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Supplementary Figure 3 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 7 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 1 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Materials and Figure Legends 1-5 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure Legends 1-8 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Methods from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 5 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 8 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 6 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 2 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Supplementary Figure 3 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure 4 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Supplementary Figure 5 from An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic HrasG12V (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer. [Cancer Res 2008;68(14):5699–705]

The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, ΔNp63α, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that ΔNp63α functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing ΔNp63α. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of ΔNp63α decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that ΔNp63α maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of ΔNp63α together with p53 inactivation in SCCs. Cancer Res; 70(10); 4034–44. ©2010 AACR.

Supplementary Figure 1 from ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic HrasG12V (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer. [Cancer Res 2008;68(14):5699–705]