Changes in <i>ERα</i> and <i>ERβ</i> expression in skin carcinogenesis <i>in vitro</i> and <i>in vivo.</i>

<p>(A) Immunohistochemical expression of ERα is enhanced in serial section of a spindle cell carcinoma in comparison with skin papilloma derived from chemical induction of carcinogenesis in athymic mice. (B) Immunohistochemical staining of ERβ is faint in spindle cell carcinomas in comparison with skin papillomas. Nuclear staining of the positive control ERα-HEK293 cells and cytoplasmic staining of the ERβ-HEK293 cells, which are used as positive controls validate the efficiency of anti-ERα and anti-ERβ, correspondingly, in this experimental setup. <i>Symbols:</i> Block arrows indicate positively stained nuclei, while arrowheads indicate positively stained cytoplasm. The asterisks show macrophages. (C) Western blot with anti-ERα in total extracts of C5N, P1, B9, A5 and CarB cell lines reveals that ERα is enhanced during transition from the immortalized C5N cell lines to the most aggressive CarB cell lines. (D) Western blot with anti-ERβ reveals slight changes of ERβ during transition from the immortalized C5N cell lines to the most aggressive CarB cell lines. GAPDH protein expression was used as a loading control. (E) Quantification of ERα and ERβ levels in C5N, P1, B9, A5 and CarB cell lines (F) ERα/ERβ ratio is gradually increased during transition from the immortalized C5N through most aggressive CarB cell line.</p

<i>In vivo</i> effect of S554fs-ERα on the biologically aggressive behaviour of mouse spindle cells.

<p> (A) Injection of C-ER3 and C-ER4 cells in BalB/c SCID mice resulted to a reduction in the number of the positive sites compared to the control CarB-V-injected mice, as well as to smaller tumours. Tumour sites are circled in black. (B) Tumorigenicity (positive sites/mouse) of control and dnERα transfected CarB cell lines. (C) Latency period (weeks) of tumours in dnERα-CarB injected mice as compared to parental and mock-transfected cells. Corresponding summary graphs (means±SEM from 7 to 8 mice) derived by comparison of mice injected with CarB-V against CarB, C-ER3, C-ER4 and C-ER7 using t-test. Results are flagged with no asterisk when P-value is more than 0.05, with a single asterisk when the P-value is less than 0.05, with two asterisks when the P-value is less than 0.01, and three asterisks when the P-value is less than 0.001.</p

S554fs-ERα suppresses oncogenic properties of CarB-transfected cells and alters cytoskeletal and cell adhesion molecules’ levels.

<p>(A) Phenotypic characteristics of dnERα-transfected clones (C-ER3, C-ER4) in comparison with untransfected and vector-transfected CarB cells. The dnERα-transfected cells appear flattened and acquire a more epithelial morphology, whereas untransfected and vector-transfected cells present a spindle morphology. (B) Growth rate assay reveals reduction in the growth rate of C-ER3 and C-ER4 cells in comparison with control vector-transfected CarB cells. (C) Cell growing of vector-transfected CarB, C-ER3 and C-ER4 cells is expressed in OD values at 595 nm, in 24 h, 48 h and 72 h. All the results are plotted as mean ± standard deviations from three independent experiments. (D) Representative illustrations of anchorage-independent growth of CarB-V, C-ER3 and C-ER4 cells. (E) Western blot analysis for determination of levels of actin, vinculin and integrin α1 proteins in parental, CarB-V cells and S554fs-transfected CarB cells. Actin and vinculin protein expression increased, whereas integrin α1 expression decreased in all three transfected clones compared to the Car-B and CarB-V cells. GAPDH protein expression was used as a loading control. (F) Summary graph (means±SEM from triplicates) for soft agar cloning efficiency of parental control, vector-transfected and dnERα transfected spindle cell lines. Colonies were scored after 3 to 4 weeks. CarB-V is compared against CarB, C-ER3, C-ER4 and C-ER7 using t-test, and results are flagged with no asterisk when P-value is more than 0.05, with a single asterisk when the P-value is less than 0.05, with two asterisks when the P-value is less than 0.01, and three asterisks when the P-value is less than 0.001.</p

Additional file 3: of ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

List of genes whose expression was changed 24 h after induction of ∆Np63α or TAp63α expression in MDA-MB-468 cells. (PDF 503 kb

Additional file 3: of ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

List of genes whose expression was changed 24 h after induction of ∆Np63α or TAp63α expression in MDA-MB-468 cells. (PDF 503 kb