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

Evaluation of claspin as a proliferation merker in human cancer and normal tissues

Claspin is a nuclear protein involved in DNA replication and DNA damage response. It may represent a potentially useful proliferation marker. A novel monoclonal antibody against human Claspin was created and its expression was investigated in normal and neoplastic cell populations both in vivo and in vitro. Material-Methods: Tumour and adjacent normal tissues were obtained from patients with non-small-cell-lung carcinoma (49), colonic adenocarcinoma (18) ,transitional cell carcinoma of the bladder (31) and ductal breast carcinomas (52). The samples were analysed by immunohistochemistry and immunofluorescence. Ki67 expression was also examined in these tissues. Additionally, Claspin and cyclin A expression was assessed by immunoblotting in normal lung fibroblasts (DLFs) and various cancer cell lines (Saos2, A549, HT-29 and HCT116). Finally, functional assays were performed in synchronized DLFs and in a tetracycline-inducible Saos2-p21 variant Results-Conclusions: Immunoblot analysis confirmed the specificity of the generated antibody. In addition, immunohistochemistry was shown to be effective in archival material demonstrating a clear nuclear signal. In normal cells Claspin expression was weak, whereas increased levels were observed in cancer. Claspin labeling index was higher in tumors (p<0.001). Claspin expression strongly correlated with Ki67 expression in normal (p<0.001) and tumor tissues (p<0.001). Claspin LI was consistently lower than Ki67 LI in both normal (p=0.001) and tumor tissues (p<0.001), suggesting that its expression during the cell cycle may be limited to a narrower time-frame. Indeed, co-localization assays with cyclin A and cell synchronization experiments indicated that Claspin expression coincides with the early period of S-phase. Finally, the relative increase of the Claspin LI in the tumor samples compared to the normal ones was significantly higher (14-fold) than that of Ki67 (5-fold) implying a role of the former in the response to replication stress developed during carcinogenesis. These findings suggest that Claspin expression, as assessed by the use of a newly created monoclonal antibody, may serve as a potentially useful S phase-specific marker, as well as a marker of replication stress in both neoplastic and normal tissues.Η κλασπίνη είναι μία πυρηνική πρωτεΐνη που συμμετέχει στην φυσιολογική αντιγραφή του DNA. Συμβάλλει ακόμη στην κυτταρική ανταπόκριση σε βλάβες του DNA. Ενδέχεται να αποτελεί ένα χρήσιμο και ειδικό δείκτη κυτταρικού πολλαπλασιασμού. Στη παρούσα μελέτη συνετέθη ένα νέο μονοκλωνικό αντίσωμα έναντι της κλασπίνης και διερευνήθηκε για πρώτη φορά η έκφρασή της σε πολλαπλασιαζόμενα φυσιολογικά και καρκινικά κύτταρα. Υλικό-Μέθοδοι: Εφαρμόστηκε ανοσοϊστοχημεία και έμμεσο ανοσοφθορισμό για την ανοσοεντόπιση της κλασπίνης σε τομές παραφίνης από 49 δείγματα μη-μικροκυτταρικού καρκίνου του πνεύμονα, 18 αδενοκαρκινώματα παχέος εντέρου, 31 καρκινώματα μεταβατικού επιθηλίου της ουροδόχου κύστεως, 52 πορώδη καρκινώματα του μαστού και στα αντίστοιχα φυσιολογικά τους. Στα ίδια δείγματα εκτιμήθηκε και ο κυτταρικός πολλαπλασιασμός με ανοσοϊστοχημεία έναντι του Ki67. Επιπλέον, χρησιμοποιήθηκαν πρωτογενείς φυσιολογικοί πνευμονικοί ινοβλάστες (DLFs) και ανθρώπινες καρκινικές κυτταρικές σειρές (HCT-116, HT-29, A549 και Saos-2), για τη μελέτη της έκφρασης της κλασπίνης και της κυκλίνης Α με μεθόδους ανοσοεντόπισης. Τέλος, πραγματοποιήθηκε λειτουργική ανάλυση σε συγχρονισμένους DLFs και το επαγώγιμο κυτταρικό σύστημα Saos2-p21 Tet/ON. Αποτελέσματα-Συμπεράσματα: Η εφαρμογή στυπώματος Western επιβεβαίωσε την ειδικότητα του αντισώματος έναντι της κλασπίνης, με την ανίχνευση μιας ξεκάθαρης ζώνης στο ύψος των 250 kDa. Η ανοσοϊστοχημεία στις τομές παραφίνης ήταν επιτυχής, και παρατηρήθηκε μια σαφής, αμιγώς πυρηνική ανοσοχρώση. Η έκφραση της κλασπίνης στους φυσιολογικούς ιστούς ήταν ασθενής, ενώ στους όγκους ήταν σημαντικά υψηλότερη (p<0.001). Η σχετική αύξηση του δείκτη σήμανσης στους όγκους ήταν σημαντικά εντονότερη για την κλασπίνη (14 φορές) απ’ότι για το Ki67 (5 φορές), παρατήρηση που είναι πιθανό να οφείλεται στην απάντηση της κλασπίνης έναντι του αντιγραφικού στρες που διέπει τα καρκινικά κυττάρα. Το ποσοστό των σεσημασμένων για κλασπίνη κυττάρων βρέθηκε να έχει ισχυρή, γραμμική συσχέτιση με το ποσοστό των σεσημασμένων για Ki67 κυττάρων τόσο στους φυσιολογικούς (p<0.001), όσο και στους καρκινικούς ιστούς (p<0.001). Συνολικά, το ποσοστό σήμανσης της κλασπίνης ήταν σταθερά χαμηλότερο από το ποσοστό σήμανσης του Ki67 τόσο στους φυσιολογικούς (p=0.001), όσο και στους καρκινικούς ιστούς (p<0.001), υποδεικνύοντας έτσι την περισσότερο ειδική έκφρασή της σε μέρος του κυτταρικού κύκλου. Πράγματι, η έκφραση της κλασπίνης σε πειράματα κυτταρικού συγχρονισμού συμβαδίζει με τα πρώιμα στάδια της φάσης S του κυτταρικού κύκλου.Τα αποτελέσματά μας αναδεικνύουν την κλασπίνη σε χρήσιμο δείκτη πολλαπλασιασμού με χαρακτηριστική ειδικότητα για τη φάση S στους φυσιολογικούς και στους νεοπλασματικούς ιστούς

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

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

Peptide–drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανβ3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases

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

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