S. Sommerville

E2F7 Can Regulate Proliferation, Differentiation, and Apoptotic Responses in Human Keratinocytes: Implications for Cutaneous Squamous Cell Carcinoma Formation

The E2F family of transcription factors plays a crucial role in the regulation of genes involved in cell proliferation, differentiation, and apoptosis. In keratinocytes, the inhibition of E2F is a key step in the control and initiation of squamous differentiation. Because the product of the recently identified E2F7a/E2F7b gene has been shown to repress E2F-regulated promoters, and to be abundant in skin, we examined its role in the epidermis. Our results indicate that E2F7b mRNA expression is selectively associated with proliferation-competent keratinocytes. Moreover, E2F7 was able to antagonize E2F1-induced proliferation and apoptosis. In contrast, although E2F7 was able to inhibit proliferation and initiate differentiation, it was unable to antagonize the differentiation suppression induced by E2F1. These data indicate that E2F7-mediated suppression of proliferation and apoptosis acts through E2F1-dependent pathways, whereas E2F7-induced differentiation acts through an E2F1-independent pathway. These data also suggest that proliferation, differentiation, and survival of primary human keratinocytes can be controlled by the relative ratio of E2F1 to E2F7. Because deregulated proliferation, differentiation, and apoptosis are hallmarks of cancer, we examined the expression levels of E2F1 and E2F7 in cutaneous squamous cell carcinomas (CSCC). We found that both genes were overexpressed in CSCCs compared with normal epidermis. Furthermore, inhibition of E2F7 in a SCC cell line sensitized the cells to UV-induced apoptosis and doxorubicin-induced apoptosis. Combined, these data suggest that the selected disruption of E2F1 and E2F7 in keratinocytes is likely to contribute to CSCC formation and may prove to be a viable therapeutic target.

Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

Osteosarcoma is characterised by reduced expression of markers of osteoclastogenesis and antigen presentation compared with normal bone.

Background:Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Patients who respond poorly to chemotherapy have a higher risk of metastatic disease and 5-year survival rates of only 10–20%. Therefore, identifying molecular targets that are specific for OS, or more specifically, metastatic OS, will be critical to the development of new treatment strategies to improve patient outcomes.Methods:We performed a transcriptomic analysis of chemo-naive OS biopsies and non-malignant bone biopsies to identify differentially expressed genes specific to OS, which could provide insight into OS biology and chemoresistance.Results:Statistical analysis of the OS transcriptomes found differential expression of several metallothionein family members, as well as deregulation of genes involved in antigen presentation. Tumours also exhibited significantly increased expression of ID1 and profound down-regulation of S100A8, highlighting their potential as therapeutic targets for OS. Finally, we found a significant correlation between OS and impaired osteoclastogenesis and antigen-presenting activity. The reduced osteoclastogenesis and antigen-presenting activity were more profound in the chemoresistant OS samples.Conclusion:Our results indicate that OS displays gene signatures consistent with decreased antigen-presenting activity, enhanced chemoresistance, and impaired osteoclastogenesis. Moreover, these alterations are more pronounced in chemoresistant OS tumour samples.

Abstract LB-298: The bone marrow microenvironment increases osteosarcoma tumour cell migration by signaling through uPA/uPAR

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and can occur in up to 50% of cases, resulting in just 10–20% long-term survival for these patients. We have shown that the bone microenvironment contributes to the metastatic potential of osteosarcoma (Endo-Munoz et al. Cancer Res 70:7063–72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS in vitro and in vivo , and bone marrow cells (BMC) increase the migration of OS cells in vitro. However, the factors mediating this increase in migration has not been elucidated. Using a combined transcriptomic and proteomic (SILAC) approach, we found very high levels of mRNA and protein expression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) in metastatic, but not in non-metastatic OS. Furthermore, examination of the cytokines secreted in the conditioned medium of metastatic and non-metastatic OS cells in an antibody array showed high and selective secretion of uPA by metastatic OS cells only. Addition of this conditioned medium to metastatic OS cells significantly ( P in vitro migration to levels similar to those observed with recombinant human uPA. No effect was observed for non-metastatic cells. Overall migration was significantly ( P uPA to be the most significantly upregulated gene after BMC treatment (FC = 7.97, B = 18.82), suggesting that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. Finally, we explored the possibility that BMC may also secrete uPA into the microenvironment to further increase migration of OS tumours in close proximity. Immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. These data indicate that BMC increases OS cell migration not only by increasing uPA gene expression in the tumour, but also by secreting uPA which serves to amplify the migration-inducing signaling of tumour-secreted uPA. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2011-LB-298