Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Differential calcium channel gene expression in TNBC TP53 mutant patient samples and cell line versus TP53 wildtype. ( A ) Differential expression analyses (DEAs) carried out using CCLE breast cancer cell line microarray data and TCGA patient sample RNA-sequencing data. (i) The CCLE volcano plot illustrates the DEGs (Dots) when comparing TNBC TP53 MUT (n = 20) vs. all BC subtype WT (n = 13) with 372 significantly upregulated (lfc ≥ 2, BH-adj. p -value ≤ 0.01) and 168 significantly downregulated (lfc ≤ −2, BH-adj. p -value ≤ 0.01) genes. and (ii) the TCGA volcano plot illustrates the DEGs when comparing TNBC TP53 MUT (n = 142) vs. all BC subtypes WT (n = 658) samples with 2282 significantly upregulated and 1389 significantly downregulated genes. ( B ) (i) Differentially expressed genes from the CCLE BC and TCGA BC analyses were cross-analysed using a Venn diagram to identify overlapping targets between datasets. (ii) Calcium channel genes were searched for in the overlapping targets, with only CACNA1D identified as significantly differentially expressed, with details displayed in a table with relevant lfc, p -value, and p-adj (BH-adjusted). ( C ) Bar graph displaying relative fold change of calcium channel gene expression in TP53 mutant TNBC cell lines HDQ-P1 (red) and MDA-MB-157 (blue) with respect to (w.r.t.) to TP53 wild-type TNBC cell line CAL-51 using qRT-PCR. ( D ) shows the gene expression of CACNA1D in expanded panel of TNBC cell lines include missense mutants, MFM223 and MDA-MB-468 (n = 4). Kruskal-Wallis with Dunn’s multiple comparisons was used to determine significance. ( E ) Table outlining the cell lines used in this study, including their corresponding TP53 mutation status and type, as well as associated p53 protein expression levels retrieved from the CCLE.

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Gene Expression, Mutagenesis, Quantitative Proteomics, Microarray, RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers17101614

Figure Lengend Snippet: SOC and tumour biology is altered in TP53 mutant TNBC cell lines compared to WT. ( A ) Traces of SOC were measured in TNBC cell lines, either TP53 WT CAL-51 (black, n = 7) or mutant HDQ-P1 (red, n = 8), MDA-MB-157 (blue, n = 7), MFM-223 (Purple, n = 4) and MDA-MB-468 (Cyan, n = 4). ER Store release was induced by 4 μM TG and SOCE by the addition of 2 mM Ca 2+ . Trace analysis measured ( B ) basal calcium in 0 mM Ca 2+ , ( C ) TG peak and ( D ) SOCE peak between cell lines. ( E ) Presents the rate of proliferation read-out from the acid-phosphatase assay in TP53 mutant TNBC cell lines compared to the TP53 wild-type TNBC cell line (n16, n = 4). ( F ) Displays percentage growth inhibition after treatment with ER stressor thapsigargin (100nM, TG) for 5 days compared to DMSO control as measured by acid-phosphatase assay. For all above Kruskal-Wallis with Dunn’s multiple comparisons was used to determine the significance.

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis, Acid Phosphatase Assay, Inhibition, Control

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Impact of CACNA1D manipulation through COTI-2 treatment or siRNA on calcium channel expression and SOC in TP53 mutant TNBC cell lines. ( A ) Effect of COTI-2 treatment on relative fold change gene expression of calcium channels using qRT-PCR in HDQ-P1 cells (purple) and MDA-MB-157 cells (pink) compared to untreated control. ( B ) Individual graphs of CANCA1D relative fold change gene expression in COTI-2 treated TP53 mutant cells vs untreated. SOC measured by Fura-2AM ratiometeric in ( C ) HDQ-P1 or ( D ) MDA-MB-157 as displayed as a trace over time(s) (i) compared to DMSO control, with individual analysis displaying (ii) average baseline 0 mM calcium, (iii) max TG peak and (iv) SOCE. Mann-Whitney was used for all above comparing two groups. ( E ) SOC trace for CAL-51 following (Ei) CACNA1D siRNA compared to siNegative or untreated control with individual analysis displaying (ii) average baseline 0 mM calcium, (iii) max TG peak and (iv) SOCE. Kruskal-Wallis with Dunn’s multiple comparisons was used for analysis in ( E ).

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Expressing, Mutagenesis, Gene Expression, Quantitative RT-PCR, Control, MANN-WHITNEY

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Impact of COTI-2 and TG treatment on Apoptosis. TP53 wildtype CAL-51 and TP53 mutant MDA-MB-157 cells were analyzed for apoptosis following TG and COTI-2 treatment along with DMSO and untreated controls in duplicates. Using the caspase 3/7 dye, apoptosis was recorded every 4 h over a period of 5 days. ( A ) Shows a trace of apoptotic induction in CAL-51 and MDA-MB-157 in the presence and absence of COTI-2. ( B ). Responses to (An) at 120 h where graphed and analysed by Mann-Whitney. Apoptosis traces in CAL51 ( C ) and MDA-MB-157 ( D ) cells under control (black), DMSO (blue), thapsigargin (TG; green), COTI-2 (red), and combined TG+COTI-2 (purple) treatments are shown. Analysed by Two-way ANOVA Post hoc Tukey. Cell proliferation rates after treatment with TG (green), COTI-2 (red) and TG+COTI-2 (black) measured by acid phosphatase in ( E ) TP53 WT CAL-51 and ( F ) T53 mutant MDA-MB-157, with bar graph analysis at 3.125nm. Analysed by One-way ANOVA with post hoc.

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis, MANN-WHITNEY, Control

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Schematic representation of TP53 mutation-dependent regulation of Ca 2+ homeostasis and apoptosis via CACNA1D /Cav1.3 modulation in TNBC. Left (TP53 WT): In wild-type TP53 cells, cellular stress (e.g., chemotherapy, hypoxia) activates p53, which upregulates CACNA1D (Ca V 1.3), leading to increased basal Ca 2+ levels. Functional SERCA pumps and IP 3 Rs ensure proper ER Ca 2+ handling and mitochondrial Ca 2+ transfer, resulting in mitochondrial Ca 2+ overload and apoptosis. Middle (TP53 mutant): In TP53 mutant cells (frameshift/stop), impaired p53 function leads to downregulation of Cav1.3 and SERCA activity, reducing basal and ER Ca 2+ levels and limiting mitochondrial Ca 2+ uptake. This dampens Ca 2+ -dependent apoptotic signaling, promoting apoptotic resistance. Right (TP53 mutant + COTI-2): Treatment with the p53 reactivator COTI-2 restores p53 activity in TP53 mutant cells, increasing Cav1.3 expression and basal Ca 2+ levels. Enhanced Ca 2+ transfer to mitochondria reinstates mitochondrial Ca 2+ overload and apoptotic signaling, reversing apoptosis resistance.

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis, Functional Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: APR-246 & COTI-2 increase chemoradiotherapy sensitivity via ROS-induced DNA damage and ferroptosis in p53 mutant HPV negative head and neck squamous cell carcinoma

doi: 10.1101/2024.12.23.630069

Figure Lengend Snippet:

Article Snippet: APR-246, Buthionine sulfoximine (BSO), cisplatin, COTI-2 and erastin were purchased at Bioconnect, Netherlands.

Techniques: Standard Deviation