Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Gamma subunit CACNG4 of L-type VGCCs is variably expressed in breast cancer cells (a) VGCCs and their subunits; gamma subunits are unique to L-type VGCCs (b) Protein expression of CACNG4 relative to beta-actin in 8 breast cancer cell lines of various invasive potentials, and a non-malignant breast epithelial cell line (MCF10A), single band is observed at 36kDa (c) Immunofluorescence staining of rat cardiomyocytes and breast cancer cell lines MCF7 and MDA-MB-231 show cytoplasmic/perinuclear and some membrane localization of CACNG4 protein (d) Immunohistochemical staining of the breast cancer cell line MDA-MB-468 (wildtype versus overexpressing CACNG4) show cytoplasmic/perinuclear and membrane localization (e) IHC staining of primary breast tumours shows a range of low, intermediate and high expression of CACNG4 protein in the cytoplasm and membrane of tumour cells.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Immunohistochemical staining, Immunohistochemistry

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Down regulation of CACNG4 via shRNA or siRNA results in decreased proliferation, motility and cell adhesion of breast cancer cells in vitro (a) A decrease in cell proliferation over 5 days in shCACNG4 MCF7 breast cancer cells versus scrambled control (b), (c) A decrease in cell migration through Boyden chamber inserts in shCACNG4 MCF7 and siCACNG4 MDA-MB-231 breast cancer cells versus scrambled controls (d) A decrease in cell invasion through Matrigel™ coated Boyden chamber inserts in shCACNG4 MCF7 breast cancer cells versus scrambled control.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: shRNA, In Vitro, Control, Migration

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Cell–cell interactions are altered upon regulation of CACNG4 in breast cancer cells. An increase in MDA-MB-231 cell-cell aggregation in the siRNA1 clone, only (down regulation of CACNG4 was observed in siRNA1 but not siRNA 2 or 3), as measured by aggregate size (number of positive pixels in area selected).

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques:

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: 3D culture of MCF10A non-malignant breast epithelial cells shows tumourigenic morphogenesis upon over-expression of CACNG4 (a) A significant increase in size was observed in cells over-expressing CACNG4 compared to controls at day 10 and 15 (increase in size of acini were measured as number of pixels in selected area, 4x magnification) (b) Multiacinar structures which are a feature of increased proliferation and loss of polarity in MCF10A cells over-expressing CACNG4 (day 10, 10× magnification) (c) Immunofluorescence staining of MCF10A acini showing a cross-section with filled lumens and disorganisation of cells (loss of E-cadherin: red) in MCF10A cells over-expressing CACNG4 (bottom panel) compared to controls (top panel) between day 10–16 (d) IHC staining for H/E and cleaved Caspase-3 marker for apoptosis in a section of MCF10A acini grown in 3D culture shows mostly non-apoptotic cells filling the lumen of acini between day 10–16.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Immunohistochemistry, Marker

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Over-expression of CACNG4 leads to increased metastasis to lungs in vivo (a) Schematic of chick embryo CAM model for metastasis. Micrometastases that form in the chick embryo lungs, liver or lower CAM are analysed for changes in metastatic potential of primary tumours produced from human tumour cells (b) There was no significant change in primary tumour size between CACNG4 over-expressing cells and controls (weighed on day 20) (c) Average number of cells that metastasise to the chick embryo lungs is higher in the metastasis assays that over-express CACNG4 in primary tumours compared to controls. Data are represented as mean ± SEM; Control labeled 513B1-A (d) Non-metastatic PyMT RhoC-/- cells were transduced with CACNG4 over-expression vectors (cacng4 oex) compared to empty vector controls (EV). Kaplan Meier data illustrates, 4 of 4 mice were positive for lung metastases, and 3 of 4 mice died on day 45 post injection compared to controls.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Over Expression, In Vivo, Produced, Expressing, Control, Labeling, Transduction, Plasmid Preparation, Injection

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: CACNG4 modulates calcium influx and VGCC antagonist binding kinetics (a) and (b) L-type calcium channel antagonists verapamil and amlodipine inhibit MCF7 and MDA-MB-231 breast cancer cell proliferation in a dosage dependent manner at 48hrs. Data are represented as mean of triplicate values (c) shRNA knockdown of CACNG4 results in rescue of inhibition of proliferation by amlodipine (0,1,5 and 10μM) in MCF7 breast cancer cells (d) Knockdown of CACNG4 increases serum induced Ca2+ influx and also leads to resistance to influx inhibiting concentrations of amlodipine in MDA-MB-231 breast cancer cells. Data for c and d are represented as mean ± SEM for triplicate values. (e) Down regulation of CACNG4 in breast cancer cells affects EGFR and calcium signaling pathways; AKT2, pAKT, and HDAC3 are down regulated while p-PKCZ is up regulated as confirmed by protein expression levels in MCF7 cells validating the mRNA results (f) Serum induced calcium influx in MDA-MB-231 cells is reduced when cells are treated individually with EGFR specific inhibitor Tryphostin AG148 or L-type VGCC antagonist amlodipine. The level of calcium influx is most strongly reduced when cells are treated with both calcium influx antagonists combined.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Binding Assay, shRNA, Knockdown, Inhibition, Protein-Protein interactions, Expressing

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: CACNG4 expression in metastatic breast cancer. (a) Two spatially separate blocks of primary tumours A and B (B1 and B2) show heterogeneous staining of CACNG4. Matched lymph node metastases show highest expression of CACNG4 (b) distant metastases to liver and a local breast recurrence show absent to moderate expression of CACNG4, comparable to normal breast tissue that does not show expression. (low = +, moderate = ++, high = +++, absent = neg).

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Expressing, Staining

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Model for tumour growth and dissemination regulated by CACNG4. Under normal conditions of CACNG4 expression, VGCCs are regulated in an open/closed state, to maintain low intracellular calcium conditions for a state of homeostasis. In breast cancer, tumour cells with gains of chromosome region 17q24 (encompassing the CACNG4 gene), have an increased expression of CACNG4 which results in closed VGCCs and a preferential low intracellular calcium level for tumour cells which allows for survival and other aggressive functions leading to dissemination and metastasis. Targeting CACNG4 would keep VGCCs in an open state, therefore increasing intracellular calcium levels, leading to decreased tumour growth and metastasis as we have demonstrated. Alternatively, anti-CACNG4 is a combinatory option to overcome acquired drug resistance of parallel signaling pathways such as EGFR.

Article Snippet: For immunofluorescence and IHC staining, the CACNG4 antibody was purchased from Novus Biologicals and used at a dilution of 1:50 and 1:800, respectively.

Techniques: Expressing, Protein-Protein interactions

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Role of Epstein-Barr Virus and Human Papillomavirus Coinfection in Cervical Intraepithelial Neoplasia in Chinese Women Living With HIV

doi: 10.3389/fcimb.2021.703259

Figure Lengend Snippet: Sequences of RT-PCR primers used for validation.

Article Snippet: Briefly, sections were blocked for 1 h, incubated overnight at 4°C with a rabbit primary antibody against human CACNG4 (Immunoway, YM3404), and subsequently incubated with a secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Abcam, ab175696) for 1 h. Slides were mounted with mounting medium containing DAPI and imaged by a Leica SP8 laser scanning confocal microscope system.

Techniques: Biomarker Discovery

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Role of Epstein-Barr Virus and Human Papillomavirus Coinfection in Cervical Intraepithelial Neoplasia in Chinese Women Living With HIV

doi: 10.3389/fcimb.2021.703259

Figure Lengend Snippet: Selected mRNAs with significant changes and overexpression of CACNG4 in EBV-HPV-associated CIN lesions. Expression levels of the selected, significantly altered mRNAs of the mRNA-seq analysis (A) and RT-PCR validation (B) . Each value was normalized to the GAPDH value and is presented as the mean ± SD, * p <0.05, compared with the HPV group. (C) A red fluorescence signal indicating the presence of CACNG4 was detected in cervical tissues. (D) Protein expression of CACNG4 relative to β-actin expression in cervical tissues. (E) Densitometric analysis of the immunoblots. The intensity of each band was normalized to the β-actin intensity. Due to the limited amount of clinical material, the EBV-HPV sample consisted of 4 pooled biological replicates and the HPV sample consisted of 3 pooled biological replicates in (D, E) . (F) The expression of CACNG4 between cervical cancer samples and normal tissue samples taken from TCGA and the GTEx databases. TPM, transcripts per million.

Article Snippet: Briefly, sections were blocked for 1 h, incubated overnight at 4°C with a rabbit primary antibody against human CACNG4 (Immunoway, YM3404), and subsequently incubated with a secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Abcam, ab175696) for 1 h. Slides were mounted with mounting medium containing DAPI and imaged by a Leica SP8 laser scanning confocal microscope system.

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Fluorescence, Western Blot

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Gamma subunit CACNG4 of L-type VGCCs is variably expressed in breast cancer cells (a) VGCCs and their subunits; gamma subunits are unique to L-type VGCCs (b) Protein expression of CACNG4 relative to beta-actin in 8 breast cancer cell lines of various invasive potentials, and a non-malignant breast epithelial cell line (MCF10A), single band is observed at 36kDa (c) Immunofluorescence staining of rat cardiomyocytes and breast cancer cell lines MCF7 and MDA-MB-231 show cytoplasmic/perinuclear and some membrane localization of CACNG4 protein (d) Immunohistochemical staining of the breast cancer cell line MDA-MB-468 (wildtype versus overexpressing CACNG4) show cytoplasmic/perinuclear and membrane localization (e) IHC staining of primary breast tumours shows a range of low, intermediate and high expression of CACNG4 protein in the cytoplasm and membrane of tumour cells.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Immunohistochemical staining, Immunohistochemistry

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Down regulation of CACNG4 via shRNA or siRNA results in decreased proliferation, motility and cell adhesion of breast cancer cells in vitro (a) A decrease in cell proliferation over 5 days in shCACNG4 MCF7 breast cancer cells versus scrambled control (b), (c) A decrease in cell migration through Boyden chamber inserts in shCACNG4 MCF7 and siCACNG4 MDA-MB-231 breast cancer cells versus scrambled controls (d) A decrease in cell invasion through Matrigel™ coated Boyden chamber inserts in shCACNG4 MCF7 breast cancer cells versus scrambled control.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: shRNA, In Vitro, Control, Migration

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Cell–cell interactions are altered upon regulation of CACNG4 in breast cancer cells. An increase in MDA-MB-231 cell-cell aggregation in the siRNA1 clone, only (down regulation of CACNG4 was observed in siRNA1 but not siRNA 2 or 3), as measured by aggregate size (number of positive pixels in area selected).

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques:

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: 3D culture of MCF10A non-malignant breast epithelial cells shows tumourigenic morphogenesis upon over-expression of CACNG4 (a) A significant increase in size was observed in cells over-expressing CACNG4 compared to controls at day 10 and 15 (increase in size of acini were measured as number of pixels in selected area, 4x magnification) (b) Multiacinar structures which are a feature of increased proliferation and loss of polarity in MCF10A cells over-expressing CACNG4 (day 10, 10× magnification) (c) Immunofluorescence staining of MCF10A acini showing a cross-section with filled lumens and disorganisation of cells (loss of E-cadherin: red) in MCF10A cells over-expressing CACNG4 (bottom panel) compared to controls (top panel) between day 10–16 (d) IHC staining for H/E and cleaved Caspase-3 marker for apoptosis in a section of MCF10A acini grown in 3D culture shows mostly non-apoptotic cells filling the lumen of acini between day 10–16.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Immunohistochemistry, Marker

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Over-expression of CACNG4 leads to increased metastasis to lungs in vivo (a) Schematic of chick embryo CAM model for metastasis. Micrometastases that form in the chick embryo lungs, liver or lower CAM are analysed for changes in metastatic potential of primary tumours produced from human tumour cells (b) There was no significant change in primary tumour size between CACNG4 over-expressing cells and controls (weighed on day 20) (c) Average number of cells that metastasise to the chick embryo lungs is higher in the metastasis assays that over-express CACNG4 in primary tumours compared to controls. Data are represented as mean ± SEM; Control labeled 513B1-A (d) Non-metastatic PyMT RhoC-/- cells were transduced with CACNG4 over-expression vectors (cacng4 oex) compared to empty vector controls (EV). Kaplan Meier data illustrates, 4 of 4 mice were positive for lung metastases, and 3 of 4 mice died on day 45 post injection compared to controls.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Over Expression, In Vivo, Produced, Expressing, Control, Labeling, Transduction, Plasmid Preparation, Injection

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: CACNG4 modulates calcium influx and VGCC antagonist binding kinetics (a) and (b) L-type calcium channel antagonists verapamil and amlodipine inhibit MCF7 and MDA-MB-231 breast cancer cell proliferation in a dosage dependent manner at 48hrs. Data are represented as mean of triplicate values (c) shRNA knockdown of CACNG4 results in rescue of inhibition of proliferation by amlodipine (0,1,5 and 10μM) in MCF7 breast cancer cells (d) Knockdown of CACNG4 increases serum induced Ca2+ influx and also leads to resistance to influx inhibiting concentrations of amlodipine in MDA-MB-231 breast cancer cells. Data for c and d are represented as mean ± SEM for triplicate values. (e) Down regulation of CACNG4 in breast cancer cells affects EGFR and calcium signaling pathways; AKT2, pAKT, and HDAC3 are down regulated while p-PKCZ is up regulated as confirmed by protein expression levels in MCF7 cells validating the mRNA results (f) Serum induced calcium influx in MDA-MB-231 cells is reduced when cells are treated individually with EGFR specific inhibitor Tryphostin AG148 or L-type VGCC antagonist amlodipine. The level of calcium influx is most strongly reduced when cells are treated with both calcium influx antagonists combined.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Binding Assay, shRNA, Knockdown, Inhibition, Protein-Protein interactions, Expressing

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: CACNG4 expression in metastatic breast cancer. (a) Two spatially separate blocks of primary tumours A and B (B1 and B2) show heterogeneous staining of CACNG4. Matched lymph node metastases show highest expression of CACNG4 (b) distant metastases to liver and a local breast recurrence show absent to moderate expression of CACNG4, comparable to normal breast tissue that does not show expression. (low = +, moderate = ++, high = +++, absent = neg).

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Expressing, Staining

Journal: EBioMedicine

Article Title: Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis

doi: 10.1016/j.ebiom.2020.102646

Figure Lengend Snippet: Model for tumour growth and dissemination regulated by CACNG4. Under normal conditions of CACNG4 expression, VGCCs are regulated in an open/closed state, to maintain low intracellular calcium conditions for a state of homeostasis. In breast cancer, tumour cells with gains of chromosome region 17q24 (encompassing the CACNG4 gene), have an increased expression of CACNG4 which results in closed VGCCs and a preferential low intracellular calcium level for tumour cells which allows for survival and other aggressive functions leading to dissemination and metastasis. Targeting CACNG4 would keep VGCCs in an open state, therefore increasing intracellular calcium levels, leading to decreased tumour growth and metastasis as we have demonstrated. Alternatively, anti-CACNG4 is a combinatory option to overcome acquired drug resistance of parallel signaling pathways such as EGFR.

Article Snippet: Antibodies against CACNG4 were purchased from Aviva Systems Biology and used at a dilution of 1:5000 for western blotting.

Techniques: Expressing, Protein-Protein interactions