Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: CDCA3 protein is upregulated in EGFR mutant NSCLC. ( A ) Representative images of CDCA3 low (left panel) and CDCA3 high (right panel) staining by immunohistochemistry in NSCLC cohort. CDCA3 levels determined by stratifying on median H-score. Scale bar = 50 µm. ( B ) Stacked bar chart showing distribution of CDCA3 low (blue) and CDCA3 high (grey) levels quantified from immunohistochemistry staining of non-oncogene-driven NSCLC (EGFR-WT) cases and EGFR mutant cases (chi-square test, * p = 0.02). ( C ) Representative endogenous CDCA3 Western blot analysis from lysates of NSCLC adenocarcinoma cell lines evaluating a small panel of non-oncogene-driven (EGFR WT) and EGFR mutant (EGFR MUT) cell lines. Phosphorylated and total ERK and mTOR Western blot analysis to assess constitutive signalling in EGFR mutant cell lines. Tubulin used as a loading control. ( D ) Densitometry quantification of ( C ), with dot points representing relative CDCA3 levels from three independent experiments. Blue lines indicate median values.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Staining, Immunohistochemistry, Western Blot, Control

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulation of CDCA3 protein levels in EGFR mutant NSCLC is dependent on RTK signalling. ( A ) Violin plots showing relative CDCA3 mRNA levels in EGFR wild-type and EGFR mutant adenocarcinoma from The Cancer Genome Atlas (TCGA) RNAseq datasets. ( B ) RNAseq analysis of CDCA3 mRNA levels in cell lines examined in C comparing EGFR wild-type and EGFR mutant adenocarcinoma. ns, not significant. ( C ) Endogenous CDCA3 Western blot analysis of A549 (EGFR WT), HCC827 (EGFR exon 19 deletion) and H1975 (EGFR T790M) cells treated in the absence or presence of EGF over 24 h. Phosphorylated (S473) and total Akt Western blot analysis to assess EGF-induced signal transduction. Actin used as a loading control. ( D ) Endogenous CDCA3 Western blot analysis of HCC827 lysates assessing protein turnover by treating with cycloheximide over 6 h in the presence or absence of erlotinib. Tubulin used as a loading control. ( E ) Densitometry quantification of ( D ), showing average log 2 of relative CDCA3 protein levels relative to 0 h from three independent experiments.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Western Blot, Transduction, Control

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: CDCA3 correlates with sensitivity to EGFR TKI. ( A , B ) Scatter plots showing linear regression analysis of The Cancer Genome Atlas (TCGA) RNAseq datasets assessing the correlation between CDCA3 levels and WikiPathways of TKI resistance in ( A ) adenocarcinoma (LUAD) and ( B ) EGFR mutant LUAD. R and p values determined according to Spearman’s rank correlation. ( C ) Left panel, Dose–response curves for five EGFR mutant NSCLC cell lines treated with escalating doses of erlotinib. Cells were treated with erlotinib for 72 h before assessing cell viability. Right panel, Cell lines ranked by CDCA3 protein levels where erlotinib potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 4. ( D ) Box and whisker plot showing erlotinib potency (log IC 50 value) in CDCA3 low and CDCA3 high EGFR mutant cell lines (unpaired Student’s t test, * p = 0.0187).

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Whisker Assay

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulating CDCA3 protein levels enhances TKI sensitivity in CDCA3 low H1975 cells. ( A ) Western blot analysis of lysates from H1975 cells transfected with empty vector or CDCA3-FLAG treated in the absence (vehicle) or presence of erlotinib or osimertinib for 24 h. CDCA3 Western blot analysis was performed to detect ectopic (arrowhead) and endogenous (asterisk) CDCA3. Phosphorylated (Y1068) and total EGFR Western blot analysis was performed to confirm TKI response. Tubulin was used as a loading control. Representative Western blot analysis from three independent experiments. ( B ) Dose–response curves for CDCA3 low H1975 cells either vector transfected or ectopically overexpressing CDCA3-FLAG treated with escalating doses of erlotinib for 72 h before assessing cell viability. ( C ) Cellular proliferation analysis of vector transfected or CDCA3-FLAG ectopically expressing H1975 cells over 96 h using the Incucyte Zoom live-cell imaging system. ( D ) Beeswarm plot showing the mitotic index determined by histone H3 pS10 staining and high throughput immunofluorescence microscopy of vector transfected or CDCA3-FLAG ectopically expressing H1975 cells. Data points represent an average percentage of mitotic nuclei per field of view from a minimum of 1100 nuclei ( n = 23 fields total). Blue lines indicate median values. ( E ) Endogenous CDCA3 Western blot analysis of H1975 cells treated in the absence or presence of CX-4945 (5 µM) over 24 h. Phosphorylated CK2 substrate probe to assess impact of CK2 inhibition with CX-4945. Tubulin was used as a loading control. ( F ) Dose–response curve for H1975 cells treated with escalating doses of CX-4945. Cells were treated with CX-4945 for 72 h before assessing cell viability. CX-4945 potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 3. ( G ) Dose–response curves showing the impact of combining IC 25 or IC 50 CX-4945 concentrations with osimertinib in H1975 cell. Potency values (IC 50 ) were calculated using GraphPad Prism. n = 3.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Expressing, Live Cell Imaging, Staining, High Throughput Screening Assay, Immunofluorescence, Microscopy, Inhibition

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulating CDCA3 protein levels in models of isogenic EGFR TKI resistance cells enhances TKI sensitivity. ( A , B ) Dose–response curves for vector transfected or ectopic CDCA3-FLAG overexpression in isogenic parental and resistant ( A ) HCC827 and ( B ) PC-9 cells treated with escalating doses of osimertinib for 72 h before assessing cell viability. ( C ) Plot showing erlotinib potency (log IC 50 value) comparing vector transfected and ectopic CDCA3-FLAG overexpression in isogenic parental and TKI-resistant cell lines determined in from four independent experiments. Lines connect respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.029, ns = not significant). ( D ) Plot showing osimertinib potency (log IC 50 value) comparing vector transfected and ectopic CDCA3-FLAG overexpression in isogenic parental and TKI-resistant cell lines determined in ( A , B ) from four independent experiments with lines connecting respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.0339, ns = not significant). ( E ) Dose–response curves for HCC827 and PC-9 isogenic parental and TKI-resistant cell lines treated with escalating doses of CX-4945. Cells were treated with CX-4945 for 72 h before assessing cell viability. CX-4945 potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 3. ( F ) Plot showing potency (log IC 50 value) of osimertinib alone or in combination with CX-4945 (IC 50 ) comparing isogenic parental and TKI-resistant cell lines from three independent experiments. Lines connect respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.0331, ns = not significant).

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Plasmid Preparation, Transfection, Over Expression