Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: The expression of TROP-2 in PSCC tissues and PSCC cells. ( A ) TROP-2 staining in normal tissue and tumor tissue. ( B,C ) TROP-2 protein and mRNA were higher in tumor tissues compared to normal tissues. ( D,E ) TROP-2 expression in different PSCC cell lines. ( F ) The standard staining intensity score of TROP-2 in tumor fissue in the member was no staining was scored with 0, weak staining was scored with 1, moderate staining was scored with 2, and strong staining was scored with 3. ( G ) Proportion of TROP-2 expression in different subgroups. **** p < 0.0001, n.s., No significant. Statistics are expressed as the means ± SDs of three independent experiments. ENE: Extranodal extension; PSCC: Penile squamous cell carcinoma

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: Expressing, Staining

Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: Survival analysis between TROP-2 expression and clinicalopathological factors in PSCC patients. ( A ) Kaplan-Meier survival analysis indicated that high expression of TROP-2 was associated with reduced OS and DFS. ( B ) Survival analyses were also performed in the pT subgroup, ( C ) pN subgroup, and ( D ) the G subgroup. DFS: 5-year disease-free survival; OS: Overall survival

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: Expressing

Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: TROP-2 promotes cell proliferation in PSCC in vitro and in vivo . After being transfected with silencing or over-expressing plasmids, the following experiments were conducted to determine whether TROP-2 affects cell proliferation. ( A ) WB for TROP-2 expression in TROP-2 knockdown/over-express PSCC cells. ( B ) Knockdown and overexpression of TROP-2 in PSCC cells regulate cell proliferation in the CCK-8 experiment in vitro . ( C ) Knockdown and overexpressed of TROP-2 in PSCC cells regulate cell proliferation in the cell colony formation experiment ( D ) and regulate the growth of the tumor in vivo . ( E ) PSCC cells with TROP-2 knockdown exhibit an increased percentage in the G2 phase. ( F ) The knockdown of TROP-2 decreased the G2/M checkpoint proteins expression, such as cdc2 and cyclin B, while G1/S regulators remained unchanged. Significance levels are indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistics are presented as the means ± SDs of three independent experiments. NC: Negative control; OE: Overexpression; WT: Wild type

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: In Vitro, In Vivo, Transfection, Expressing, Knockdown, Over Expression, CCK-8 Assay, Negative Control

Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: TROP-2 up-regulated PSCC proliferation through PKCa and AKT pathway. ( A,B ) The GO analysis and heatmap of transcriptome sequencing. ( C ) The expression of protein in the PKCa and AKT pathways in PSCC cells after using PKCa inhibitors. ( D,E ) Cell proliferation was rescued in TROP-2 over-expressed PSCC cells after using PKCa inhibitors. ( F,G ) PSCC cells were arrested in G2/M stage after using PKCa inhibitors. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistics are presented as the means ± SDs of three independent experiments. FC: Fold change; OE: Overexpression

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: Sequencing, Expressing, Over Expression

Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: TROP-2 ADC drug shows a significant efficacy in PSCC cells in vivo and in vitro . ( A ) TROP-2 over-expressed PSCC cells showed a lower IC 50 dose to ADC drug than the negative control group. ( B,C ) TROP-2 ADC drug down-regulated the cell proliferation of PSCC cells more significantly in the TROP-2 over-expressed group. ( D–F ) TROP-2 ADC drug also showed better efficacy than cisplatin in TROP-2 over-expressed cells in vivo . **** p < 0.0001. Statistics are presented as the means ± SDs of three independent experiments. IC50: Half maximal inhibitory concentration; OE: Overexpression

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: In Vivo, In Vitro, Negative Control, Concentration Assay, Over Expression

Journal: Oncology Research

Article Title: TROP-2 Promotes Cell Proliferation via the AKT-Mediated PKC α Pathway and Is a Novel Target for Antibody-Drug Conjugates in Penile Carcinoma

doi: 10.32604/or.2025.066184

Figure Lengend Snippet: The toxicity and targeting test of TROP-2 ADC drugs (IMMU-132). IMMU-132 didn’t show serious toxicity to major organs of mice, and its targeting function was also verified. ( A,B ) After injecting PBS, cisplatin or IMMU-132 for 3 weeks, the major organs and blood of mice were obtained. Results of H&E staining and biochemical examinations showed that IMMU-132 didn’t cause obvious toxicity to mice compared with PBS, while cisplatin caused kidney toxicity. ( C ) TROP-2 protein was fixed on the ELISA plate, and different concentrations of IMMU-132 were used to examine its binding ability to TROP-2 protein. ( D ) IMMU-132 was labeled with CY-5 fluorescence. Immunofluorescence was performed at different time points after IMMU-132 was used in TROP-2 OE Penl2 cells. **** p < 0.0001, n.s., No significant. Statistics are expressed as the means ± SDs of three independent experiments

Article Snippet: Given the association of TROP-2 to adverse outcomes and heightened proliferation in PSCC, we performed drug experiments to evaluate the efficacy of the TROP-2 ADC (IMMU-132, MCE) in vivo and in vitro .

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Fluorescence, Immunofluorescence

Journal: PLoS ONE

Article Title: The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells

doi: 10.1371/journal.pone.0178375

Figure Lengend Snippet: (A) Basal protein expression level of endogenous MTH1 in all four NET cell lines. The expression of MTH1 is evaluated by Western blot analysis. A representative blot out of three independently performed experiments is shown, together with densitometry quantification of 3 independent Western blots. (B) The effects of different concentrations of TH588 (100 nM to 10 μM) on cellular survival in neuroendocrine pancreatic BON1, pancreatic islet QGP1, bronchopulmonary H727 and ileal GOT1 cells are displayed after 144 h of incubation. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either single substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***. (C) 20% inhibitory concentration (IC 20 ) of TH588 (100 nM to 10 μM) in four different NET cell lines after 144 h of incubation.

Article Snippet: The human pancreatic neuroendocrine BON1 tumor cell line [ ] was kindly provided by Prof. R. Göke, Marburg, Germany and the pancreatic islet tumor cell line QGP1 [ ] was acquired from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank).

Techniques: Expressing, Western Blot, Incubation, Standard Deviation, Concentration Assay

Journal: PLoS ONE

Article Title: The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells

doi: 10.1371/journal.pone.0178375

Figure Lengend Snippet: (A) FACS analysis of BON1 and QGP1 cells after 72 h of incubation with TH588. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either sinlge substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***. (B) Western blot analysis of PARP and Caspase 3 cleavage in NETs. A representative blot out of three independently performed experiments is shown. (C) Caspase 3/7 activity in BON1 and QGP1 cells upon 72 h of incubation with TH588. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either sinlge substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***.

Article Snippet: The human pancreatic neuroendocrine BON1 tumor cell line [ ] was kindly provided by Prof. R. Göke, Marburg, Germany and the pancreatic islet tumor cell line QGP1 [ ] was acquired from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank).

Techniques: Incubation, Standard Deviation, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells

doi: 10.1371/journal.pone.0178375

Figure Lengend Snippet: (A) Effect of TH588 on cell survival. Human neuroendocrine pancreatic BON1 and pancreatic islet QGP1 cells were incubated with TH588 (5 μM and 10 μM) alone and in combination (TH588 (5 μM)) with 5-FU (5 μM) and everolimus (10 nM) for 96 h and 144 h. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either sinlge substance treatment are shown, considering p<0,05 = *; p<0,01 = **; p<0,001 = ***. (B) Western blot analysis components from PI3K-Akt-mTOR pathway and the apoptotic cell apparatus were analyzed with TH588 alone (5 μM and 10 μM) alone and in combination with 5-FU (5 μM) and everolimus (10 nM) after 96 h. A representative blot out of three independently performed experiments is shown, together with the densitometry quantification.

Article Snippet: The human pancreatic neuroendocrine BON1 tumor cell line [ ] was kindly provided by Prof. R. Göke, Marburg, Germany and the pancreatic islet tumor cell line QGP1 [ ] was acquired from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank).

Techniques: Incubation, Standard Deviation, Western Blot

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: RSUME mRNA and protein level ( A ) were stimulated during hypoxia (1% O 2 ) for the indicated time points in BON1 cells. As expected RSUME mRNA and protein was down-regulated and less sensitive to hypoxia in BON1 cells with RSUME knock-down (BON1 RSUME-KD ) compared to scramble siRNA trasfected cells (BON1 Scramble ) ( B ). HIF-1a mRNA and in particular hypoxia-induced HIF-1a protein production was strongly impaired in BON1 RSUME-KD cells ( C ). Normoxic and hypoxic mRNA synthesis and secretion of VEGF-A was suppressed in BON1 RSUME-KD cells ( D ) whereas IL-8 mRNA and protein production was enhanced. ( E ). All experiments were performed three times and in B to E treatment time was 3 h for mRNA expression and 12 h for protein production studies, respectively. Results are expressed as mean ± SEM of triplicates for mRNA and quadruplicate for ELISA. * P < 0.05, ** P < 0.01.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Knockdown, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: BON1 cells were transfected with IL-8-LUC ( A , left) or IL-8 (NF-κB-mut)-LUC (A, right) reporter vector, RSUME or IκBα super repressor (I-κBα-SR) and β-gal plasmid. After 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured in the cell extracts. ( B ) BON1 cells were co-transfected with I-κBα, I-κBα mutated at the SUMO1 conjunction target sites lysine 21 and 22 (K21, 22R), and the indicated expression vectors (including the SUMO1/sentrin specific peptidase 1 -SENP1), to analyze I-κBα sumoylation status in BON1 cells. After 24 h, cell extracts were subjected to WB with anti-I-κBα antibody. β-actin was used as the loading control. ( C ) BON1 cells were transfected with NF-κB-LUC reporter vector, RSUME, HA-SUMO1 or I-κBα expression vectors. After 24 h, cells were stimulated with 10 ng/ml TNF-α for 6 h, and LUC activity was measured. ( D ) NF-κB-Luc activity in BON1 RSUME-KD and BON1 Scramble cells was measured with or without TNF-α (10 ng/ml, 6 h). All values were normalized to β-gal activity (mean ± SEM of 3 different experiments, t test). * P < 0.05, ** P < 0.01 ( E ) Western blot experiments were performed with cell extracts were RSUME was knockdown and with Immunoblotting for NF-κB subunits (p65, p-p65(Ser536), I-κBα and pI-κBα (Ser32/36) in BON wild type, BON1 RSUME-KD and BON1 Scramble cells lysates. β-actin was used as the loading control. ( F ) Immunofluorescence for total and phosphor-p65 (Ser536) in BON1 RSUME-KD and BON1 Scramble cells. Antibodies used were indicated above each image with corresponding colors. DAPI staining was used to visualize the nucleus. Scale bars are equal to 10 μm. Each image is representative of three independent experiments with similar results.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Transfection, Plasmid Preparation, Activity Assay, Expressing, Control, Western Blot, Knockdown, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: ( A ) PTEN mRNA expression was measured in RSUME overexpressing (right) and RSUME silenced (left) BON1 cells. β-actin was used for normalization. Immunoblot for PTEN was analyzed in RSUME silenced (left) and RSUME overexpressing (right) BON1 cells. β-actin was used for normalization. High-migration PTEN (H-PTEN). ( B ) Immunoprecipitation was performed with a PTEN antibody in BON1 cells followed by western blot using antibodies against PTEN and SUMO2. ( C ) GST-PTEN was incubated using an in vitro sumoylation assay mixture containing SUMO2 with or without RSUME. After incubation, reactions were stopped by adding loading buffer and subsequently an immunoblot was performed with anti-PTEN antibody. ( D ) COS7 cells were co-transfected with HA-PTEN, wild type V5-RSUME or the mutated V5-RSUME (Y61A, P62A), His6-SUMO2 and V5-Ubc9. 48 h post-transfection, cells were lysed, purified by Ni-NTA and immunoblotted with indicated antibodies. ( E ) COS7 cells were co-transfected with HA-PTEN expression or sumoylation-deficient PTEN mutants (K254R, K266R, K254/266R), His6-SUMO2 and V5-RSUME plasmids. 48 h post-transfection, cells were lysed, Ni-NTA purified and immunoblotted with the indicated antibodies. ( F ) COS7 cells were transfected with HA-PTEN with or without V5-RSUME. 48 h post-transfection, cells were lysed with RIPA buffer (10% for Input), precipitated with PTEN antibody and subsequently immunoblot was performed with antibodies indicated. For all experiments, one representative experiment from two independent experiments with similar results is shown.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Expressing, Western Blot, Migration, Immunoprecipitation, Incubation, In Vitro, Transfection, Purification

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: ( A ) COS7 cells were transfected with HA-PTEN, His6-ubiquitin and V5-RSUME or a backbone construct pCEFL. 48 h post-transfection, cells were incubated with 5 μM MG132 or vehicle for 6 h. His-ubiquitinated (His-Ub) proteins were isolated from denatured whole-cell extracts and pulled down by nickel beads. Purified proteins and input samples (whole-cell extracts) were analyzed by western blotting with anti-PTEN and anti-V5 (RSUME) antibodies. Signal in His-Ub pull-down lanes corresponds to ubiquitinated PTEN. b-actin was used as a loading control. ( B ) COS7 cells were transfected with HA-PTEN, His6-ubiquitin and 10 μM siRSUME. 48 h posttransfection, cells were incubated with MG132, purified and immunoblotted with anti-PTEN and anti-RSUME. β-actin was used as a loading control. ( C ) COS7 cells were transfected with HA-PTEN or different mutants (K254R, K266R, K254/266R), V5-RSUME and His6-Ubiquitin. 48 h post-transfection, cells were incubated with MG132, purified and immunoblotted with anti-PTEN and anti-RSUME. One representative experiment from two independent experiments with similar results is shown. ( D ) BON1 RSUME-KD and BON1 Scramble cells were treated with cycloheximide (CHX) for the time indicated. Cell lysates were collected and subjected to immunoblot using antibodies against PTEN and RSUME. β-actin was used as the loading control. ( E ) COS7 cells were co-transfected with wild type or sumoylation-deficient PTEN mutant (K254/266R) with or without V5-RSUME. 48 h post-transfection, cells were treated with cycloheximide for the indicated time points and immunoblotted with the indicated antibodies. β-actin was used as the loading control.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Transfection, Ubiquitin Proteomics, Construct, Incubation, Isolation, Purification, Western Blot, Control, Mutagenesis

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: ( A ) Immunofluorescence studies showed that in the normal pancreas, PTEN (red) is predominantly localized in the nuclei of insulin-producing cells (green) (A, left) whereas in PanNETs PTEN is present in the cytoplasm of tumor cells (A, middle) or completely absent (A, right). PTEN showed both nuclear and cytoplasmic expression in BON1 cells transfected with GFP-PTEN (green) whereas the PTEN sumoylation deficient double mutant (K254/266R) (green) is localized only in the cytoplasm ( B , left). Over-expression of RSUME (red) in GFP- or K254/266R-PTEN expressing BON1 cells strongly increased nuclear PTEN (green) expression in cells with GFP-PTEN whereas PTEN remained in the cytoplasm of cells expressing the K254/266R mutant (B, right). Cell nuclei were visualized using DAPI (blue). Scale bar: 10 μm.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Immunofluorescence, Expressing, Transfection, Mutagenesis, Over Expression

Journal: Oncotarget

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

doi: 10.18632/oncotarget.11081

Figure Lengend Snippet: Orthotopic tumors from BON1 cells without (scramble) or with RSUME knockdown (RSUME-KD) were generated by injecting the cells into the pancreas of athymic nude mice. In comparison to scramble tumors, RSUME-KD tumors showed reduced chromogranin A staining (brown) indicative of a loss of neuroendocrine differentiation ( A ). RSUME-KD tumors are significantly less vascularised (CD31 staining) than scramble tumors ( B ) but show strongly enhanced spread of metastases into the liver as shown morphologically ( C ) and immunohistochemically by detecting CgA-positive neuroendocrine tumor tissue in the nude mouse liver ( D ). Tumors with RSUME-KD show reduced PTEN expression and signs of epithelial-mesenchymal-transition (EMT) such as enhanced TGF-β, N-Cadherin and Snail protein levels as well as reduced E-Cadherin protein expression ( E ) indicating that loss of RSUME promotes EMT in PanNETs. Microvessel density in the right panel in B was determined by counting of the number of vessels in one field under 100× magnification. Results were obtained from 6 independent pictures for each condition and are expressed as mean ± SEM. * P < 0.05 vs. scramble tumors. Scale bar 100 μm.

Article Snippet: Human pancreatic endocrine tumor BON1 cells used in the study were authenticated by Eurofins (Ebersberg, Germany).

Techniques: Knockdown, Generated, Comparison, Staining, Expressing