Progression of mouse skin carcinogenesis is associated with increased ERα levels and is repressed by a dominant negative form of ERα.

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα

Progression of mouse skin carcinogenesis is associated with increased ERα levels and is repressed by a dominant negative form of ERα.

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα

Progression of Mouse Skin Carcinogenesis Is Associated with Increased Er alpha Levels and Is Repressed by a Dominant Negative Form of Er alpha

Estrogen receptors (ER), namely ER alpha and ER beta, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ER alpha in skin cancer, using chemicallyinduced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ER alpha is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ER beta levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ER alpha gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ER beta levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ER alpha (dnER alpha) resulted in reduced ER alpha levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ER alpha promoter through which dnER alpha decreased endogenous ER alpha levels. The dnER alpha-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ER alpha in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnER alpha

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