 cell extracts.

Article Snippet: Second, we tested anchorage-dependent and -independent proliferation and observed that loss of VANGL2 decreased the proliferation rates of SUM149 ( and ) and HCC1806 cells ( ).

Techniques: Mass Spectrometry, Sequencing

Journal: Nature Communications

Article Title: Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2–JNK signalling in breast cancer

doi: 10.1038/ncomms10318

Figure Lengend Snippet: ( a ) Endogenous interaction between VANGL2 and p62/SQSTM1 is revealed by western blot analysis after immunoprecipitation using SKBR7 cell extracts. TL is total lysate. Crtl ab is an isotype-matched antibody. ( b ) The VANGL2–p62/SQSTM1 interaction occurs independently of LC3. Proteins were extracted from SUM149 cells and co-immunoprecipitations were carried out with the indicated antibodies. LC3 co-precipitates with p62/SQSTM1 but not with VANGL2. Reciprocally, VANGL2 co-immunoprecipitates with p62/SQSTM1 but not with LC3. IP control antibodies (IP crtl ab) are a polyclonal rabbit (for IP LC3) and a monoclonal rat antibody (for IP VANGL2). IgHs are immunoglobulin heavy chains. ( c ) GST pulldown assays of in vitro translated GFP–VANGL2 (full length: WT, N-terminal 1–102: NT, C-terminal 242–521: CT) showed that VANGL2 WT and CT directly bind to GST–p62/SQSTM1 (GST-p62) but not to GST. Asterisks indicate in vitro translated VANGL2 (top panel) and GST (bottom panel) proteins. AR, autoradiography; CBB, Coomassie Brilliant Blue. ( d ) A p62/SQSTM1 peptide (p62 DN ) disrupts the endogenous VANGL2–p62/SQSTM1 complex. SUM149 cell protein extracts were incubated with the indicated peptides p62 DN or scrambled control peptide (Ctrl peptide) at 100 μM. VANGL2 was then immunoprecipitated (IP VANGL2) and bound proteins were immunoblotted with the indicated antibodies. TLs showed that equal amounts of proteins were present in each condition.

Article Snippet: Second, we tested anchorage-dependent and -independent proliferation and observed that loss of VANGL2 decreased the proliferation rates of SUM149 ( and ) and HCC1806 cells ( ).

Techniques: Western Blot, Immunoprecipitation, Control, In Vitro, Autoradiography, Incubation

Journal: Nature Communications

Article Title: Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2–JNK signalling in breast cancer

doi: 10.1038/ncomms10318

Figure Lengend Snippet: ( a ) Downregulation of VANGL2 in SUM149 cells using a specific shRNA led to reduced JNK phosphorylation induced by Wnt5a (100 ng ml −1 for the indicated times). JNK is represented by two isoforms: p54 and p46. Wnt5a led to p46 (phospho-p46) and not p54 (phospho-54) phosphorylation. Relative quantification of immunoblots (phosphorylated JNK/total JNK) is representative from three independent experiments and use of two different shRNAs. ( b ) Expression of GFP-p62 DN , but not GFP, in SUM149 cells led to decreased p46 JNK phosphorylation (phospho-46) induced by 100 ng ml −1 of Wnt5a at the indicated times. Relative quantification of immunoblots (phosphorylated JNK/total JNK) is representative from three independent experiments. ( c ) SUM149 cell extracts were added with the control peptide (Ctrl peptide) or the p62/SQSTM1 peptide (p62 DN ) that inhibited recruitment of JNK and p62/SQSTM1 to VANGL2. ( d ) Proteins extracted from SUM149 cells treated or not with serum were immunoprecipitated with anti-VANGL2 antibody and blotted with the indicated antibodies. p62/SQSTM1, JNK and phosphorylated JNK (phospho-p54 and phospho-p46) were present in the VANGL2 complex.

Article Snippet: Second, we tested anchorage-dependent and -independent proliferation and observed that loss of VANGL2 decreased the proliferation rates of SUM149 ( and ) and HCC1806 cells ( ).

Techniques: shRNA, Phospho-proteomics, Quantitative Proteomics, Western Blot, Expressing, Control, Immunoprecipitation