Journal: Cancer Research

Article Title: Tipifarnib Potentiates the Antitumor Effects of PI3Kα Inhibition in PIK3CA - and HRAS -Dysregulated HNSCC via Convergent Inhibition of mTOR Activity

doi: 10.1158/0008-5472.CAN-23-0282

Figure Lengend Snippet: Tipifarnib blunts mTOR and RSK reactivation following alpelisib treatment and induces cell-cycle arrest and apoptosis in PIK3CA -mutant, PIK3CA -amplified, and HRAS-high cell lines. A, Immunoblot of the indicated signaling proteins in PIK3CA -mutant CAL33 cells treated with 250 nmol/L alpelisib for 0, 1, 2, 6, or 24 hours in the presence or absence of 1 μmol/L tipifarnib. Cells were treated with tipifarnib for 48 hours. Images are representative of three biological replicates. B, Apoptosis (Annexin V) and cytotoxicity (loss of membrane integrity, nuclei exposure) over time in CAL33 cells treated with DMSO, 250 nmol/L alpelisib, 1 μmol/L tipifarnib, or the combination for 72 hours measured via Incucyte live cell imaging. Staurosporine (100 nmol/L) was used as a positive control. Data are means ± SD of three biological replicates. C and D, Immunoblots of indicated proteins in PIK3CA copy gain BICR22 cells ( C ) or HRAS-high SCC9 cells ( D ) treated with 250 nmol/L alpelisib for 0, 1, or 24 hours in the presence of absence of 1 μmol/L tipifarnib (48-hour treatment). E, Graphical overview of role of mTORC1 in regulating assembly of the eIF4F translation initiation complex. mTORC1 phosphorylates 4EBP1, impeding its binding to eIF4E, allowing for complex assembly. F, eIF4G (or IgG control) was immunoprecipitated from CAL33 cells treated with DMSO, 250 nmol/L alpelisib (24 hours), 1 μmol/L tipifarnib (48 hours), or the combination. Immunoblots indicate the levels of eIF4G and eIF4E coimmunoprecipitated and in the input cell lysate. Images are representative of two biological replicates.

Article Snippet: Cell lines were obtained from ATCC (SCC25, SCC9, FaDu, and Detroit 562), JCRB (SAS, HSC2, HSC3), DSMZ (CAL33), or Sigma (PECAP15J) and maintained in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Mutagenesis, Amplification, Western Blot, Membrane, Live Cell Imaging, Positive Control, Binding Assay, Control, Immunoprecipitation

Journal: Cancer Research

Article Title: Tipifarnib Potentiates the Antitumor Effects of PI3Kα Inhibition in PIK3CA - and HRAS -Dysregulated HNSCC via Convergent Inhibition of mTOR Activity

doi: 10.1158/0008-5472.CAN-23-0282

Figure Lengend Snippet: Depletion of the FTase targets RHEB and HRAS phenocopies tipifarnib treatment in PIK3CA/HRAS–dysregulated HNSCC cell lines. A, Graphical overview of the effect of tipifarnib on the membrane localization of HRAS and RHEB. Inhibition of FTase activity leads to defarnesylation of HRAS and RHEB and loss of membrane localization and activity. B–D, Immunoblots of HRAS, RHEB, LAMP1, and β-tubulin in the cytosol and membrane fractions of CAL33 ( B ), BICR22 ( C ), and SCC9 ( D ) cells treated with DMSO, 250 nmol/L alpelisib (24 hours), 1 μmol/L tipifarnib (48 hours), or the combination. E–G, Immunoblots of indicated signaling proteins in CAL33 ( E ), BICR22 ( F ), and SCC9 ( G ) cells treated with siRNAs to knock down RHEB and HRAS expression (vs. control nontargeting pool) for 48 hours prior to addition of 250 nmol/L alpelisib. Cells were collected and lysed after 0, 1, or 24 hours alpelisib treatment and immunoblot analysis performed.

Article Snippet: Cell lines were obtained from ATCC (SCC25, SCC9, FaDu, and Detroit 562), JCRB (SAS, HSC2, HSC3), DSMZ (CAL33), or Sigma (PECAP15J) and maintained in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Membrane, Inhibition, Activity Assay, Western Blot, Knockdown, Expressing, Control

Journal: Cell Death & Disease

Article Title: circRNA_0000140 suppresses oral squamous cell carcinoma growth and metastasis by targeting miR-31 to inhibit Hippo signaling pathway

doi: 10.1038/s41419-020-2273-y

Figure Lengend Snippet: a The exonic information of circ_0000140 was illustrated as indicated. The specific primers of circ_0000140 were validated by Sanger sequencing. The length of circ_0000140 was 585 bp. The red arrow indicates the backsplice site. b The relative RNA levels were examined by qRT-PCR after treatment with actinomycin D at the indicated time points in HOK cells. c The relative RNA levels were examined by qRT-PCR after treatment with RNase R or mock in total RNAs derived from HOK cells. d The cellular distribution of circ_0000140 was analyzed by cellular RNA fractionation assays. GAPDH and U6 were used as cytoplasmic and nuclear positive controls, respectively. e The cellular distribution of circ_0000140 was analyzed by fluorescence in situ hybridization (FISH). Green indicates circ_0000140. Nuclei were stained with DAPI. Scale bar, 50 μm. The levels of KIAA0907 ( f ) and circ_0000140 ( g ) in 56 paired OSCC and matched adjacent normal tissues were examined by qRT-PCR. h Kaplan–Meier method with the log-rank test was used to analyze the overall survival of OSCC patients in high and low circ_0000140 expression groups. i The relative expression levels were examined by qRT-PCR after treatment with shKIAA0907. All the results were shown as mean ± SD. * P < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human oral keratinocyte (HOK) cells and four human OSCC cell lines (Cal-27, SCC-25, SCC-9, and HSC-3) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Sequencing, Quantitative RT-PCR, Derivative Assay, Fractionation, Fluorescence, In Situ Hybridization, Staining, Expressing

Journal: Cell Death & Disease

Article Title: circRNA_0000140 suppresses oral squamous cell carcinoma growth and metastasis by targeting miR-31 to inhibit Hippo signaling pathway

doi: 10.1038/s41419-020-2273-y

Figure Lengend Snippet: a The expression level of circ_0000140 and miR-31 in Cal-27 and HSC-3 cell lines transfected with circ_0000140 overexpression vector. b The expression level of miR-31 in Cal-27 and HSC-3 cell lines transfected with miR-31 mimics, miR-31 inhibitors, and negative control. c The targeting sequence of circ_0000140 and miR-31. d Dual luciferase assay of miR-31 mimics, miR-31 inhibitors, and negative control on circ_0000140 wild type and mutated type. e Anti-AGO2 RIP assays were used in Cal-27 and HSC-3 cells to determine circ_0000140 and miR-31 RNA enrichment in IP complexes. Anti-IgG was used as a control. f , g Protein levels of the indicated Hippo pathway in circ_0000140-overexpressing OSCC cells, as detected by Western blotting. h Expression levels of YAP1 in circ_0000140-overexpressing OSCC cells as detected by immunofluorescence analyses. Scale bar, 50 μm. All the results were shown as mean ± SD ( n = 3), which were three separate experiments performed in triplicate. * P < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human oral keratinocyte (HOK) cells and four human OSCC cell lines (Cal-27, SCC-25, SCC-9, and HSC-3) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Negative Control, Sequencing, Luciferase, Control, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: circRNA_0000140 suppresses oral squamous cell carcinoma growth and metastasis by targeting miR-31 to inhibit Hippo signaling pathway

doi: 10.1038/s41419-020-2273-y

Figure Lengend Snippet: a The effect of miR-31 mimics and inhibitor on LATS2 mRNA level in Cal-27 and HSC-3 cells, as determined by qRT-PCR. b The targeting sequence of LATS2 mRNA and miR-31. c Dual luciferase assay of miR-31 mimics, miR-31 inhibitors, and negative control on LATS2 wild type and mutated type. d , e The effect of miR-31 on p53 and Hippo pathway-related proteins by Western blotting. f Expression levels of YAP1 in miR-31-overexpressing or miR-31-underexpressing OSCC cells as detected by immunofluorescence analysis. Scale bar, 50 μm. All the results were shown as mean ± SD ( n = 3), which were three separate experiments performed in triplicate. * P < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Human oral keratinocyte (HOK) cells and four human OSCC cell lines (Cal-27, SCC-25, SCC-9, and HSC-3) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Sequencing, Luciferase, Negative Control, Western Blot, Expressing, Immunofluorescence

Journal: Cell Death & Disease

Article Title: circRNA_0000140 suppresses oral squamous cell carcinoma growth and metastasis by targeting miR-31 to inhibit Hippo signaling pathway

doi: 10.1038/s41419-020-2273-y

Figure Lengend Snippet: a Typical OSCC tumors from tumor-bearing BALB/c mice after subcutaneous injection of Cal-27 and HSC-3 cells stably overexpression circ_0000140. b The volume of OSCC tumors from tumor-bearing BALB/c mice after treatment. c The weight of OSCC tumors from tumor-bearing BALB/c mice after treatment. d The expression level of circ_0000140, miR-31, and LATS2 in xenograft tumor tissues as determined by qRT-PCR. e The protein level of Ki-67, MMP-9, LATS2, and YAP1 in xenograft tumor tissues as determined by immunohistochemistry. Scale bar, 50 μm. f , g The protein level of Hippo pathway and EMT markers in xenograft tumor tissues as determined by western blotting. h Typical lung tissues with visualized metastatic nodules from tumor-bearing BALB/c mice after tail vein injection of Cal-27 and HSC-3 cells stably overexpressing circ_0000140. i The number of metastatic nodules of lungs from tumor-bearing BALB/c mice after treatment. j H&E for metastatic nodules of lungs in circ_0000140 overexpression group and vehicle group. Scale bar, 200 μm. All the results were shown as mean ± SD. * P < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human oral keratinocyte (HOK) cells and four human OSCC cell lines (Cal-27, SCC-25, SCC-9, and HSC-3) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Injection, Stable Transfection, Over Expression, Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot